1��SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

We carried out a series of incubations with sediment from three different rivers and synthetic river water . Tests were carried out at an initial concentration of 100-200 µg L-1 for approx . 30 days; samples were filtered and subsequently analyzed by UPLC-qTOF-MS . The high-resolution mass spectrometric data were processed with a workflow combining the tools mzMine (http://mzmine .sourceforge .net/) and enviMass (http://www .eawag .ch/forschung/uchem/software/enviMass1) . By comparing incubations containing pharmaceuticals to control samples without pharmaceuticals, this yields a list of peaks identified by exact mass and retention time for each time step of the incubation experiment . Using these time series data, peaks showing a temporal trend expected for transformation products (e .g ., not present at the beginning of the incubation, increasing concentrations with time) can be located and processed further . Through application of this time series approach, the number of peaks to be processed further is substantially reduced . The first results obtained by this approach are promising and provide a solid basis for the evaluation of the workflow, but also for scrutinizing its precision and robustness in detecting unknown peaks at low concentrations . This is necessary to increase confidence in the applied analytical and in silico methods for the specific aims of our study . We will present results of this evaluation, and we will discuss the tentatively identified transformation products with respect to their formation/elimination kinetics and their applicability as indicators for in situ micropollutant transformation .

MO 203Evaluation of the contamination of aquatic micro-organisms by micro-QuEChERS-nano-LC-MS/MSM .T . Tournier1, J . Camilleri2, R .B . Baudot1, L .W . Wiest1, A .B . Buleté1, E .V . Emmanuelle1, J .G . Garric3, C .C . Cren-Olivé1

1CNRS UMR 5280 ISA, Villeurbanne, France2SCA, Solaize, France3CEMAGREF, Lyon, FranceIt is currently accepted that the main route for pharmaceuticals and Endocrine Disruptors Chemicals (EDCs) to the aquatic environment is via sewage treatment plants receiving wastewater from households, industries and hospitals . In a concern to follow their fates and their impacts in the environment, we developed analytical methods to quantify endogen and synthetic hormones, pharmaceuticals and chemicals in benthic species . We established a molecule list in accordance with prioritisation lists [1] and identification of contamination sources around the wastewater treatment plant (industries, hospital, villages): estrone, 17α-ethinyl-estradiol, lorazepam, oxazepam, acetaminophen, carbamazepine, ibuprofen, 4-methylbenzylidene camphor, fluoroquinolones and sulfonamides for example . We decided to evaluate the contamination of three different species which are well-known bio indicators of water quality . More precisely, invertebrates like gastropods (Potamopyrgus antipodarum), amphipods (Gammarus) and chironomidae larvae (bloodworms) are exposed to effluents from treatment plants . . In the interests of handling a few milligrams of biological matrices and of being accurate, we chose an adequate analytical technique: the innovative nano-LC coupled with tandem mass spectrometer . After cryo-grinding, we extracted samples with a salting-out assisted LLE followed by a purifying dSPE . The extract was evaporated then reconstituted in a proper mobile phase . Only a 1-µl injection is done on the pre-concentration cartridge and the capillary column . Thus, we can reach limits of detection of a few injected femtograms . In conclusion, the comprehensive method was applied to the study of the fate and bio-accumulation of preoccupying micro-pollutants in aquatic micro-organisms .

MO 204Occurrence and seasonal variations of preservatives and UV filters used in cosmetic products in Japanese riversK .K . Kimura1, K .Y . Kameda2, W .S . Watanabe3, M .S . Masunaga1

1Yokohama National University, Yokohama, Japan2Center for Environmental Science in Saitama, Kazo, Japan3Saitama City Institute of Health Science and Research, Saitama, JapanPreservatives and UV filters are widely used in many cosmetics . These compounds may enter the aquatic environment from showering, wash-off, and so forth via wastewater treatment plants or sewage treatment tank . Most of these preservatives are included in order to suppress microorganisms growing . UV filters are used to prevent skin damage from UV radiation, however some of them are hormonally active to aquatic organisms . Their ecological adverse effect on aquatic organisms should be concerned after discharge . But there is little published data of their occurrence in aquatic environment . In this study, we determined twelve preservatives (2-Phenoxyethanol, Resorcinol, Chroloxylenol, 4-Isopropyl-3-methylphenol, Chlorphenesin, Triclosan, Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben) and four benzophenone-type UV filters (BP-1, BP-2, BP-3, BP-6) using solid phase extraction and GC/MS . Surface water samples were collected monthly at six sites in four Japanese rivers from December 2010 . 2-Phenoxyethanol, Resorcinol, 4-Isopropyl-3-methylphenol and Triclosan were found in most of the samples in all seasons . 2-Phenoxyethanol was detected at much higher concentration in winter . Isopropylparaben, Isobutylparaben, BP-2 and BP-6 were not detected at any sampling sites . The highest concentration in all samples was for 2-Phenoxyethanol at 1μg/L level . Further investigation is necessary to evaluate the ecological effect of high concentration preservatives including 2-Phenoxyethanol .

MO 205Solid phase extraction and LC-MS/MS analysis of pharmaceuticals in the Irish aquatic environmentG .L . Mceneff1, W . Schmidt2, B . Kelleher3, L . Barron4, B . Paull5, B . Quinn2

1Irish Separations Science Cluster/Irish Centre for Environmental Toxicology, Dublin, Ireland2Irish Centre for Environmental Toxicology, Galway-Mayo Institute of Technology, Galway, Ireland3School of Chemical Sciences, Dublin City University, Glasnevin, Dublin, Ireland4Analytical and Environmental Science Division, King’s College London, London, United Kingdom5Australian Centre for Research on Separation Science, University of Tasmania, Hobart, AustraliaPharmaceutical and personal care products (PPCP’s) are resisting water remediation techniques carried out currently in wastewater treatment plants and are thus being released into receiving waters . In previous studies PPCP presence has been detected in Irish effluent1 and soils2 in measurable quantities . In the work presented herein, 24 hour composite effluent samples were collected from two large Irish Wastewater Treatment Plants and analysed for a total of six pharmaceuticals from a range of therapeutic classes including non-steroidal anti-inflammatories, anti-biotics, lipid regulators and anti-epileptics . The complexity of these sample matrices requires a clean up and extraction step (Phenomenex Strata-X cartridges - 200mg, 6ml) before chemical analysis . Samples were eluted with 50:50 v/v ethyl acetate/acetone and reconstituted in 0 .5mls of starting mobile phase . Qualitative and quantitative analysis was carried out using reversed phase LC separation (Waters Sunfire C18 3 .5 µm 2 .1 x 150 mm column with a 45 minute mobile phase gradient of 20 % acetonitrile with 13 mM ammonium acetate, pH6 .2, to 80 % acetonitrile) with UV detection followed by MS/MS detection . Analytes were identified by comparing their retention times and precursor and product ions to those of a known standard . References: [1]Lacey, C ., McMahon, G ., Bones, J ., Barron, L ., Morrissey, A . and Tobin, J ., (2008) An LC-MS method for the determination of pharmaceutical compounds in wastewater treatment plant influent and effluent samples, Talanta, 75 (4), 1089-1097 [2] Barron, L ., Nesterenko, E ., Hart, K ., Power, E ., Quinn, B ., Kelleher B ., Paull, B ., (2010) Holistic visualisation of the multimodal transport and fate of twelve pharmaceuticals in biosolid enriched topsoils, Analytical & Bioanalytical Chemistry, 397 (1), 287

MO 206Use of LC-Orbitrap accurate mass spectrometry in risk assessment after a chemical incident.in the NetherlandsA .P . van Wezel, T . van Leerdam, L . PuijkerKWR Watercycle Research Institute, Nieuwegein, NederlandEarly 2011, a big incident took place in a chemical storage site in Moerdijk, the Netherlands . There was an extensive fire up to 500 meters high, with formation of soot . The column of smoke went in the northeastern direction within a large area . On the chemical storage site, many different inorganic, organic solid and liquid chemicals were stored . The fire extinguishing caused a vast volume of contaminated water . Several Dutch water utilities have surface-water related raw drinking water in large storage reservoirs in the area under the plume . After storage, the raw water is treated by different treatment processes on the drinking water production location . These reservoirs were possibly influenced by the Moerdijk incident . For precautionary reasons, the reservoirs relatively close to Moerdijk were directly taken out of production and intake of possibly contaminated surface water was stopped . The quality of the drinking water produced by the utilities in the period during and after the incident has never come into play . Despite these precautionary measures, it was relevant to know if the storage reservoirs were negatively influenced by this Moerdijk incident . Therefore, several drinking-water related surface waters were sampled and analyzed using broad-screening analytical-chemical GC-MS and LC-MS accurate mass screening . The high sensitivity, the high resolving power and accurate mass measurement make this a valuable technique for the screening of trace levels of organic contaminants . In contrast to most current environmental monitoring campaigns, this approach does not target a predetermined set of (known) chemicals . Also, the developed approach does not aim at the detection of all organic chemical compounds present in a sample as in general unknown screening approaches . Instead the data was screened for the presence of 249 known chemicals and 386 unidentified chemicals . The list of 386 unidentified compounds was the result of broad screening campaigns in our laboratory in several Dutch water samples for the last 5 years . In most of the storage reservoirs, no different compounds were found than normal . An exemption was a storage reservoir that was located 15 km from the incident .

MO 207Polybrominated diphenyl ethers, alternate brominated flame retardants and Dechloranes in sediments from German Bight and Laizhou Bay, ChinaS . Quade1, Z . Zhao1, J . Tang2, G . Zhong1, R . Sturm1, A . Möller1, Z . Xie1, G . Zhang3, R . Ebinghaus1

1Helmholtz-Zentrum Geesthacht, Geesthacht, Germany2Yantai Institute of Coastal Zone Research, Yantai, China3Guangzhou Institute of Geochemistry, Guangzhou, ChinaBrominated flame retardants (BFRs) have been commercially used since the 1970s . Since then more and more polymeric materials have been introduced for textiles, electronic equipment, upholstery, automobiles and building materials . Equally the production and usage of chemical additives for fire resistance of the mostly easy inflammable products have been increasing as well . Due to their physicochemical properties i .e . low volatility, low water solubility and high Kow-values the BFRs tend to adsorb on particles . Therefore they were detected preferably in sediment samples . Benthic organisms and other contaminated sediment consuming animals can be the first step for bioaccumulation in the marine food web .

1�6 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

In this study 73 surface sediment samples were collected in Germany and China to investigate the levels, distribution, possible sources and different profile patterns of flame retardants (FRs) in the two countries . 25 German surface sediment samples were taken in the German Wadden Sea, the rivers Elbe and Weser and the German Bight in 2011 . 48 Chinese samples were collected in Laizhou Bay and from discharging rivers in 2009 . The samples were Soxhlet extracted with dichloromethane and then purified by 10% water deactivated silica before measured by GC-ECNCI-MS . Quantification of 27 different FRs was obtained using 5 mass labelled internal standards ([13C]HBB, [13C]BDE-77, [13C]BDE-138, [13C]syn-DP, [13C]BDE-209) . The dominating compound in Laizhou Bay was Decabromodiphenyl ethane (DBDPE) and BDE-209 in German Bight, respectively . The alternate BFR DBDPE showed higher concentrations than BDE-209 in most Laizhou Bay samples and slightly lower concentrations in German Bight . Dechlorane Plus (DP) was detected in 71 out of 73 samples . DP concentrations were higher in German Bight except some Chinese river samples . Dibromopropyltribromophenyl ether (DPTE), Dechlorane (Dec) 602 and 603 were found in German samples only . Generally, the concentrations of FRs in German Bight and discharging rivers were quite comparable, while the Chinese samples showed big differences between marine and riverine sediment contamination .

MO 208Water quality in the Three-Gorges Reservoir (China) - First results from the Yangtze Project WATERUSEA . Wolf1, A . Bergmann1, T . Flöhr2, X . Gao3, H . Chen4, H . Hollert2, T . Schiedek5, C . Schüth5

1IWW Rhenish-Westfalian Institute for Water Research, Mülheim an der ruhr, Germany2Institute for Environmental Research, RWTH Aachen University, Aachen, Germany3Faculty of Urban Construction & Environmental Engineering, Chongqing University, Chongqing, China4Inst . of Water Environment Research, Chin . Res . Academy of Environmental Scs ., Peking, China5Department of Hydrogeology, University of Technology Darmstadt, Darmstadt, Germany To provide the public with high quality drinking water is a technological challenge, particu-larly in emerging countries . In the catchment area of the Three-Gorges Reservoir (TGR, China) productive and protected groundwater resources are very rare . Surface water is often used as a raw water source, but bank filtration as a first purification step is not feasible at TGR area . Thus, water works usually produce purified tab water directly from surface water, using standard filtration and disinfection techniques . The presented study focuses on the quality of surface water from Yangtze river and its tributaries in order to assess its suitability as drinking and irrigation water . Hydrogeology and water chemistry of the 630 km long TGR are strongly influenced by the geology (red sandstones and clay stones, limestones) and a subtropical climate (mean temperature 19°C, precipitation ca . 1 .138 mm/a) . Soil erosion is leading to a high particulate matter load in the relevant rivers . Anthropogenic input of pollutants originates mainly from sewage, industrial discharge, garbage dumping and from agricultural activities . Surface water, suspended solids and river bed sediments from TGR area were analysed for in-organic (anions, cations, heavy metals, contrast agents) using IC and ICP-MS and organic emerging pollutants (industrial chemicals, pesticides, pharmaceuticals, x-ray contrast media) . Additionally, acute toxicity and Ah receptor mediated toxicity are assessed using the Neutral red and the EROD assays with the cell line RTL W1 obtained from rainbow trout . First results of the inorganic river water analyses reflect the geogenic background . Concentrations of heavy metals and organic trace substances were usually well below international river water regulations . Particularly, pharmaceutical substances, which showed increased water concentrations in Germany in recent years, are almost absent in TGR water samples . Summer monsoons are controlling the highly varying river discharge . Thus, pollutant loads show a high seasonal variability with eco-toxicological impact . Further sampling campaigns will focus on varying hydrological, geographical, and anthropogenic settings (e .g . high/low river water level) and the anthropogenic impact on the TGR chemistry .

MO 209EDA of extracts from two different passive sampling devices in a polluted Norwegian riverM . Grung, C . Harman, I .J . Allan, K . Langford, S . Ranneklev, M . Muusse, K .V . ThomasNIVA, Oslo, NorwayThe Alna river running through Oslo has historically received pollution from a wide range of different industrial sources located nearby . Earlier investigations of the occurrence of contaminant in the river has shown high levels of PAHs, brominated flame retandants and PCBs in the water and sediments from the river . The river represents a significant contribution to the overall input of contaminants in the receiving Oslofjord . We wanted to investigate whether compounds other than those previously investigated contribute to the environmental problems in the river . We achieved this by using a combination of passive sampling devices exposed throughout the length of the river in combination with EDA (effects-directed analysis) and target chemical analyses to elaborate on the pollution status . Six different stations downstream the river were investigated using two different passsive samplers (silicone and LDPE - low density polyethylene) deployed for 6 weeks during summer 2011 . Extracts from the passive samplers were analysed for environmental effects by using DR-CALUX assay, and were also subjected to both target chemical analyses (PAH, PCB, PBDE) and screening for non-target compounds by GC/MS-HR ToF . The environmental effect of the extracts measured as DR-CALUX increased considerably (x100) downstream of the river source . The highest effects were measured in a tributary receiving run-off from a waste site upstream . GC/MS HR-ToF analyses revealed a difference in pattern of absorbed compounds downstream the river, and between the two different samplers . Initial results indicate that silicone seem to sample more of each compound, and a broader range of compounds, both in the relatively non-polluted upstream station, and the in the tributary . These results will be presented in more detail, along with a comparison of the pattern of target chemical analyses downstream the river and between the two samplers, and with sampling rate estimates from performance reference compounds used with both sampler types . The results will be discussed in light of the different properties of the two passive samplers . Examples of identified compound and their occurrence downstream the river will also be discussed .

MO 210Target and non-target screening of lake sediments using HR-MS/MSA .C . Chiaia-Hernandez1, H . Singer2, J . Hollender3

1Eawag/ETH, Dübendorf, Switzerland2Eawag - Swiss Federal Inst . of Aquatic Science and Technology, Dübendorf, Switzerland3Eawag, Dübendorf, SwitzerlandSediments are excellent archives of environmental contamination if the chemicals persist over time under mostly anaerobic conditions . After a suitable extraction of sediment samples, the new technology LC combined with high resolution mass spectrometry (HR-MS) enables the performance of target screening of compounds which are known to be emitted in the aquatic environment in large amounts, suspect screening of compounds which are expected to be emitted, and non-target screening that up to now are unknown or unexpected aquatic contaminants . Besides the complexity in the ionization and detection of organic compounds, there are additional challenges to be addressed such as the lack of reference standards available for confirmation of many potential contaminants or their transformation products . Results of target screening analysis show the detection of musk fragrances, personal care products (PCP), pesticides and biocides . The temporal resolution of selected organic pollutants in Lake Greifen were obtained with concentrations ranging from pg/gdw to ng/gdw . HR-MS/MS was also used to demonstrate the suitability of the method for the identification of compounds of interest which are expected to be present in the sediments based on consumption, discharge to the environment, and physical-chemical properties . Due to their high production volume and chemical characteristics, quaternary ammonium surfactants (QAs) might sorb strongly to soils and sediments and therefore deserve more attention . Several QA congeners were tentatively identified based on their retention times in accordance with their log Kow values and accurate masses . The identification of the QAs was confirmed by comparing the findings with purchased reference standards . Non-Target screening is being explored by subtracting chromatograms of extracts of different layers from a sediment core . The top 5 cm of the core, which represent the condition of the lake from the past 10 years, was subtracted with a “blank sample”which corresponds to a sediment core layer from 100 years ago . The finding shows that around 41% of the detected peaks are unknown . The most abundant non-targets in the sediment correlate with the total phosphorus concentration in the lake . Automated exact mass filtering and peak detection in combination with in silico fragmentation for computer assisted identification of mass spectra is being used to identified possible non-target contaminants in the lake sediment .

MO 211Characterization of surface water by multi-target screening of organic micropollutants combined with toxicological assessmentR . Bloch1, C . Hug2, M . Krauss2, T . Schulze2, P .C . Von der Ohe2, E . Fetter2, J .W . Einax1, W . Brack2

1Institute of Inorganic and Analytical Chemistry, Jena, Germany2UFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyIn the past decades, several environmental and toxicological studies focused on persistent organic pollutants in domestic and industrial waste water . Due to incomplete retention by waste water treatment plants (WWTPs), tons of chemicals are yearly discharged to surface water . Although occurring in low concentration, these micropollutants might harm aquatic organisms . Especially mutagenic and endocrine substances have the potential to damage ecosystems . The aim of this study was to link the complex chemical composition of surface water samples with their multifactorial effects on organisms . Therefore, surface waters downstream of WWTP outlets associated to different sources including big cities, health resorts and chemical industry were examined . Water extracts were analysed by liquid chromatography-high resolution mass spectrometry for almost 300 environmental relevant substances, such as pharmaceuticals, pesticides, industrial chemicals or ingredients of personal care products . For biological characterization, the samples were tested for mutagenic and estrogenic effects by an Ames-test II and a zebrafish egg test, respectively . Obtained data were evaluated by chemometric tools such as partial least-squares regression . Chemical fingerprints and toxicological endpoints were correlated by a multivariate regression model . Groups of chemicals causing similar effects on organisms, as well as possible marker substances for waste water sources will be presented .

MO 212Known unknowns: strategies for identification of toxicants in Effect Directed AnalysisE . Simon, J .M . Weiss, T . Hamers, P .E .G . Leonards, M .H . LamoreeInstitute for Environmental Studies (IVM) - VU Amsterdam, Amsterdam, NederlandTechnological innovations in the field of analytical chemistry have contributed significantly to the potential to find, identify and quantify unknown compounds present in the environment . Potentially, the application of accurate mass spectrometric techniques (e .g . time-of-flight, Orbitrap, FTMS) in environmental analysis can facilitate the

1��SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

identification of unknown toxicants . One of the research areas that would benefit enormously from improvements regarding the identification of compounds is the field of Effect Directed Analysis (EDA) . Our current work is dedicated to the development of a generic workflow aimed at the identification of compounds in extracts that are responsive in a toxicological/biological assay . In this presentation, we demonstrate our approach using a biotic sample that showed elevated thyroid hormone like activity . Using accurate mass LC-ToF-MS in the electrospray ionization mode, we were able to confirm the presence of a number of compounds that had been quantified in the samples using target analysis and that had very typical mass spectra: hydroxylated metabolites of PCBs . For compounds that have one or even a few halogens in their molecular structure, the typical isotopic patterns greatly enhance compound identification/confirmation . Using compound lists (e .g . list of pharmaceuticals, bioaccumulating compounds) target screening was performed on LC-ToF-MS data with Bruker Compass TargetAnalysis 1 .2 software . Identities were established based on accurate mass, mSigma value (rate for the agreement of the theoretical and measured isotopic pattern of the mass peak of interest), molecular formula and elemental composition . In order to otain significant progress regarding the identification of biologically active compounds in the framework of EDA, further development of mass spectral libraries are of major importance . Initiatives such as the NORMAN Mass Bank for sharing mass spectrometric data are expected to contribute to the advancement of identification of unknown compounds . In future work, more attention needs to be paid to the use of already available toxicological data, or software to derive theoretical compound characteristics .

MO 213Silicone-based agricultural adjuvants: trace analysis in aqueous environmentA . Michel1, E . Worch2, H .J . Brauch1, F .T . Lange1

1DVGW-Technologiezentrum Wasser, Karlsruhe, Germany2TU Dresden, Fakultät Forst-, Geo-, Hydrowissenschaft, Institut für Wasserchemie, Dresden, GermanyPesticides fate and ecotoxicology have been an issue for the last decades but agricultural adjuvants are much less studied yet . Among adjuvants, trisiloxane surfactants are increasing in popularity because of their high efficiency compared to traditional carbon-based surfactants . They are often referred as superspreaders or superwetters because of their ability to promote a rapid spreading on the hydrophobic surface of leaves . This effect makes the silicone-based surfactants toxic for terrestrial insects . Trisiloxane surfactants may reach surface or ground waters after their application in agriculture but no data are available on their environmental fate . A method for trace analysis of trisiloxane surfactants in aqueous environment was therefore developed and validated . It allows the quantification of two important trisiloxane surfactants in different aqueous matrices with a limit of quantification in the ng/L range . The strength of the method is to consider every homologue as a single molecule and to obtain information on the oligomeric distribution of the surfactant .

MO 214Tentative risk assessment of the UV filtersC .P . Perazzolo1, N .C . Chèvre2, E .S . Saouter1

1Science et Environnement, Geneva, Switzerland2University of Lausanne, Lausanne, SwitzerlandIn the last years, the impact of personal care products (PCP) and pharmaceutical on the environment has gained considerable attention . Still, little is known on specific classes such as the UV absorbing chemicals (UV filters) . UV filters are hydrophobic, bioaccumulative chemicals; few of them have been shown to have endocrine disrupting properties . UV filters are widely used and enter in the composition of sunscreens, cosmetics and in the protection of goods . Currently about 30 products are allowed by FDA and are present in the European cosmetic directive . UV filters can enter in the environment directly as the result of recreational activities and through the WWTP . Although their use is widely spread, the information on their fate and ecotoxicity is scare and sparse . After last REACH registration deadline, the amount of information has not increased as expected . About half of the allowed substances should have been registered in 2010 and the data of only 5 chemicals is available on ECHA website . To understand their potential impact in the environment, we performed a risk assessment; unfortunately due to their properties, it is highly probable that the classical risk assessment underestimates the risk . Whenever possible, the hazard was evaluated based on experimental data, with priority for information on REACH dossiers . We also used structure activity relationship methods to estimate missing ecotoxicity values . To estimate the exposure, priority was given to measured quantities . When no data were available, the REACH deadline was used as an estimation of the European tonnage . A reliability score was given to the risk characterisation ratio (RCR); RCR calculated using experimental data obtained higher reliability score . At the current usage, some the UV filters have and RCR above one thus being potentially dangerous for the environment . In many cases, a refinement of the data, both hazard and exposure, is necessary and will improve our understanding of the potential danger of these chemical . In a few cases either it was not possible to calculate the RCR due to the absence of data, or it cannot be considered as reliable due to the high number of estimated data .

MO 215Potential toxicity of sucraloseA .K .E . Wikklund, M . Breitholtz, K . Adolfsson-EriciStockholm University, Stockholm, SwedenSucralose is an intensively sweet food additive derived from sucrose in a process where three hydroxyl groups are substituted for three chlorine atoms, producing a very stable substance and has become a popular substitute for sugar . Its exceptional stability in combination with high water solubility and popularity has resulted in measurable concentrations not only in recipient waters but also further out in the oceans . Animal and human studies have concluded that sucralose is safe for human use and earlier studies in aquatic organisms indicate low bioaccumulation potential and negligible acute/chronic toxicity . The close structural resemblance with sucrose in combination with the importance of sugar in nature implies that other assessment techniques than traditional methods might be of interest . The aim of this investigation was therefore to combine traditional methods with behavioral studies in crustaceans and algae . Our results show that no effects were obtained by traditional toxicological tests, but both physiology and locomotive behaviour were affected by exposure to sucralose . In Daphnia magna, the behavioural response was manifested as altered swimming height and increased swimming speed . In gammarids, effects on the time to reach food and shelter as well as activity responses measured by a Multispecies Freshwater Biomonitor[TRADEMARK] were observed . It is unclear whether these behavioural responses were initiated via traditional toxic mechanisms or stimulatory effects, but we imply that they still should be considered as a warning, since divergence from normal behaviour, may ultimately have ecological consequences .

MO 216Antibiotic activity as a tool to identify and prioritize relevant point sourcesS .A .E . Kools1, E .B . Burger2, H . Kooman2, B .J . Pieters2

1Grontmij (Aquasense), Amsterdam, Nederland2Grontmij, Amsterdam, NederlandThe research effort on pharmaceuticals in the last two decades has raised concern that pharmaceuticals, including antibiotics may enter the environment still as active substances . The route of entry of pharmaceuticals to the environment is largely via sewage treatment works . More and more is known that these systems are not always capable of removing these compounds to full extent . Next, too little is known on the effects of the substances and last, less is known on the effects of the mixture of compounds . In our research, we have calculated both emission data on loads to identify these mixtures and used available chemical analytical methods to identify known substances . However, the capacity to measure these emerging compounds is yet limited . For that reason, we have adopted a new bioassay that can be included as screening tools or in WET (Whole Effluent Test) procedures . The bioassay is capable of identifying the activity of 5 types of antibiotics . We present the latest results using this bioassay and discuss the potential as an initial screening step to prioritize effluents, one of the research needs as identified in the European Water Framework Directive . MO 217Chronic aquatic toxicity of sucralose to aquatic invertebrates and fishB . HuggettUniversity of North Texas, Denton, United States of AmericaThe intense artificial sweetener sucralose has been detected in municipal wastewater effluent and surface waters at concentrations ranging from ng/L to low ug/L . Few chronic ecotoxicological data are available in the peer reviewed literature with respect to sucralose . To address this data gap, 21 d Daphnia magna, 28 d Americamysis bahia (mysid shrimp) studies and a fish early-life stage study (ELS) were conducted to assess the effects of sucralose on the survival, growth and/or reproduction of these organisms . Concentrations ≤ 1800 mg/L resulted in no statistically significant reduction in D . magna survival or reproduction . Survival, growth and reproduction of mysid shrimp were unaffected by ≤ 93 mg/L sucralose . Collectively, these data suggest that the concentrations of sucralose detected in the environment are well below those required to elicit chronic effects in freshwater or marine invertebrates . Data will also be presented with respect to findings in the fish ELS study .

MO 218Rapid acute toxicity screening of commercial and potential green plasticizers using bioluminescent bacteriaP .A . Segura, P . Kaplan, H . Erythropel, D .G . Cooper, V . YargeauMcGill University, Montreal, CanadaThe Vibrio fischeri bioluminescence inhibition assay (Microtox®) using an in-house diluent containing 2% DMSO was used to perform a rapid toxicity screening of 24 commercial and potential green plasticizers . This bioassay allowed us to better understand the impact of a key structural feature, alkyl chain size, on the toxicity of the target plasticizers . Results indicated that dibenzoate compounds with lower number of C atoms in their alkyl chains, such as 1,3 propanediol dibenzoate (PrDDB) and 1,4-butanediol dibenzoate (BDDB), were more toxic (EC50 < 2 µM) than commercial glycol dibenzoates such as DEGDB and DPGDB (EC50 = 14-31 µM); however dibenzoates with higher number of C atoms in their alkyl chains such as 1,5-pentandiol dibenzoate (PDDB) and 1,6-hexanediol dibenzoate (HDDB) had similar toxicities to those commercial plasticizers . Dibutyl and dihexyl compounds of the dialkyl esters (fumarates, maleates and succinates) were more toxic (EC50 = 2-6 µM) than the commercial plasticizers di(2-ethylhexyl) phthalate (DEHP), 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) and di(2-ethyl hexyl) adipate (DEHA), which did not inhibit the bacterial luminescence . Nevertheless diethyl, dioctyl and di(2-ethyl) hexyl compounds are weakly toxic (EC50 = 130-1292 µM) or non toxic at all to the luminescent bacteria . These data and previous studies on biodegradation and plasticizing properties indicate that 1,5-pentandiol dibenzoate (PDDB), 1,6-hexanediol dibenzoate (HDDB), dioctyl fumarate (DOF), dioctyl maleate (DOM) and dioctyl succinate (DOS) might be good candidates to replace current commercial plasticizers .

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MO 219Contribution of PPCPs and surfactants on the sub-chronic toxicity of effluent-dominated urban creeks to aquatic organismsH . YamamotoGraduate School of Integrated Arts and Sciences, The University of Tokushima, Tokushima, JapanJapan Ministry of Environment started research on possible introduction of the bio-assay-based management system of wastewater and receiving water similar to USEPA’s WET to Japan but information had been limited in Japan . Thus, our research group has examined the aquatic toxicity of some industrial and municipal wastewaters in Tokushima, Japan using the short-term chronic test mainly used in USEPA’s WET Test Methods with slight modification and found significant adverse effects on the reproduction of Ceriodaphnia dubia and hatching/survival of eggs of Japanese medaka or zebrafish for several samples . On the other hand, we have been investigated on ecological risk of pharmaceuticals and personal care products and found some PPCPs including some antimicrobial agents and UV filters might be a possible threat for the aquatic organisms .

In the present study, the growth inhibition test of green algae (Pseudokirchneriella subcapitata), the Ceriodaphnia dubia reproduction test, and the zebrafish (Danio rerio) embryo/sac fly stage test was conducted to evaluate the sub-chronic toxicity of waters sampled in 12 urban streams of Tokushima, Kyoto, and Saitama, Japan, contaminated by treated or untreated domestic wastewater . The reproduction and survival of Ceriodaphnia dubia was adversely affected in the waters sampled in most of the sites at least once . The growth of green algae was significantly inhibited in those sampled mostly in summer season in at least three sites . Some potential toxicants, mainly PPCPs and surfactants, were measured for the water sampled using GC-MS or LC/MS/MS for the water samples in addition to the toxicity tests for approximately twenty individual PPCPs and surfactatns . Toxicity unit (TU) for each individual PPCP was calculated based on NOEC values and compared with the TU of the whole river waters . Relatively higher concentrations of most of PPCPs were found in Tokushima compared to Kyoto and Saitama . Surfactants, triclosan and other antifungal agents slightly or moderately contribute the whole toxicity of the water samples for algae but the factors other than PPCPs may play important role for Ceriodaphnia and zebrafish .

ET08P - Extrapolation within wildlife toxicology

MO 241Variability in the outcome of outdoor mesocosms with three-spined Stickleback (Gasterosteus aculeatus) populations in control conditions: data from 20 replicates in two annual experimentsG .K . de Kermoysan, A .R .R . Péry, S . Joachim, V . Martz, P . Miguet, J .M . Porcher, R . BeaudouinIneris, Verneuil-en-halatte, FranceEcotoxicology aims at protecting ecosystems and populations . Population level is rarely studied in ecotoxicity tests . However, the population level can be monitored with high realism relative to ecosystems through experiments in outdoor mesocosms . At Ineris, we focus on the three-spined Stickleback, Gasterosteus aculeatus, because it is a well-studied species, autochthonous in French rivers, and a promising species to assess the impact of contaminant . All studies already performed in mesocosms suffer from an important variability between replicates for a same treatment (more than 50% of variation is generally observed on biological variables) . Temporal replicability of mesocosms’ outcomes is also an issue . In our study, we aimed at identifying the major causes of inter and intra annual variability in control conditions . Thus 10 populations per year, for two years experiments were monitored between March and October 2010 and 2011 . At the beginning of experiments, each compartment of the ecosystem (weight of macrophytes, number and masse of invertebrates, number, length and sex ratio of fish[3DOTS]) were introduced in standardized conditions . At the end of the experiments, all populations were defined by characteristics such as total number of fish, sex ratio, and percentage of mature organisms . We found that, in 2010, the proportion of males and females fish introduced in the system was the most influent parameter to account for the population characteristics (total number of fish and structure of the population) at the end of the experiment . Inter-annual variability was mainly due to differences in water temperature profiles . Intra-annual variability can be reduced through a better sex determination of the fish introduced at the beginning of the experiment . We developed an original methodology to achieve that (See another poster of de Kermoysan et al .: A non-invasive method based on head morphology to sex mature three-spined Stickleback (Gasterosteus aculeatus) in rearing conditions) . Inter-annual variability, in link with temperature, could be quantified provided the influence of temperature on the main components of G . aculeatus population dynamics are understood and modelled .

MO 242Maternal transfer of lead in mallards under field conditions: biochemical and immunological effects on ducklingsM .E . Ortiz-Santaliestra1, A . López-Antia1, M . Martínez-Haro1, S . Espín2, D . Vidal1, R . Mateo1

1IREC (CSIC-UCLM-JCCM), Ciudad real, Spain2University of Murcia, Murcia, SpainThe ingestion of Pb shot used for hunting constitutes the main cause of lead poisoning in waterfowl . Females may transfer some of the accumulated Pb to their eggs and, although most Pb is expected to accumulate in the eggshell, it can ultimately affect embryonic and hatchling health and development . We collected eggs of mallard ducks from the Ebro delta (NE Spain), an area with high Pb shot prevalences in waterfowl, and incubated them artificially . Blood samples were collected at days 3 and 28 post hatching to measure Pb levels, δ-ALAD activity, oxidative stress indicators, biochemical parameters, vitamins and carotenoids . Cellular immune function was quantified at day 14 as the intensity of swelling produced in response to an intradermal injection of phytohemagglutinin (PHA) . Humoral immune function was measured at day 28 as the amount of antibodies synthesized against an intraperitoneal injection of sheep erythrocytes . Ducklings with blood Pb levels above 180 ng/ml died during the first 7 days post hatching . Eggshell Pb levels were significantly correlated with blood Pb levels in ducklings at day 3 (r=0 .549, p=0 .002), but not at day 28 . δ-ALAD activity and cellular immune function were negatively correlated with Pb levels in eggshell (δ-ALAD : r=-0 .383, p=0 .040; PHA reaction: r=-0 .471, p=0 .048) and blood of mallard ducklings (δ-ALAD : r=-0 .468, p=0 .009; PHA reaction: r=-0 .639, p=0 .003) . Neither the humoral immune function nor the rest of blood parameters were affected by blood or eggshell Pb concentrations . δ-ALAD activity decreased linearly at blood Pb concentrations above 60 ng/ml, as it has been described for adult mallards [6] . The results suggest that some of the Pb present in eggshells was accumulated in target tissues (i .e . liver, spleen, bone), potentially contributing to the observed immunotoxic . Because range of Pb levels is wider in eggshells than in blood samples, eggshell contents may be used to estimate in ovo Pb exposure of the ducklings, being a more representative indicator than blood levels to estimate the effects of Pb intoxications .

MO 243Blood concentrations of heavy metals (Ba, Cd, Hg and Pb) and Metalloid (As) in raptor species (Buteo buteo, Milvus migrans and Gyps fulvus) from PortugalM .A . Carneiro1, B .J . Colaço1, R . Brandão2, C . Ferreira3, N . Santos4, V . Soeiro5, A . Colaço1, M .J . Pires1, P . Oliveira1, S . Lavin6

1Universidade de Trás-os-Montes e Alto Douro, Vila real, Portugal2Centro de Ecologia, Recuperação e Vigilância de Animais Selvagens, Gouveia, Portugal3Centro de Recuperação e Investigação de Animais Selvagens da Ria Formosa, Olhão, Portugal4Centro de Recuperação de Fauna Selvagem do Parque Nacional da Peneda do Gerês, Braga, Portugal5Centro de Recuperação do Parque Biológico de Gaia, Gaia, Portugal6Servicio de Ecopotatologia de Fauna Selvage . Universidad Autónoma de Barcelona, Barcelona, SpainHeavy metals are ubiquitous elements present in the environment and they can easily enter the food chain and cause possible toxic effects on humans and animals, being a serious threat for the stability of ecosystems and living organisms . Birds of prey can be useful as sentinel species to monitor the quality of the environment, once they forage over a large geographical area and as top consumers in food chain it would be expected that they accumulate environmental contaminants, like heavy metals . The purpose of this study was to evaluate the exposure to arsenic (As), barium (Ba), cadmium (Cd), mercury (Hg) and lead (Pb) in three raptor species from different areas of Portugal . Birds of this study were brought alive, but injured or debilitated for various reasons, to four Portuguese Wildlife Rehabilitation Centers . Blood samples were collected from 72 common buzzards (Buteo buteo), 22 black kites (Milvus migrans) and 33 griffon vultures (Gyps fulvus) and concentrations of toxic elements were measured by inductively coupled plasma mass spectrometer (ICP-MS), after blood wet digestion . Results obtained showed the presence of various toxic elements in all samples analyzed . Cd means concentrations were the lowest in the three species and no statistically significant differences were found between species (p = 0 .758) . However, statistically significant differences were observed between species for As (p<0 .001), Hg (p<0 .001) and Pb (p<0 .05), showing griffon vulture and black kite the highest blood Pb concentrations (respectively 21 .278 ± 13 .588 and 20 .536 ± 32 .662 µg/dl w .w .), common buzzard the highest Hg concentrations (17 .156 ± 22 .439 µg/dl w .w .) and results concerning As, black kite presents the highest values (4 .607 ± 5 .090 µg/dl w .w .) . The area of origin seems to influence blood Hg concentrations in common buzzard, once were found statistically differences between Braga and other five districts (p<0 .05), presenting the animals of Braga the highest levels of Hg . Results concerning Hg and obtained in this study suggest that common buzzards are exposed to high levels of this toxic metal, when compared with other elements . Black kite and griffon vulture are exposed to high levels of Pb, possible to cause subclinical or clinical toxicity . Although concentrations could not be directly related to lethal effect, they might constitute a serious environmental factor affecting the survival, and special attention should be given at species in decline .

MO 244A multi-species approach for assessing lead bioavailability in the ecological risk assessment of a shooting rangeS .R . Bowman, J .L . Bryant, R .P . LannoThe Ohio State University, Columbus, United States of AmericaEcological risk assessment is an important tool for evaluating potentially hazardous contaminants on a site-specific basis . Few methods exist for evaluating population and community level effects of contaminants on terrestrial invertebrates, though such assessments may aid in site evaluation and subsequent decision-making with regard to site remediation . In the current study, we investigated the utility of ground beetles (Carabidae) as indicators of elevated metal concentrations originating from lead-based shot at a shooting range . Ground beetles were collected in 2008 and 2009 using pitfall traps in shotfall and reference areas . Forty-five species were collected and overall species richness did not differ between sites . Abundance of the most common species was not significantly different between sites or was more abundant at the reference site . Collection of ground beetles is on-going and additional years of data may reveal different trends in ground beetle distribution and abundance . In addition to ground beetle data, bioassays using earthworms (E . fetida) were used to estimate bioaccumulation and sublethal effects of the metals in shooting range soil . Twenty-eight day bioaccumulation

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and reproduction tests were conducted with earthworms . Cocoon production was lower in shotfall soil compared to reference soil and Webster (laboratory standard soil) . No mortality was observed and Pb bioaccumulation increased with increasing Pb concentration in soil . Pb bioavailability will also be estimated using a modified in vitro gastrointestinal method (mimicking the short-tailed shrew, Blarina brevicauda) . Field soils and earthworms exposed to field soils will be analyzed for bioaccessible Pb . In the future, we hope to collect earthworms and shrews from the shooting range for the determination of Pb concentrations in their tissues . If a correlation is established between field Pb levels in shrews and the relative bioaccessibility, then these modified methods may be a useful tool for Pb exposure estimates in ecological risk assessment .

MO 245Effects of lead shot ingestion on constitutive immune function of red-legged partridgesN . Vallverdú, M .E . Ortiz-Santaliestra, J .M . Pérez de la Lastra, R . MateoIREC, Ciudad real, SpainThe effects of toxic levels of Pb are diverse and represent several functional systemic alterations . Although the effects of exposure to Pb on the immune system of mammals, and especially in humans, have been extensively studied, little is known about how this heavy metal affects the constitutive immune function in birds . Pb intoxication may be a major problem for birds as they accidentally feed on Pb shots that are confounded with grit particles . We present a preliminary study based on different tests to assess changes in innate immune response of red-legged partridges (Alectoris rufa) subjected to Pb shots . We used four experimental groups (0-9 Pb shots) with six replicates each . A marginally significant lethal effect was detected for the highest Pb treatment (60% mortality; p=0 .057) . Among survivors, decrease in body mass after Pb exposure affected especially to females . Blood samples were obtained from the survivors 25 days after exposure . We compared blood Pb levels, analyzed by Atomic Absorption Spectrophotometry with graphite furnace, with different parameters indicative of the innate immune response . The ratio between lymphocytes expressing CD4 and CD8 membrane receptors, analyzed by flow cytometry, was affected by the interaction between treatment and sex in a less than significant manner (p=0 .060) . Haematocrit, serum lysozyme concentrations, bactericidal activity of the whole fresh blood and the ratio heterophils/lymphocytes were unaffected by the exposure to Pb . Our results highlight the necessity to analyze several parameters to asses the immunotoxicity of a substance . Nonetheless, these and other tests should be repeated with a larger sample size to obtain meaningful results . Analyzing constitutive immune function is important in wild populations as it represents the organism’s ability to display a quick response against opportunistic pathogens .

MO 246Assessment of fungicide- and insecticide-coated seed risks for red-legged partridgesA . López-Antia, M .E . Ortiz-Santaliestra, R . MateoIREC, Ciudad real, SpainThe use of agrochemicals is suspected to be a major cause of population declines of farmland birds from Western Europe . Apart from indirect effects like reduction of available food and optimal habitat, ingestion of pesticide-coated seeds constitute a potential way of direct intoxication . Although the most toxic chemicals are being legally restricted, some of the pesticides currently used for seed coating can cause adverse effects when consumed by birds . The aim of the present project is to test the effects of coated seed ingestion on red-legged partridge (Alectoris rufa) physiology, fitness and reproduction . We tested two insecticides (natural pyrethrins and imidacloprid) and two fungicides (maneb and thiram) . Each experiment consisted in two groups of 16 pairs of partridges exposed to two doses: a high dose corresponding to recommended for seed coating and a low dose being the 20% of the recommended one . Expsoure was splited in two periods, corresponding to the two seasons of cereal sowing; 25 days in autumn and 10 days in late winter . Imidacloprid was the most toxic compound, killing all patridges at the recommended dose in 21 days . All pesticides affected hematocrit (controls: 42 .3%; treated: 38 .3-41 .0%), whereas maneb at high dose reduced body condition after the first exposure period (10 .6% lower than controls) . Reproduction was affected especially by the fungicides, and at low doses rather than at high ones . The two fungicides reduced the fecundation rate (34-54% vs . 62% in controls) and thiram caused a significant decrease in chick body condition 24 days after hatching (18% lower than controls) that ultimately caused a marginally significant mortality (p=0 .07) . At high dose, this fungicide also caused an immunosupressive effect, as estimated by the PHA test to quantify the cellular immune response . The use of coated seeds is a widespread practice, and cereal seeds consitute a major portion of partridge diet during autumn and winter, when alternative food sources are scarce . On the other hand, colour, taste or texture of the coat can make the seed unpalatable for birds, reducing the risk of ingestion . Upcoming analyses will include an assessment of the environemntal exposure of partridges to coated seeds that will be achieved by the study of crop contents of field collected partridges provided by hunters .

MO 247Long-term effects of Pb pollution on mineralization and biomechanical properties of bone in wild ungulates: relationship with vitamins A, D and EJ . Rodríguez-Estival1, P . Álvarez-Lloret2, A .B . Rodríguez-Navarro3, R . Mateo1

1Instituto de Investigación en Recursos Cinegéticos, IREC, Ciudad real, Spain2Departamento de Geología, Facultad de Geología, Universidad de Oviedo, Oviedo, Spain3Departamento de Mineralogía y Petrología, Facultad de Ciencias, Universidad de G, Granada, SpainLong-term effects of environmental lead (Pb) exposure in bone could play an unknown role threatening some populations of wildlife . Certain physiologic disorders attributed to Pb exposure are related to the generation of oxidative stress, which may be involved on the depletion of antioxidant vitamins . The aim of this work was to study the occurrence of abnormalities on bone tissue composition and turnover mechanisms through the Pb-mediated disruption of vitamins (A, E and D) homeostasis in wild ungulates living in a Pb-polluted mining area . Red deer (Cervus elaphus) and wild boar (Sus scrofa) from the mining area had significantly higher liver and bone Pb levels than in the control area, which were associated with the depletion of liver retinyl esters and the corresponding increase of free retinol levels both in deer and boar from the mining area . The most characteristic trait in bone mineralization of Pb-exposed adult deer was a lower carbonate content in bone mineral that was associated with the increased free retinol percentage, which in turn had consequences on bone biomechanics . Although Pb effects on wild boar have been less significant, the degree of bone mineralization was also positively associated with higher burdens of retinyl esters . In conclusion, we suggest that changes in antioxidant vitamins found in deer may be involved in the toxic effects of Pb on bone composition .

ET09P - Global climate change: implications for environmental toxicology and chemistry

MO 249The influence of global climate change on the scientific foundations and applications of environmental toxicology and chemistry: summary from a SETAC international workshop (July 2011)S .J . Moe1, J . Balbus2, W .H . Clements3, A . Fritz4, T . Gouin5, R . Helm6, C . Hickey7, M . Hooper8, W .G . Landis9, R .G . Stahl, Jr .10

1Norwegian Institute for Water Research (NIVA), Oslo, Norway2National Institute of Health, Bethesda, maryland, United States of America3Colorado State University, Fort collins, colorado, United States of America4NOAA, Seattle, washington, United States of America5Unilever, Sharnbrook, United Kingdom6US Fish & Wildlife Service, Arlington, virginia, United States of America7NIWA, Hamilton, New Zealand8US Geological Survey, Columbia, missouri, United States of America9Western Washington University, Bellingham, washington, United States of America10DuPont Company, Wilmington, delaware, United States of AmericaThis is an introduction to and summary from a SETAC international ‘Pellston’ workshop titled “The Influence of Global Climate Change on the Scientific Foundations and Applications of Environmental Toxicology and Chemistry” . Held July 16-21, 2011 it involved some 36 scientists from 11 countries . The central question addressed was: How will global climate change influence the environmental impacts of chemicals and other stressors; and, the way we assess and manage chemical contamination and other stressors in the environment? While more detail is found in the complete series of papers, some key consensus points are: 1 . human actions (adaptation, mitigation, etc .) may have as much or more influence on the fate and effects of chemical contaminants as does GCC, and modeled predictions should be interpreted cautiously; 2 . effects of GCC may be slow, variable and difficult to detect though some species and populations of high vulnerability (polar, coral reefs) may exhibit responses sooner and more dramatically than others; 3 . future approaches to human and ecological risk assessments will need to incorporate multiple stressors, cumulative risks (positive and negative) considering the wide spectrum of potential impacts stemming from GCC; 4 . baseline / reference conditions for estimating resource injury and restoration / rehabilitation will continually shift due to GCC and represent significant challenges to practitioners . MO 250Consequences of metal adaptation on sensitivity to future climate change stressors in Daphnia pulex populationsD . De Coninck1, M . Fischer2, S . Glaholt2, J . Asselman1, C .R . Janssen1, J . Shaw2, K .A .C . De Schamphelaere1

1Ghent University, Gent, Belgium2Indiana University, Bloomington, in, United States of AmericaGenetic variability may allow for natural populations to adapt to a stressor, which is manifested by an increased tolerance to the stressor . As a consequence of this genetic adaptation, populations may also become more (co-tolerance) or less tolerant (cost-of-tolerance) to other stressors . In this research we investigated the cost-of-tolerance hypothesis in the context of climate change, i .e . that populations of the waterflea Daphnia pulex that adapted to cadmium (i .e . more tolerant to sublethal cadmium concentrations) are less tolerant to exposure to two climate change stressors, cyanobacteria and hypoxia . Cyanobacteria are a common stressor to most zooplankton species and are predicted to play an increasingly prominent role in aquatic ecosystems as their occurrence in harmful algal blooms is predicted to increase due to climate change . Increasing deposition of atmospheric nitrogenous gasses and phosphorus loading from agricultural fertilization and fossil-fuel emissions into the world’s surface waters increases eutrophication of these waters, eventually resulting in an increased amount of sinking organic matter . Hypoxia is caused by an increase in microbial, oxygen requiring processes which cope with digestion of this organic matter . Furthermore, higher water temperatures tend to increase the duration and strength of thermal stratification by reducing the mixing of oxygen rich water layers with oxygen poor layers, resulting in a prolonged hypoxia zone . To test the cost-of-tolerance hypothesis, animals were exposed to either a diet containing 50% Microcystis aeruginosa, 2 mg∙L-1 O2, or 0 .5 µg Cd∙L-1 to assess cyanobacterium, hypoxia, and cadmium tolerance, respectively . Control animals were maintained at an oxygen level of 9 mg∙L-1 O2 and were fed 100% Ankidostresmus falcatus . Tolerance was assessed as control normalized

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reproduction and growth . Contrary to the cost-of-tolerance hypothesis, we found significant correlations between cadmium and both M. aeruginosa and hypoxia tolerance, suggesting that co-tolerance rather than a cost-of-tolerance occurred . These findings suggest that cadmium adapted Daphnia populations are better suited to face some future climate change stressors than non-cadmium adapted populations .

MO 251Toxicity interaction of salinity and copper on the development of early life stages of ZebrafishB .Á .R .B .A Santos1, T .S . Andrade2, I . Domingues3, R . Ribeiro4, A .M .V .M . Soares5, I . Lopes3

1University of Aveiro, Aveiro, Portugal2Universidade de Aveiro, Aveiro, Portugal3Departamento de Biologia & CESAM, University of Aveiro, Aveiro, Portugal4IMAR - Instituto do Mar, Department of Life Sciences, University of Coimbra, Coimbra, Portugal5University of Aveiro & CESAM, Aveiro, PortugalClimate changes are expected to modify environmental parameters in several aquatic ecosystems . Within these scenarios, an increase in salinity, due to the sea level rise, is predicted to occur in coastal freshwater ecosystems . According with this scenario, a set of works has already addressed the sensitivity of several freshwater species to increased salinity in order to evaluate the potential risks associated with the salinization of such coastal ecosystems . However, as many of these coastal freshwater lagoons are already impacted with metal contamination, it is pertinent and important to assess and understand the combined effects of metals and increased salinity to freshwater species . Accordingly, the present work intended to evaluate the possible interactions of copper and NaCl (as a measure of increasing salinity) on the development of zebrafish . To achieve this goal, larvae (96hpf) were exposed to several concentrations of copper (0 .44 - 7 .38 µM) and NaCl (5 - 7 .93 g/L), both individually and combined (using a full factorial design), during 96h . During exposure several lethal and sublethal endpoints (mortality, swim-bladder size, edemas, malformations, and activity) were monitored . Preliminary results showed that in the lowest copper concentrations (without NaCl), mortality was low or null, but, when combined with NaCl, mortality increased during the four days of the experiment . Furthermore, the highest copper concentrations combined with NaCl caused higher mortality rates, but only after 48h of exposure . These data suggest a possible synergism between copper and NaCl, which may lead to higher rates of mortality than those expected from their individual toxicity . Additionally, in treatments with NaCl alone, no significant rates of malformations or edemas were observed and when the same concentrations where combined with copper there was an increased of incidence of these two parameters . This same scenario was observed with copper alone . It is then expected that this synergistic effect between compounds may affect as well other significant sublethal endpoints . These preliminary results suggest that attention must be paid to the effects of salinization on aquatic ecosystems impacted with metals as interactions are likely to occur, and already chemical-impacted populations may be at a higher risk comparatively with non-exposed ones .

MO 252In vitro testing of biofuels - Verifying the environmental friendliness of biofuels in comparison with fossil fuelsS . Heger1, K . Bluhm1, T .B . Seiler2, A . Schäffer1, H . Hollert1

1Institute for Environmental Research, RWTH Aachen University, Aachen, Germany2RWTH Aachen University, Aachen, GermanyIn the attempt to reduce the emission of greenhouse gases and simultaneously decrease the amount of fossil fuels that is used in the transport sector, fuels produced from biomass, so called ‘biofuels’, are becoming more and more popular . In this context, the amount of biofuels in the transport sector will be increased during the next decades . In 2022, 10% of the total fuel consumption in the European Union should be obtained from biomass . The United States of America, on the other hand, plan to increase the amount of biofuels up to 30% until 2030 . While the negative effect on the global climate due to decreased carbondioxide emission from fossil fuels will be reduced, significant effects on the environment can be expected! However, there is nearly no data available on (eco)toxic effects these biofuels could have on the environment or the human health . In this study, we want to investigate the use of in vitro biotests for the (eco)toxicological investigation of biofuels on the basis of the three potential biofuels ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran . These potentials biofuels are currently investigated by the excellence cluster “Tailor-made fuels from biomass”of the German excellence initiative . We want to identify relevant endpoints and test organisms that could be used for a comprehensive hazard assessment of biofuels . There are, however, some points that need to be addressed due to the nature of the test substances . The particular physical-chemical properties of these potential biofuels and the also investigated fossil fuel reference substances require some adaptations of the test designs . We investigated three in vitro biotests and ecotoxicological endpoints . Acute Cytotoxicity was determined by means of the Neutral Red Retention assay and ah-receptor mediated activity, so called dioxin-like activity, was detected using the mechanism-specific 7-Ethoxy-o-resorufindeethylase (EROD) assay, both using cells from the permanent cell line RTL-W1 (Onchoryhnchus mykiss) as a test organism . Furthermore, genoxtoxicy was determined by means of the micronucleus assay using the cell line V79 (Chinese hamster) (ISO21427-2) . Besides the direct dosing approach, which is more or less a standard method, a passive dosing approach using Polydimethylsiloxan-Rings was also applied, in order to investigate the suitability of this method for these substances .

MO 253How important is chemical toxicity for biodiversity conservation?F . de Laender1, D . Verschuren1, R . Bindler2, C .R . Janssen1

1Ghent University, Ghent, Belgium2Umea University, Umea, SwedenIn the early 1990’s, biodiversity was recognized as an important factor determining ecosystem functions . Ecotoxicology approaches biodiversity conservation from a chemical toxicity perspective using time scales that match those of the exposed organisms’ life cycles (hours to months) . However, the contribution of chemical toxicity to centennial trends in biodiversity remains unknown . Here, we propose a combination of paleo-ecological approaches with statistical modelling to increase our insight into the quantitative importance of chemical toxicity to biodiversity changes over long time periods . Specifically, we analysed if and to what extent fluctuations of two biodiversity indicators (richness and evenness) during the last 2000 years in a copper mining region in central Sweden were steered by copper toxicity, relative to land-cover changes and continent-wide temperature anomalies . Historical trends of copper, biodiversity and land-cover were reconstructed from copper concentrations, counts of fossil diatoms, and counts of terrestrial pollen - all measured in sediment cores - respectively . Temperature anomalies were obtained from climate models . Using additive modelling, we found that 76% and 50% of the variability in evenness and richness can be explained by lake identity and the degree of land disturbance . Addition of more predictors (including Cu concentrations) did not improve model fit . However, the fitted lake-specific effects indicated that the two lakes located next to metal smelters (Cu up to 3000 µg/g) had communities that were less rich (30 to 40 taxa less) and had a lower evenness (0 .1 lower than lakes located further away from mining activity) . The absence of significant effects of Cu concentrations between 10 and 100 mg/kg on biodiversity could be explained by the replacement of sensitive taxa by tolerant taxa . The beneficial effect of land disturbance on biodiversity was explained by increasing abundances of r-strategist species with increasing disturbance . Our findings illustrate that - even in a metal polluted area - land-cover was the major driver for biodiversity fluctuations .

MO 254Temperature-dependent effects of the fungicide pyrimethanil on the reproduction of Physella acutaA .S . Seeland, R . Müller, J . OehlmannGoethe University, Frankfurt, GermanyThe environmental impact of climate change is meanwhile not only discussed in the scientific community but also in the general public . However, little is known about the relationship between pesticides and climate change . A combination of multiple stressors may lead to severe alterations for organisms such as changes in time of reproduction, reproductive success and growth performances, mortality and geographic distribution . Recent research revealed that aquatic model organisms tend to react more sensitive towards contaminants under climate change conditions . To understand the sublethal impact of contaminants and how temperature influences sensitivity of molluscs as non-model organisms we assessed an embryo toxicity test as well as a whole life-cycle test (F1 and F0-generation) with the snail Physella acuta . Therefore embryos and adult snails were exposed towards the fungicide pyrimethanil and three different temperatures (15, 20 and 25°C) . Independent of pyrimethanil concentrations, more embryos hatched the higher the temperature was . Furthermore pyrimethanil caused high mortality at the highest concentration of 1 .0 mg L-1 (chronic EC10 for Daphnia magna = 0 .95 mg L-1) at all temperatures . In the life-cycle test more egg masses were produced the higher the temperature was in the F0-generations and the snails reacted more sensitive towards pyrimethanil the lower the temperature was . Due to high mortality no snails were available for the F1-generation at the highest concentrations (0 .5 and 1 .0 mg L-1) . Compared to the F0-generation, overall more egg masses were produced in the F1, while all egg masses were fertile and no mortality occurred . For the F1-generation the strongest pyrimethanil effects were detected at 15°C . A comparison of effect concentrations between both generations showed that the F1-generation is more sensitive than the F0-generation . These results indicate that an exposure over more than one generation may give a better overview of the impact of xenobiotics . With the establishment of an embryo and reproduction test under different temperatures and various concentrations of pyrimethanil with P . acuta we could successfully show that molluscs can respond more sensitive than model organisms and that both, chemical and thermal stressor strongly influence the behaviour of the snails .

MO 255Health condition of Minho and Lima Corbicula fluminea populations in relation to summer mass mortality eventsC .A . Oliveira, P .V . Vilares, J .R . Rocha, C .G . Gravato, L .M . GuilherminoCIIMAR, Porto, PortugalThe Asian clam Corbicula fluminea is one of the most important non-indigenous invasive species (NIS) in aquatic ecosystems that has been causing considerable negative ecological and economic impacts, especially in Europe and North America . In the last decade, massive mortalities of C . fluminea have been recorded periodically at the end of the summer in the Minho River (NW Iberian Peninsula) which has been related with extreme events (draught conditions) and variations in water quality possibly including chemical stress effects . To go further on the causes contributing to such die-offs, a monitoring study investigating the health status of the population in relation to water quality and other abiotic variables has been carried out . Several biomarkers and other parameters indicative of the health condition of the animals were investigated monthly between July and October . Results indicate changes in the health condition of animals (e .g . oxidative stress) in relation to the variation of abiotic conditions, and the multivariate analysis of data associates a decrease of health condition with individual abiotic factors . These findings are discussed in relation to the reduction of the diversity of the molluscan community of the Minho River estuary and ecosystem functioning .

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This study was carried out in the scope of the project “NISTRACKS - Processes influencing the invasive behaviour of the non indigenous species Corbicula fluminea (Mollusca: Bivalvia) in estuaries - identification of genetic and environmental key factors”funded by the Portuguese Foundation for the Science and the Technology (FCT) (PTDC/AAC-AMB/102121/2008) and FEDER COMPETE funds (FCOMP-01-0124-FEDER-008556) . C . Oliveira had a PhD grant from FCT (SFRH/BD/68423/2010) funded by national funds of the MCTES and FEDER European funds (POPH-QREN-Tipology 4 .2 .) .

MO 256Differential proteomic responses of larvae of the marine medaka Oryzias melastigma upon cadmium exposure at thermal extremesP .T .Y . Leung, A .J . Li, K .M .Y . LeungThe University of Hong Kong, Hong kong, HongkongUpon facing unpredictable extreme temperature events under the human-driven climate change, marine ectotherms would generally be more susceptible to additional stressors like toxicants . This study aimed to reveal the stress responses in marine medaka larvae Oryzias melastigma upon exposure to cadmium (Cd) under different thermal conditions including extreme temperatures . Larval fish were thermally treated by elevating or decreasing temperatures at a rate of 1ºC per hour followed by 7 days of acclimation to the test temperatures, before commencement of 4 days of Cd treatments . The associated toxic stress responses were elucidated using two-dimensional gel electrophoresis (2-DE) coupled with multivariate statistical analyses . Proteins of fish larvae from temperature treatment groups (i .e ., 10ºC, 25ºC and 32ºC) were successfully separated using IEF strip with pH 3-10 and gradient SDS-PAGE with gel percentage of 4-12 .5% . The total- and phospho-proteins were visualized using Sypro Ruby and Pro-Q Diamond stains, respectively . Similar numbers of total protein spots (from 206 to 225) were detected in the three temperature treatments . Higher percentage number of phosphoprotein spots was however detected at 10ºC (~25%) than at 25ºC (~12%) and 32ºC (~12%) . This observation indicated that alteration of physiological pathways can take place at the low extreme temperature, i .e ., 10ºC . In view of the progressive decrease in whole-organism aerobic scope and shift towards anaerobic metabolism under the extreme temperatures, and needs for extra energy for detoxification and cellular repairing processes under chemical exposure, adverse toxic responses could be expected under the extreme temperatures . Proteomic response of the fish larvae under combined Cd and thermal stresses is under investigation . The results of this study could provide certain essential and fundamental information to further advance our understanding on specific toxic effects of trace metals under temperature extremes .

MO 257The influence of fluctuating temperature on the effect of copper toxicity in Caenorhabditis elegansN .J . Norhave1, D . Spurgeon2, C . Svendsen2, N . Cedergreen1

1University of Copenhagen, Frderiksberg c, Denmark2Centre for Ecology and Hydrology, Wallingford, United KingdomClimatic variables like humidity and temperature have been shown to affect the toxic effect of some chemicals . Temperature especially has a direct effect on the toxicity of metals and pesticides, generally causing toxicity to rise with increasing temperature, in particular when approaching the temperature tolerance limit of the test organism . Studies on the effect of temperature differences on toxicity have so far been done at static temperature regimes, which ignore any additional stress which may be caused by fluctuating temperature . Since daily temperature changes in the terrestrial environment can fluctuate with more than 10 degrees, it is questionable whether information from static conditions can be extrapolated to describing the effect of temperature on toxicity in the field . We hypothesize that fluctuating temperatures will prove to be a stress factor in itself, which may increase the effect of a toxicant, as compared to the equivalent static scenario at the average temperature . In the present study we expose the nematode Caenorhabditis elegans to a range of copper concentrations at three static temperatures (12, 16 and 20°C), as well as in a non-static temperature scenario with a daily temperature fluctuation from 12 to 20°C and an average daily temperature of 16°C . The temperature exposures were chosen to be within the approximate optimal temperature range of C . elegans . The study use full life cycle exposures, with daily measurements of reproduction and survival during the entire lifespan . To our knowledge this is the first study to compare the effect of static and fluctuating temperature on toxicity of copper .

MO 258Effect of temperature on embryotoxicity of the antifouling biocide zinc pyrithione to Sphaerechinus granularisM .I . Imre, R .O . OralEge University, Faculty of Fisheries, Izmir, TurkeyThe present study investigated combined effects of the antifouling biocide zinc pyrithione and different temperature (17, 23 and 26 oC, control at 20 oC) on embryonic development of sea urchin, Sphaerechinus granularis . S. granularis embryos were reared in zinc pyrithione contaminated seawater at concentrations ranging from 10-8 to 10-6

M for 48, 72 or 96 h, respectively depending on the temperature . Embryological analysis was carried out to determine percent developmental anomalies and/or embryonic mortality . The median effective concentrations (EC50) were 65,9±8,3nM at 17°C, 83,1±15,2nM at 20°C, 81,5±10,8nM at 23°C and 55,9±15,9nM at 26°C for S. granularis embryos . Within the tested temperature range, increased developmental anomalies and/or embryonic mortality occured particularly at concentrations of 50 .0 and 500 nM zinc pyrithione . Our results verified that toxicities of the zinc pyrithione, were highly temperature-dependent . The EC50 values of S. granularis exposed to zinc pyrithione decreased with increasing temperature between 20-26°C, whereas the EC50 values at 17°C were significantly lower than 20°C . To conclude, higher temperatures increased the sensitivity of S. granularis embryos to zinc pyrithione stress .

MO 259Temperature-dependent toxicity of DDT to larvae of the marine medaka Oryzias melastigma: pollutant responses at thermal extremesJ . Li, P .T .Y . Leung, K .M .Y . LeungThe Swire Institute of Marine Science, Hong kong, ChinaIn light of recent predictions of an increase in average sea surface temperature (e .g ., a rise of 3°C by the end of this century) and incidences of temperature extremes involved in global climate change, it is important to study the temperature-dependent chemical toxicities on marine organisms and elucidate toxic mechanisms associated with the interacting effect of thermal stress and pollutant exposure . The vast majority (>95%) of aquatic species are ectothermic, making temperature a key environmental factor in controlling their fundamental physiological processes . This study aimed to examine the combined effects of temperature and dichlorophenyltrichloroethane (DDT) pollution to larvae of the marine medaka fish Oryzias melastigma with a focus on their molecular and physiological responses . A stepwise acclimation was applied for the fish larvae at a rate of 1°C/h from 25°C to 13, 15, 20, 28 and 32°C, followed by 7 days of acclimation and another 4 days of exposure to DDT or the control . Cumulative mortality was under 50% for all selected thermal points at the end of acclimation period . Growth performances, in terms of length and weight, varied across different temperature treatments, having the lowest growth at 13 and 15°C . Active metabolic rate in terms of oxygen consumption was significantly higher at 32°C but was the lowest at 13 and 15°C . After 96h exposure to DDT, thermal treatment of 32°C did exert a significant influence on cumulative mortality and LC50 . Further studies on enzyme activities (e .g ., lactate, malate and succinate dehydrogenases) and expression of proteins (e .g . heat shock proteins) through advanced proteomic analysis are ongoing and the results may yield a mechanistic explanation on the observed synergistic effect of temperature and DDT .

ET12P - Soil ecotoxicology

MO 261Are natural biocides used in agriculture safe for soil ecosystem? Case study of non-target bioactive compounds produced by rhizosphere soil Streptomyces sioyaensis TM 822 N . Nakaew1, R .U .N .G .R Sungthong2, C . Rangjaroen3

1Naresuan University, Phitsanuloke, Thailand2Instituto de Recursos Naturales y Agrobiologia de Sevilla, Seville, Spain3Chiang Mai University, Chiang mai, ThailandStreptomyces sioyaensis TM 822 was isolated from turmeric rhizosphere soil at Chiang Mai, Thailand . Crude bioactive compounds produced by strain TM 822 showed antagonistic activity against Gram-positive bacteria, filamentous fungi and yeast, moreover, they were used directly for biological control of white root disease in rubber tree causing by Rigidoporus sp . This work, we aim to assess the ecological risk of emerging natural biocides used in agriculture . Impact in ecotoxicology of the crude bioactive compounds derived from the strain was tested with both eukaryotic and prokaryotic organisms . Lethal effect of the crude bioactive compounds against normal cell line; mouse fibroblast (3T3 cells) revealed less in cytotoxicity with a mean lethal dose (LD50) of 2 .5 µg mL-1 within 48 h of incubation, however, no lethal effect was observed in all concentrations tested during incubation for 12 h . The crude bioactive compounds exhibited stronger antifungal with a median effective dose (ED50) of 5 .7% (v/v) against mycelia growth of Rigidoporus sp ., compared to positive control (metalaxyl) . Microbial numbers in agricultural soils that applied with the crude bioactive compounds varied according to the concentrations of the crude used . We suggest that suitable dose of either natural or synthetic biocides should be evaluated before use for agricultural purposes with avoiding side effect to biological components in soil ecosystem .

MO 262Copper toxicity in soils amended with copper containing fungicidesK .J . Oorts1, S . Ruyters2, E . Smolders3

1ARCHE, Gent, Belgium2KULeuven, Heverlee, Belgium3K .U . Leuven, Heverlee, BelgiumThe long-term application of copper containing pesticides can cause a significant accumulation of Cu in soils . In order to assess the potential toxic effects of these increased Cu concentrations, a comprehensive comparison of Cu toxicity in a set of soils with high Cu concentrations due to the application of Cu pesticides and in corresponding reference soils was performed . Soils with a long history of application of Cu containing fungicides were sampled at 11 vineyards across Europe including an uncontaminated control soil outside each vineyard . Standard ecotoxicity tests for plant growth, root elongation, nitrification and invertebrates (Enchythraeus albidus) reproduction were performed on each soil of both the vineyard gradient and the corresponding control soil spiked with CuCl2 . For all vineyard gradients, the increased Cu concentration (maximum 349 - 689 mg Cu/kg) did not negatively affect the response of any test compared to the corresponding control soils . Spiking these control soils with CuCl2 however resulted in clear dose-response curves and toxicity data for these freshly spiked control soils are within the range of toxicity data for these endpoints reported in the European Voluntary Risk Assessment Report (VRAR) on Cu and its compounds . Soil limits (Predicted No Effect Concentration, PNEC) calculated according to this VRAR are a factor 2 .2 to 5 .7 below the Cu concentrations observed in the sampled vineyard soils and therefore seem to be over-protective for soils contaminated with Cu due to

1�2 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

the long term use of Cu containing pesticides . It is assumed that long-term accumulation of Cu in soils due to the annual application of limited amounts of Cu, as is the case for Cu containing plant protection products, may result in stronger attenuation of Cu availability with time and hence a lower toxicity of Cu in soils compared to the default assumptions in the VRAR on the effect of ageing on toxicity . The effect of ageing is commonly taken into account in the effect assessment through the application of a so-called leaching-ageing factor . Therefore, a larger scenario-specific leaching-ageing factor is proposed for the effects assessment of Cu in soils affected by long-term application of Cu containing pesticides .

MO 263Toxicity of Tributyltin (TBT) on terrestrial invertebrates and plantsA .R . Silva1, P .V . Silva1, S . Mendo1, S . Loureiro2

1Departament of Biology & CESAM, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, Portugal Tributyltin compounds are highly toxic organotins that were deliberately introduced into the marine environment by antifouling paints since 1960s . Their slow degradation rate and high adsorption to sediments and suspended matter contributed to the high levels of TBT found in sediments . Their disposal on land as well as the disposal of contaminated sewage sludge and application of TBT-based biocide products on agricultural fields constitute an important source of TBT contamination in soils . Due to their adverse effects to non-target organisms at very low concentrations (ng .l-1) and bioaccumulative properties, TBTs are a hazard to soil flora and fauna . This work aimed to assess the effects of TBT on invertebrates and plants species . An agriculture soil collected from Portugal was used for the TBT exposure . For this evaluation several bioassays were performed: two feeding inhibition assays (with contaminated soil and with contaminated food) and one avoidance behaviour test were carried out with the isopod Porcellionides pruinosus . Likewise, a reproduction test with the collembolan Folsomia candida and seed germination and plant growth test with the rapid cycle turnip Brassica rapa and common wheat Triticum aestivum were also performed . Feeding parameters (consumption, assimilation and egestion ratios) and isopod survival decreased when exposed to TBT . P. pruinosus was able to detect very low concentrations of TBT in soil and thus could possibly escape from the contaminated sites . A dose related response between the decrease in the number of juvenile springtails and adult survival with the increase of TBT exposure was also observed for the springtail exposure . For the plants’ exposures a dose-response relationship was observed, showing a delay and decrease on seed germination in both plant species B. rapa and T. aestivum . With increasing tributyltin concentrations, a decrease on growth parameters, including biomass production and plants’ length, was also observed .

MO 264Effects of zinc oxide nanoparticles on developmental stages of soybean in standard soilY .J . An1, S .J . Yoon1, J .I . Kwak1, M .I . Lee1, P .A . Holden2

1Konkuk University, Seoul, South-Korea (Rep)2University of California, Santa barbara, United States of AmericaLong-term effects of zinc oxide nanoparticles to soybean (Glycine max (L .) Merrill) growths were evaluated in the OECD standard soil . The OECD standard soil was amended as 0, 50, and 500 mg/kg soil with ZnO nanoparticles (< 50 nm) . Plant experiments were conducted in a greenhouse . The growth and developmental stages of soybean were measured during exposure period of 8 to 9 weeks . The results showed that development of soybean was significantly affected at 500 mg/kg dry soil . This developmental delay led to a failure in the formation of seeds . The results demonstrated that ZnO nanoparticles affected negatively the life cycle of soybean .

MO 265Soil risk assessment with terrestrial model ecosystems (TME): Population dynamics of microarthropods under field (grassland) and semi-field (TME) conditions B . Theiβen, U .F . Frommholz, L .M . Larnaudie Lopez, R .A . Richter, T .L . LeicherBayer CropScience, Monheim, GermanySince the PERAS workshop in 2007 Terrestrial Model Ecosystems (TME) are discussed as a higher tier option in soil Risk Assessment . In comparison with standard laboratory tests they maintain an intact and complex soil community with a lot of potential faunistic endpoints from e .g . the taxa Collembola or Oribatida under natural climatic conditions . Until now several experiments have been conducted to study the TME community under different management [Theißen et al . 2010, Toschki et al . 2011] or pesticide application regimes [e .g . Scholz-Starke et al . 2011] . The results did either show no detrimental effect (management) or just short term effects (pesticide application) indicating, that the system itself can be resilient due to external impacts . Nevertheless these studies were conducted under artificial conditions testing a TME community potentially being under disturbance in the new test facility situation . There is no doubt that TME entail some limits inherent to the system . In contrast to the field the horizontal mass flow, the growth of plant roots as well as the emigration/immigration of animals is inhibited . Thus life within TME is constrained to a small scale, potentially selecting organisms e .g . with restricted spatial requirements . In our study we wanted to examine the resilience of the former meadow community after its transfer into TME steel cylinders . We present data on the population dynamics of springtails and mites in TME and the meadow, where the TME we cored from . Results shall contribute to a discussion to what extent TME can reflect natural conditions . Theißen, B ., Scholz-Starke . B, Hammers-Wirtz, M ., Schäffer A ., Kölzer, U ., Leicher T ., Roß-Nickoll M . (2010): Does “sequentially sampling”influence population dynamics of soil mesofauna in TME? Poster, SETAC Europe 20th Annual meeting 23 . - 27 . May 2010, Sevilla, Spain . Toschki, A ., Hammers-Wirtz, M ., Hoelscher, C ., Ross-Nickoll, M ., Theissen, B . (2010): Requirements for Terrestrial Model Ecosystems (TME) in Environmental Risk Assessment - influence of coring sites and management on microarthropod communities . Poster SETAC Europe 21st Annual meeting 15 .- 19 . May 2011, Milano, Italy . Scholz-Starke, B ., Nikolakis, A ., Leicher, T ., Lechelt-Kunze, C ., Heimbach, F ., Theißen, B ., Toschki, A ., Ratte, H . T ., Schäffer, A ., Roß-Nickoll, M . (2011): Outdoor Terrestrial Model Ecosystems are suitable to detect pesticide effects on soil fauna: design and method development . Ecotoxicology, 20(8):1932-1948 .

MO 266Phosphogypsum as a soil fertilizer: evaluation of the impacts on soil habitat and retention functionsH .O . Hentati1, A .L . Ana2, B .S . Bouguerra1, A .N . Abrantes2, G .F . Gonçalves2, R .J . Rombke3, P .R . Pereira2

1High Institute of Biotechnology of Sfax, Sfax, Tunisia2Dep . de Biologia, Fac . Ciências, Universidade do Porto & CESAM, Univ . Aveiro, Aveiro, Portugal3ECT Oekotoxikologie GmbH, Böttgerstrasse, Flörsheim, Germany, Flörsheim, GermanyPhosphogypsum (PG) is a metal and radionuclides-rich by-product of phosphoric acid production . For every tonne of phosphoric acid made, from the reaction of phosphate rock with acid, commonly sulfuric acid, about 3t of PG are created . There are three options for managing PG: (i) disposal or dumping (ii) stacking and (iii) re-use in agriculture, construction, or landfill . Despite some authors have suggested the use of PG as a fertilizer, to the best of our knowledge, till now any evaluation was made about the real impacts of soil amendment with PG, on soil habitat and retention functions . In this work reproduction assays with earthworms (Eisenia andrei), collembolans (Folsomia candida), enchytraeids (Enchytraeus cryticus) and mites (Hypoaspis aculeifer) and seed germination and growth assays with plants (Zea mays and Lactuca sativa) were performed to assess the effect of soil amendment with 25, 16 .6, 11 .1, 7 .4, 4 .9% of PG, following standard protocols . The standard OECD soil was used as test substrate for these amendments . Further soil elutriates were obtained from OECD soil amended with 6 .25, 12 .5 and 25% of PG, preparing soil suspensions with water in a proportion of 1:4 (m/v) . Assays with the bacteria Vibrio fischeri, the cladoceran Daphnia magna, the green algae Pseudokircheneriella subcapitata and the aquatic plant Lemna minor were performed to assess the toxicity of elutriates, testing a range of dilutions as suggested by standard guidelines . As far as assays with soil invertebrates are considered a similar hormetic pattern in reproduction inhibition was observed for all the species . The number of juveniles produced was significantly lower in soils with the lowest and highest concentrations of PG . No toxic effects were recorded for plants . Results obtained for the aquatic assays, showed no toxicity of all elutriates for L minor, as well as only a slight inhibition in the growth of P . subcapitata . V . fischeri and D . magna were more sensitive . Only the elutriate obtained from soil amended with 6 .25% of PG was not toxic to the bacteria . However, all the elutriates were acutely toxic to D . magna, despite an EC50 close to 100% has been obtained . The results gathered in this study raise concerns about the potential contamination of freshwater resources caused by amendment of soil with PG for agricultural purposes . However, it stills precocious to advice the use of PG for such purpose before assessing cumulative effects of amendment within other aspects .

MO 267Modeling effects of NaCl and of its interactions with organic matter and pesticides towards soil faunaG . Duarte1, S . Chelinho2, R . Pereira3, J .P . Sousa2

1IMAR-CMA, Coimbra, Portugal2IMAR-CMA, Department of Life Sciences, Apartado 3046, EC Univ . Coimbra, Coimbra, Portugal3Dep . of Biology, Fac . of Sciences, Univ . of Oporto and CESAM, Univ . of Aveiro, Aveiro, PortugalSoil salinization consists in the accumulation of salts (mainly NaCl) in the upper layers of the soil by natural processes and human activities, which can cause various problems, including soil desertification . This threat to the soil system become a growing concern in the recent years, mainly in the Pannonian and Mediterranean regions . To complement the published information on the effect of salt as a stressor for soil fauna, avoidance and/or reproduction tests were performed with three soil organisms: Folsomia candida, Eisenia andrei (avoidance tests only) and Enchytraeus crypticus (reproduction tests only) . The effect of NaCl alone (1, 2 and 4g/Kg soil), as well as the interaction between salt and soil organic matter content (1%, 2 .5% and 5%), was tested in these endpoints . Furthermore, the interaction of NaCl with a commonly used agricultural pesticide (Lambda-cyalothrin) was also tested using reproduction tests with F . candida . Generalized Linear Modeling (GLM) techniques were used to predicted avoidance and reproductive responses due to NaCl, its interaction with soil organic matter and also to evaluate the shift in toxicity of the Lambda-Cyalothrin when tested on saline soils . Results showed an increased avoidance response with increasing salinity on both test organisms, with earthworms showing a much higher sensitivity than collembolans . Reproduction was impaired starting at medium salinity (2g NaCl/Kg soil) in F . candida and at low salinity (1g NaCl/Kg soil) in E . crypticus, and ceased completely at high salinity (4g NaCl/Kg soil) . Models for the avoidance response were able to explain only 45% of the variation on both test organisms, and were able only to predict the response up to a certain level of salinity . In opposition, models on reproduction data explained over 70% of the variance in reproduction data . Although NaCl was the major driver of the responses obtained, all models showed a significant response of the OM content and of the interaction between these two factors . Lambda-Cyalothrin caused a dose-response decrease in the reproductive output of F . candida in all NaCl treatments tested but the models showed the positive interaction with salt, with EC50 values increasing from low to medium salinities .

1��SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

MO 268Using the comet assay to determine species sensitivity of earthworms to copperA . Reinecke, F . Fourie, A .J . ReineckeStellenbosch University, Stellenbosch, South AfricaThe endpoints normally used to construct Species Sensitivity Distributions (SSDs), comparing the various sensitivities of species for toxicants, are predominately whole body or life-cycle characteristics . Biomarkers are easy and rapid tests . This study investigated whether a biomarker, the comet assay, could be used to compare species sensitivity between earthworms . This assay, measuring DNA damage in the form of single strand breaks, gives an indication of the genotoxicity of a toxic substance for an exposed organism . Four earthworm species from three different families were experimentally exposed to copper in the form of the fungicide, copper oxychloride . The species used were a lumbricid species, Eisenia andrei, two megascolecid species Perionyx excavatus and Amynthas diffringens and Chilota spp ., an endemic South African species of the family Acanthodrilidae . At toxic concentrations, Cu can be damaging on various levels and, as shown by the results of the present study, also acts as a genotoxic agent . Adult specimens were exposed to the fungicide in artificial OECD soil, with benomyl as positive control . An exposure concentration series of 0, 20, 80, 160, 320 and 640 mg/kg Cu was used . Exposures were done in four replicates for a duration of 14 days . After the exposure period coelomocytes were harvested from the organisms and the cells prepared for the comet assay according to the method of Singh et al (1988) as adapted by Reinecke & Reinecke (2004) . The Tail DNA % parameter was used to determine the comet values . The highest mean damage to the DNA at all exposure concentrations was observed in A. diffringens and the lowest in P. excavatus . EC10, LOECs and NOECs were determined and SSDs constructed using EC10 and LOEC values . These showed that A. diffringens was the most sensitive and P. excavatus the least sensitive species of the four tested . We conclude that a biomarker, such as the comet assay, can give reliable and useable data from which to infer sensitivity differences between species .

MO 269Determining chemical effects on earthworms at the population level through the use of energy budget and individual based modellingA .S . Johnston1, R .M . Sibly1, M .E . Hodson1, P . Thorbek2, T . Alvarez3

1University of Reading, Reading, United Kingdom2Syngenta Ltd ., Bracknell, United Kingdom3EcoRisk Solutions, Norwich, United KingdomStandard OECD test methods for measuring the toxicity of pesticides on earthworms use optimal laboratory conditions to record specific endpoints e .g . mortality, growth and reproduction . These data are compared to exposure values in accordance with EU guidelines for registration of agrochemicals, and the resulting risk quotients are compared to arbitrary safety factors for acute and sublethal risks to earthworms . When these triggers are not passed, field trials are required . Field trials are expensive, time-consuming and variable, providing coarse information about population-level effects . Mechanistic population modelling of organism responses to chemicals has the potential to act as a reliable intermediate stage in higher tier risk assessments and can provide greater resolution to help identify key parameters driving population-level effects . Energy budget models relate individual life cycle processes to one another through energy and mass conservation . Organisms uptake resources from their environment and expend assimilated energy on maintenance, growth and reproduction . The allocation of energy to these different subsystems depends on a combination of environment- and organism-specific conditions . Here we present a general energy budget model, based on broadly accepted fundamental principles . The model is applied within an individual-based model (IBM) framework, which allows individuals to interact with their simulated environment, giving an insight into life cycle processes affected under variable environments and chemical exposure . This enables ecotoxicological inferences to be made about population dynamics under realistic field conditions, as the biology of individuals relates to the ecology of populations through metabolism . Standard toxicity data, including NOEC and EC50 values, can be used as input data to the model, highlighting its usefulness as a method in higher tier risk assessments . We use data for the OECD recommended earthworm species Eisenia fetida to validate the model for use in predicting ecotoxicological effects . We discuss the potential of the model in decision-making for pesticide regulation and environmental management, particularly in comparison to alternative modelling techniques . Future studies aim to utilize the flexibility of the model to extrapolate population-level ecotoxicological effects between the varied ecological niches of different earthworm species .

MO 270Ecotoxicity of phenanthrene and its nitrogen-containing analogues to the earthworm eisenia fetida in soilI .N . AnyanwuLancaster University, United Kingdom, Lancaster, United KingdomMajor sources of these homocyclic and N-heterocyclic polycyclic aromatic hydrocarbons (PAHs) are found in the environment being discharged from petrogenic, pyrogenic and biogenic sources . The toxicity of soil amended with phenanthrene a three ringed PAH and its nitrogen heterocyclic aromatics (NHAs) analogues were assessed on the earthworm Eisenia fetida . The effects were studied over a 21-day incubation, during which health and mortality were assessed . During the 60 day incubation, LC50 and EC50 ranged from nd-606 mg/kg and 4 .8-574 mg/kg respectively . NOEC levels were derived from EC10 values: estimated NOECs ranged from 0 .01 to 204 mg/kg . LC50 and EC50 results were compared to other studies from literature and our results differed significantly . The results of this study showed that NHAs were more toxic in soil than homocyclic PAHs (with aging) . This can be attributed to their higher solubilities and lower Kows . Based on visible pathological changes, NHAs were not only carcinogenic, mutagenic, teratogenic and genotoxic, they may also have been clastogenic to soil organisms especially benzo(h)quinoline . These differences might be useful in soil contamination / risk assessment procedures and also provides basis for regulatory action .

MO 271Toxicity to the earthworm Eisenia fetida of two transformation products of amines used in carbon capture technologyC . Coutris1, M .D . Woldehawariat1, D .H . Oughton1, K .E . Tollefsen2

1Norwegian University of Life Sciences, Aas, Norway2Norwegian Institute of Water Research, Oslo, NorwayThe aim of carbon capture and storage technology is to minimize greenhouse gas emissions through a reaction between amines and effluents from gas power plants . During the overall process of CO2 capture, amines and their transformation products might escape to the environment through emissions, leakage, and as solid waste . Although amines in some applications are known to represent a health risk, there is still a lack of knowledge on environmental hazards and risks related to amines and derivatives used in CO2 capture . The aim of this study was to assess the acute and chronic toxicity to the earthworm Eisenia fetida of two transformation products of amines, dimethylnitramine (DMNA) and 2-(nitroamine) ethanol (NAE), following the OECD guidelines 207 and 222 . Acute tests demonstrated that neither DMNA nor NAE induced acute toxicity in earthworms, even at a maximum concentration of 1000 mg/kg dry soil . Therefore we conducted a reproduction test at 1000 mg/kg only, as recommended by the OECD guideline 222 . At 1000 mg/kg, both DMNA and NAE impaired the reproduction of Eisenia fetida, although NAE was more toxic than DMNA . In comparison to control worms, the production of cocoons was reduced by 30% and 70% by DMNA and NAE, respectively . The production of juveniles was reduced by 20% and 80% by DMNA and NAE, respectively . NAE induced a reduction in hatchability by 35% . In contrast, DMNA did not reduce hatchability but delayed hatching, as shown by the smaller size of juveniles . Finally, the number of juveniles per cocoon was slightly higher (although not statistically significant) when worms were exposed to DMNA . Evidence of reproductive toxicity at a single high concentration suggests assessing the effects of these compounds at several lower concentrations in order to get the ECx required for risk assessment .

MO 272New earthworm endpoints based on coelomocitesW .D . Di Marzio, S . Curieses, M .E . SáenzCONICET - UNLU, Luján, Argentina Earthworms are used as test organisms in environmental pollution assessment . Used in studies of acute and chronic toxicity determining how response variables such as mortality, growth and reproduction . In addition the group offers ability to make assessments of biomarkers of oxidative stress and genotoxicity . In the last case coelomocites isolated by extrusion of the coelomic liquid are frequently used . Among them there are three main groups with different functions: eleocitos, amebocytes and granulocytes . The relative proportions of these cell types in the body are related to nutritional and physiological status . The cultivation of these animals in the laboratory is relatively simple and is a means to obtain cells in a non-invasive or destructive method . In this work we explored a set of indices that allow us to recognize the trophic status of individuals prior and during their use in ecotoxicological studies . This set of endpoints include: Whole Body Coelomocites Index (WBCI): log(10) (Cn/wwf) where Cn is total coelomocites number average per individual / mL of celomic fluid; and wwf is wet weight without feces . Absolute Trophic Index Earthworm (ATIE): (En/Cn) where En is total eleocites number average per individual / mL of celomic fluid . Relative Trophic Index Earthworm (RTIE): ATIE/wwf .

MO 273Effects of the use of onshore drilling cuttings as soil amendment on reproduction of Eisenia andreiM .O . Bianchi1, H .M . Machado2, M .E .F . Correia1, M .B . Amaral Sobrinho2

1Embrapa, Seropédica-rj, Brazil2Soil Department, Universidade Federal Rural do Rio de Janeiro, Seropédica-rj, BrazilThe onshore procedures of an oil drilling well generate a great amount of drilling cuttings that are composed of rocks, sand, oil residues and drilling fluid . The cuttings volume produced by an oil well, estimated form 0 .6 to 6 barrels, depends on reservoir depth, diameter, geological features and drilling fluid type . The drilling cuttings are treated to recover the fluid, using different equipment, as cuttings dryer and centrifuge . A common drilling fluid in Brazil contains barium sulfate (Ba SO4), what requires an appropriate disposal or safely utilization . One alternative use is as soil conditioner to increase pH in acidic soils . However, the cuttings have other potential contaminants as arsenic, lead, molybdenum, zinc and sodium that can act in a synergistic harmful way to soil organisms . The objective of this study is to test mixtures of drilling cuttings and soil to provide information about the potential toxic effects of this residue to soil non-target organisms . Reproduction assays with Eisenia andrei were performed using two types of cuttings: from the drier and from centrifuge, with artificial OCDE soil (10% organic matter content) . The barium doses tested were: 300, 900, 1500, 2100, 2700 e 3000 mg Ba .soil kg-1, which correspond to critical values determined by Brazilian law . Results demonstrated that the waste from the cuttings drier (EC 50 = 114 mg Ba .soil kg-1) had a more deleterious effect to E. andrei reproduction than the waste originated in centrifuge (EC 50 = 797 mg Ba .soil kg-1) . A more detailed ecotoxicological study has to be carried out to ensure the environmental safety of the cuttings as soil conditioner in agriculture .

1�4 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

MO 274Methodological improvements in the study of pollutant-induced toxicity on the earthworm Eisenia andrei: the effects of chromium(VI) on coelomocytes physiologyS . Sforzini, M . Boeri, S . Olivieri, A . ViarengoUniversity of Piemonte Orientale, Alessandria, ItalyOver the last decades, the widespread use of chromium by industries has led to numerous cases of environmental contamination by this element . In mammal cells, Cr(VI) is reduced to Cr(III), a process during which reactive Cr(V/IV) intermediates and reactive oxygen species (ROS) are generated . Cr(VI) is widely known to cause toxic and genotoxic effects in vertebrates . Relatively less investigated are the effects of Cr(VI) on invertebrates, particularly on the edaphic organisms . In vitro and in vivo studies were performed on Eisenia andrei to investigate the biological effects of environmentally relevant concentrations of Cr(VI) . In this study, we show the results obtained on coelomocytes of earthworms exposed for 1 and 3 days to different sublethal concentrations of Cr(VI) using the filter paper test . Coelomocytes are the cells circulating in the coelomic fluid and playing an important physiological role in the immune defence of earthworms . Cr(VI)-induced oxidative stress was investigated by evaluating the radical production using fluorescent probes, the lysosomal accumulation of lipofuscin and the oxidative damage to DNA . Moreover, to highlight changes in cellular physiology, mitochondrial activity and lysosomal membrane stability were analysed . Pathological reactions involving the lysosomal system are often linked to augmented autophagic sequestration of cellular components . In situ FITC diacetate labeling of intracellular proteins was performed to assess the transport into autophagic degradative compartments of these proteins . In addition, the leakage of lactate dehydrogenase in the coelomic fluid was determined to reveal cell membrane damage . Finally, the immune response of coelomocytes was studied assessing the phagocytic activity of the cells . The results demonstrated significant changes in the different parameters evaluated on coelomocytes of treated earthworms . Cr(VI) provoked, just 1 day after the exposure to lower doses, an increase in ROS levels, generating in the cells oxidative stress conditions leading to lipid peroxidation with lysosomal lipofuscin accumulation and oxidative damage to DNA . Cr(VI) also caused alterations of intracellular compartments till to reduce, at the highest dose and at the longer exposure time, the immune response of the cells . Overall, the data demonstrated that oxidative stress is an important factor involved in Cr(VI)-induced toxicity and genotoxicity in E. andrei coelomocytes .

MO 275An exposure regime that brings more realism in the testing of plant protection products in soil?G . Ernst1, M . Bergtold2

1Bayer CropScience, Monheim, Germany2BASF SE, Limburgerhof, GermanyA modified exposure regime for Plant Protection Products (PPP) in soil eco-toxicological test systems (e .g . earthworm, collembola, or predatory mite reproduction test) is proposed in order to achieve more realism in the risk assessment . Beside active substances, the toxicity of PPP is often influenced by highly volatile solvents in formulations (i .e . in emulsion concentrates), which degrade fast and occur only in the top soil layer . In the risk assessment the Predicted environmental concentrations in soil (PECsoil) are compared to the endpoint in the laboratory test and a safe use is indicated if an assessment factor of 5 is surpassed . For persistent active substances (a .s .) the PECsoil consists of the plateau concentration (only the a .s .) and the initial concentration (the a .s . within the product) . However the potential effects of the formulation inerts within the end use product are limited in space and time . Thus, the risk of PPP to soil organisms may not be correctly reflected if a) the NOEC is related to a PECsoil calculated for 0-1 cm soil depth and b) if the end use product is evenly distributed in the soil layer to establish the test concentration in the laboratory test . In this study the effects of a PPP (fungicide; EC formulation with 2 persistent a .s .) on the reproduction of Eisenia fetida (OECD, 222) will be determined according to the currently valid test design and compared to a more realistic exposure scenario . The realistic exposure regime considers a plateau concentration (adjusted with the technical grade a .s .) at 0-5 cm soil depth . The test organisms are inoculated and the end use product is applied by overspray application . Finally, the total concentrations of the a .s . in the treatment groups can be calculated as the sum of two components: a) the plateau concentration and b) the calculated concentration from the overspray application, considering a soil bulk density of 1 .5 g/cm3 and a soil depth of 0-2 .5 cm or 0-1 cm, depending on the KOC-values of the a .s . This modified test design considers two aspects improving the realism of exposure of PPP in soil eco-toxicological test systems: 1) a more realistic exposure of soil organisms to formulation inerts and 2) a more realistic distribution of the PPP in the uppermost top soil layer, considering the specific sorption properties of the a .s . This modification would improve the realism without losing the conservativeness necessary in a tier I test system in the registration process for PPP .

MO 276Introducing the ‘ResOrgRisk’ Project: environmental risk assessment of the use of organic residues as soil amendmentsP . Alvarenga1, P . Palma2, A .C . Cunha-Queda3, E . Duarte3, A .P . Chung4, P . Morais4, T . Natal-Da-Luz4, J .P . Sousa4

1Instituto Politécnico de Beja, Escola Superior Agrária, Beja, Portugal2Polytechnic Institute of Beja - Agricultural Superior School, Beja, Portugal3DCEB - UIQA - Unidade de Investigação Química Ambiental, ISA (UTL), Lisboa, Portugal4IMAR - CMA, Department of Life Sciences, University of Coimbra, Coimbra, PortugalThe amount of organic residues (ORs) generated worldwide is increasing and environmentally sound strategies for their disposal and recycling must be developed and optimized . Council Directive 1999/31/EC has set down strict mandatory targets for reduction in the amount of ORs reaching landfill; by keeping organic matter away from landfills, their nutrients can be recycled, less methane is emitted at the landfill, and leachates produced are less polluted . Recycling ORs (e .g . sewage sludge, compost, agro-food industrial wastes) in agriculture is considered a way of maintaining or restoring the quality of soils, enlarging the slow cycling soil organic carbon pool . However, a wide variety of undesired traits, such as heavy metals and organic contaminants, can have adverse effects on the environment . In fact, the Proposal for a Soil Framework Directive recognized that “soil degradation or soil improvements have a major impact on other areas, ([3DOTS]) such as surface waters and groundwater, human health, climate change, protection of nature and biodiversity, and food safety” . Taking that in account, the aim of the Project “ResOrgRisk”is to assess the environmental risk of the use of ORs as soil amendments, evidencing their benefits and constraints, and emphasizing which tests are more trustworthy to assess their quality and the environmental risk of their application . The study will rely on integrated results obtained by a multidisciplinary team based on: (1) Chemical, biochemical, microbial and ecotoxicological characterization of the ORs, of soils amended with ORs, and of their leachates/extracts; (2) Ecotoxicological and ecological evaluation of effects and fate of pollutants derived from the application of different ORs to agricultural soil using TME’s (Terrestrial Model Ecosystems) experiments; (3) Evaluation of chemical, biochemical, microbial, ecotoxicological and ecological effects of the application of ORs to soils in field experiments . Classical and instrumental chemical analysis will be used to quantify physico-chemical characteristics of the materials . Ecotoxicity tests will be used to provide an integrated measurement of both environmental and toxicological bioavailability of the contaminants, and their detrimental effects in the ecosystem . The effects in soil microbial communities will be assessed evaluating microbial indicators, and using molecular methodologies .

MO 277Validation of the Czech approach to dredged sediments ecotoxicity assessmentJ . Vasickova1, J . Hofman1, M . Sanka1, P . Cupr1, R . Vacha2, J . Priessnitz1, M . Dymak1, J . Lana1, L . Skulcová1, L . Sindelarova1

1Research Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech Republic2Research Institute for Amelioration and Soil Protection, Prague, Czech RepublicResearch is focused on the actual environmental issue - a dredged sediment application on soils and preceding evaluation of their ecotoxicity to prevent possible ecological risks . High volumes of sediments are removed from rivers and ponds routinely . Although they may have a lot of positive properties (e .g . high nutrient levels) there are reasonable suspicions about contaminants present accumulated sediments . Therefore, an assessment scheme is necessary which helps to decide, if the land application will be allowed for every dredged sediment . This assessment cannot be based only on chemical analyses and should be accompanied by ecotoxicological testing . In the Czech Republic, novel directive 257/2009 Coll . determines the limits for the dredged sediments to be applied on agricultural soils . There are also 4 ecotoxicological bioassays suggested in the directive . In our study, we verified practicability and reasonability of ecotoxicological testing of dredged sediments using thirty-six sediment samples and wide battery of ecotoxicity bioassays, both for solid and eluate samples . The aim was to compare directive bioassays with other soil bioassays (for example, faster and more practical ones) and to compare bioassays of eluates with the soil bioassays .

MO 278Selection of relevant ecotoxicity tests to assess the effects of biosolids used in agricultureP . Huguier1, N . Manier1, L . Chancerelle1, P . Pandard1, P . Bauda2

1INERIS, Verneuil-en-halatte, France2LIEBE, Metz, FranceIn modern agriculture and in accordance with environmental sustainability, biosolids are applied on fields . These materials are used as fertilizers, to maintain a high production rate of plants . But if they are a source of nutrients, they can also be potentially a source of pollutants . As a consequence they can affect plants, soil fauna and finally humans . In this study, environmental effects of different biosolids (sewage sludge, manure, composted materials [3DOTS]) were studied using ecotoxicity tests on soil and aquatic organisms . The aim of this work was to establish a test battery to assess the effects of biosolids, by comparing their respective sensitivities towards these materials . Organisms composing the biotest battery and their respective acute and/or chronic endpoints are the followings: - algal growth inhibition of Pseudokirchneriella subcapitata (NF EN ISO 8692) - inhibition of growth and reproduction of the nematode Caenorhabditis elegans for solid and liquid media (ISO 10872) - inhibition of the mobility of the crustacean Daphnia magna (NF EN ISO 6341) - inhibition of population growth of the rotifer Brachionus calyciflorus (NF ISO 20666) - inhibition of light emission of the bacteria Vibrio fischeri (NF EN ISO 11348-3) - inhibition of root growth, emergence and growth of higher plants (ISO 11269-1 and ISO 11269-2) - avoidance and reproduction inhibition of the earthworm Eisenia fetida (ISO 17512-and ISO 111268-2) All of these bioassays were conducted using their respective standardized protocol, excepted for C. elegans soil tests which were carried out using the method detailed in another work (in Huguier et al., Applicability of the C. elegans survival, growth and reproduction test to assess the effect of solid wastes; same conference topic) . Concerning samples preparation, standard soils (ISO and/or LUFA) were mixed with biosolids at defined doses (application rate, and e .g . 2 times, 5 times, 10 times, 20 times the

1��SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

application rate) to determine toxicity thresholds . For liquid samples, water extracts of the mixtures were obtained following the standard EN 14735 . Results of a limed sludge, a cow manure, ashes and a compost obtained from domestic wastes will be presented and discussed in details .

MO 279Effects assessment of biosolids soil amendments on terrestrial organismsG . Carbonell1, M .A . Martinez2, M . Torrijos1, E .M . Beltran1, A . de la Torre2, I . Navarro2, M .P . Garcia1, C . del Rio1, J .L . Pareja1, J . Pro1, M .A . Porcel1, C . Fernandez1

1INIA, Madrid, Spain2CIEMAT, Madrid, SpainUse of municipal solid waste (MSW) and sewage sludge composts is a common practice to improve the chemical and biological characteristics of impoverished soils by supplying soil organic carbon content and nutrients . Nonetheless, they not only influence soil fertility, but may also be a route of entry of emerging pollutants to consequently affect soil organisms, plants and microorganisms . Consequently, there is increasing concern for the presence of these pollutants and the need to assess their environmental risk . This work aims to identify the effects of biosolids directly or indirectly on terrestrial organisms and plants, and aquatic organisms, respectively, in order to assess environmental risks and to provide information which may be useful for decision-making support systems on both the national and international regulatory scale . A terrestrial microcosm was carried out with four biosolids (MSW composts, and sewage sludge: compost or thermal drying sludge) obtained from different Spanish solid urban waste treatment plants to assess the effects on organisms . Biosolids application rates were calculated by considering the N requirement of plants and were added to control the soil used to fill the microcosm columns . After 21 exposure days, the effects on soil invertebrates, plants and microorganims were assessed . Acute toxicity tests (Daphnia magna, Chlorella vulgaris) were also performed on leachates . This study was funded by Spanish projects CTM2010-19779-C02-01/02 and RTA2010-00004 .

MO 280Analytical and ecotoxicological characterisation of the biosolid samples collected from Spanish wastewater and municipal solid waste treatment plantsC . Fernandez1, M .A . Martinez2, E .M . Beltran1, M . Torrijos1, I . Navarro2, A . de la Torre2, P . Garcia1, G . Carbonell1

1INIA, Madrid, Spain2CIEMAT, Madrid, Spain Biosolids are end products of wastewater and municipal solid waste (MSW) treatments processes and are, therefore, rich in nutrients and organic matter . Regulations on the presence of pathogens, nutrients and metals in biosolids for use as agricultural soil amendments are clear; however, the contents of organic compounds, such as pharmaceutical, personal care products (PCPs), endocrine disrupting compounds (EDCs) or persistent organic pollutants (POPs), are not yet regulated . Indeed, Spain and other EU Member States have considering reviewing the two European Directives on applications of composted MSW (Directive 86/278/EEC) and sludge WWTPs (2nd Draft of Biological Treatment of Biodegradable Waste, 2001) in soil for agricultural purposes to establish new limits for the pollutants first considered and to increase the number of analysed substances as regards new emerging organic compounds . To assess the potential environmental risk of biosolids, it is necessary to know their chemical composition and their effects . This study aims to obtain information about the analytical and ecotoxicological assessment of 16 biosolids collected from different Spanish wastewater and MSW treatment plants as a previous step for use in microcosms and field studies . The results of the analysed samples show similar ranges to those found in references from other works using biosolids collected in other countries .

This study was funded by Spanish projects RTA2010-00004 and CTM2010-19779-C02-01/02

MO 281Ecotoxicological fate and effects of German river sedimentsS . Gartiser1, C . Hafner1, C . Hercher1, M . Garcia-Kaeufer1, S . Peddinghaus2, S .H . Keiter2, H . Hollert2

1Hydrotox GmbH, Freiburg i .br ., Germany2RWTH Aachen University, Aachen, GermanySediments are well-known to act as a sink and secondary source for environmental pollutants . The joint research project DanTox, funded by the German Federal Ministry of Education and Research (http://www .bio5 .rwth-aachen .de/DanTox, 2009 - 2012) aims to develop combined vertebrate-based sediment contact assays to investigate teratogenicity, neurotoxicity, genotoxicity, mutagenicity and Ah-receptor agonists as well as to analyze the gene expression (microarray and qRT-PCR) in embryos of the zebrafish (Danio rerio) . For this purpose, three model sediments were selected, two from the Rhine River (Altrip and Ehrenbreitstein) and one from the outer Vering Canal in Hamburg . First, the sediments have been ecotoxicologically characterized with standardized routinely applied bioassays . This is important for ranking the results achieved with the newly developed methods in the context of routinely applied assays . The poster presents results from the bioassay battery applied . Toxicity of sediment soxhlet extracts transferred into DMSO (20 g mL-1) has been determined in the Algae growth inhibition test (OECD 201, 2006), the Daphnia acute toxicity test (OECD 202, 2004), the Fish acute toxicity test (OECD 203, 1992), the Fish egg test (ISO 15088, 2007), the Lemna growth inhibition test (ISO 20079, 2005), the Luminescent Bacteria test (ISO 11348, 2007), the Ames test (ISO 16240, 2005) and the umuC test (ISO 13829, 2000) . Freeze dried sediments have been investigated in the Fish egg sediment contact assay . Genotoxicity was evaluated in the Comet assay with zebrafish eggs exposed for 48h post fertilization . In addition, toxicity of freeze dried sediments towards bacteria was investigated using the Arthrobacter contact test (ISO/DIS 10871, 2009) . Algae turned out to be the most sensitive species . EC50 values for all three sediments were in the range of 1 mg SEQ/L (sediment equivalent) . Daphnia and fish were at least 10 times less sensitive . Two sediment extracts (Altrip and Vering Canal) were found to be mutagenic in the Ames test . One sediment (Ehrenbreitstein) turned out to be genotoxic in the umuC test . The results obtained so far do not indicate genotoxic effects with the Comet assay performed with zebrafish eggs . For several assays, DMSO concentration in the test solution was limiting the determination of ECx values . Here, only limit tests with the highest possible DMSO concentration could be performed . Analytical data are available for all sediments tested .

MO 282State of the art of soil ecotoxicology standards in Brazil: challenges and perspectivesL .S . SisinnoSoloTox Consultoria, Rio de janeiro, BrazilSoil ecotoxicology in Brazil had a conceptual mark in 1988 when it was published by the extinguished Special Office of Environment (SEMA) the Handbook of Tests for the Ecotoxicity Evaluation of Chemicals . In that document it was described a single method for soil organism toxicity evaluation: a method (Norm X31-250, 1984) using earthworm (Eisenia fetida) based on AFNOR (ASSOCIATION FRANÇAISE DE NORMALIZATION) . This method was applied during several years for pesticides registry and the study of pesticides ecotoxicity was also the major interest in soil ecotoxicology of the Brazilian researches during the last years . In 2002 it was created the Special Committee of Ecotoxicological Analysis of ABNT (Brazilian Association of Technical Standards), an organization member of ISO - International Organization for Standardization - responsible for the development of Brazilian Standards in many areas . The Committee of Ecotoxicological Analysis was created based on the increasing demand related to quality and new tests, for example, using soil organisms . In 2004 an increasing in soil ecotoxicological studies was noted in Brazil mainly because of the rising of academic and research groups in Brazil together with Germany and Portugal . So, in 2006 ABNT Committee wrote the first Brazilian Standard related to terrestrial ecotoxicology and soil quality (ABNT NBR 15537:2007, acute toxicity test with earthworm), based on the translation of ISO 11268-1:2003 . Nowadays there are six Brazilian Standards published (tests with earthworms, collembolan, and soil flora; guidance for ecotoxicological characterization of soil and soil materials) based on ISO Standards (ISO 11268-1:2003, ISO 17512-1:2008, ISO 11267:1999, ISO 11269-2:2005, ISO 17616:2008, ISO 15799:2003) . The work will continue and the group intends to translate ISO Standards that could be applied on contaminated sites, an increasing problem in Brazil . These standards will be the basis for the development of new researches and tropical findings that would be incorporated in the next revisions . The members of ABNT Committee of Ecotoxicological Analysis believe that this is just the first step because a lot need to be done but the availability of standards written in portuguese and acquired with local money could be an important way for the development of soil ecotoxicology studies in Brazil .

MO 283Derivation of soil screening levels (SSLs) for Uranium, Cooper and Cadmium using a natural reference soilA .L . Caetano1, C . Gavina1, F .P . Carvalho2, J .P . Sousa3, F .J . Gonçalves1, E . Ferreira Da Silva4, R . Pereira5

1Departamento de Biologia & CESAM, Universidade de Aveiro ., Aveiro, Portugal2Instituto Tecnológico e Nuclear (ITN), Sacavém, Portugal3IMAR-CMA, Departamento de Ciências da Vida, Universidade de Coimbra ., Coimbra, Portugal4Departamento de Geociências, Universidade de Aveiro, GeoBioTec Research Center, Aveiro, Portugal5Departamento de Biologia da Faculdade de Ciências da Universidade do Porto, Porto, PortugalAimed in evaluating and managing land contaminated with metals, many countries have been adopting generic quality standards, the soil screening values (SSV) . SSVs are particularly useful for the first tier of Ecological Risk Assessment processes applied to contaminated sites, allowing decision-making in this initial stage of assessment, based on the chemical line of evidence . Due to the influence of soil properties in the toxicity of metals, SSVs derived based on specific soils could not be appropriate for site-specific assessments . Hence, a Portuguese natural soil (PTRS1), representing one of the dominant types of soil from a granitic region (cambisol), was characterized and used as substrate for ecotoxicological tests aimed at deriving SSVs for national assessments of contaminated sites . An extensive chemical characterization was initially done in terms of pseudo-total metals, PAHs, PCBs and pesticide contents . The performance of invertebrate (Eisenia andrei, Folsomia candida, Enchytraeus crypticus) and plants species (Avena sativa, Lycopersicon esculentum, Zea mays, Lycopersicon esculentum) was also tested to evaluate the performance of test species in this natural soil . After this deep characterization, the soil was used to perform sub-lethal ecotoxicological tests with the same species of invertebrates and plants aimed at deriving NOEC, LOEC, EC20 and EC50 values for uranium, copper and cadmium . Eisenia andrei proved to be less sensitive than Folsomia candida to uranium contamination . For plants, since concentrations till 1000 mg Kg-1 were tested, we can say that uranium does not yield a toxic effect in the endpoints evaluated in these species . NOEC values of 101 .69 mg Kg-1 and EC50 values of 131 .23 mg Kg-1, were recorded for copper in the reproduction of the E. Andrei. Folsomia candida showed to be less sensitive than E. andrei to copper when reproduction was assessed (EC50 163 .3 mg Kg-1) . All species revealed to be high sensitive to cadmium contamination . The EC20 values estimated using the logistic model were remarkably lower than the NOEC values, hence they were selected for the derivation of more protective soil screening values, which were determined integrating all the ecotoxicological data obtained .

MO 284Environmental risk assessment in the São Domingos Mine area (Portugal). Tier I: screening phase

1�6 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

L .C .I . Oliveira Filho1, C .S .R . Pereira2, A . Petrucci2, D . Baretta1, J .C .P . Santos1, R . Pereira3, M . Moreira-Santos2, T . Natal-Da-Luz2, A . Ferreira2, R . Ribeiro2, J .P . Sousa2

1UDESC, Lages, Brazil2IMAR-CMA, Department of Life Sciences, University of Coimbra, Coimbra, Portugal3Dept . Biologia, Univ Porto & CESAM, Univ Aveiro, Porto, PortugalThe present study constitutes the Tier 1 of the site-specific ecological risk assessment (ERA) of the São Domingos Mine (South of Portugal), integrating information from the chemical line of evidence (metal analysis and calculation of toxic pressures) and the ecotoxicological line of evidence (Vibrio fischeri solid-phase test, Folsomia candida avoidance test and Avena sativa seed germination test), which were integrated to calculate risk values . A total of 62 soil samples were collected along six transepts perpendicular to a central axis (CA) which was coincident with the inactivated railway line that was used for ore transport across the area; at each transept samples were taken at 0, 20, 50, 150, 400, and 1000 m distant from the CA, both on left and right sides . First, a multivariate factor analysis was run on various soil properties (silt/clay, fine sand, coarse sand, and organic matter contents, conductivity, water holding capacity, and pH) to group the soils . Four groups of soils were defined indicating the need to use a multi-reference approach . To comply with the latter three additional soil samples were collected (at 15 to 20 Km from de CA), to be used as reference material, together with a soil from one of the transept points . Total metal and arsenic concentrations showed high heterogeneity among samples with most points along the CA exceeding the benchmark values . Results showed that the ecotoxicological tests used were adequate to assess the ecotoxicological potential of the soils collected . As predicted, integrated risk values decreased with increasing distance from the CA . The importance of using a multiple test battery to screen the toxicity of soil as Tier 1 of a site-specific ERA was demonstrated since it allowed clarifying some false positives or false negatives revealed by chemical analysis on its own . However, the risk uncertainty still persisting at some points highlighted the need for a more detailed evaluation, which will constitute the next stage of the risk assessment (Tier 2) .

MO 285The impact of red mud on reproduction of the collebolan Folsomia candidaM .O . Bianchi1, M .E .F . Correia1, A .S . Resende1, E .F .C . Campello1, J .P . Sousa2

1Embrapa, Seropédica-rj, Brazil2IMAR-CMA, University of Coimbra, Coimbra, PortugalRed mud is a residue generated from the refining of bauxite for alumina production (Al2O3) by the Bayer process . The main features of this residue is the high alkalinity, high salt contents and the clay texture . Brazil is the third largest producer of alumina, resulting in an estimated red mud production up to 16 million tons each year . The disposal of this residue is done in large sealed reservoirs that become inappropriate for any kind of use . The treatment and utilization of a high volume of the red mud waste as an alternative to conventional disposal has been a challenge for the alumina industry around the world . In Brazil there is a proposal to process the waste to reduce its alkalinity and sodium contents, what enables it´s return to the original mine sites . This will require a variety of tests, including soil ecotoxicological evaluation to ensure environmental safety . The objective of this study is to test mixtures of red mud and soil to provide information about the potential toxic effects of this residue to soil non-target organisms . Reproduction assays with Folsomia candida were performed using two types of mixtures: with artificial OCDE soil (5% organic matter content) and with a natural soil from Poços de Caldas municipality, Minas Gerais state, Brazil . The concentrations tested were: 0, 2 .5, 5, 10, 20, 30 and 40% of residue (w:w basis) . Results demonstrated that the red mud strongly inhibited the reproduction of F. candida . No juveniles were observed at concentrations of 30% and higher for artificial and natural soils . The high sodium content (Na+ = 49 .19 cmolc dm-3) is probably the most deleterious factor to Collembola reproduction . The EC50 was 9 .36% for OCDE and only 1 .36% for natural soil . This difference indicates that the properties of the natural soil increase the toxicity of the red mud and that strong protective measures should be taken when encapsulating this residue .

MO 286Assessing biological activity of soil organisms in no-till soils under different agricultural practices using the bait lamina methodS . Campiche1, C . Maurer-Troxler2, I . Werner3

1Ecotox Centre Eawag-EPFL, Lausanne, Switzerland2Amt für Landwirtschaft und Natur des Kantons Bern, Bern, Switzerland3Schweizerisches Zentrum für angewandte Oekotoxikologie Eawag/EPFL, Dübendorf, SwitzerlandIn agriculture, application of fertilizers and phytosanitary products varies depending on the cultivation methods used . Sustainable farming practices such as zero tillage increase the amount and variety of life in the soil but may require the use of herbicides . In the present work, the impact of different fertilizer and herbicide treatments on the biological activity of soil organisms of a no-till soil was compared . Using the bait lamina test, the changes in overall feeding activity of the soil biocenosis were measured in an agricultural brown soil (15% clay, 3% humus) with and without application of glyphosate for two types of fertilizers . The experiment was conducted at different exposure times, immediately and several weeks after the herbicide application, at different application rates and with different crops (corn, winter barley) . The results of the bait lamina test show that according to the exposure parameters and application rates of the herbicide, the biological activity of the soil organisms tends to be higher in soil where glyphosate was applied . The bait lamina results were completed by physico-chemical analyses and by reproduction tests with Collembolans on the collected field samples .

MO 287Effects of contaminated soils from North Africa on enchytraeids and predatory mites in standard laboratory tests. II. The case of a Phosphogypsum plant near Sfax (Tunisia)J . Roembke1, C . Bandow1, N . Märker1, A . Haddioui2, M . Ksibi3, R . Pereira4, J .P . Sousa5

1ECT Oekotoxikologie GmbH, Floersheim, Germany2University of Sultan Moulay Slimane, Beni mellal, Morocco3Laboratory of Water, Energy & Environment, Sfax, Tunisia4University of Porto & University of Aveiro, Porto, Portugal5University of Coimbra, Coimbra, PortugalMining activity is an important economic activity in several NATO and Mediterranean Dialogue Countries . Within their territory derelict or active mining explorations represent serious risks to surrounding ecosystems; nevertheless engineered-based remediation processes are usually too expensive to be an option for the reclamation of these areas . Thus, in order to find a more eco-friendly solution for mining areas, mainly related with uranium and phosphorus exploration, experts in Ecotoxicology, Chemistry, Plants Physiology, Microbiology and Molecular Biology from Portugal, Germany, Tunisia and Morocco, have joined together in a project funded by North Atlantic Treaty Organization (NATO) . As part of this project the risk of contaminated soils to selected soil organisms has been assessed . In this contribution, it is asked: Does the contaminated soil of a still active phosphogypsum plant located in Sfax (Tunisia) affect the reproduction of enchytraeids (Enchytraeus bigeminus) and predatory mites (Hypoaspis aculeifer)? For this purpose soil samples were taken at 15 plots along five transects encircling the main storage area of phosphpgypsum . These soils were characterized pedologically (e .g ., the pH of all soils was in the range between 6 .0 and 8 .5) and chemically . Few plots showed meaningful metal contamination (mainly copper) but all soils contained high concentrations of hydrocarbons and salt . The reproduction tests with the enchytraeids and mites were performed following standard laboratory test protocols according to OECD guidelines 220 (2004) and 226 (2008) . The predatory mites were less affected (at 8 plots the reproduction was almost similar to the control), while no or very few juveniles were found at the remaining plots . The number of juveniles of E . bigeminus was lower than the control at all plots but almost zero at 11 out of 15 plots . These results are probably caused by a complex interaction between soil parameters, contaminants and the salt content .

MO 288Assessing pesticide use impacts on soil compartment at a Brazilian potato and strawberry producer regionM .E .T . Nunes1, E . Espíndola2, B .K . Rodrigues3, N .C .D .E . Lima4, T .G .T . Decina1

1Escola de Engenharia de São Carlos/Universidade de São Paulo, São carlos, Brazil2University of São Paulo, São Carlos, Brazil3Universidade Federal de São Carlos, São carlos, Brazil4Petrobras, São paulo, BrazilInterviews with potato and strawberry producers from Bom Repouso, MG, Brazil, showed intense and inappropriate utilization of pesticides . Local soil samples presented residue of aldrin, hepthaclor, hepthaclor epoxide, chlorpyriphos and carbofuran (potato soils); aldrin, hepthaclor epoxide, endosulfan-I, carbofuran and abamectin (strawberry soil); and aldrin, hepthaclor, hepthaclor epoxide, endosulfan-II, λ-BHC, chlorpyriphos, abamectin and carbofuran (preserved forest soils) . Retention function tests with D . rerio showed low acute toxicity of soils percolates, but organisms presented lower growth and gill histopathological alterations . Direct effects on soil organisms of a commercial formulation of abamectin, the most mentioned product, were also analyzed, through acute, chronic (reproduction) and behvioral (avoidance) tests with E . andrei in a tropical artificial (TAS) and a natural (NS) soils, in addition to an multi-species assay . The LC50; 14d for E . andrei in TAS tests was 7 .64 mg abamectin/kg of soil . Chronic test showed significant reduction of earthworms survival (28 days) with 10 .5 mg abamectin/kg of TAS . Morphological (threadlike and colorless posterior region; constrictions on different body regions; fragmentation and loss of segments) and behavioral alterations (lethargy or slowness to respond to mechanical stimuli) and weight loss occurred, and were correlated to the increasing pesticide concentrations . Average number of juveniles was 33 (Control); 3 (0 .875 mg of abamectin/kh of TAS) and 0 (other treatments) . Earthworms avoided contaminated soils, with significant difference from control, from 1 .75 mg abamectin/kg of TAS . There was no significant response of the earthworms to any parameter in SN tests . Multi-species tests on SN soil showed significant effects on density of organisms, higher in control, which showed higher diversity and richness indexes for edaphic fauna, and small dominance index, compared to treatment with abamectin .

MO 289Collembola community response to petroleum hydrocarbon contaminants in sub-Antarctic soilsT .J . MooneyUNiversity of New England and Australian Antarctic Division, Kingston, tas, AustraliaA series of fuel spills (diesels and lubricating oils) have occurred on sub-Antarctic Macquarie Island associated with fuel storage and power generation for the Island’s research station . The Australian Antarctic Division is currently bioremediating these sites through the addition of air and nutrients to the soil . To contribute to the development of appropriate remediation targets, this study examines responses of Collembola to Special Antarctic Blend (SAB) diesel . As organic carbon plays a crucial role in determining bioavailability of organic contaminants in soils, Collembola community response to SAB was examined in 30 plots (1 m²) across a natural organic carbon gradient . Seven pairs of soil cores (75 mm wide x 70 mm deep) were sampled from each plot and spiked with SAB by injection (7 concentrations, 0 - 10,000 mg/kg) and incubated under field-simulated conditions (8°C, 16 h light / 8 h dark) for 15 days . At the end of the exposure period, surviving Collembola were extracted using a temperature gradient

1��SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

extraction cabinet . Collembola were counted and identified, and matched cores were analysed for physical and chemical properties . Community assemblage response to SAB contamination and interactions with TOC and other physical and chemical variables were determined . The Collembola species richness decreased with increasing SAB concentration, with the Shannon-diversity index decreasing from 1 .5 to 0 .02 . Sensitive and tolerant species within the community assemblage were able to be identified for future research . EC50 values for the Collembolan community response to SAB were calculated . This work contributes to a multinational project investigating hydrocarbon contamination in cold region soils aimed at developing site specific clean-up and remediation guidelines .

MO 290Spatial distribution of micro-arthropods in agricultural ecosystemsS . KnaebeEurofins Agroscience Services EcoChem GmbH, Niefern-oeschelbronn, GermanyInformation on species distribution and abundance of individual groups of micro-arthropods is available . The best researched group are the collembola . However, even for this group there is not much knowledge on the spatial distribution on small plots . For the semi field study approach (terrestrial model ecosystem TME) a grass site is used with the smallest sampling area possible . In field studies individual plot sizes of 3 m x 7 m or bigger are used . The aim of this publication is to compare different sampling schemes and their potential for the evaluation of micro-arthropod communities in field trials . Sampling approaches are presented that differ in the mean distance between individual soil core samples . The species abundance, diversity and heterogeneity are evaluated . The results are discussed with respect to field study test designs, sampling schemes and the minimum number of samples that are required to yield a robust dataset .

MO 291Seasonal dynamics of soil micro-arthropod populations in agricultural ecosystemsS . KnaebeEurofins Agroscience Services EcoChem GmbH, Niefern-oeschelbronn, GermanyThe structural and functional protection goals for soil are given in the EU directive for the registration of plant protection products . In the past the impact of a plant protection product on the soils’s ability to turn over organic matter was tested with litter bag studies . Recently the structure of the micro-arthropod community has been the focus for a future directive . The main challenge for field tests with micro-arthropods is the agroecosytem were the tests take place . Reasons for this are the low content of organic matter in the soil, the high degree of soil disturbance (tillage) and the reduced diversity in cultivated fields . Collembola species in the agroecosystem are adapted to the changing environment through a high reproductive potential . Since the abundance of micro-arthropods is a primary endpoint in field studies it is important to have an idea about seasonal population changes over the period of a year . In this publication the seasonal changes in abundance and structure of micro-arthropod communities in two fields are presented over one year . Results are discussed in terms of test design and sampling scheme .

MO 292Effects of different land uses on soil microarthropod biodiversity in a Mediterranean urban contextL . Santorufo1, J . Cortet2, G . Maisto1

1University of Napoli Federico II, Napoli, Italy2Université de Lorraine - UMR INPL/INRA 1120, Nancy, FranceThe majority of human activities frequently lead to the conversion of natural habitats to anthropogenic land uses . Land-use change is an important form of global pressure affecting biodiversity . Unfortunately, little is known about causes and consequences of soil biodiversity loss, although soil biodiversity affects the main regulatory services of the ecosystem . The present study aimed to analyse biodiversity of soil microarthropods in soils with different human impact, in order to highlight how different land-uses affect soil arthropod communities . In addition, the soils were characterized for metal and PAH contents, to evaluate if different land uses cause differences in kind and degree of pollution . Finally, the content of metals and PAHs were related to different taxa of soil arthropods to individuate the taxa more sensitive/tolerant to different pollutants . To this purpose, 18 soil samples, grouped in 4 different typologies (natural, residential, industrial and agricultural), were collected in Naples and surroundings (Southern Italy) . The soils were analysed for physical-chemical properties (pH, water holding capacity, bulk density, porosity, organic matter content, content of nutrients, metals, PAHs) . To evaluate the arthropod biodiversity, the organisms were extracted using Macfadyen method . Abundance of Mites (identified at the sub-order level) and abundance and diversity of Collembola (identified at the species level) were assessed . Preliminary results highlighted that two taxa (Collembola and Acarina) were extremely widespread and abundant in all the investigated soils . Arthropod communities largely differ among the soil typologies .

MO 293Year-round responses of soil invertebrate communities to pesticide applications in mediterranean apple orchardsC .J . Mazzia1, M . Hedde2, M . Richert2, Y . Capowiez2

1University, Avignon, France2INRA, Versailles, FranceApple orchards are highly manipulated crops in which large amounts of pesticides are applied . The aim of the present work is a better understanding of soil invertebrate responses to pesticide use taking account year-round dynamics . The originality of this work is the study of both (i) the within-year gradient and (ii) the between pest management strategies gradient, i .e . abandoned, organic, IPM (integrated pest management) or conventional . Pest managements are characterised by difference in the nature of pesticide (organic orchards) and in the intensity of application (IPM vs conventional) . Arthropods were sampled during fall, spring and summer in 20 apple orchards with different management strategies using six pitfalls in each orchard . In spring, orchards with organic practices had the highest abundance and abandoned orchards the highest taxonomic diversity . Some taxonomic groups present a negative response (isopoda, homoptera, arachnea) or a positive response (carabidae) with the increasing use of synthetic pesticides . These differences were less marked in autumn, which could be a sign of resilience (last pesticide applications occurred in August) . We concluded that not all arthropod families have the same sensitivity to pesticide management . The next step is to determine if difference in sensitivity occurred as well for natural enemies and other kinds of arthropods species .

MO 294Effects of the nitramine explosive CL-20 on the soil invertebrate community in a sandy loam soilR .G . Kuperman, R .T . Checkai, C .T . Phillips, M . SiminiEdgewood Chemical Biological Center, Aberdeen proving ground, United States of AmericaWe investigated the effects of nitramine explosive CL-20 on soil invertebrates in Sassafras sandy loam (SSL) soil using a 12-week soil microcosm assay with the indigenous soil microinvertebrate community . In these studies, freshly-collected SSL soil was amended with CL-20 and thoroughly mixed to prepare individual analytically determined treatment concentrations 0 (carrier control, acetone), 108, 479, 870, 2355, 4635, 7020, and 10300 mg/kg . The selected concentration range of CL-20 adequately assessed the concentration-response relationships for total microarthropods, as well as for individual microarthropod groups in SSL soil, and allowed us to determine group-specific ecotoxicological benchmarks for CL-20 . The overall composition of microarthropod community in SSL soil was not affected by exposure to CL-20, based on the number of taxonomic groups present in the individual treatments after 12 weeks . However, community structure analysis revealed greater sensitivity to CL-20 by predatory mesostigmatid mites . Microarthropod and nematode communities showed contrasting sensitivities to CL-20 in SSL soil . Total numbers of nematodes were either unaffected or significantly (p<0 .05) increased in CL-20 treatments compared with control . Only predator group among nematodes was consistently adversely affected by exposure to CL-20 . The abundance of predatory nematodes decreased in a concentration-dependent manner throughout the 12-week exposure . Overall toxicity data for soil microarthropods and predatory nematodes established in this microcosm assay were comparable with mortality data established in our standardized single-species toxicity tests with the soil invertebrates earthworm Eisenia fetida (ISO 11268-2:1998), potworm Enchytraeus crypticus (ISO 16387:2004), and collembolan Folsomia candida (ISO 11267:1998) exposed to CL-20 in similar SSL soil . Microcosm assay with corresponding community structure analysis can provide the means for validating the ecotoxicity data from standardized laboratory tests, both complimenting and expanding upon the ecotoxicological significance of data from standardized single-species toxicity tests .

MO 295Microarthropod communities in lead contaminated forest soils at active and abandoned shooting rangesS . Selonen, M . Liiri, H . SetäläUniversity of Helsinki, Lahti, FinlandAs a part of a larger ecosystem-scale investigation, microarthropod communities at two shooting range sites with different contamination history and at one uncontaminated site were studied . The three study sites locate in a same pine forest ecosystem; one site situates right behind an active shotgun shooting range and another site locates in a position where shotgun shooting terminated over 20 years ago . The amount of lead pellets in the soil of both of the contaminated study sites is equal, reaching up to four kilograms of pellets per 1 m2 . The overall toxicity of the organic soil layer was higher at the active shooting site than at the abandoned site . In the active shooting range, lead posed negative effects on Collembola, Oribatida, Mesostigmata, Prostigmata and total microarthropod abundances, whereas at the abandoned site the effects were clear in total number of microarthropods, Prostigmata and some separate groups at lower taxonomical level . The microarthropod community structures of the uncontaminated and abandoned shooting sites were quite similar, whereas the microarthropod community at the active shooting site was different from the others . These findings suggest the recovery of the soil system due to accumulation of litter on soil surface after shooting is closed . However, when the microarthropods were sampled separately in fermentation layer and humus layer of the soil, the humus proved to be more toxic at abandoned shooting site than at active shooting site . Total microarthropod, Oribatida and Mesositgmata abundances were lower in the humus layer at the abandoned shooting site than at the uncontaminated site, whereas the abundances of any higher microarthropod taxa in the humus at the active shooting site did not differ from those at the uncontaminated site . Thus, although the soil biota at the upper soil layer appear to recover after cessation of shooting, the dissolution of Pb from the pellets deeper in the soil continues to pose a risk for microarthropod communities .

MO 296What to do when there are too many specimens: two strategies for sub-sampling in Off-crop studies for non-target arthropods

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S . Knaebe, P . MackEAS EcoChem GmbH, Niefern-oeschelbronn, GermanyOver the last four years protocols have been developed to test the influence of plant protection products on arthropods in off-crop areas . However, for some sampling methods, especially suction sampling, in off-crop areas high numbers of arthropods together with high amounts of undesired material (i .e . fine plant material, seeds, litter and soil) are sampled at the same time . This leads in turn to a tremendous work load for sorting and taxonomical determination of the specimens, which rapidly exceeds an acceptable time period for practical and economical purposes . Therefore, procedures need to be developed to efficiently and effectively use the samples with high specimen numbers and a high pollution . Here, two different sub-sampling methods were used for Vortis suction samples from an off-crop field study with high individual numbers (e .g . > 15 000 individuals per sample) . The first method uses a sample divider in four steps . The divider is originally constructed for creating sub-samples of plankton samples . The second method was additionally to set a time limit for sorting the arthropods that are not micro arthropods out of the sample . The aim was to take out at least 80% of the arthropods from the sample before sub-division . As a following step the subsamples were divided by the sample divider before counting the numerous collembolans and mites . To assess the performance of both methods the coefficient of variance for different taxa identified was calculated and compared . There was also an assessment of the sample time for the two methods .

MO 297Computer controlled high gradient extractor for soil organismsG . Henkes, K . Henkes, T . StädtlerRIFCON GmbH, Hirschberg, GermanySoil micro-arthropods, such as Oribatida and Collembola, are important components of the soil decomposer system . This makes the soil micro-arthropod community a valuable indicator of soil quality . Therefore, there will be probably a higher focus on possible impact of plant protection products on soil micro-arthropod communities in the future . For different field study types soil micro-arthropods have to be removed from the soil in order to determine species . The most common method according to ISO 23611 for extraction of soil micro-arthropods from respective samples is the high gradient extractor according to Macfayden (1961) .

The principle of this extraction method is to create an artificial temperature gradient between the canister in which the samples have been placed (hot) and the collecting device below (cold) . The presentation will show a highly sophisticated computer controlled extractor constructed by RIFCON GmbH . In this modified high gradient extractor the temperature can be regulated stepless and monitored in all different compartments, which enables a repeatable and adaptable extraction of soil micro-arthropods . The efficiency of the extractor is verified with Collembola species (Folsomia candida and Heteromurus nitidus) in different soil types and soil humidity regimes . At least 80% of F. candida and 96% of H. nitidus were recovered from soil cores .

MO 298Use of anaerobic digested-based composts as soil organic amendments: effect on the soil biological statusP . Grenni1, I . Nogues1, M .A . Bustamante2, F . Loreto1, R . Moral2, M .P . Bernal3, A . Barra Caracciolo1

1National Research Council, Monterotondo, rome, Italy2Miguel Hernandez University, Alicante, Spain3CSIC, Murcia, SpainClimate changes together with organic matter and plant nutrient loss, low water infiltration and water holding capacity accelerate soil degradation . This is a serious problem for farmers and food suppliers and also for the reclamation of landscapes for nature conservation . Moreover, intensive livestock production has led to a generation of great amounts of animal manures and slurries that can constitute an environmental risk, if they are not managed properly . Consequently, there is a need for more environmentally sound methods for the treatment and utilisation of animal manure . Anaerobic digestion constitutes one of the main alternatives for managing it and is based on the anaerobic conversion of organic matter, obtaining biogas and a digested substrate called digestate . Although the latter presents a high fertilising value, this material shows some characteristics that make necessary to treat it prior to its direct soil application . One option is to separate the digestate into a liquid and a solid fraction, the latter can be composted to obtain valuable and marketable end-products, which can be used with agricultural purposes or for soil restoration . Within this context, different studies have shown that compost application increases soil organic matter content, improving physical, chemical and biological soil properties and thus, preventing soil erosion . The general aim of this work was to evaluate if the addition of two anaerobic digestate-based composts to a soil with a low C content could improve the overall microbiological activity and the C pool conservation . For this purpose, two composts (C1, mainly composed by cattle manure anaerobic digestate and C2, mainly composed by pig slurry anaerobic digestate), at two different rates (30 t/ha and 60 t/ha respectively) were incorporated into a semiarid soil from central Italy . Two other additionally treatments were also considered: the unamended soil (control) and a mineral fertilised soil . Subsequently, several plants of rosemary (Rosmarinus officinalis) were planted on these soils . The efficiency of the treatments was evaluated by analyzing soil physico-chemical and microbiological properties, such as soil respiration, microbial biomass and soil bacterial community activities, such as dehydrogenase and bacterial viability .

MO 299Evaluation of interactive toxic effects of binary metal mixtures to the bioluminescent fungus Gerronema viridilucensF .F . Ventura, L .F . Mendes, E .L . Bastos, C .V . StevaniUniversidade de São Paulo, São paulo, BrazilFungal-based assays can be used to assess the toxicity of environmental samples of polluted areas . Toxicity studies with binary mixtures of metals are more realistic than those with single metals as interactions between the species can interfere with the toxicities . Currently, there is no work on the combined toxic effect of metal binary mixtures to fungi, despite the importance of these organisms in soil nutrient cycling . This work reports the use of a fungal-based luminescent assay using the Brazilian bioluminescent fungus Gerronema viridilucens to assess the toxicity of binary mixtures of metal cations (Ca+2, Cd+2, Cu+2 and Zn+2) . The toxic unit concept was used to calculate the EC50 values of binary mixtures (EC50mixC) . Experimental EC50 values (EC50mixE) were obtained from bioluminescence inhibition vs . log(cation concentration) curves and were compared with EC50mixC to determine the nature of interactions (antagonistic, additive, greater than additive, or synergistic) . The single metal cation toxicity was found to be Cd+2 > Cu+2 > Zn+2 > Ca+2 . The interactive effects were as following: synergistic for Cu+2+Cd+2, between synergistic and greater than additive for Cu+2+Zn+2, between additive and greater than additive for Zn+2+Ca+2, between additive and antagonistic for Cu+2+Cd+2 and Zn+2+Cd+2, and antagonistic for Ca+2+Cd+2 .

MO 300Soil adenosine triphosphate (ATP) as a marker for assessing ecotoxicology of crude oil pollution in soilI .N .E . Onwurah, S . UbaniUniversity of Nigeria, Nsukka, Nsukka, NigeriaCrude oil pollution on land can cause serious damage to soil by affecting both its biophysical and biochemical properties . Microbial populations (and microbial diversity) in the soil provide adequate nutrients and stored energy (ATP) for primary productivities . Hence, in this study, the effect of terrestrial oil pollution on the ATP content of the soil and changes in microbial diversity in our terrestrial agricultural soil ecosystem were studied in soil macrocosms polluted with Bony light crude oil at different concentrations . In order to add value to this evaluation method, the mark-up of ATP content was assessed by evaluating the ameliorating effect of some bioremediation strategies exemplified by the use of cow and poultry dung and inorganic fertilizer (NPK) . The results of this study show that both the physicochemical properties and ATP content as well as microbial diversity of the soil were negatively altered significantly (p< 0 .05) . Soil pH in water decreased from 6 .55 ± 0 .05 to 5 .85 ± 0 .05 and 5 .75 ± 0 .05 at 0 .1 % and 0 .5 % contamination respectively . Similarly, in KCl medium, the soil pH decreased significantly (p < 0 .05) from 5 .95 ± 0 .05 to 4 .95 ± 0 .05 and 4 .75 ± 0 .05 at 0 .1 % and 0 .5% crude oil contamination respectively . Contamination of soil by crude oil (0 .1 %) decreased the ATP content from 0 .44 mg/100g to 0 .22 mg/100g after 7 days of exposure; from 0 .55 mg/100g to 0 .33 mg /100g after 14 days and from 0 .61 mg/100g to 0 .44 mg/100g after 28 days of exposure . Addition of cow dung, poultry dung and NPK to the polluted soil increased the ATP level as against the untreated polluted soil samples . The cow and poultry dung with high microbial biomass and diversity marked up the ATP content of the amended soil . Hence soil ATP can be used as a reliable marker to assess the impact of crude oil pollution on soil ecosystem productivity and microbial diversity of terrestrial agricultural soil ecosystem .

MO 301Adverse effects of fungicides on ectomycorrhizal mycelium growth - impact of quinoxyfen, boscalid, tebuconazole and azoxystrobinI . Zunker, W .U . Palm, W .K .L . RuckLeuphana University Lüneburg, Lüneburg, GermanyEctomykorrhizal fungi (ECM) are symbionts of many tree species such as European beech (fagus sylvatica L .) . In Germany about one third of the landscape is forested, the European beech dominating large areas . Mycorrhized plants have an increased and thereby more efficient water and nutrient uptake and are better adapted to the specific challenges of climate change, such as drought stress [1] . Studies have shown that fungicides on the one hand stimulate and on the other hand inhibit mycorrhization [2,3] . However, the impact of fungicides on the mycorrhizal symbiosis is poorly understood . This is especially true for new and modern active compounds . First results of laboratory experiments presented on the poster were performed to choose fungicides to be used in field experiments . The impact of authorized fungicides used in agriculture and forestry on the ECM fungi pisolithus arhizus, hebeloma crustuliniforme, lyophyllum and cenococcum geophilum was investigated . In a first step active ingredients quinoxyfen, boscalid, tebuconazole and azoxystrobin were examined in in-vitro experiments under laboratory conditions . In a consecutive step corresponding commercial fungicide formulations were used . For this purpose, serial dilutions were prepared from 0 .1% to 200% of the application rate used in agriculture and forestry together with controls at each dilution step . Tests were conducted in Petri dishes as well as with sterilized soils to approximate natural conditions . For the plate tests mycelium was taken and placed on the treated medium . The test tubes were incubated for 29 days at 25°C in the dark . The evaluation and quantification was performed by determination of the radial growth of mycelium corrected by results from the control group . In all disk tests tebuconazole caused the strongest inhibition whereas the lowest inhibition of mycelial growth was found for quinoxyfen . For Pisolithus arhizus an inhibition was found even at 0 .1 % application rate for three fungicides . For Cenococcum geophilum an inhibition was found at 10% application rate using tebuconazole . Interestingly an irreversible damage of ECM was found . Hence, ECM with inhibited growth after 29 days shows adverse effects on mycelial growth even after re-inoculation on untreated culture medium . [1] Chakraborty et al .(2000) Environmental Pollution 108,[3]317-326

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[2] Diedhiou et al .(2004) Journal of Plant Diseases and Protection,545-556 [3] Laatikainen,et al .(2002) Microbiol . Res . 157,127-137

MO 302Selective removal of high molecular weight PAHs in crude oil and tar contaminated soils using biodieselT .O . Oriaku, D .M . JonesNewcastle University, Newcastle upon tyne, United KingdomAn observation that biodiesel amendment can enhance the selective removal of benzo(a)pyrene has been made in soils spiked with crude oil and coal tar . Results from laboratory microcosm experiments of tar spiked soils showed that after 60 days, this pollutant was reduced by 91% in biodiesel amended samples as compared to 17% and 29% reductions in the control and nutrient amended microcosms, respectively . Perylene was reduced by 70% and anthracene was almost completely mineralised in the biodiesel amended experiments . However, there was no detected depletion in phenanthrene in the biodiesel amended soils as opposed to that in the control (91%), and nutrient amended (85%) experiments, even up to a 180-day degradation period . Fluoranthene and pyrene were also not depleted in the biodiesel amended experiments . A similar trend was observed for the crude oil spiked experiments which showed 52% reduction of benzo(a)pyrene in the biodiesel amended samples after 60 days . However, the degradation of fluoranthene, pyrene, and phenanthrene was substantially inhibited in these samples as compared to the other treatments . The six ring compounds indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene and benzo(g,h,i)perylene were observed to be more degraded in the treatments with biodiesel for both crude oil and tar contaminated soils . These findings suggest that biodiesel can preferentially degrade higher molecular PAHs by a selective degradation mechanism during bioremediation . Further work to investigate this process and also assess soil toxicity changes with treatments are being conducted .

MO 303The contribution of bacterial cell wall fragments to the formation of soil organic matter - a case study from a glacier forefieldC . Schurig1, R . Smittenberg2, J . Berger3, F . Kraft1, S .K . Woche4, M .O . Göbel4, H .J . Heipieper1, A . Miltner1, M . Kästner1

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2Stockholm University, Stockholm, Sweden3Max Planck Institute for Developmental Biology, Tübingen, Germany4Leibniz University Hannover, Hannover, GermanyRecently, stocks of soil organic matter (SOM) have been shown to decrease in European soils and also worldwide, which compromises soil fertility and enhances emissions of carbon dioxide and other, even worse green-house gases, to the atmosphere . However, the general structure of SOM, and thereby the mechanisms behind its genesis and loss, remain unclear . In this framework, microbial biomass is generally regarded to be of low importance for SOM formation . In particular on freshly exposed surfaces, however, bacteria colonize barren mineral surfaces faster than fungi or higher plants . Moreover, recent results indicate that bacterial cell wall fragments frequently occur on soil mineral surfaces and also accompany the microbial colonization of previously clean and sterile activated carbon surfaces after incubation in groundwater . Hence, we hypothesized that, at least, in the initial stages of soil formation bacteria and their fragments may play an important role in particulate SOM formation bearing in mind that most dead organic matter entering the soil is processed by bacteria . This hypothesis was proven by tracing the development of SOM in a chronosequence with samples from the forefield of a receding glacier (Damma-glacier, Canton Uri,Switzerland) by scanning electron microscopy and other methods . The initially barren mineral surfaces have been shown to be rapidly covered with microbial residues as soil age increases . Moreover, this data compares well to growing C/N-ratios, water contact angles and fatty acid contents in earlier deglaciated samples .

MO 304Bioavailability and effects of cadmium, copper and zinc contaminations on microbial communities and respiration activity of soils in SE-GeorgiaH . Kaplan1, S . Ratering2, T . Hanauer3, P . Felix-Henningsen3, S . Schnell2

1Justus Liebig University Giessen, Germany2Justus Liebig University, Institute of Applied Microbiology, Giessen, Germany3Justus Liebig University, Institute of Soil Science and Soil Conservation, Giessen, GermanyThe study area is located about 80 km south of Tbilisi in southeast Georgia in the Mashavera valley . Intensive agricultural land use is limited by low annual precipitation (504 mm) and hot, arid summer months . Accordingly, vegetable gardens, vineyards and orchards as well as arable land are irrigated with water of the Mashavera River . The water is heavily burdened with suspended particles, which show very high concentrations of sulfidic metals (Cu, Zn, Cd) and come from slag heap erosion and a flotation plant of a non-ferrous metal mine . For this reason the irrigated soils in the Mashavera valley are highly contaminated with these trace metals . Aim of the study was to test the use of soil microbial parameters for the evaluation of the habitat function of soils contaminated with trace metals . Overall 51 soil samples were collected of the Mashavera valley in March 2010 . Habitat quality was evaluated by determination of pH-value, organic matter, soil texture and total as well as bioavailable (aqua regia, EDTA-, NH4NO3-exctraction) trace metal concentrations . Total contents ranged between 284 mg kg-1 and 1193 mg kg -1 for Cu, between 303 mg kg-1 and 975 mg kg-1 for Zn, and between 1 .4 mg kg-1 and 5 .9 mg kg-1 for Cd . Basal and substrate induced respiration of the microorganisms were assessed by carbon dioxide production determined with an infrared gas analyser . Low respiration rates were measured in samples with high trace metal contents Indicating a reduced microbial activity in soils irrigated with contaminated Mashavera water . The analysis of microbial community structure by polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) shows differences in the structural diversity of soil bacteria between contaminated and uncontaminated soils . Due to the exposure a shift of the native microbial community structure is shown by a decrease in microbial diversity . Cloning for the phylogenetic classification is still in process . First results will be presented .

MO 305Assessing the effect of metal pollution on soil functioning in forest and grassland ecosystems in Antwerp, BelgiumM .C . Boshoff, R . Blust, L . BervoetsUniversity of Antwerpen, Antwerp, BelgiumIn the suburb of Hoboken (Antwerp-Belgium), UPMR (Umicore Precious Metal Refining) runs one of the world’s largest precious metal recycling units . Although the plant has implemented substantial modernization since the 1970’s, the legacy of 122 years of historic pollution is still present; Pb, As and Cd levels in the soil increase with proximity to the factory . In highly polluted smelter locations the decomposition of litter and thus nutrient cycling is impaired due to the almost complete absence of litter decomposers in the soil and litter layer . Endemic soil microbial communities vary from site to site; therefore a set of tests with different assessment endpoints is needed . The bait lamina test developed by Von Törne, (1990) measures the feeding activity of the decomposer community and consists of vertically inserting 16-hole-bearing plastic strips filled with a bait substrate into the soil . Bait removal is directly linked to decomposition activity . An alternative method based on substrate induced respiration will also be applied to support the results of the bait lamina test . The MicroResp[TRADEMARK] system consists of a 96 well ‘deepwell’ plate in which moisture corrected soil samples (at the soil’s field capacity for water) amended with various carbon substrates are placed . The plate is separated from a 96 well microtiterplate containing a colorimetric CO2 detection gel by a PTFE lined porous rubber seal . The system is incubated at 25°C for 6 hours followed by spectrophotometry readings carried out at 570nm . The results will be interpreted in relation to the soil physicochemical parameters of each test site .

MO 306Biodiversyty of soil microfungi and the role of melanin pigment in biomonitoring B .S . Sosak-SwiderskaUniversity of Cardinal Stefan Wyszynski,, Warsaw, PolandWe known numerous examples of fungal structures such as spawn and spores that are extremely resistant to harsh environmental conditions and it is significant that most of these resistant structures contain black pigments which have been described as melanin . Microfungi were isolated from soils in the Karkonosze and Bieszczady Mountains and the Upper Silesia Region in Poland . Among dominant soil microfungi was isolated such species with genera Aspergillus as: A . flavus, A . fumigatus, A . humicola and A . versicolor; among Cladosporium such as C . cladosporoides, C . brevi-compactum and C . herbarum; among Penicillium such as: P . canescens, P . frequentans, P . lanosum, P . nigricans and P . ochraceum; among Trichoderma - T . album and T . viride . Isolated melanin polymers were of high molecular weight, were decolorized by oxidizing agents, were insoluble in organic solvents, and gave a positive reaction for polyphenols . On the basis of their chemical and spectral properties, the melanin from this fungi have been described as similar to “indole”melanin and belonged to eumelanin group . The location of granules with melanin was investigated in the Cladosporium cladosporoides cells . Cadmium, cuprum and zinc form discrete black granules and electron microscopy analysis indicated that these metals were also associated with cell walls and intracellular structures . Localization of lead granules was clearly evident, which were found in cell walls and at intracellular structures, most likely-vacuoles, which have an important role in the regulation of the cytosolic concentration of metal ions . Explaining the role of melanin would certainly contribute to understanding the protective function of dark-pigmented fungi on soil pollutants . It is a problem of a great importance especially as far as degraded soil by heavy metals is contaminated . In the natural soil environment are another dark-pigmented soil fungi species such as Aspergillus sp . and Penicyllium sp ., too . Their wide occupation in all of soil environments may be used in biomonitoring of heavy metals and melanin pigment is the useful ecotoxicological biomarker .

MO 307Understanding binding mechanisms of active pharmaceutical ingredients to sewage sludge using a surrogate solid phase extraction approachL .M .C . Berthod1, G .C . Roberts2, A .D . Sharpe2, G .A . Mills3, R . Greenwood3, D .C . Whitley1

1University of Portsmouth / AstraZeneca, Brixham, United Kingdom2AstraZeneca, Brixham, United Kingdom3University of Portsmouth, Portsmouth, United KingdomOver the past decade there has been a growing concern over the fate of active pharmaceutical ingredients (APIs) that are released into the aquatic environment . Some APIs can be effectively biodegraded in waste water treatment plants but others can be sorbed onto sludges that are often subsequently used as fertilisers or disposed of as landfill . In order make an accurate risk assessment it is important to know how a given API will be distributed between the aqueous or solid phases within a treatment plant . A sorption test to determine the partitioning (determination of the Kd value) of an API in sewage sludge is already available (OPPTS 835 .1110), but this method is long and fastidious .

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Predictions of Kd based on Log Kow have been attempted, but these are not very reliable, especially for ionisable compounds, such as many APIs of interest . Therefore, better predictive models are urgently needed that include binding mechanisms in addition to the hydrophobic interaction . As activated sewage sludge is a complex matrix and hard to fully characterise we used ten commercially available solid phase extraction (SPE) cartridges with different chemistries to try to reproduce these binding processes . Various eluents with different organic modifiers and different buffers at a range of pH were tested . The results of these experiments, where the sorption mechanism is known, were compared to results obtained using ‘in house’ prepared cartridges of sludge at different sludge/silica ratios .

MO 308Risk assessment of pesticides to soil organism: exposure analyses of two insecticides in soil layers during a sampling period of one yearC . Possberg, B . Schmidt, A . SchaefferRWTH Aachen University, Aachen, GermanyThe spatial and temporal behavior of pesticides in the soil environment depend on properties of the substance (e .g . degradation, leaching, sorption), the soil and the climate . To study the effects of the chemicals on the soil fauna, the spatial and temporal distribution of soil organisms is also important . Within the project “Risk assessment of pesticides to soil organism under real conditions” investigations to the metabolism and leaching of pesticides are synchronized with studies on their potential effect on soil organisms . Two insecticides, imidacloprid and lindane, both persistent in soil, but differing in the leaching behavior were separately applied to Terrestrial Model Ecosystems (TMEs) in two concentration each: 7,5 kg/ha and 20 kg/ha (lindane) and 0,75 kg/ha and 2 kg/ha (imidacloprid) . The distribution of the pesticides in the soil profile (0-2,5 cm, 2,5-5 cm, 5-10 cm and 10-20 cm depth) during one year was analyzed . We here present the analytical work . Lindane was extracted with ethyl acetate and the residues were determined by gas chromatography with electron-capture detection . Imidacloprid samples were extracted by microwave digestion in acetonitrile/water; after centrifugation the extract was analyzed with LC-MS/MS . Results of the currently performed chemical analyses will be presented . In another part of the project the effect of the insecticides to soil organisms on the population level are investigated and shown in a platform presentation .

MO 309Understanding the relationship between extraction technique and bioavailabilityC .V . Eadsforth1, M . Galay Burgos2, M .S . Holt2, D .J . Letinski3, H . Noble4, R . Oliver5, M . Telscher6

1Shell International Ltd ., Chester, United Kingdom2ECETOC, Brussels, Belgium3ExxonMobil Biomedical Sciences Inc ., Annandale, United States of America4AstraZeneca, Brixham, United Kingdom5Syngenta, Guildford, United Kingdom6Bayer CropScience, Monheim, GermanyIn addressing the significance of bound residues in environmental risk assessment, ECETOC concluded previously that non-extractable residues were characterized by a pragmatic extraction approach for determining whether they are extractable or not . There is now a need to develop a standard framework for soil and sediment extraction methods and to associate extractable fractions (such as freely dissolved, readily desorbed or reversibly desorbed) with a level of bioavailability and with appropriate test organisms for these compartments . A Task Force has reviewed extraction methodologies and how such methods correlate with bioavailability, identified those ‘destructive’ extraction techniques which result in loss of sample matrix integrity and also considered the state of knowledge regarding mechanisms of binding and their forces which would result in either extractable or non-extractable residues . The aim was to develop new approaches to screen the bioavailable fractions and validate such an approach . This supports a consistent interpretation of data and provides a transparent basis for assessing the potential risk of non-extractable residues . The Task Force will evaluate a framework for an intelligent extraction strategy and make recommendations for future research topics .

MO 310Plant uptake of some commonly used pharmaceuticalsL . Nei1, M . Lillenberg2, E . Haiba1

1Tallinn University of Technology, Tartu, Estonia2Estonian University of Life Sciences, Tartu, EstoniaPresence of different pharmaceuticals in sewage sludge is apparent, but there is still a lack of knowledge about the fate of pharmaceutical residues in the environment . Still, remarkable amounts of pharmaceuticals enter the soil via fertilizing with sewage sludge . As long as such a disposal practice is widely applied, a better knowledge on the fate and effects of chemicals in sludge treated soils is needed . Only a relatively small number of investigations have been published on the mobility and bioavailability of pharmaceuticals in the environment . Uptake of antimicrobials into vegetation is a major route for these substances into the food chain . In the current study uptake of ciprofloxacin, norfloxacin, ofloxacin, sulfadimethoxine and sulfamethoxazole was demonstrated in the case of lettuce, potato, carrot and wheat . Measureable residues of the studied compounds occured in food plants . The uptake of fluoroquinolones and especially sulfonamides by plants like potato and carrot might pose health risk, as the detected levels of the studied pharmaceuticals were of considerable magnitude, if compared to their soil concentrations . As a rule, the uptake of antimicrobials was more efficient from sandy soil into underground parts on plants - roots and tubers . The concentrations of antimicrobials in plants exceeding the MRL values for meat and milk were seen only at antimicrobial soil concentrations equal to 1 mg/kg and 10 mg/kg . Such concentrations might be present in soil if sewage sludge treated by anaerobic digestion is used as a fertilizer . In Estonia the soil concentrations of antimicrobials are presumably lower, as only sewage sludge compost may be used for fertilization .

MO 311Degradation of pharmaceuticals in sewage sludge compostE . Haiba1, M . Lillenberg2

1Tartu College of Tallinn University of Technology, Tartu, Estonia2Estonian University of Life Sciences, Faculty of Food Hygiene, Tartu, EstoniaSewage treatment facilities do not remove pharmaceutical residues completely . Several antimicrobials do not decompose during aerobic activated sludge process or anaerobic digestion, and release in sewage effluents or adsorb to sewage sludge . The sewage sludge containing pharmaceutical residues is used as a fertilizer . Pharmaceuticals reach the soil where they can affect microorganisms and accumulate in plants . Our recent study showed that some fluoroquinolones and sulfonamides were frequently present in Estonian sewage sludge and the composting technology played the major role of their degradation . In Tallinn, where composting with peat was used and mixing of the compost stacks lasted 12 months, all studied antimicrobials were degraded after a year . In Tartu, where composting with tree bark was used and mixing of the compost stacks took place during 8 months, the highest contents of ciprofloxacin, norfloxacin, ofloxacin after a year storage were 70, 64 and 8 µg/kg (dm) respectively . Fluoroquinolones have been described as photodegradable, therefore we suppose that exposure to the light due to frequent and long lasting mixing could be one of the reasons of their faster decomposition in Tallinn . Plant experiments in greenhouse showed that potato, carrot and lettuce can uptake fluoroquinolones from soil with antimicrobial concentration 10 µg/kg (dm) (the trigger value for antimicrobial content in soil recommended by EMEA/CVMP) . The mixture of sulfonamides and fluoroquinolones in soil can be toxic for wheat and lettuce plants, if antimicrobial concentration in soil reaches 10 mg/kg (dm) . Antimicrobial content in plants grown at soil antimicrobial concentration 10 mg/kg exceeded the MRL for meet and milk (100 µg/kg) . To prevent such a high antimicrobial concentration in soil, the sewage sludge composting has a remarkable importance . To compare the influence of different sewage sludge composting technologies to the degradation rate of the fluoroquinolones and sulfonamides in it, some new experiments were performed . The sewage sludge was mixed with different materials as tree bark, peat, oil shale ash, litter and sawdust . The antimicrobial decomposition rate dependance on added material was estimated . Antimicrobials were extracted from compost samples using PLE, extracts were cleaned up by SPE and analyzed by LC-MS . The results will be presented . This work helps to optimize the sewage sludge composting technology to produce safe agricultural fertilizer .

MO 312Prospects of miscanthus x giganteus for the rhizoremediation of PAH contaminated soilsD .E . Técher1, P .H .I .L .I Laval-Gilly1, S .O .N .I .A Henry1, A .M .A .R . Bennasroune1, C .L .A .U .D Martinez-Chois1, M .A .R .I .E d’Innocenzo1, J . Falla2

1Université Paul Verlaine Metz, Yutz, France2Université de Metz, Yutz, FrancePhytoremediation of large surface area using crop cultivation appears as an attractive strategy to allow for long-term but economically viable site rehabilitation programs . The aim of this study was to characterize the phytoremediation potential of Miscanthus x giganteus for PAH polluted soils . For this purpose, three factors affecting bioremediation, namely the nature of soil contamination (spiked-phenanthrene and pyrene each at 100 mg/kg dry soil), bioaugmentation (with a PAH-degrading microflora) and planting (with Miscanthus x giganteus), were arranged according to a full factorial design in a pot experiment . Their effects (alone or in combination) were assessed on soil bacterial communities (total and degrading microflora enumeration, 16S rDNA molecular diversity analysis) and PAH dissipation . Results showed that contamination was the main factor positively influencing the development of PAH degraders . Phenanthrene disappearance was observed regardless of the presence of plants or initial bacterial inoculum whereas remaining pyrene contents were negatively affected by bioaugmentation and to a lesser extent by planting . The ability of plants to maintain a degrading rhizosphere microflora, thus favouring dissipation of the recalcitrant compound would deserve further investigations .

MO 313Nano, bulk or complexed iron: what to use for soil remediation?J . Filser, E . Lesnikov, I . Burfeindt, U . UebersUniversity of Bremen, Bremen, GermanyZero-valent Iron has a long tradition in soil remediation, due to its reductive properties and its sorption capacity . The latter substantially increases when iron is present in nanoparticular form . Since at the same time the costs of remediation are considerably reduced, nano-iron has been used for remediation purposes . We are using an array of standard test systems for prospective hazard assessment of new substances, including nanoparticles . In an earlier study we found that nano-iron had a limited capacity to reduce the toxicity of cadmium to plants (Lepidium sativum, Arabidopsis thaliana) and caused avoidance by earthworms (Eisenia fetida), even when applied in uncontaminated soil . The tests also rendered A . thaliana way more sensitive than L . sativum . Currently we are conducting follow-up experiments in which we test the impact of iron powder and iron fertilizer on both plant species, earthworms and the toxicity of cadmium to them . First results clearly show that highly concentrated fertilizer is highly

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toxic to plants and thus cannot be used for remediation purposes . The experiments will be finished in early spring so that we expect to answer our title question during the conference .

MO 314Time-course changes of PAHs and their oxygenated derivatives in a PAH contaminated soil during full scale biological remediationM .E . Larsson, O . Westman, M . EngwallSchool of science and technology, Örebro university, Örebro, SwedenPolycyclic aromatic hydrocarbons (PAHs) are fairly common in contaminated industrial areas, especially at old gasworks sites, gas stations and former wood impregnation facilities . U .S . Environmental Protection Agency (EPA) has listed 16 PAHs as priority pollutants . Because of their toxicity, PAH-contaminated areas are highly prioritised for remediation . Today, the generic guideline values of PAHs in contaminated soils are usually based on chemical analysis of the 16 priority PAHs, even though often hundreds of PAHs and PAH derivatives exist in the soils . Oxygenated PAH derivatives (oxy-PAHs) originate from combustion sources and are also formed during biological remediation of polycyclic aromatic hydrocarbons (PAHs) . Oxy-PAHs are mutagenic, can induce aryl hydrocarbon receptor (AhR) mediated toxicity and are in addition more mobile than their parent PAHs and therefore of environmental concern . In contrast to the well monitored 16 priority PAHs, only little information is available regarding toxicity, levels and distribution of oxy-PAHs in the environment . In the present study we will analyse levels of 26 PAHs and 12 oxy-PAH derivatives during a full scale biological remediation of a PAH contaminated soil using GC-MS . Our objectives are (1) to examine the changes in PAH and oxy-PAH levels during the remediation process (2) study the availability of the compounds over time using a mild methanol extraction and (3) study if the toxicity in the soil is reduced in proportion to the putative reduction of the PAHs, using the H4IIE-luc dioxin bioassay, based on rat hepatoma cells . Mass balance analysis will show how much of the AhR-mediated activities in the soil the GC-MS analysed compounds account for . The results will give information of levels of PAHs and oxy PAHs found in PAH contaminated soils, the degradability of these compounds, formation of oxy PAHs and changes in availability during a biological remediation .

MO 315Ecoethic problems of erosion, salinization and alcalination of soils in AzerbaijanG .M . Mammadov1, S .Z . Mammadova2

1State Land and Cartography Committee of Azerbaijan Republic, Baki, Azerbaijan2Institute of Soilscience and Agrochemistry of ANAS, Baki, AzerbaijanThe exposure soil to different level of erosion is one of characteristic ecological problems of our Republic . But real disaster of land management in republic and in the world is the water erosion (31% of land) and wind erosion (defilation) which actively influence 34% of surface land . Relief of the territory plays an important part in erosion process and intensively influences to erosion process and gradually changes under its influence . At the result of agricultural activities 30 th . ha of land cover destructed, but 40% of land cover exposed to erosion processes, result of wrong land reclamation work shows its negative influence, in results underground water level and salinization of arable land increases . In arable regions of the country more harm gives irrigational erosion . In appropriation of mountain mass under not follow antierosional activities washout of the soil will decrease which shows negative influence on productivity and often causes take out of the plant from agricultural cultivation . Very often antierosional processes causes washout of the 100 t/ha soil, especially in irrigation season . Mountain and plain regions of Azerbaijan exposed to irrigational erosion . At the result of wrong irrigation activities productivity of the land decreases . Research works shows that total territory of lands exposing to erosion is 31444,7 th . ha in our Republic, which means 36,4% territory of Azerbaijan . Problem of salinization and alcalination are the most important problems among the ecoethic problems . Nowadays approximately 37000 th . ha soils from all existing land (8641506 ha) in Azerbaijan exposed to degradation . Soil salinization is wide spread in Azerbaijan . 60% of Kur-Araz plain occupying 2,2 million ha territory consists of average and full saline soil . Generally in the territory of our Republic total area of average and full saline soil is 1,3 mln ha . That is why these lands considered useless from the point of view of land reclamation and ecology . Knowing its negative dynamics is necessary to take otherwise actions, such as taking certain ecoethic measures (derange, washout, chemical melioration, necessary administrative and legal activities and etc) . In the way of struggle against erosion, salinization and alcalination which became one of the important ecoethic problems for our soil and plant cover, complex measures are needed to be carried out .

MO 316Ecological characterization of Mountain Shirvan Regions of AzerbaijanT .G . GasimzadeInstitute of Botany of ANAS, Baki, AzerbaijanThe composition of Mountain Shirvan economic and geographic region includes Agsu, Ismailli, Gobustan and Shamakha districts . Total area of the region is 613 thousand ha, population-273,4 thousand persons . The relief of the area is divided into upland and plain territories . On the plain territory climate is soft-hot and dry subtropical; on the upland places - the summer is some cool, the winter is rather cold and arid . Annual middle temperature of foothill-plain territory is 14,10C (Agsu), in upland territory - 10,50C . Temperature of the coldest month in Agsu is 3,30C, in Shamakha - 0,60C . Annual rainfall is 800 mm (Ismayilli) and 379mm in Maraza (Shamakha) . Evaporation is 762mm in Ismailli, 872mm in Maraza, and 970mm in Agsu . 428 thousand hectare (69,8%s) of general territory is upland and 185 thousand hectare (30 .2%s) is foothill-plain . Composition of the land cover: 37,2%-mountain chestnut; 11,4%-chestnut; 11,8% brown and brown mountain-forest; 3,3%-black; 8,2%-grey; 8,0%-alluvial-meadow;2,7%- meadow-forest; 5,7%- mountain-meadow . Composition of land supply: useful lands for agriculture - 376769 ha (67,1%), from them arable - 37,5% (141,3 thousands hectares), parennial plants -2,5% (9,2 thousands hectares), hayland-1,2% (4,6 thousands hectares), pastures -57,1% (212,5 thousands hectares), Garden plot - 2,5 % (9191ha), forest -13,3% (81596ha) . Irrigated lands useful for agriculture is 52,8 thousand hectare (14,2%) . From vegetation - cereal-growing, potato-growing, vegetable-growing, vine-growing, melon-growing, fruits-growing, cotton-growing, feed-growing . In subalp are meadows: moderate-damp-cereal, mix-cereal, damp- forb meadow, mezophyl-bean and sedge, superfluous-humidified, subalpine criophyte and subalpine steppe meadows . The vegetation of the Alpine belt widespread within 2400-3200m above s .l . also is expressed by meadow and carpet elements . The vegetation at these heights is formed, mainly, on gentle slopes, dome-shaped tops, saddles . Data of the spent researches give representation about spatial laws of ecosystem distribution of Mountain Shirvan with different disturbs level and also data on change of typical structure of soils and specific structure of vegetation accordingly .

MO 317Influence of anthropogenic factors on landscape complexes in mountains areasZ .R . MammadovMardakan Arboretum of ANAS, Baki, AzerbaijanIn modern age which the society is developing rapidly and has great technical strength, the relation between nature and human brought and dangerous state was beginning . Even recently the getting the nature is so strong that natural resource begins to exhaust more hastly . Now the guarding of natural resources, the use of them with suitable way, especially the guarding of animate resources and soils are considered as very important national and international problem . From this viewpoint, the solition of ecological problems pretends increasing of soils fertility with scientific bases to register them, estimating and monitoring . Part of our Republic regarding to Small Caucasus is one of the region which has natural complex and increasing the anthropogenic influences to the cover of soil . This district has according to soil climate condition or economical, geographical position to number suitable sides . Landscape complex of this area and cover of soil annually suffer to change under influence of anthropogenic factor (the weakening of agriculture’s soil as for quality, expanding of the settlements, forest breaking) in last ten years . In Small Caucasus driving works such as “The ecological evaluating of the North-East slope of Small Caucasus”assume scientific and practical importance . So, general area of the investigation on the North-East slope of Small Caucasus is 433642,86 hectars and present large territory . There is a special place the soil erosion among the problems of the North-East slope of Small Caucasus . 21 soils subtype are calculation suffer to erosion have been determined . 25% s of the soil general area are undergone to erosion in the weak degree . The soils which suffer to erosion in the middle degree is 21 % s, it is less than the soils which suffer to erosion weakly . 75% soil spreading in the North-East slope of the Small Caucasus have been undergone or erosion apparently . This is the main ecological problem for the area which has been investigated not only for our Republic and also for region generally . Thus, on the weaken bases of fertility indicator of investigated soils taking into consideration the natural resources of district, for the purpose of restoration and extension of fertility and preventing negative process, complex agromeliorative, agrotechnical measures (shift sowing system, planting parennial herbs, correct pasture system) must be organized .

MO 318Organochlorine pesticides in soils from the agricultural valley of Mexicali, Baja California, MexicoJ .L . Sánchez-Osorio1, J .V . Macías-Zamora2, N . Ramírez-Alvarez2

1Universidad Autónoma de Baja California, Ensenada, baja california, Mexico2Instituto de Investigaciones Oceanológicas, UABC, Ensenada, baja california, Mexico Mexicali valley is one of the most important agricultural areas in northwest Mexico . Is located in the borderline between Mexico and the State of California, in the United States . Due to the highly technified agriculture; organochlorine pesticides were extensively used in 60s, 70s and early 80s to enhance productivity . Even though most of the organochlorine pesticides are prohibited or restricted in their use in Mexico, there is little research about their current concentration levels, and how much they have spread in the area . In 2008, soils samples were collected for analysis of 24 pesticides (α-HCH, β-HCH, γ-HCH, δ-HCH, Heptachlor, Hectachlor epoxy, Aldrin, Dieldrin, Endrin, Endrin aldehyde, 4,4-DDE, 4,4-DDD, 4,4-DDT, 2,4-DDE, 2,4-DDD, 2,4-DDT, α-Chlordane, γ-Chlordane, Endosulfane I (alpha), Endosulfane II (beta), Endosulfane sulfate, Metoxychlor, Mirex, and total Toxaphene . Because several population centers are spread all over the valley, and are surrounded by crop land, human health risk was of the main concern . So, soils samples were collected in public areas located inside 25 of those towns . Concentration results indicated that all the organochlorine pesticides analyzed are widely spread in Mexicali valley, with DDTs accounting for the highest contribution to the total pesticides, mainly the 4,4-DDE (50-80%) . The highest concentration for total DDT was 151 .6 ng/g d .w ., 8 .1 ng/g d .w . for total HCHs and 1334 ng/g d .w . for total toxaphene . For most pesticides, surficial distribution pattern showed the mayor concentrations in the central area of the valley . Conventional ratios for DDTs indicate ancient application for this compound whereas HCH ratios indicate probable use of technical-HCH as the main source of this compound to the environment . Toxaphene results are important because basically there is lack of information about these compounds in the area . This work presents an overview of the most relevant results in order to enhance the interest of the government in this valley . Concern is raised because these and maybe other pollutants are being released constantly to the atmosphere as it has been confirmed by our passive samplers in the area .

MO 319Environmental fate of heavy metals at small arms firing rangesM .J . Brochu1, A . Laporte-Saumure1, A . Martel2, Mercier3

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1DRDC Valcartier, Quebec, Canada2INRS ETE, Quebec, Canada3INRS-ETE, Quebec, CanadaCanadian military small arms firing range (SAFR) backstop berm soils contain high concentrations of Pb, Cu, Sb, and Zn, as a consequence of the bullet compositions consisting of a Pb-Sb core, and a Cu-Zn jacket . In order to characterize the vadose zone pore water total Pb, Cu, Sb, and Zn concentrations, three Teflon® gravimetric lysimeters (GL) were installed in the vadose zone of the backstop berm, at 30 cm and 150 cm depths . This was done in order to assess the potential for vertical migration of the metals from the berm to groundwater . The GL consisted of Teflon® cylindrical buckets filled with the in-place soil, in the bottom of which Teflon® tubes where installed . The tubes were connected to LDPE sampling bottles located in an access well . Results indicated that the Cu and Zn concentrations were below the drinking water thresholds prescribed by Health Canada (1 mg/L for Cu, and 5 mg/L for Zn) and that in many cases, Pb and Sb concentrations were above the thresholds (0 .01 mg/L for Pb, and 0 .006 mg/L for Sb) . This indicates that a metal vertical migration occurred in the vadose zone, at levels over the acceptable drinking water criteria for Pb and Sb . Decreasing order of metal concentrations was Zn>Pb>Sb>Cu . Water infiltration rates were also monitored with a drain gauge, and used along with metal concentrations to calculate the annual total metal fluxes in the first 150 cm of the berm, and the annual metal leaching rates . Results indicated on one hand that only a small portion of the metals in the soil were mobilised in the vadose zone, as annual metal leaching rates were 0 .0001% for Pb, 0 .0005% for Cu, 0 .003% for Sb, and 0 .09% for Zn, and on the other that metals could remain in the backstop berms for extremely long period of times; e .g ., several hundreds of thousands years for Pb to several thousands of years for Zn, highlighting the importance of finding ways to remediate such soils in order to limit metal mobility in SAFR backstop berms . The effects of various amendments on the environmental fate of Zn, Cu, Sb and Pb will also be described .

MO 320Quantitative determination of PAHs in biochar - a prerequisite for its quality and safe applicationI . Hilber1, H .P . Schmidt2, F . Blum1, T .D . Bucheli1

1Agroscope, Zurich, Switzerland2Delinat Institute for Ecology and Climate Farming, Arbaz, SwitzerlandBiochar (BC) is pyrolyzed organic feedstock . It has become more and more important as soil conditioner and received broader attention as a possible measure against climate change . Polycyclic aromatic hydrocarbons (PAHs) are produced during BC production due to incomplete combustion . These PAHs will enter the environment when the BC is applied as soil conditioner to avoid leaching of nutrients, to increase microbial biomass and activity, to remediate the soil of organic pollutants, etc . Such positive effects are undermined if PAH exposure is concomitantly increased . Methods specifically adapted for PAH analysis in BC are hardly available . Different solvents and extraction techniques have been used to determine the total concentration of PAHs in BC such as dichloromethane, methanol, acetone, hexane with Soxhlet, liquid extraction, pressurized fluid extraction, etc . Such approaches were often based on certified analytical procedures (e .g . ISO 38 414 using cyclohexane, DIN EN 15527 using petroleum ether) originally established for other matrices such as soil, and may lead to negligible or low total concentrations of PAH in BCs, or low recoveries of PAH internal standards . Hence, traditional methods may fall short to determine PAH in BC quantitatively . The aim of this study is therefore to provide an easy and validated method for the quantitative determination of PAHs in BCs to researchers, practitioners, and legislators . Four BCs of different feedstocks were extracted with Soxhlet with two different extraction durations and with accelerated solvent extractor . Different solvents were used such as toluene, hexane, dichloromethane/acetone (1:1, v/v) and others and different cleanup steps were applied . Total concentrations were measured by gas chromatography-mass spectrometry (GC-MS) . The extraction results will be presented which include the best extraction solvent and technique, the optimal extraction duration, linearity, detection limits, method precision, sample representativeness and absolute and relative recoveries . Some implications concerning the amendment of BC will be critically evaluated . We have developed a simple, robust and sensitive extraction method to quantitatively determine total concentrations of PAHs in BCs . This method may serve researchers, practitioners and legislators to optimize BC production with a view to minimize its PAH content, and to properly assess the environmental benefits and risks of this overall promising material . MO 321Fate of persistent organic pollutants in soils amended with sewage sludge: sampling and modelling resultsA . Passuello1, I . Valor-Herencia2, J . Llorca-Porcel2, E . Martínez-Soriano2, P . Navalón-Madrigal2, M . Nadal3, M . Schuhmacher4

1Environmental Analysis and Management Group, ‘’Rovira i Virgili’’ University, Tarragona, Spain2LABAQUA S .A, Barcelona, Spain3TecnATox - ‘’Rovira i Virgili’’ University, Reus, Spain4Universitat Rovira i Virgili, Tarragona, SpainSewage sludge (SS) application on agricultural soils is a managing practice of increasing use because of its benefits to soil and crops . However, SS has an extensive range of persistent organic pollutants (POPs) that may be transferred from soil to crops and reach the food chain . The main objective of this work was to evaluate POPs fate in soils amended with sewage sludge . To accomplish this objective, the work was divided in two parts: samples evaluation and model development . First, 200 SS samples from 125 wastewater treatment plants all around Spain were collected and the concentrations of the POPs proposed in the directive (PAH, NP+NPEs, PCDD/Fs, PCBs, LAS) and other emergent organic contaminants (PBDEs, PCNs, PFOS+PFOA) were measured . Also, controlled parcels with different soil texture, organic matter content, crops, and sewage sludge application rates were chosen . In each parcel, soil samples were measured for POP’s . Then, a probabilistic multimedia model to predict the accumulation of POPs in soils, as a consequence of SS application on agricultural soils, was developed . This dynamic model predicts the concentration of POPs in amended soils for different time spans, considering the following processes: volatilization, diffusion between compartments, leaching, reaction and deposition . The model input parameters are: sludge characteristics and application dose, soils characteristics (density, bioavailability, volume fraction of soil compartments air, water, organic matter and mineral matter) and pollutant properties (solubility, KOW, KOC, vapour pressure, H, persistency, and half life in soil) . Two layers of soil were considered . This model was validated with data from the sampling campaign, considering SS characteristics and application rates, soil characteristics and POPs concentration in soil and SS . Results showed a good agreement between model predictions and field values .

MO 322Alteration of the reproductive toxicity of Cadmium in the soil living Nematode C.elegans upon potential chemosensitization by HHCBA .A . Abbas1, B . Hegemann1, W . Ahlf2

1Technical University Hamburg-Harburg, Hamburg, Germany2TU-Hamburg-Harburg/Inst . of Energy and Environmental Techniques, Hamburg, Germany1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[g]-2-benzopyran (HHCB or Galaxolide) is a bioaccumulative and environmentally prevalent synthetic musk compound contained in various consumer products (such as perfumes and detergents) . From first toxicity studies on HHCB the European Commission (2008) concluded with no or negligible risks for humans and the ecosystem . Mechanistically HHCB could be characterized as a chemosensitizing compound and as a possible substrate and inhibitor of cellular efflux pumps (Luckenbach and Epel, 2005) . Cellular efflux systems were shown to co-regulate the intracellular bioavailability of xenebiotics and other contaminants (Bard, 2000; Epel, 1998; Kurelec et al, 2000) . Seizing the scientific debate on the potential toxicological implications of chemosensitization this study presents novel ecotoxicological data on the chemosensitization potential of HHCB on Caenorhabditis elegans (C .elegans) towards the heavy metal cadmium . The reproductive toxicity of the LOEC of cadmium (0 .1 mg/L) was herewith potentiated by 27% under simultaneous exposure to the LOEC of HHCB (0 .1 mg/L) . Remarkably, a post-exposure chemosensitization effect was observed 12 to 96 h after a 12 h pre-exposure to 0 .1 mg/L HHCB, reflected in a 17% to 60% higher reproductive toxicity of Cadmium at 0 .1 to 10 .0 mg/L . We assume that HHCB at low concentrations had potently inhibited relevant efflux transporters of C .elegans (Broeks et al ., 1995 and 1996) in a competitive manner, coinciding with an elevated intracellular bioavailability of Cadmium . The obtained results support related observations by Luckenbach and Epel (2005) and by Cheng and Zhou (2009) . Overall the results appreciate the mechanistic complexity of mixtures of prevalent aquatic and soil contaminants and the manifold factors involved in their bioavailability and toxicity .

ET13P - Natural toxins and bioactive compounds

MO 326Tetrodotoxin expansion in the temperate waters of the Atlantic Ocean new vectors and impact on marine ecosystemsM . Silva1, J . Azevedo1, P . Rodriguez2, A . Alfonso2, L .M . Botana2, V . Vasconcelos1

1CIIMAR, Porto, Portugal2University of Santiago de Compostela, Lugo, Spain Tetrodotoxin (TTX) is a potent low weight marine toxin found in the warm waters of the Indic and Pacific Oceans . Intoxications are usually linked to the consumption of the puffer fish although TTX was already detected in several different taxa . The rise of water temperature due to climate change, and anthropogenic interventions are pointed as the main causes of the toxin spreading . Benthic organisms such as mollusks and echinoderms, with different eating habits were collected monthly along the Portuguese coast since the summer of 2009 till the end of 2010 . The extraction and analysis techniques were optimized and TTX and analogues were detected for the first time in two intertidal gastropod species -Gibbula umbilicalis and Monodonta lineata by UPLC-MS/MS and LC-MS/MS . These findings and their possible impact on marine communities are discussed taken into account the dynamics of the ecosystem and human health . This work was made under the ATLANTOX project .

MO 327Hydroxylated and methoxylated brominated diphenyl ethers: natural products or Anthropogenic Contaminants?X .Y . Wei1, J .P . Giesy2, R .S .S . Wu3

1PRC/ HK/ The University of Hong Kong, Hong kong, Hongkong

20�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

2University of Saskatchewan, Saskatchewan, Canada3The University of Hong Kong, Hong kong, HongkongBrominated Diphenyl Ethers (PBDEs) have been widely used as flame-retardants in the last few decades . Since their chemical structure is similar to PCBs, these chemicals may pose a significant threat to the environment and have emerged as contaminants of concern . In recent years, however, structural analogs of PBDEs, including hydroxylated- and methoxylated- BDEs (OH-/MeO-BDEs) have been detected the biota at different trophic levels . The wide spreading high concentration of OH-/MeO-BDEs in marine biota leads to the postulation that these compounds may be natural metabolite products of PBDEs of marine organisms rather than anthropogenic contaminants . . In this study, four species of plankton algae (two diatom species: Thalassiosia pseudonan and, Skeletonema costatum; and two dinoflagellate species Prorocentrum dentatum and Prorocentrum minimum) were cultured in (a) Br free artificial sea water and (b) artificial seawater with isotope 81Br under laboratory conditions . Levels of 9 common MeO-BDEs analogues, 10 OH-BDEs analogues and 12 Brominated Phenol (BRPs) analogues were determined by GC-MS .

Mass balance of 81Br in the algal and water samples was constructed to test the hypothesis that OH-/MeO-BDEs are synthesized by the algae . Result of this study will be discussed and help us to differentiate and understand the anthropogenic and natural sources of hydroxylated and methoxylated BDEs found in the marine environment .

MO 328Assessing the toxicity of two natural bioactive compounds applied in aquaculture using zebrafish embryosR . Oliveira1, P .S . Galindo2, A .M .V .M . Soares3, A .J .A . Nogueira2, I . Domingues1

1Universidade de Aveiro, Aveiro, Portugal2CESAM & Department of Biology, University of Aveiro, Aveiro, Portugal3University of Aveiro & CESAM, Aveiro, PortugalLosses in aquaculture production are often associated with predation by wild fish species in culture ponds . The rotenone (RO) and saponin (SA) are two piscicides plant-derived widely used to remove undesirable fish species in the ponds in aquaculture activities . In the present study the effects of RO and SA in zebrafish embryos were studied . A 144-h test was performed following the draft guideline Fish Embryo Toxicity (FET) test and several endpoints were analysed . Lethal endpoints included egg coagulation, non-development of somites, non-detachment of the tail, and absence of heart beat . Sub lethal endpoints included hatching, lack of body and eyes pigmentation, oedemas (yolk sac, pericardial) and tail malformation . The results show clear dose response effects of rotenone on zebrafish development with a 96 h-LC50 value of 16 .8 µg/l . After 96 h of exposure to concentrations >10 μg/L of RO the embryos showed abnormal body pigmentation, loss of equilibrium, and spine deformities and at 20 μg/L embryos showed cardiac oedema . For SA a 96 h-LC50 value of 27900 µg/l was determined and no sub lethal effects were found for the concentrations tested . Thus, SA was much less toxic than RO for zebrafish embryos . Nowadays, the available data on RO and SA for fish control purposes indicate that it can be used safely . However due the very high toxicity of RO and uncertainties beyond the intended target fish to both compounds, effects on non-target organisms can be expected .

MO 329Environmental responsibility, nematode management and collaboration between academia and industryC . McgawleyLouisiana State University Agricultural Center, Baton rouge, louisiana, United States of America Plant-parasitic nematodes account for annual losses in world agriculture that exceed (U .S . dollars) 11 billion . An extensive arsenal of tactics for their management exists; but, unfortunately the use of ozone-depleting, soil-contaminating, mammalian-toxic, broad-spectrum and expensive organophosphate, carbamate and fumigant nematicides remains the most widely utilized tool . This chemical management tactic choice is economics-driven in an era hypersensitive to escalating agricultural production costs . Widespread public awareness of the negative environmental consequences of over- and/or mis-usage of pesticides, especially nematicides, has resulted in legislation in America and abroad prohibiting or severely curtailing their production . A decade of university-based research with a novel colloidal mixture, formulated with near-inert sodium alginate as the primary ingredient, has produced an environmentally -responsible, low-rate and inexpensive material effective against a range of the most damaging phytoparasitic nematode species . Research to date had documented significant efficacy of this material against populations of reniform, root-knot, soybean cyst, sting, stunt, lesion, lance, spiral, ring and stubby-root nematodes . Subsequent yield enhancement resulting from management of these nematodes ranged from 23-60 percent on sugarcane, soybean, cotton and a wide range of vegetables including tomato, cucumber, eggplant, bell pepper, cabbage and carrot . Unlike most traditional broad-spectrum nematicides that are lethal to both pest and beneficial rhizosphere-inhabiting microorganisms, this material is nematode-specific . The activity of this as yet non-commercialized formulation results in large measure from the physical blockage of natural body openings of the nematode and also from interference with the tactile responses that lead to host-finding . Supporting evidence to be presented will include data from field, microplot and greenhouse trials comparing the density and distribution of phytonematodes and crop yields in soils treated with this alginate-based material versus traditional fumigant and non-fumigant nematicides . Additionally, data from in-vitro laboratory trials and light and electron microscopy studies will verify the mechanism of action . Collaboration between university and commercial scientists in the development of eco-responsible chemical management tools is essential for plant protection in twenty-first century agriculture .

MO 330Chronic effect of boiling green tea on Swiss albino miceM .H . HaebaGaryounis, Benghazi, LibiaBoiling green tea is a common hot drinking in Libya . Usually, it boils for 20 to 30 minutes, to have it concentrated . Most people who drinking the green tea, have it three times daily . In this study we investigated the effect of boiling green tea on Swiss albino mice for chronic effect, for 21 days . The concentration were used daily, 0 .1, 0 .2 and 0 .4 ml, orally to adult mice body weight, 26 ± 3 g which is three time more than amount that people drink daily . During the study, body weight, blood parameters (PCB), Cholesterol, triglyceride, sugar, and liver, heart, kidney and pancreas weight were measured . These doses have shown no effect on mortality . However, Body weights were decreased at 0 .4ml comparison to control . RBC, PLI number as well as HGB and Cholesterol, triglyceride, sugar have no different with control group . However, WBC were increased at 0 .2 and 0 .4 ml . This study was first in Libya to show effect of boiling green tea, and can be repeated to show effects on reproduction for longer period as well as to investigate its effect on short time, on some biomarker .

MO 331The Sacaca´s study on the treatment of Hypertension, Rio Branco, Acre, Amazonia, BrazilM .C . Oliveira, L .P .M . Barros, M .M . AbreuUFAC, Rio branco, BrazilINTRODUCTION: Modern science has gradually awakened interest in plants of the Amazon . The Croton cajucara, Benth (Euphorbiaceae), known popularly as sacaca seems to have different pharmacological properties . It is known that the poorest segments of society and older have a higher prevalence of hypertension (SH), and have as the only alternative medicines supplied by the Unified Health System, so, invest on the potential of medicinal plants is essencial because it corroborates the ethical principles and international commitments, while promoting the generation of wealth with social inclusion . The study aimed to evaluate the effects of its use in the treatment of hypertension according to reports in the study population . MATERIAL AND METHODS: The study focused on people living in the city of Rio Branco, Acre . The questionnaire was used in 125 people in this population, and 25 people each SPS to carry out ethnobotanical survey of Sacaca . We used the randomizer program for the selection of each of the 25 people who were interviewed . All respondents signed a consent form Statistical analysis was performed SPSS, version 13 .0 for Windows . The work was approved by the Ethics Committee at the Federal University of Acre . RESULTS: From the survey it was possible to profile the population studied, most of whom were aged over 60 years, female, attended high school, had incomes of up to three minimum wages, lived with more than four people, worked, did not consume alcohol, not smoke and were part of some religious group . It was observed that 60% of those interviewed knew the sacaca, 80% of these had their blood pressure controlled and monitored . No respondent indicated the use of medicinal plants through medical advice . Most received indication from friends . About 30% used sacaca for the treatment of illness . The population was unable to report on side effects or contraindications, which drew attention because cytotoxic and hepatotoxic effects have been proven . CONCLUSION: We did not observe the use of sacaca in the treatment of hypertension . However, there are therapeutic effects that can be used as adjuvants in the treatment of this disease . We emphasize the need for more scientific research in the north of this plant and its uses especially in the treatment of heart disease, since in 2025 Brazil will be the sixth country in the world in the elderly population (WHO) .

KEY WORDS: Hypertension, Medicinal Plants, Sacaca .

MO 332Ecotoxicological effect studies with mycotoxins of possible environmental concernJ . Schenzel1, K .L . Chue2, J .F . Müller2, C . Otto3, K . Schirmer3, T .D . Bucheli1

1Agroscope Reckenholz-Tanikon ART, Zurich, Switzerland2National Research Centre for Environmental Toxicology; University Queensland, Coopers plains, Australia3Department of Environmental Toxicology, Eawag, Dubendorf, SwitzerlandMycotoxins are naturally occurring secondary metabolites of fungi colonizing a variety of cereals, fruits, vegetables and organic material in the soil, but can also arise due to moist conditions during storage . They regularly contaminate our food and feed and, hence, their toxicity to humans and husbandry animals has been studied in detail .

Recently published data showed that the aquatic environment can also be exposed to mycotoxins . The identified main input sources of mycotoxins into the aquatic environment include 1) run-off and drainage water from fields cultivated with cereals, like wheat or corn, 2) manure application and excretion from grazing livestock and 3) human excretion via sewer systems . However, current ecotoxicological data on mycotoxins are scarce . Therefore, we applied an array of ecotoxicological tests to quantify their effects . The baseline toxicity was quantified with the bioluminescence inhibition test using the marine bacterium Vibrio fischeri . The specific modes of toxic action that were targeted with four additional bioassays included aspects of dioxin-like activity (AhR-CAFLUX-test), genotoxicity (umuC-test), phytotoxicity (IPAM-test) and glucocorticoid like activity (GR-test) . Data from these tests will be presented and used for a first preliminary evaluation of the ecotoxicological relevance of mycotoxins .

MO 333Occurrence of mycotoxins in the effluent of a waste water treatment plant

204 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

J . Schenzel1, T .D . Bucheli1, K . Hungerbühler2

1Agroscope Reckenholz-Tanikon ART, Zurich, Switzerland2Safety and Environmental Technology Group; ETH Zurich, Zurich, SwitzerlandMycotoxins are naturally occurring secondary metabolites of fungi colonizing a variety of cereals, fruits, vegetables and organic material in the soil, but can also arise due to moist conditions during storage . Mycotoxins have been studied intensively for decades due to their occurrence in food and feed and, hence, their potential threat to human and animal health . Recently published data support the presumption that the aquatic environment can also be exposed to mycotoxins via human excretion due to the consumption of mycotoxin contaminated food stuff . For instance, deoxynivalenol (DON) was frequently observed in urine and was significantly associated with cereal intake . Accordingly, DON proved to be omnipresent in the primary effluent of several waste water treatment plants (WWTPs) . Based on comparable or even higher amounts produced in wheat and maize, and similar or even higher aqueous solubilities, a larger number of mycotoxins is likely to enter the aquatic environment . This hypothesis was tested by application of a newly developed multi-residue screening method to quantify 33 mycotoxins in WWTP effluent samples . Data from this campaign will be presented .

MO 334Occurrence of mycotoxins in Swiss surface watersJ . Schenzel1, T .D . Bucheli1, K . Hungerbühler2

1Agroscope Reckenholz-Tanikon ART, Zurich, Switzerland2Safety and Environmental Technology Group; ETH Zurich, Zurich, SwitzerlandMycotoxins are naturally occurring secondary metabolites of fungi colonizing a variety of cereals, fruits, vegetables and organic material in the soil, but can also be formed due to moist conditions during storage . Mycotoxins have been studied intensively for decades due to their occurrence in food and feed and, hence, their potential threat to human and animal health . More recently, it has been shown that deoxynivalenol (DON) and zearalenone (ZON), two important mycotoxins produced by Fusarium spp ., are emitted from fields cultivated with Fusarium-infected cereals or maize, or via human excretion and wastewater treatment plants, thereby acting as micropollutants in the aquatic environment, specifically surface waters . This far, no such data is available for any other mycotoxin, although, based on comparable or even higher amounts produced in wheat and maize, and similar or even higher aqueous solubilities, a larger number is likely to enter the aquatic environment . Therefore, we applied an earlier established analytical screening method to quantify 33 of the most prominent mycotoxins at trace level concentrations (i .e . ng/L) in different aqueous matrices to surface water . For this, flow-proportional samples were gathered weekly to biweekly at twelve sites from ten rivers across Switzerland since December 2009 till end of October 2011 . Throughout the investigation period, four mycotoxins were detected regularly, with nivalenol being the most prominent mycotoxin in terms of frequency of occurrence, closely followed by DON which showed the highest mean concentration . Other compounds detected occasionally include beauvericin, and 3-acetyl-deoxynivalenol . Different approaches to elucidate the main contributors of mycotoxins in surface waters will be presented .

MO 335Lipid soluble acyl conjugates of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in blue mussels from the Baltic SeaD .C . Lindqvist, S . Jensen, L .T . AsplundStockholm University, Stockholm, SwedenAlgae and cyanobacteria in the Baltic Sea have been reported to produce high quantities of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) (1) . These are released into the surrounding ecosystem at the end of the algae’s life cycles . Some of these compounds have been proven to disrupt the oxidative phosphorylation in exposed organisms (2), which causes a decreased production of adenosine triphosphate (ATP) . This may lead to serious effects on the energy balance in the Baltic ecosystem . These compounds have also shown negative effects of the endocrine system through e .g . competitive binding to transthyretin (TTR) (3), and interactions with estrogen receptors (4) . The present study was undertaken to investigate the possible conjugation of OH-PBDEs, with lipophilic substrates (e .g . fatty acids), in mussels under natural conditions . Such conjugation could have the potential to work as a detoxification mechanism in mussels suffering from intense exposure . The study revealed that as much as 50% of the total amount of OH-PBDEs in blue mussels can be bound as esters, and individual congeners can be conjugated in concentrations of up to 65 ng/g lipids . The discovery that halogenated phenols, at least in mussels, can occur as lipid soluble neutral conjugates in high amounts, under natural conditions, is new . The repercussions of these findings may be that conventional analyses to assess the total amount of halogenated phenols, in e .g . mussels, systematically have underestimated the true levels . Although the conjugation may relieve the toxic effects of the OH-PBDEs within the mussels, it is probable that these conjugates are hydrolysed in the digestive tract of predators feeding on these mussels, which will release the OH-PBDEs back to their free biologically active form . The blue mussel has a high ecological value in the Baltic Sea where it is the dominating specie and the main source of food for many other species, e .g . marine birds . Long-tailed ducks, with a body mass of about 1 kg, have to eat about 1 kg of blue mussels per day, and eiders up to 2 .5 kg per day, just to stay alive in winter (5) . This means that these birds are exposed to high amounts of both bound and free OH-PBDEs through their diet .

(1) Malmvärn et al . Chemosphere 2008, 72 (2) van Boxtel et al . Environ . Sci . Technol . 2008, 42 (3) Ucan-Marin et al . Toxicol . Sci . 2009, 107 (4) Meerts et al . Environ . Health Perspect . 2001, 109 (5) Bustnes and Systad Waterbirds 2001, 24

MO 336Spectrophotometric determination of free gossypol in cottonseed by-productsA .G . Dantas1, E .A . Pereira1, F . Amorim1, J .A . Sousa1, H .D . Louvandidni2, F .L .F . Soares1

1Universidade de Brasília, Brasília, Brazil2Centro de Energia Nuclear na Agricultura, CENA/USP, São paulo, Brazil Gossypol is a natural herbicide found in the seeds of the cotton of the genus Gossypium, which is used as a protein source in diets for monogastric animals . This study aims to determine the level of gossypol in cotton matrices . The method used for the analysis procedure consists of a liquid-liquid extraction using organic solvents and spectrophotometric quantification using the second derivate spectra . The results obtained so far suggest that in the three matrices studied, the highest percentage of free gossypol is in cottonseed . The determination of free gossypol is important for purposes of food inspection .

MO 337Novel tumour promoting metabolites in complex cyanobacterial samples (biomasses and exudates) characterized by in vitro assaysK . Novakova, J . Kohoutek, K . Hilscherova, L . BlahaResearch Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech RepublicRecently, production of novel cyanobacterial metabolites with tumour promoting activity had been reported (Toxicon 2009, 53:519-524) . Goal of our present study was to investigate the tumour promoting activity of complex cyanobacterial samples including intracellular (extracts of biomass) and extracellular mixtures (exudates/spent-media) . Biomasses and exudates from 11 different cyanobacterial cultures forming toxic water blooms (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii, Anabaena flos-aquae, Cylindrospermopsis raciborskii etc .) were collected . Tumor promoting potencies of the cyanobacterial samples were assessed using the assay for gap-junctional intercellular communication (GJIC) in rat liver stem-like cells WB-F344, where inhibition of GJIC is established as a marker of tumor promotion . Significant down-regulation of GJIC was caused by both intracellular and extracellular mixtures but effects differed among tested cyanobacteria and were not related to the content of known cyanotoxins such as microcystins or cylindrospermopsin . Exudate of C . raciborskii was selected for further characterization because of its strong GJIC-inhibition activity . Semipreparative HPLC (C-18 column) with water/methanol gradient elution was used for fractionation . Two fractions with hydrophobic compounds strongly down-regulated GJIC and the LC-MS/MS analyses were used for detailed separation and characterization of the causative agent . A new small compound with molecular weight 320 appears to be responsible for observed tumor promoting potencies . The research was supported by the Czech National Science Foundation grant No . 524/08/0496 and by the project CETOCOEN (CZ .1 .05/2 .1 .00/01 .0001) from the European Regional Development Fund .

MO 338Influence of light, cultivation and growth phase on cyanobacterial and algal production of metabolites with specific activityK . Novakova, E . Sychrova, T . Prochazkova, K . HilscherovaResearch Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech RepublicCyanobacteria are known producers of wide spectrum of compounds with various effects such as skin irritation, gastrointestinal tract and respiratory distress, neurotoxicity, immunotoxicity or hepatotoxic, cancerogenic, genotoxic and mutagenic effects . Recent studies have shown also endocrine disruptive potential of specific cyanobacterial and algal strains, reported for natural strains as well as for laboratory cultures . Our previous papers reported activation of estrogenic receptor (intracellular nuclear receptor) using in vitro assays after exposure to both intracellular and extracellular mixtures of several cyanobacterial and algal samples . This estrogenic activity of compounds produced by cyanobacteria and algae could contribute to endocrine disruptive potential in surface waters . Aim of our study was to characterize impact of external factors such as light, cultivation conditions and growth phase on production of and estrogenic metabolites in selected cyanobacterial (Microcystis aeruginosa PCC 7806 and Aphanizomenon gracile RCX 06) and algal strains (Scenedesmus quadricauda CCALA 463) by use of in vitro reporter gene trans-activation assay with the ER-linked luciferase gene under control of the ERE (estrogen responsive element) . At the same time, the production of well known cyanotoxin microcystin was assessed . Our results characterize dynamics and variability of production of endocrine disruptive compounds and microcystin during growth phase . The detailed characterization of the cultures enabled to quantify the production of studied metabolites relative to the cell count and dry weight of the biomass .

MO 339The effects of oral administration of toxic cyanobacteria on multiple physiological , biochemical, hematological and immunological parameters in ratsO . AdamovskyResearch Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech RepublicCyanobacteria are known to produce numerous bioactive compounds including widely studied peptide hepatotoxins - microcystins (MCs) . Aims of the present study were to provide a model simulation of a simple food chain for evaluation of impacts cyanobacterial toxins on the mammal (rat) physiology under different exposure scenarios .

20�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

Experimental rats were fed for 28 days with food containing fish meat with external additions of isolated microcystins, biomass of toxic cyanobacteria Microcystis, nontoxic cyanobacteria Arthrospira and also green alga Chlorella . Subgroups of the animals were also challenged with a model antigen KLH to investigated immune-related parameters . Although considerable amounts of microcystins were orally administered to rats, levels of MCs in the tissues were below the detection limit (1 ng/g fresh weight; based on the tandem LC/MS) . Only traces of the conjugates of microcystins with cystein and glutathion were detected in the rat’s liver exposed to Microcystis biomass . The feed containing cyanobacterial biomass was found to have negative impacts on the weight gain and food conversion ratio of experimental rats . Interestingly, rats exposed to MCs had stimulated immune system (higher antibody responses to administered KLH antigen) . Also modulations of certain lymphocyte subpopulations was recorded with the most interesting observation of stimulated numbers of NK cells in the groups exposed to isolated toxins . Biomarkers of the oxidative stress (glutathione levels, lipid peroxidation) did not show any differences , while several hematological parameters of blood were significantly modulated . Our study demonstrates that oral exposure to microcystins or cyanobacterial biomass may have different impacts on various less explored biochemical and immune parameters in experimental mammals .

MO 340Protective role of N-Acetylcysteine against the oxidative damage induced by cylindrospermopsin in tilapia (oreochromis niloticus)D . Gutiérrez-Praena1, A . Jos2, S . Pichardo2, A .I . Prieto2, V . Vasconcelos3, A . Cameán2

1University of Seville, Seville, Spain2Area of Toxicology, University of Seville, Seville, Spain3Biology Department, Faculty of Sciences, University of Porto, Porto, PortugalCylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of cyanobacteria . CYN consists of a tricyclic alkaloid with a high solubility in water, where it can be present approximately the 90% of the total CYN produced by cyanobacteria . Aquatic animals and plants can be exposed to CYN, and it is known that CYN is a potent synthesis inhibitor of protein and reduced glutathione (GSH) . Moreover, recent studies have established that CYN produces oxidative damage through the generation of reactive oxygen species (ROS) . As the presence of CYN in freshwater systems could have negative effects on fish it would be interesting to find compounds able to counteract the toxicity observed . In this sense, N-Acetylcysteine (NAC) is a GSH precursor which can protect organs against the oxidative damage through two different mechanisms: by the stimulation of the GSH synthesis and by direct association to ROS . In the present work, we aimed to investigate the effects of pure CYN and CYN from a lyophilized Aphanizomenon ovalisporum culture on fish, and the protective role of a pre-treatment (7 days) with different doses of NAC . Both products (CYN and NAC) were administered together with the food . Fish were euthanized after 24 hours of the toxin exposure, and liver and kidney were extracted . For this purpose, we used different oxidative stress biomarkers such as lipid peroxidation, γ-glutamylcysteine synthetase (GCS) activity, and GSH levels . Results showed that CYN induced oxidative stress, as evidenced by the increase of lipid peroxidation, the decrease in GSH/GSSG, and the alteration of the GCS activity . Moreover, the exposure to cyanobacterial cells containing CYN induced higher toxic effects in comparison to pure CYN . NAC supplementation was effective in reducing the CYN induced toxicity, particularly at the highest dose employed (45 mg/fish/day) . In conclusion, NAC can be considered a useful chemoprotectant in the prophylaxis of CYN-related oxidative stress induction in fish . Acknowledgements: the authors wish to thank Junta de Andalucía (P09-AGR-04672) and the Ministerio de Ciencias e Innovación (AGL2009-10026) for the financial support of this study .

MO 341Preliminary study of biomarkers in mussel haemolymph after exposition to toxins extracted from Microcystis aeruginosa and Lyngbya wolleiM .G . Gelinas1, M .G . Gust2, M .F . Fournier1, F .G . Gagné2

1INRS-IAF, Laval, Canada2Environnement Canada, Montréal, CanadaThe freshwater cyanobacterium Lyngbya wollei is a dominant primary producer in many lakes and reservoir, wherease Microcystis aeruginosa can become a dominant cyanobacteria commonly associated with eutrophic conditions . Both cyanobacteria produced toxin, M. aeruginosa (MCYST) produced a potent specific hepatotoxin, wheareas L. wollei (LYNGTX) produced an analogue to saxitoxin which is a neurotoxin . Selected immune parameters investigating haemolymph integrity and immunocompetency were analyzed after exposure of the two toxins by flow cytometry of non cytotoxic low concentrations of MCYST and LYNGTX . Exposure of freshly isolated mussel haemolymph to low concentrations of 2, 5, 10, 25, 50ng/ml for 18h periods resulted in a significant interaction term (concentration x type of toxins) of the two-ways ANOVA of haemocyte viability . Haemocytes exposed to MCYST tended to decrease their viability at the highest concentration whereas heamocytes exposed to LYNGTX tended to increase their viability at the highest concentration . However, the type of toxin was significantly different for phagocytosis activity . Haemocyte exposed to MCYST had a lower phagocytosis activity than haemocyte exposed to LYNGTX . Furthermore, the present study will investigate the effects of these toxins on the intracellular thiols production, reactive oxygen species (ROS) production, cyclooxygenase activity (COX) and nitrate production in mussel haemolymph . Preliminary results showed that haemocytes integrity and immunocompetence exhibited opposite reaction of these two types of toxins . A decrease of the phagocytic index is usually observed response to in vivo or in vitro exposures to toxic concentration of xenobiotics where MCYST seemed to have a similar effect . This preliminary study will require additional biomarkers to support the hypothesis of a toxic effect associated with the mats of filamentous L. wollei .

MO 342Phosphatases and Esterases of Limnoperna fortunei (golden mussel) in presence of Microcystis aeruginosaM .M . Oliveira1, E .S . Silva2, P .C . Bastos3, S .H . Calazans4, F .C . Fernandes4, M .H .C . Baeta Neves4, M .V . Silva Filho5, V .L .F . Cunha Bastos2, J .C . Cunha Bastos2

1Instituto Federal Fluminense, Cabo frio, Brazil2Universidade do Estado do Rio de Janeiro, Rio de janeiro, Brazil3Universidade Veiga de Almeida, Cabo frio, Brazil4Instituto de Estudos do Mar Almirante Paulo Moreira, Arraial do cabo, Brazil5Fundação Oswaldo Cruz, Rio de janeiro, Brazil Toxic blooms of cyanobacteria are reported in freshwater sources around the world, and the effects of these events in environments where there are filter feeding organisms can involve the accumulation of toxins and their transfer in the trofic chain . In order to study possible changes in mussels from some of the enzymatic activity and bioaccumulation of microcystins, we performed in vivo experiment with the species Limnoperna fortunei that were exposed to cells of Microcystis aeruginosa (strain NPLJ-4) . After 48 hours of exposure, the cells were counted and assayed the total phosphatase and esterases, these at a fraction obtained by centrifugation at 10,000 x g . Specific activities of total phosphatase were inhibited by 90%, while those of carboxylesterase and acetylcholinesterase by 50% in this experiment of effect in vivo . Mussels exposed to higher concentrations in vivo (5,140,000 and 2,570,000 cells .mL-1) accumulated microcystin . In vitro assays of enzymes soluble fractions L .fortunei with extracts of M . aeruginosa strain NPLJ-4, obtained with 100% methanol (Me100 - extract with microcystin) and 10% (Me10 - without microcystin extract) show that inhibition of phosphatases occurs with both extracts, whereas esterases are inhibited only with the Me10 . The esterases of L . fortunei are similar to other species of molluscs as demonstrated in their IC50 in the presence of eserine . The inhibitions caused by the in vitro fraction without microcystin present new possibilities on the effects of exposure to Microcystis aeruginosa .

MO 343Do mixture effects of metal stress (Cu) and natural stress (cyanobacterial toxins) add up in Daphnia magna?J .D . Hochmuth1, J . Asselman2, K .A .C . De Schamphelaere2

1Ghent University (UGent), Ghent, Belgium2Ghent University, Ghent, BelgiumCurrent risk assessment of chemicals does not deal with mixture toxicity, which contrasts with ecology reality . The combined effect of stressors is not necessarily bound to the generic pattern of additivity but can instead result in their combined effect being either more or less toxic than would be expected from their individual toxicity (synergy or antagonism) . We investigated the combined and interactive effects of dissolved copper (Cu) and cyanobacteria in two Daphnia magna clones (Xinb3 and Iinb1) . Several cyanobacterial species (Microcystis aeruginosa, Cylindrospermopsis raciborskii, Anabaena sp, Nodularia sp ., Oscillatoria sp ., Aphizomenon-flosaque) were investigated, ensuring toxins with different Modes of Action (MoA) . Semi-static 21-day life-table experiments were performed according to an extended second-order two factor central composite design, consisting of 24 treatments . Several life history parameters were recorded (survival, daphnid size, time to maturity, clutch sizes, total reproduction, and intrinsic rate of increase rm) . The experiment was performed . Significant deviations from additivity determined using the MIXTOX model (Jonker et al . 2005 Environ Toxicol Chem 24:2701-2713) . The fitted model can be used to make predictions on the magnitude of increase or decrease of Cu toxicity in the presence of cyanobacteria .

MO 344Interactive effects of combined chemical and natural stressors on Daphnia pulexJ . Asselman, K .A .C . de SchamphelaereGhent University, Gent, BelgiumIn aquatic ecosystems Daphnia are exposed to a wide variety of both natural and chemical stressors . These stressors can cause interaction effects resulting in an increased impact on aquatic ecosystems . We exposed Daphnia pulex to binary combinations of cyanobacteria and insecticides . We selected three cyanobacteria differing in habitat and toxin production: Cylindrospermopsis raciborski (cylindrospermopsin producer), Nodularia (nodularin producer) and Oscillatoria (anatoxin-a producer) . These stressors were combined with 8 insecticides differing in mode of action : acetamiprid, carbaryl, chlorpyrifos, deltamethrin, endosulfan, fenoxycarb, tebufenpyrad and tetradifon . Daphnia were exposed for a period of 21 days to an insecticide, a cyanobacteria and their mixtures based on a modified central composite design . This design allowed for simultaneous testing of individual and combined effects as well as testing for different doses and ratios . Results were analysed with the mixtox model developed by Jonker (Jonker et al . 2005 Environ Toxicol Chem 24:2701-2713 .) .The significance of model deviations for synergistic and antagonistic effects were determined through maximum likelihood, while regression analysis was performed to select the best reference model . The analysis resulted among others in additive effects for combinations of carbaryl with Cylindrosepermopsis, while combinations of tebufenpyrad and Cylindrospermopsis demonstrated antagonistic effects on reproduction of Daphnia . Results suggest complicated interaction patterns between insecticides and harmful cyanobacteria potentially related to mode of action patterns .

MO 345Physiological effects of a marine algal toxin on a primary consumer: tales of the unexpected

206 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

D . Deruytter, M .B . Vandegehuchte, C .R . JanssenGhent University, Ghent, BelgiumHarmful algal blooms can cause serious health risks and economical losses due to the production of toxins . Okadaic acid (OA), a marine toxin produced by different species of dinoflagellates including Prorocentrum lima, accumulates in filter feeding shellfish . OA is known to inhibit the protein phosphatase enzymes in humans consuming contaminated shellfish, resulting in the diarrheic shellfish poisoning syndrome . Although the physiological effects of OA on mammals are relatively well understood, this is not the case for the effects on the main vector (i .e . shellfish) . In this study, the effects of OA on the lysosomal membrane stability (LMS) of the haemocytes and on the protein phosphatase (PPase) activity of the hepatopancreas in the mussel Mytilus edulis were studied . In the first phase the mussels were dissected and the enzymes of the hepatopancreas were exposed to okadaic acid in vitro (conc . up to 672 µg OA/g hepatopancreas WW) . This resulted in a significant reduction (4%) of the PPase activity, starting at a concentration of 26 .2 ng OA/g hepatopancreas WW, with a maximum inhibition of approximately 15% at 168 ng/g hepatopancreas WW and higher . In the second phase, the mussels were exposed in vivo to toxin producing algae (P. lima) . Two concentrations were tested: 2 .85 µg OA/l (500 cells/ml) and 9 .55 µg OA/l (1500 cells/ml) . Okadaic acid accumulated in the mussels’ hepatopancreas with a mean concentration of 220 ng/gWW (500 cells/ml) and 740 ng/gWW (1500 cells/ml) . Based on the in vitro results a 15% reduction in PPase activity was expected . However, there was no significant reduction of the LMS or the PPase activity . Two conclusions can be drawn from these results . First, in vitro there is a 15% reduction of the PPase activity this means that these enzymes are sensitive to OA exposure . Secondly, in vivo OA has no negative effect on the PPase activity or LMS . Apparently M. edulis is, at the concentrations tested, able to prevent the inhibition of protein phosphatase enzymes by OA .

MO 346Oxidative stress responses produced in liver of tilapia by exposure to repeated doses of cylindrospermopsin from Aphanizomenon ovalisporumR . Guzmán-Guillén1, A .I . Prieto1, C . Fernández-Blanco1, V .M . Vasconcelos2, A .M . Cameán1

1University of Seville, Seville, Spain2Centro Interdisciplinar de Investigação Marinha e Ambiental, CIIMAR/CIMAR, Porto, PortugalCylindrospermopsin (CYN) is a tricyclic alkaloid, possessing a tricyclic guanidine moiety combined with hydroxymethyluracil . It is produced by several genera of cyanobacteria, growing worldwide in eutrophic freshwaters and it is easily transferred among different trophic levels causing damages to humans, animals and plants . It is well established its mechanism to block protein synthesis and, more recently, its ability to cause oxidative stress is being studied . The aim of this work was to study the oxidative stress responses in liver of tilapia (Oreochromis niloticus) exposed to repeated doses of CYN by exposure to both a culture and lyophilized cells of Aphanizomenon ovalisporum (LEGE X-001) . These parameters were: lipid and protein oxidation, and the enzymatic activities of Glutathione-S-transferase (GST), Glutathion Peroxidase (GPx), Catalase (CAT) and Superoxide dismutase (SOD) .

In this study, nine groups of tilapia with 8 fish per group were established: 3 control groups, 3 groups exposed by immersion to an A . ovalisporum culture (10 μg CYN/L), and 3 groups exposed by immersion to lyophilized A . ovalisporum cells (10 μg CYN/L) . The exposure to the toxin was carried out every two days within the periods of 8, 14 and 22 days, respectively . Afterwards, fish were sacrificed and the liver extracted for further determinations . In general, major changes were observed in tilapia treated for 8 days and the alterations were more significant when tilapia were exposed to CYN produced by the culture cells, in comparison to the lyophilized cells of Aphanizomenon ovalisporum . Acknowledgements The authors wish to thank the Spanish Ministerio de Ciencia e Innovación (CICYT, AGL2009-10026ALI) and Junta de Andalucía (P09-AGR-4672) for the financial support for this study .

MO 347Development of a method for determination of CYN in lyophilized Aphanizomenon ovalisporum cells by LC-MS/MS: validation and application to real samplesR . Guzmán-Guillén1, A .I . Prieto1, I .M . Moreno1, A .G . González2, M .E . Soria3, V .M . Vasconcelos4, A .M . Cameán1

1University of Seville, Seville, Spain2Department of Analytical Chemistry, University of Seville, Spain ., Seville, Spain3Mass spectrometry Facility, Centro de Investigación Tecnológica e Investigac, Seville, Spain4Centro Interdisciplinar de Investigação Marinha e Ambiental, CIIMAR/CIMAR, Porto, PortugalHarmful cyanobacterial blooms are occurring in eutrophic freshwater lakes and reservoirs throughout the world and can present a public safety hazard through contamination of drinking water supplies . There are over 40 species representing 20 genera from three cyanobacterial orders known to produce cyanotoxins which include both cyclic peptides and alkaloids such as cylindrospermopsin (CYN), produced by Aphanizomenon ovalisporum among other species . Therefore it is important to develop and validate a method to determine CYN at trace levels . The aim of this work was to develop an analytical procedure based on solvent extraction followed by a purification step with graphitized cartridges and LC-MS/MS for CYN determination from lyophilized cultures of A . ovalisporum (LEGE X-001) . The extraction and purification steps were optimized using a two-level full factorial design with replications . This report presents a sensitive, reproducible, accurate, and robust method for extraction and determination of CYN in lyophilized cells . The recoveries (83-94%) and intermediate precision values obtained (5 .6-19 .3 %), as well as the robustness of the method for the three factors considered, permit its validation . This method provides acceptable detection and quantification limits for environmental studies and proves its utility for monitoring CYN in lyophilized natural blooms samples, and also for routine human health assessment purposes in relation to the provisional guideline and TDI, representing an available instrument to advance in these studies . Acknowledgements The authors wish to thank the Spanish Ministerio de Ciencia e Innovación (CICYT, AGL2009-10026ALI) and Junta de Andalucía (P09-AGR-4672) for the financial support for this study .

MO 348Screening for cylindrospermopsin, anatoxin-a and saxitoxins in FranceM . Dechesne1, F . Pitois2, C . Pagotto3, S . Lenoir1, J . Fastner4

1Veolia Environnement Recherche & Innovation, Rueil-malmaison, France2Limnologie SARL, Rennes, France3Veolia Eau Direction Technique, Saint maurice, France4Federal Environmental Agency (UBA), Berlin, GermanyMicrocystins are the most intensively studied toxins and numerous reports on their occurrence exist all over the world . A WHO guideline value of 1 μg/L Microcystin-LR exists for drinking water, which has been adopted in several European guidelines (e .g . France) . In contrast, little is known about the distribution of other hepatotoxins such as cylindrospermopsin (CYN), and the neurotoxins anatoxin-a (ATX) and saxitoxins (STX) in Europe . These are primarily produced by cyanobacteria of the Nostocale order . As they may be a problem for the safety of drinking water, Veolia carried out a research program on these cyanotoxins and their potential cyanobacteria producers in France from 2007 to 2011 . Ten freshwater reservoirs used for drinking water production were studied during the summers of 2007, 2008 and 2010 . Mixed water samples from the water column of the epilimnion were collected . They were analysed for nutrients, chlorophyll-a, phytoplankton, CYN, ATX and STX . Cyanobacteria were detected in 98% of the samples . WHO level 3 for raw water used for drinking water production (>100 000 cell/mL) was reached for 20-27% of the samples . In 2007, Oscillatoriales, dominated by Planktothrix agardhii, largely prevailed in deep water whereas Nostocales were more present in pre-reservoirs; Chrococcales were minor . In 2008, Oscillatoriales prevailed over all other orders in the pre-reservoirs or in deep waters . In 2010, Nostocales were primarily present in deep waters and Oscillatoriales in pre-reservoirs . Among the species observed in the water samples, the following potential CYN, ATX-a, STX producers were observed: Anabaena flos-aquae, Anabaena planctonica, Aphanizomenon flos-aquae, Aphanizomenon gracile and Aphanizomenon issatschenkoi . ATX, CYN and/or STX were measured in 41% of the samples in 2007 and 2008 . In 2010, 60% of the samples were positive for at least one of these toxins . Nevertheless, total concentrations remained low in the freshwater samples analysed (<< 1µg/L) . The present investigation showed that cyanotoxins not yet investigated thoroughly in France, especially anatoxin-a, are widespread in French reservoirs . The raw water concentrations of CYN, ATX-a, and STX encountered in the 2007-2010 samples did not present any risk for drinking water production . Nevertheless, the presence of cyanobacteria can be highly variable over time and space and it is recommended to have specific monitoring strategies in reservoirs used for drinking water production .

MO 349Sensitive determination of microcystins using online SPE from environmental watersR .F . Jack, X .Q . Xu, R .J . RohrerThermo Fisher Scientific, Sunnyvale, United States of AmericaWaterblooms of cyanobacteria (blue-green algae) can produce potent toxins that have become a severe problem for eutrophic aquatic environments . Hepatotoxins are among the primary toxins produced by these species growing in lakes, ponds, and rivers used as drinking water sources . Microcystin contamination of drinking water at low nanomolar concentrations is considered a risk factor for cancer, and microcystin-LR has been associated with most of the incidents of toxicity involving microcystins . Therefore, the World Health Organization (WHO) has proposed a provisional guideline concentration of 1 .0 μg/L for microcystin-LR in drinking water . The authors have reported a simple, fast, and effective target-cut on-line SPE method followed by HPLC with UV detection . This on-line SPE method is different from the typical one . The bound analyte on the SPE column is selectively eluted from the SPE column using a mobile phase gradient, just like the first dimension of a two-dimensional chromatography system . This reduces the number of interferences for sample analysis . Here, the target-cut on-line SPE method followed by HPLC with UV detection was applied to the determination of three microcystins (-LR, -RR, and -YR) in drinking, tap, and lake water . The three target analytes were co-eluted from the first column using chromatographic conditions that eliminated as many interferences as possible; then the analytes were sent to the analytical flow path and separated on the second column using the same type of stationary phase under different chromatographic conditions . This design takes advantage of the separation power of both columns and may eliminate interferences more efficiently than typical on- and off-line SPE methods . Sub-μg/L concentrations of microcystins-LR, -RR, and -YR spiked in water samples were determined, which exceeds the WHO requirement .

20�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

MO 350Hazard assessment of microcystins produced by filamentous cyanobacteria in urban lakes using molecular and biological methods. A case study in Mexico CityF .F . Martinez-Jeronimo, R .M . Pineda-Mendoza, R . Olvera-RamirezEscuela Nacional de Ciencias Biologicas, IPN, Mexico city, MexicoFormation of cyanobacterial blooms in water bodies with a high nutrient content (mostly P and N) is a frequent occurrence affecting freshwater quality at all latitudes . Bloom development is of great concern since cyanobacteria can biosynthesize a large number of secondary metabolites, some of which are toxic to aquatic biota and mammals, including humans; of these, microcystins (MCs) are the most abundant and well known . MCs are potent inhibitors of the protein phosphatases 1 and 2A . Zooplankton filter-feeders such as cladocerans are directly affected by MCs as a result of ingestion of cyanobacteria or trough contact with intracellular products when cyanobacterial cells break up during and after blooms . In present study a total of 17 strains of filamentous cyanobacteria isolated from three eutrophyzed urban lakes in Mexico City were characterized using the microcystin synthetase region mcyA-Cd . Acute 48-h toxicity was evaluated in the aqueous crude extracts of different strains using the cladoceran Daphnia magna and total microcystin content was determined by ELISA . The mcyA-Cd region was amplified in 16 microcystin-producing strains but microcystins were only identified in eight strains with values ranging from 0 .1422 to 2 .772 µg L-1 . Nevertheless, all aqueous crude extracts induced acute toxicity in D. magna neonates, with LC50 values ranging from 363 .91 to 741 .8 mg L-1 (dry weight) . The strains with the highest acute toxicity were Pseudanabaena mucicola, Planktolyngbya sp . and Spirulina sp . The toxicity observed in non-microcystin-producing strains may be induced by cyclic peptides other than microcystins (anabaenopeptins, microviridins and cyclamides) . The analytical methods used in the present study (PCR and ELISA) were useful and reliable for determining MC production and its potential contribution to the acute toxic effects observed in D. magna The results obtained warn of the toxigenic potential of filamentous cyanobacteria, since though Microcystis spp . are frequently predominant in blooms, other toxins and intracellular metabolites released by filamentous cyanobacteria may induce toxicity on aquatic organisms and potentially also in humans .

MO 351Occurrence of harmful algae and related toxins in the Po River Delta (Northern Adriatic Sea, Italy)D . Bilanicova1, G . Pojana1, C . Facca1, E . Sebastiani2, A . Sfriso1, A . Marcomini1

1University Ca’ Foscari Venice, Venice, Italy2SRA Instruments, Milan, ItalyAn integrated approach for the structural identification and quantification of algal toxins in water and mussel samples from the Po river delta (Northern Adriatic Sea) was developed by applying a combination of analytical techniques such as Optical and Scanning Electron Microscopy (OM, SEM), and Liquid Chromatography coupled with High Resolution Time of Flight Mass Spectrometry (HPLC-HR-TOF-MS) . The proposed approach was applied to the investigation of potential occurrence and distribution of harmful algae and their related toxins in the above mentioned coastal area . The performed sampling sessions showed that potentially harmful algae such as Dinophysis caudata, D. mitra, D. sacculus, P. minimum, Protoceratium sp., etc . were present during the summer period in the investigated area . Nevertheless, quantitative observations demonstrated that their abundances were always significantly below the conventional limit (~200 cells/L) for which poisoning events could occur . On the other hand, the HPLC-HR-TOF-MS analysis of collected mussels revealed a strong (over regulatory limits) contamination by free Okadaic Acid (OA), while concentration levels of Pectenotoxins (PTXs) and Yessotoxins (YTXs), concurrently analyzed, were instead below these limits . The adopted extraction/purification procedures permitted to distinguish the contributions of free toxins from those due to their various esterified forms identified to mussel contamination, and to determine the concentration levels in total edible tissue, as well in the hepatopancreas . The high resolution of the Mass Spectrometer permitted also to identify new toxins esters no previously reported by literature .

MO 352Two new microginins congeners in Brazilian strains of Microcystis sp. (CYANOBACTERIA)F . Dörr, R .L . Carneiro, F .A . Dörr, E . PintoUniversity of São Paulo / Faculty of Pharmaceutical Sciences, São paulo, BrazilMicrocystis species are the most common bloom-forming cyanobacteria in several countries and are related to significant health problem due to the production of over 60 chemical forms of microcystins . Numerous new bioactive compounds known as cyanopeptides (CNPT) were identified as cyanobacteria secondary metabolites . Among these compounds there are aeruginosins, anabaenopeptins and nostophycins, commonly described as protein inhibitors . More than 20 variants of microginins, which comprise a peptide group, were already found in Microcystis strains and natural samples . Despite several works have been published about CNPT produced by cyanobacteria, few of them are related to by Brazilian strains production . In this study microginins and microcystins diversity and their variation during growth of three Microcystis sp . strains (LPTNA01, LTPNA08 and LTPNA09) isolated from a southeast Brazilian reservoir were investigated . The strains were maintained in ASM-1 medium, 24±2 oC, with a 14:10h of light:dark cycle and photon flux of 40 µmol photons .m-2 .s-1, during 15 days, in triplicate . Samples were collected every 3 days for cell counting and CNPT analysis . Compounds were extracted from dried cells using methanol 90% and analyzed by HPLC-DAD and LC-MS/MS . Microcystins and microginins were monitored at 238 nm and 225 nm, respectively . Standard solutions of Mcyst-RR and Mcyst-LR were employed for quantification . Compound identity was confirmed by high accuracy mass spectrometry . New Microginins were identified by the characteristic gas phase fragmentation behavior for this class of peptides . Cells of Microcystis sp . grew exponentially until the 6th day of culturing (LPTNA01, R2=0 .9834±0 .002; LTPNA08, R2= 0 .9897±0 .011; LTPNA09, R2=0 .9876±0 .051) . Growth rate of LTPNA01 (rn=0 .9183±0 .003) was higher than LTPNA08 (rn=0 .8059±0 .012) and LPTNA09 (rn=0 .8111±0 .031; Dunn test, p<0 .001) . The cell content of microginins and microcystins have not shown significant variation along the evaluated growing period (KW test, p>0 .05) . The two new microginins congeners presented the peptide sequence Adha-Val-Iso/Leu-HTy-Tyr and MeAdha-Val-Iso/Leu-HTy-Tyr and were named as FD1 and FD2, respectively .

MO 353Degradation of microcystins by polymeric nanofiber membranes with encapsulated tetraphenylporphyrineS . Jesenská1, J . Kohoutek1, O . Adamovský1, L . Bláha1, J . Mosinger2

1Research Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech Republic2Charles University in Prague, Faculty of Sciences, Prague, Czech RepublicCyanobacterias cause various problems in freshwater ecosystems such as reduction of the oxygen content and production of cyanotoxins . The most common group of cyanotoxins are microcystins . They inhibit protein phosphatase and they may act as hepatotoxic and tumor promoting agents . To degrade microcystins in drinking waters, several methods based on oxidation have been suggested (e .g . using of chlorine or ozone) . In the present study we investigated photodegradation of microcystins by polymeric nanofiber membranes with encapsulated meso-tetraphenylporphyrine(TPP) . TPP is a photosensitiser which produces singlet oxygen (1O2) with big quantum yield after irradiation by visible light . Singlet oxygen is a strong oxidizing agent, and it can effectively degrade microcystins . Nanofiber membranes have large surface that assures contact with microcystins in water, they are stable, don’t release any harmful substances into the surroundings and they can be used repeatedly without substantial decrease in efficiency . It appears to be an ideal carrier for a photosensitizer such as TPP encapsulated into the structure of nanofibers . We have tested nanofiber membranes from polystyrene, sulfonated polystyrene, polyurethane, polyamide 6 and polycaprolactone for their ability to adsorb microcystins and to produce 1O2 . The rates of microcystins photodegradation by nanofiber membranes with TPP were studied . The degradation products were characterized chemically (using LC-MS/MS) and toxicologically (testing effects in vitro) . The existing results indicate potential of studied nanomaterials to photodegrade microcystins which can be used in technological applications . The final results of the study will be discussed .

MO 356Acclimatisation strategies in gastropods to environmental pollutants: comparison of a strong invasive to a declining endogenous speciesE . Lance, M . Bormans, C . Gérard, C . WiegandUEB/ University Rennes, Rennes, FranceLong term surveys in western France (Marais de Brière, Grand Lieu and Combourg Lake), evidenced declining populations of indigenous gastropod species, e .g ., Lymnea stagnalis, whereas the invasive Physa acuta became the single occurring gastropod species . The environmental stress enforcing these alarming biodiversity decreases is hypothesised to be a combination of anthropogenic pollution (e .g ., pesticides) together with recurrent and dense cyanobacterial blooms . Due to eutrophication of freshwaters, the frequency of cyanobacteria proliferations is increasing worldwide, with from 40 to 75% of cyanobacterial blooms producing hepatotoxins [e .g . microcystins (MCs)] . MCs constitute a real threat for target organisms as gastropods (intoxication by absorption of toxic cyanobacteria or dissolved toxins) . Physiological reactions of biotransformation and oxidative defence on the other hand enable organisms to withstand and excrete harmful substances . The capacity to perform these reactions is species specific and might contribute to the persistence of the invasive Physa acuta . The lower susceptibility may be facilitated by better cellular protection mechanisms: this is including the way of storage of accumulated toxin; the efficiency of biotransformation enzymes that transform the toxic compound into a less toxic one; activity of antioxidant enzymes mending or preventing oxidative damage; and excretion mechanisms . Therefore, this physiological capacity of Physa acuta will be compared to the indigenous species Lymnea stagnalis under exposure to a mixture of non MC-producing and MC-producing cyanobacteria Planktothrix argardhii with or without a common pesticide used in Brittany (glyphosate in Round Up suspension) . It seems likely that exposure to the one affects the reaction to the other, due to similar pathways for detoxification (glutathione S-transferase) and antioxidant response (enzymes and peroxidation markers) . In the environment, exposure occurs to mixtures of pollutants, this scenario is hence focus of the proposed work . Results are in process and we only expose hypothesis . Short and long term reactions of biotransformation and antioxidant response during these periods of exposure and depuration enable assessing of differences of the two species in efficiency in protection against the ecotoxicological stressors .

MO 357Study of the pro-oxidant capacity of anatoxin-a(s)-containing extract in mice exposed to a sub-lethal doseV .R . Rodríguez, E .P . Pinto, H .S . SpinosaUniversity of São Paulo, São paulo, BrazilAnatoxin-a(s) (antx-a(s)) is a cyanobacterial neurotoxin whose principal mechanism of action is the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AChE) . Because of the molecule´s instability and the lack of an analytical standard, the occurrence of this toxin in freshwater reservoirs and the knowledge of the whole events related to its toxicity are scarce . In the last decade, research related to organophosphates insecticides capability to induce oxidative stress in humans and animals has been profuse . Considering that antx-a(s) is the unique organophosphate produced by cyanobacteria currently known; the main of this work was to investigate the biochemical mechanism related to the pro-oxidant capacity of antx-a(s)-containing extracts . In order to achieve the objective, there was determined the activity of cholinesterasic and antioxidant enzymes such as acetylcholinesterase (AChE), butirylcholinesterase (BuChE), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD) in

20� SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

Swiss mice blood . Group of 9 or 10 animals were treated intraperitoneally (ip) with 20 mg .kg-1 of antx-a(s)-containing extract and sacrificed after 24 h, 48 h, 7 and 14 days of treatment by cardiac puncture . The AChE and BuChE activities in mice treated with antx-a(s)-containing extract stayed inhibited more than 55% during 48 h (n=9, p<0,001) . Normal activity of both enzymes was observed after seven days of treatment . By the other hand, changes in the antioxidant enzymes activities only began after the second day of treatment . Initially, both CAT and GPx showed lower activity than the control group after 48 h . Among those enzymes, GPx showed the highest decreased activity (n=9, p<0,001) . After seven days, while the antx-a(s)-containing extract promoted increasing of CAT and GR activities, GPx activity remained deeply decreased (n=10, p<0,01) . SOD activity did not show any statistical difference related to the control during all the treatments . Activity of all the evaluated enzymes was completely recovered after fourteen days . From the obtained results, it can be concluded that the antx-a(s) extract demonstrated its pro-oxidant capacity in mice at sub-lethal levels . This fact was proved through the unbalance on the activity of the antioxidant enzymatic defense system .

MO 358Physiological effects of phytotoxins in plants - laboratorial studies to evaluate the impact of toxic cyanobacteria in agriculture, food quality and human healthA .M . Campos1, D . Gutierrez-Praena2, A . Cameán2, J . Azevedo1, C . Azevedo1, V . Vasconcelos3

1CIIMAR, Porto, Portugal2Area of Toxicology, Faculty of Pharmacy, University of Seville, Seville, Spain3Faculty of Sciences, University of Porto, Porto, PortugalToxic cyanobacteria outbreaks are recognized as emerging environmental threats worldwide . The process occurs in inland water systems as in some coastal areas and is becoming widespread mostly as a consequence of human activity, leading to nutrient input increases and degradation of water quality . The cyclic heptapeptide microcystin (MC) is geographically the most widely distributed phytoxin in freshwaters . This toxin has been directly related to cases of human poisoning and to a prevalence of cancer induction in populations with chronic exposure to MC . Cylindrospermopsin (CYN) is another secondary metabolite produced by cyanobacteria . It is a tricyclic alkaloid with bioactivity . These compounds have shown to produce effects in a variety of aquatic organisms, vertebrates and mammals . Reports regarding variations in the animal behavior, growth rates, mortality, oxidative stress induction is contributing to a better understanding of the impacts of these toxins in the environment and animal health . Recent investigation supports the hypothesis of accumulation of MC and CYN in plant tissues . Nevertheless a major gap lies in the physiological response of plants to phytotoxins and the mechanisms of uptake and translocation of these molecules from roots to shoot . This knowledge is essential to evaluate the impact of the use of contaminated waters in agriculture, food quality and human health . In this presentation we will discuss preliminary findings regarding the interaction of Lycopersicum esculentum and Oriza sativa plants with MC and CYN . Hydroponic cultures have been established and plants exposed to either isolated toxins or cyanobacterial cell extracts with concentrations varying between 1 µg/L and 300 µg/L . Short term exposures to both toxins does not alter the quantum yield of PSII in O . sativa and in L . esculentum after 15 days of exposure or the growth rate of plants . Nevertheless proteomic analyses displayed variations in protein expression in L . esculentum leaves suggesting for a biochemical response of plants . Analysis of the activity of antioxidant enzymes is being undertaken to estimate the oxidative stress in plants in response to the phytotoxins . Moreover HPLC and mass spectrometry techniques are being applied to quantify MC and CYN in plant tissues . We expect with this additional data to gather new insights in the toxicity of phytotoxins in these plant species and in the role of bioaccumulation in food quality and human health safety .

ET16P - Quantitative Structure Activity Relationship (QSARs) and similar models for predicting the toxicity of chemicals, mixtures and combined stress

MO 361Environmental impact analysis of 10 active pharmaceutical ingredients with QSARsL . Geerts1, I . van Ginneken2, B . Mertens2, D . Caldwell3

1VITO NV, Mol, Belgium2Janssen Pharmaceutica, Beerse, Belgium3Johnson & Johnson, New brunswick, new jersey, United States of AmericaThe environmental risk assessment of an active pharmaceutical ingredient (API) follows a stepwise approach, with a base set of studies on aquatic toxicology and fate as a start . This base set can be generated in standard tests, but at first the potential effects on aquatic organisms and the environmental fate can be calculated to screen for possible alerts . Quantitative structure activity relationships (QSAR) are methods for estimating intrinsic environmental (hazardous) properties of chemicals, while reducing time, monetary cost and animal testing . In this study QSAR models are used to calculate physical-chemical properties, acute and chronic ecotoxicity and environmental fate of 10 APIs from multiple therapeutic categories . Calculations are performed in EPISuite . Model estimations are discussed and compared with measured data . (Q)SARs show to be a reliable tool for screening environmental properties of APIs . All the test result on (non) ready biodegradability were confirmed by the predictions of the Biowin model . All the ecotoxic substances were identified by Ecosar . The acute toxicity is in line with measured values (74%) or overestimated (26%) by the model . For the chronic toxicity, the lowest chronic value determines the PNEC and hence the outcome of the risk characterisation . The calculated lowest chronic value for each API is in line with the measured value or lower . For the Ecosar classes considered for these 10 APIs, the main parameter in the algorithm is the log Kow which is inversely related to the ecotoxicity value . For the APIs with only covalent bonds the results with measured and calculated ecotoxicity are in the same order of magnitude . It can be argued for the salts to provisionally use the measured log Kow in order to refine the overestimation and avoid underestimation of the ecotoxicity by the model . However further research is needed on this subject . As part of an intelligent testing strategy (ITS) within risk assessment, QSAR predictions can deliver added value to other non-test methods such as in vitro tests, read across, grouping and the weight of evidence approach, in order to minimise animal testing by focusing on those endpoints that may be of concern and to secure a high level of safety for man and the environment . Acknowledgement - The authors thank Janssen Pharmaceutica (Beerse, Belgium) of Johnson & Johnson for sponsoring this project

MO 362Externally predictive QSAR models: thresholds of acceptance by various external validation criteria and critical inspection of scatter plotsN . Chirico, E . Papa, P . GramaticaUniversity of Insubria, Varese, ItalyThe evaluation of linear regression QSAR models performances, both in fitting and external prediction, is of pivotal importance . In the last decade different external validation parameters have been proposed: Q2

F1 (Shi), Q2F2 (Schuurmann), Q2

F3 (Todeschini), averaged r2m (Roy) and the Golbraikh -Tropsha (GT) method . Recently, the concordance correlation coefficient (CCC, Lin) has been proposed by our group as an external validation parameter to be used in QSAR studies . In our recent work, published in 2011 on JCIM, we have shown that, comparing with the commonly used acceptance thresholds (Q2

Fn=0 .6, averaged r2m=0 .5), the concordance correlation coefficient threshold value (0 .85) is usually the most restrictive in the acceptance of QSAR models as externally predictive . This fact suggested that the CCC could be used as the preferred validation parameter in a precautionary approach, if the aim of QSAR developers is to have the smallest differences among the experimental data and the predictions of the external data set . In this new work, we have studied and compared the general trends of the various criteria in dependence of different possible bias in the external data distributions (scale and location shifts), by means of a wide range of different simulated scenarios . This study highlighted, also by visual inspections of the experimental vs . predicted plots, some problems related to a few criteria; in particular, averaged r2m values, if based on the proposed cut-off, could be prone to accept also not predictive models . This analysis allowed also to propose recalibrated, and inter-comparable, new thresholds for each criteria in the definition of a QSAR model as externally predictive . Two additional relevant topics emerged from the analysis of the results: 1) the scatter plot of the external predictions must always be evaluated and 2) the root mean squared error (RMSE) must also be calculated, as it is usually done in the good QSAR practice . In fact, we have verified that the sensitivity of the various validation criteria to RMSE often differs . An additional important topic, here considered and applicable only to CCC, was to check by hypothesis test if the value of the calculated CCC is statistically significant . This procedure allowed, consequently, to determine also the minimum acceptable size of the external data set, an important point in QSAR studies, where the data set sizes are often small .

MO 363Collection screening of QSAR models for REACHC . Milan, E . Benfenati, A . Roncaglioni, R . Gonella Diaza, A . CassanoInstitute for Pharmacological Research „Mario Negri„, Milan, ItalyREACH is the Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals . This legislation promotes the use of non-testing methods (NTM), which are all the approaches used to predict the effects of chemical compounds without the use of the real chemical compound . These methods include Quantitative Structure-Activity Relationship (QSAR) models and read-across . Before making an animal experiment the industry should verify if alternative methods exist . However, so far there is a deep gap of knowledge on which methods are available and can be used in practice . In this context a LIFE project, named ANTARES, aims to reduce this gap assessing NTM as an alternative approach for the REACH legislation . Here we will present the screening study on existing implemented QSAR models, both commercial or freely available through the internet . Almost 40 REACH endpoints are in principle covered by 250 QSAR models, 70 of them freely available . The full list of the QSAR models is available on http://www .antares-life .eu/index .php?sec=modellist . The QSAR models cover endpoints for physico-chemical, environmental, ecotoxicological and toxicological properties . Within ANTARES we are evaluating in details the models for these eight endpoints: Partition coefficient n-octanol/water; Carcinogenicity study; In-Vitro gene mutation study in bacteria; Acute toxicity-by oral route; Short-term toxicity testing on invertebrates/Daphnia; Short-term toxicity testing on fish; Ready biodegradability; Bioconcentration factor . In this way we cover all the areas of the effects, giving preference to endpoints where more models and data exist . Within ANTARES we are evaluating the performances of these models with external test sets using specific criteria . Results will be discussed . The possibility to have checked and validated non-testing methods will provide a huge benefit, reducing the costs and time needed to get the information on chemicals, and saving animals . The use of these methods will reduce the animal tests not only for their use by chemical industry, but in the other sectors too . Financial support from ANTARES project (LIFE 08 ENV/IT/000435) is gratefully acknowledged .

MO 364

20�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

QSAR classification models of the terrestrial toxicity of (benzo) triazoles: predictive tools for hazard and risk assessment E . Papa, P .P . Roy, S . Kovarich, S . Cassani, P . GramaticaUniversity of Insubria, Varese, ItalyTriazoles and benzo-triazoles (TAZ/BTAZ) are potentially hazardous chemicals that adversely affect humans and other non-target species, and are on the list of substances of very high concern (SVHC) in the European regulation of chemicals REACH . TAZ/BTAZ are synthetic molecules, widely used in various industrial processes, as well as deicing agents, pharmaceuticals and pesticides . Because of their wide use they have been found distributed throughout the environment . The amount of experimental data available for these molecules is insufficient for a comprehensive characterization of their environmental and toxicological profile and they have been included among the four classes of chemicals studied in the European FP7 Project CADASTER (CAse studies on the Development and Application of in-Silico Techniques for Environmental hazard and Risk assessment) . In this study quantitative structure-activity relationships (QSAR) were used to model different endpoints of interest to define the potential toxicological profile of over 50 TAZ/BTAZ, with experimental acute toxicity data available for three key organisms of the terrestrial ecosystem . The studied end-points were: Honeybees 48 hour LD50, Earthworm 14 day LC50, Bird oral-LD50 . Different theoretical molecular descriptors were calculated by different proprietary and freely available online software (DRAGON 5 .5 and PADEL-Descriptor 2 .6) . The endpoints of interest were modeled by classification (k-Nearest Neighbors (k-NN)) and the best modeling variables were selected by Genetic Algorithm . The predictivity of the best models was validated by calculating the parameters sensitivity (Sn), specificity (Sp) and the non error rate (NER) . External validation was also performed, depending on the dimension of the studied experimental datasets . All the proposed models have good statistical performance in fitting and in external prediction with Sn%, Sp% and NER%, calculated for the three endpoints, never below 85%, 75% and 81% respectively (full models) . The reliability of the predictions was always checked by the leverage approach in order to verify the chemical applicability domain of the models . In conclusion, the proposed models, which were developed in line with the ‘OECD principles for the validation of QSARs for regulatory purposes’, are useful tools applicable to fill the data gaps and to support the activity of regulators with additional information to be used in terrestrial Hazard and Risk Assessment procedures .

MO 365The effect of molecular chain architecture on polyelectrolyte toxicityR .J . Costa1, J .L . Pereira2, J .M .F . Gomes1, I .C . Rosa2, F .J .M . Gonçalves2, D . Hunkeler3, M .G . Rasteiro1

1University of Coimbra, Coimbra, Portugal2University of Aveiro, Aveiro, Portugal3Aqua+ Tech, Geneva, Switzerland Polyelectrolytes are applied in a wide range of industrial sectors, from pulp and paper mills to waterworks, and hence risk assessment for this sort of substances is of major interest . The functional performance, for example flocculant action, of polyelectrolytes may be maximised by manipulating parameters such as the molecular weight, the polymer charge density and the molecules’ chain architecture . In this context, understanding how these variables affect the toxicity of polyelectrolytes towards aquatic organisms is very important . In fact, if reliable structure-activity relationships (SAR) become available, early risk assessment may be incorporated into the first steps of the development process instead of being postponed to later stages when the product is close to get into the market . Ultimately SAR may assist the optimisation of the overall product performance through the maximisation of the product functionality and the minimisation of the environmental impacts . The dependence of polymer toxicity on molecular weight and charge density has been fairly investigated . While the molecules’ chain architecture is a useful design variable, significantly affecting the product functionality, little is known about the way this parameter affects toxicity . In this study, preliminary data elucidating the relationship between polymer chain architecture and toxic effects on aquatic organisms are provided . The responses elicited by cationic polyelectrolytes with different branching degree on Daphnia magna and Pseudokirchneriella subcapitata were assessed in standard laboratory bioassays . The toxicity of the chemicals to the Asian clam Corbicula fluminea, a biofouling bivalve that is potentially controlled by polyelectrolytes, was also tested . The results show that the molecules’ chain architecture significantly affects polyelectrolytes’ toxicity .

MO 366Development and validation of QSARs for chronic narcosis to fishF .J . Claeys1, F . Iaccino1, C . Janssen2, M . Pavan3, P . van Sprang1, F . Verdonck1

1Arche, Gent, Belgium2Ghent University, Ghent, Belgium3S-IN Soluzioni Informatiche, Milan, ItalyVertebrate testing requirements under REACH are encouraged to be avoided by use of alternative non-testing approaches such as QSARs . However, robust QSARs predicting chronic ecotoxicity to fish are largely lacking . The Ecological Structure Activity Relationships (ECOSAR) Class Program is a computerized predictive system that estimates aquatic toxicity . ECOSAR uses a number of log Kow based QSARs to estimate the acute and chronic toxicity of organic compounds for several structural classes . In case acute data sets are used, acute to chronic ratio’s (ACR) are used to predict chronic toxicity to fish . Although ECOSAR scores good on several OECD criteria, the chronic QSARs in ECOSAR are not fully compliant with OECD criteria in the framework of REACH and/or CLP . This poster will present the development of a chronic ecotoxicity QSAR to fish for nonpolar and polar narcosis . These QSARs were build on a database of carefully screened toxicity values, considering only chronic exposure durations and relevant endpoints . After statistical multivariate diagnostic modeling, mechanistically relevant descriptors were selected to develop a multivariate regression model using the OECD Toolbox . These QSARs have been tested and were compliant with the OECD principles for the validation of a QSAR .

MO 367Extension and validation of the target lipid model for deriving predicted no effect concentrations for hydrocarbonsF . Parkerton1, A .D . Redman2, J .A . Mcgrath3, D .J . Letinski4, R .G . Manning5, E .J . Febbo6, P . Mayer7, D .M . di Toro8

1ExxonMobil Biomedical Sciences, Houston, tx, United States of America2Exxon Biomedical Sciences, Inc, Annandale, nj, United States of America3HDR/Hydroqual Inc, Mahwah, nj, United States of America4ExxonMobil Biomedical Sciences, Inc, Annandale, nj, United States of America5ExxonMobil Biomedical Sciences, Inc ., Annandale, United States of America6ExxonMobil Research Qatar, Doha, Qatar7Aurhus University, Roskilde, Denmark8University of Delaware, Newark, de, United States of AmericaThe hydrocarbon block method enables environmental risk assessment of complex petroleum substances to be performed . To implement this approach, predicted no effect concentrations (PNECs) intended to protect aquatic, marine, benthic and soil organisms need to be established for representative hydrocarbon structures that are used to simulate the complex substance composition . The target lipid model (TLM) provides a quantitative framework for deriving PNECs for hydrocarbons using the HC5 statistical extrapolation procedure . However, the TLM does not correctly predict the lack of observed chronic effects for poorly water soluble hydrocarbons . This limits the current applicability of the TLM since low solubility hydrocarbons are often constituents of many petroleum substances . To expand the TLM domain to include these hydrocarbons, chronic algal and daphnid tests were performed for selected C11-C16 aliphatic hydrocarbons . The aqueous solubility of each hydrocarbon was first determined using the slow-stir test method . Chronic ‘limit’ tests were then performed at a single exposure concentration corresponding to the aqueous solubility limit using vapor saturation and passive dosing techniques . Results indicated measured aqueous solubilities ranged from 0 .3 to 25 micrograms/liter . Chronic effects were not observed for hydrocarbons with measured water solubility below 5 micrograms/liter . To estimate aqueous PNECs and discriminate toxic from non-toxic structures, the equation used in the TLM to estimate partitioning to target lipid was modified . The modified TLM was then evaluated using test data from this study and additional chronic effects data reported in the literature for aliphatic and aromatic hydrocarbons, ethers and alcohols . This compilation yielded over 260 experimental chronic values for 92 substances and 30 test species . Comparison of chronic data to predictions derived from the modified TLM confirmed that the HC5 method provides the level of protection expected . Therefore, this work extends the range of TLM applicability to poorly soluble hydrocarbons in support of risk assessment of complex petroleum substances using the hydrocarbon block method .

MO 368QSAR models for aquatic toxicity of triazoles and benzotriazoles: WP3 results within the FP7 European Project CADASTERS . Cassani1, S . Kovarich1, E . Papa1, P .P . Roy1, M . Rahmberg2, S . Nilsson2, T . Öberg3, N . Jeliazkova4, N . Kochev5, O . Pukalov5, P . Gramatica1

1University of Insubria, Varese, Italy2Swedish Environmental Research Institute, Stockholm, Sweden3School of Natural Sciences, Linnaeus University, Kalmar, Sweden4IdeaConsult Ltd ., Sofia, Bulgaria5University of Plovdiv, Department of Analytical Chemistry and Computer Chemistr, Plovdiv, BulgariaQuantitative structure-activity relationships (QSARs) are useful tools to predict unknown activities/properties for existing and not yet synthesized chemicals . The development and validation of QSAR models to be used as support tools for the application of the EU-REACH regulation, is one of the topics of the FP7 European project CADASTER (CAse studies on the Development and Application of in-Silico Techniques for Environmental hazard and Risk assessment) . This poster summarizes the QSAR models developed by partners within the CADASTER project for the aquatic toxicity of triazoles and benzotriazoles (TAZ/BTAZ) . These chemicals are synthetic molecules with industrial and pharmaceutical uses . TAZ/BTAZ have been found distributed throughout the environment, mainly in water compartments, and are cause of concern due to their possible effects mainly on aquatic organisms . The modeling study presented in this poster involved different project partners in universities and research institutes across Europe (University of Insubria, Linnaeus University, IVL Swedish Environmental Research Institute, Ideaconsult Ltd ., and University of Plovdiv) . The presented QSARs were realized by different modeling approaches (Multiple linear regression by MLR-OLS or PLSR) starting from theoretical molecular descriptors calculated by different commercial (DRAGON) and freely available (PADEL-Descriptor, CADASTER Online) software . The studied end-points were: LC50 in Onchorhynchus mykiss, EC50 in Daphnia magna, and EC50 in the algae Pseudokirchneriella subcapitata . The proposed models were checked for their robustness, external predictivity and applicability domain, in agreement with OECD principles for the validation of QSARs for regulatory purposes . The best models have good statistical performance in fitting and in external prediction with R2 and Q2 ranges, calculated for the three endpoints, of 0 .75 - 0 .85 and 0 .70 - 0 .81

210 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

respectively, for MLR-OLS models, and 0 .93 - 0 .99, 0 .79 - 0 .88 for PLSR models . The structural applicability domain to TAZ/BTAZ in ECHA preregistration list and the comparison of some models with ECOSAR predictions was also performed for the Insubria models . The external predictivity of the models was compared by analysis of the Root Mean Squared Error (RMSE) and of other statistical parameters such as different external Q2 or the Concordance Correlation Coefficient (CCC) recently proposed, by the Insubria group, for its use in QSAR validation .

MO 369Assessment of alternative approaches to hazard identification of substancesA .M . Verdonck, F .J . Claeys, F . Iaccino, M . VangheluweArche, Gent, BelgiumThe REACH Regulation stipulates that the principles of replacement, reduction and refinement of the use of animals in procedures should be fully taken into account in the design of the test methods, in particular when appropriate validated methods become available . REACH additionally promotes the use of (Q)SARS ((Quantitative) Structure Activity Relationships) because they contribute to the reduction of tests with vertebrates . The concept and use of alternative testing and (Q)SARs are therefore strongly embedded in the REACH Regulation and guidance documents . In particular, alternative testing methods and (Q)SARS may be applied where information is scarce, as is likely to be the case for substances registered between 1 to 10 t/y . This poster will present an assessment of recent developments in the area of information generation for hazard identification, in particular focusing on alternative methods, such as (Q)SARs or in-vitro methods . The assessment is a consolidation of review papers, state-of-the-art papers and several recently finished/ongoing EU research projects on alternative approaches . Options will be presented for using the new developments to meet information requirements (including as information below or beyond the minimum information requirements) for substances registered between 1 and 10 tonnes .

MO 370Prediction of Michael-acceptor protein reactivity from quantum chemical reaction barriersD . Mulliner, G . SchüürmannUFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyThe so-called Michael acceptors, i .e . α,β-unsaturated carbonyls, are an important class of industrial chemicals with the potential to enter cells and react with endogenous macromolecules such as proteins and DNA . For this reason their toxicity toward the ciliate Tetrahymena pyriformi (logarithmic 50% growth impairment concentration after 48-h exposure, log EC50) exceeds the minimum or baseline toxicity as calculated from their hydrophobicity (logarithmic octanol-water partitioning coefficient, log Kow) . It has been shown that their reactivity toward glutathione (GSH), quantified as logarithmic 2nd-order rate constant, log kGSH, can be employed to predict the toxicity component driven by their Michael-acceptor reactivity, making log kGSH a key determinant in predicting their toxicity .

The reaction of the electrophilic Michael-acceptors with the thiol group of GSH or protein side chains can proceed via two mechanisms . Firstly, a direct addition to the olefinic double bond (subject to our previous study), or secondly, a conjugated 1,4-addition . In this work we present an extensive quantum chemical assessment of the 1,4-conjugated addition and the subsequent tautomerization step in simulated aqueous solution . For a set of 12 compounds, consisting of 4 aldehydes, 4 ketones, and 4 esters with diverse substitutions at the C

α=C

β double bond, reactant, transition-state, and product complexes with two explicit water molecules were calculated at the DFT and

MP2 levels of theory . While DFT calculations yield ambiguous results as to which of the two steps is rate-determining, MP2 calculations support the textbook view that tautomerization is fast and not rate-determining . The correlation of the intrinsic reaction barriers, ΔE“ , of the addition step with log kGSH was explored, yielding a simple linear regression model with good statistics able to predict log kGSH . In a second step, a regression model for log EC50 was developed, employing both ΔE“ and log Kow as descriptors . The derived models appear useful as in silico tools for screening the reactivity and toxicity of α,β-unsaturated carbonyls in the context of integrated testing strategies (ITS) for REACH, enabling an early waiving of animal tests for non-reactive toxicants . This work was financially supported by the European Union project OSIRIS (Optimized Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Testing Information), contract no . GOCE-CT-2007-037017 .

ET17P - Trait-based approaches in prospective and retrospective risk assessment

MO 371Relative toxicity of metal cations to the macroalga Gracilaria domingensis in synthetic mediumL . Mendes, E . Bastos, C . Stevani, P . ColepicoloInstituto de Química, IQUSP, Universidade de São Paulo, São Paulo, SP, São paulo, sp, BrazilThe potential use of Quantitative Ion-Character Relationships (QICARs) to investigate the toxicity of metals to algae has been underestimated when compared to the Quantitative Structure-Activity Relationships (QSARs) studies, commonly used for organic toxicants . In the present study, we determine the individual toxicity of mono-, bi- and trivalent metal ions (Li+, K+, Na+, Cd2+, Pb2+, Zn2+, Cu2+, Ni2+, Mn2+, Co2+, Ca2+, Mg2+, Sr2+ and La3+) to the red macroalga Gracilaria domingensis (Kütz .) Sonder ex Dickie (Gracilariales, Rhodophyta) originally from the Brazilian Coast and cultivated in synthetic seawater medium under optimized conditions . The median inhibitory concentration values (IC50) were determined from daily growth rate (DGR, μg/d) vs . concentration curves using a 48h-assay . The IC50 values were converted to free ions (ICF50) values using the software minimization of total equilibrium activity (MINTEQA2, U .S . Environmental Protection Agency), version 3 .0 . After speciation calculations of the metal ions, these were transformed for logICF50 values and the toxicity of the free metals were predicted using the QICAR approach . The following parameters were used: ionic radius (r), atomic number (AN), electronegativity (Xm), covalent index (Xm2r), first hydrolysis constant (logKOH), softness index (σp), ion charge (Z), ionization potential (ΔIP), electrochemical potential (ΔEo) and ionic index (Z2/r) . The genetic function approximation (GFA) was used to investigate the sample distribution and to identify the most favorable subsets of descriptors to QICAR models (100 random models and 5,000 interactions to evolution) . The best correlation found was with AN, Xm2r and σp [logIC50F = 1 .00 ‘ 0 .24(AN) + 0 .52(Xm2r) + 1 .30(AN x σp), adj-R2: 0 .93, pred-R2: 0 .91, LOF: 0 .87] . This mathematical expression allowed the prediction of IC50 values for mono-, bi- and trivalent metal ions (Ag+, Cs+, Ba2+, Hg2+, Fe2+, Fe3+, Cr3+ and Al3+) . Moreover, the correlation of IC50F with σp and Xm2r allowed us to infer that the toxicity mechanism might involve the interaction between the metal and sulfur-containing biological species, such as proteins and smaller organic molecules with groups -SH, RSH, RS and R2S .

MO 373Using benthic macroinvertebrate biotraits to assess specific risk of stream ecological impairment under multiple pressures scenariiC .P . Mondy, P . Usseglio-PolateraUniversity Paul Verlaine-Metz, Metz, FranceThe full accomplishment of the European WFD objectives has required from EU members three successive steps: (i) the evaluation of the actual ecological status of their water bodies, (ii) a diagnostic assessment of the risk to be impaired by different anthropogenic pressure types and (iii) the implementation of appropriate management and restoration programs based on the previous diagnostic conclusions . We already have proposed a new French WFD-compliant bioassessment index (I2M2) to replace the current IBGN index (step i) . In this paper, we aimed at designing an innovative risk assessment tool working for most of French wadeable stream types in a context of multiple anthropogenic pressures (step ii) . This tool, based on random forests, was built on macroinvertebrates biotraits, we built one random forest model for each of 10 water quality and each of 6 habitat pressure categories . We demonstrated that this approach was very promising in stream ecological risk assessment, Our predictive models gave consistent impairment risk (IR) assessment, even at moderate risk level, for several specific pressure categories, three exhibited correct classification rate (CCR) higher than 0 .70 and eight others had a CCR between 0 .60 and 0 .70 . Moreover, most of the observed relationships between trait category utilization and IR could be explained through a mechanistic action of human-induced pressures on invertebrate assemblages, that is a highly recommended feature for diagnostic tools . To our knowledge, this diagnostic tool, composed of 16 random forest models, is the first macroinvertebrate-based functional diagnostic tool that address efficiently the problem of diagnostic in multiple pressure scenarii (including water quality and habitat degradations) at large spatial scale .

MO 374Comparison of the species sensitivity distribution (SSD) and SPEAR index in the retrospective risk assessment of pesticidesS . Jesenská1, M .A . Beketov2, M . Liess2, L . Bláha1

1Research Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech Republic2UFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyVarious methods to evaluate impacts of contamination of aquatic ecosystems have been suggested including specific bioindices such as saprobic index or SPEARpesticides index, which has been shown to reflect pesticide pollution . The SPEARpesticides index uses biomonitoring data on macroinvertebrates and classifies different species as ‘at risk’ or ‘not at risk’ according to their ecological traits . On the other side, Species Sensitivity Distribution (SSD) is a statistical method, which uses toxicity data (e .g . EC50 or NOEC) of different species or taxons, and it can be used for prediction of the ‘potentially affected’ fraction (PAF) of the aquatic community exposed to a certain concentration of specific compound . It can also be used to predict PAF of mixtures, i .e . multisubstance PAF (msPAF) . In the present study we calculate msPAF values for 25 pesticides measured at localities in France, Germany and Finland (years 1998-2000), and compare the results with the bioindicaton (SPEARpesticides index) . Our objective was to compare performance of both approaches in the retrospective site-specific risk assessment of pesticides, and the preliminary results indicate complementarity of both SPEAR and SSD . The full outcomes of the exercise will be discusses in detail .

MO 375Physiological sensitivity of freshwater macroinvertebrates to heavy metalsE . Malaj1, M . Grote2, R .B . Schäfer3, W . Brack1, P .C . Von der Ohe1

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2EDF R&D, Laboratoire National d’Hydraulique et Environnement, Chatou, France3Institute for Environmental Sciences, University Koblenz-Landau, Landau, GermanyMacroinvertebrate species traits, such as physiological sensitivity have successfully been introduced in trait-based bioassessment approaches . We ranked macroinvertebrate species according to their physiological sensitivity for heavy metals, using acute toxicity data . Rankings for each heavy metal, namely Cd, Cu, Cr, Ni, Pb, Zn, and Hg were developed by normalizing each heavy metal distribution function based on all available species data in standardized laboratory conditions . Rankings for different heavy metals

211SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

on the species level were reasonably well correlated pairwise (0 .50<r<0 .73) and showed no significant difference for all heavy metals . Thus, an aggregated heavy metal ranking was developed (Smetal), which assigns physiological sensitivity values to macroinvertebrates taxa . Considering the high variation, especially for higher taxonomic levels i .e . order, it is recommended to use S-values of the genus or species level for meaningful analysis . In terms of taxonomic ranking, crustaceans were found to be the most sensitive organisms, with species in the order of Cladocera being three orders of magnitude more sensitive than the most sensitive insects of the order of Trichoptera . By contrasts, molluscs covered a wide range of sensitivities, with Bivalvia being on an average one order of magnitude more sensitive than Gastropods . Finally, physiological sensitivity represents a promising trait that would help identify the effects of heavy metal pollution in lotic ecosystems, therefore improving practices in ecological risk assessment .

MO 376Identifying traits from arthropod community responses to insecticidesF .M . Bakker1, S .A . Aldershof2

1MITOX Consultants, Amsterdam, Nederland2Bioresearch and Promotion, Amsterdam, NederlandIn earlier work we analysed NTA field studies performed with the same active substance but in different cropping systems and different regions to provide a first insight into the importance of geographical gradient for the response of non-target arthropod communities to insecticide exposure . Using a variety of statistical techniques we investigated phenomena related to effects on community structure . Here we present an extended meta-analysis that includes studies with additional active substances and additional locations . The focus of this analysis was at the population rather than at the community level . In particular we asked whether populations that respond to insecticide exposure share traits . The objective of this investigation was to assess whether traits related to population response could be expected to relate to latitude . For each study we calculated direct impact on populations sufficiently represented in the samples as the ratio of population densities in treatment and control groups at the first sampling moment after treatment . Duration of effects was categorized using the time to reach densities as in conditions of non-exposure . Analysis at the community level was performed using Principal Response Curves analyses (PRC) . This analysis involved combination of PRC-axes . Additional multivariate techniques were used to investigate which autecological traits correlate to population level phenomena as initial impact and recovery potential . Furthermore, we investigated the prevalence of traits in communities at different latitudes . Finally, the relevance of the SPEAR evaluation method (Liess & Beketov, 2011) for terrestrial arthropod ecosystems was discussed from the perspective of using ecological traits for prospective or retrospective risk assessment in terrestrial ecosystems, similar to established methods used in aquatic systems .

MO 377Application of a lentic SPEAR index to pond mesocosm experiments : why community assemblage characteristics and season matter in ERAM . Roucaute, A . Auber, L . Lagadic, T . CaquetINRA, Rennes, FranceAn adapted version of SPEAR metrics for use in lentic experimental systems, LSPEAR (Lentic SPEcies At Risk), has recently been proposed . In this study, LSPEAR was applied to two different pond mesocosm experiments to check if the invertebrate communities hosted by the systems were vulnerable and if this index could track their responses to pesticides . Data from two experiments previously run in 9-m3 outdoor tanks were used . During the first experiment, the ponds were allowed to mature for 1 year . Pond snails and Asellus aquaticus were introduced but asellids did not successfully settled . Eight ponds were then treated with deltamethrin and eight were kept as untreated controls . Four units within both the treated and untreated groups were covered with lids just after treatment to prevent external recolonization . The second experiment was performed after a six-month maturation period during which invertebrates collected from uncontaminated ponds were introduced . Among these, different crustacean taxa successfully settled in the systems . Exposure scenarios were built based upon the modelling of transfer of pesticides applied on wheat and oilseed rape crops according to two crop protection programs . Different pyrethroid insecticides were applied, including deltamethrin . Abundance data were analysed using the multivariate Principal Response Curve (PRC) procedure . LSPEAR values in control and treated ponds were compared by ANOVA followed by post-hoc Tuckey tests . During the first experiment, PRC only detected a transient effect of deltamethrin on invertebrates in open ponds, followed by a quick recovery . A strong effect of treatment was shown in lid-covered ponds but without recovery . LSPEAR index partially anticipated the effects of deltamethrin in lid-covered ponds but not in open ones due to the mere absence of vulnerable taxa when the experiment started . During the second experiment, pyrethroids induced significant negative effects on invertebrates, especially crustaceans . LSPEAR index was able to forecast these effects . It therefore appears to provide complementary information to a posteriori assessment based on statistical analysis of abundance data . It can give a good a priori idea of the vulnerability of mesocosm invertebrate communities and be a good indicator of the exposure of a community to pesticides . The results also stress out the influence of community characteristics and season of exposure on the outcome of mesocosm experiments .

MO 378Comparative sensitivity of detritivores and detritus processing to CuH .S . Kraak1, E .R . Hunting1, M . Mulder2, A .M . Breure2, W . Admiraal1

1University of Amsterdam, Amsterdam, Nederland2RIVM LER, Bilthoven, NederlandDecomposition and sequestration of organic carbon are central ecosystem processes that depend on the interactions between functionally diverse microbial and invertebrate communities . Ecosystem services are, however, under continuous pressure . Therefore the aim of the present study was to evaluate how toxicants may affect the functional links in the benthic food-web and the services it is providing . To this purpose, the effects of the model toxicant Cu on functionally distinct macrofauna species (Asellus aquaticus and Tubifex spp .) and detritus processing were compared in microcosm experiments . Freeze dried, ground stinging nettle (Urtica dioica) served as detritus . After 5 days of exposure to Cu, detritus processing was determined by measuring LOI of the sediment and DOM content of the overlaying water . Comparing the dose-response relationships for the effects of Cu on macrofauna survival and detritus processing revealed that the ecosystem process was much more sensitive to Cu than survival of the invertebrates . Spatial-temporal redox profiles of the upper 10 mm of the sediment showed that this was explained by a decreased activity of the invertebrates resulting in lower detritus processing rates at sublethal Cu concentrations . We conclude that detritus processing is a sensitive parameter for ecological water quality and ecosystem functioning .

MO 379Comparative assessment of the structural and functional effects of a fungicide in static and dynamic mesocosmsY .B . Bayona1, M . Roucaute1, C . Gorzerino1, A . Roucaute1, K . Cailleaud2, A . Bassères2, L . Lagadic1, T . Caquet1

1INRA-Agrocampus Ouest, Rennes cedex, France2Service Environnement, Total Petrochemicals, PERL, Lacq, FranceA several month-long experiment was implemented to assess the relevance of various parameters for the identification of direct and indirect effects of pesticides in aquatic ecosystems including the study or recovery periods . The same protocol was applied to outdoor dynamic and static experimental systems to assess if comparable results were obtained in both types of cosms . t was anticipated that the recovery potential of lotic communities should be higher due to drift of organisms from upstream and that lentic communities should be more resistant to toxic effects due to the selection of taxa exhibiting various traits associated with a higher tolerance to stressful environments (e .g ., drying or low oxygen concentration) . Outdoor streams (length: 40 m, depth: 50 cm) and ponds (volume: 7 m3, depth: 90 cm) were exposed to a dithiocarbamate fungicide, thiram . Two concentrations, corresponding to surface water contamination by drift from orchard treatment (nominal concentrations: 35 and 170 µg L-1), were used, with two replicates per concentration . Four untreated cosms were used as controls . Streams were continuously exposed for three weeks whereas ponds were contaminated once a week for 4 weeks . Macroinvertebrate communities were repeatedly sampled in order to assess the structural effects of exposure using various parameters such as taxa abundances and diversity indices . Alder litter breakdown rate was chosen as a functional parameter and measured using litter bags during the exposure and recovery periods . In addition, ergosterol concentration on litter was used as a proxy for hyphomycetes biomass in order to assess the effects of the treatments on fungi . A strong effect of exposure to thiram on macroinvertebrates was shown in streams with a decrease in the abundance of various taxa such as gastopods and asellids . Midge larvae were indirectly positively favoured by treatments . These structural effects of thiram were associated with a significant decrease of litter breakdown rate . No recovery was observed up to two months after the end of the exposure period for both structural and functional parameters . The effects were less important in pond mesocosms with only a slight tendency to a decrease in the abundance of some groups and no effects on litter breakdown . Results are discussed using data of thiram concentration in water and sediment and information about biological and ecological traits of the organisms inhabiting both types of experimental systems .

MO 380Life history characteristics of surrogate species used in ecological risk assessment: what is necessary for the protection of guilds?J .D . Stark1, N . Hanson2, J .E . Banks3

1Washington State University, Puyallup, United States of America2University of Gothenburg, Gothenburg, Sweden3University of Washington, Tacoma, Tacoma, United States of AmericaThe ability of a population to recover from stressors such as pollutants, is dependent not only on exposure and innate susceptibility of individuals, but on the life history characteristics of the organism . Thus, species with certain life history characteristics (short generation times, early time to first production of offspring, and large numbers of offspring) should recover faster from stress than organisms with different characteristics if exposure and susceptibility are similar . The ecological risk assessment (ERA) process involves developing toxicological data for surrogate species . Surrogate species, often chosen because they are easy to culture, are supposed to be protective for many other species . For example, some regulatory agencies use toxicity data developed for one or two fish species to protect all fish and all amphibians . Because different, even closely related species can exhibit vastly different life history characteristics, surrogate species may not be representative of other species and may in fact result in under- or over-protection of the environment from chemical toxicants . We will show how population models can be used to determine the life history characteristics necessary to protect groups of organisms . We will discuss a mathematical approach using insect biological controls and pesticides to determine whether one species can act as a predictive surrogate species for others and what characteristics would make it protective . We will also present a generic model for the ERA of fish populations . The generic model was developed from five life history types and setup to be useful in a number of different management scenarios based on different environmental protection goals . A decision framework set up for three levels of population models (continuous, time limited, and varying exposure) and three levels of environmental protection will be presented . The approaches presented here may guide future use of population models and surrogate species in ERA .

MO 381Behavioural response of juvenile rainbow trout during a short, low-dose exposure to a herbicide mixture

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H . Shinn1, G . Grenouillet2, S . Lek2

1IMAR - Instituto do Mar, Coimbra, Portugal2Laboratoire Evolution et Diversité Biologique, Toulouse, FranceFish are capable of sensing water-borne chemicals at concentrations below the lowest observed effect concentrations, which in turn are often several orders of magnitude lower than at the reported onset of mortality . In this study we exposed juvenile rainbow trout (Oncorhynchus mykiss) to an environmentally-relevant low-dose mixture of three co-occurring pesticides - atrazine, linuron and metolachlor - during a 5-day test in controlled, flow-through conditions . Our hypothesis was that fish swimming activity, the use of the water column, and interactions between individuals would be modified due to exposure to the mixture . Fish exposed to the mixture of pesticides were hypoactive and spent more time in the lower parts of the aquaria in comparison to non-exposed controls, reflecting inhibited swimming activity . Levels of aggression were comparable between the two treatments . In natural conditions, inadequate behavioural responses, such as excessive hypoactivity, could result in increased predation risk, as well as susceptibility to malnutrition through reduced foraging activity, with serious implications for survival . Studies on the effect of pesticide mixtures are currently lacking, especially regarding the impact of environmentally realistic low doses on ecologically important and highly sensitive biological endpoints . Laboratory studies as the one presented here can contribute to the definition of the suitability of different behaviours for use in routine risk and hazard assessment . The behavioural endpoints chosen here were easily observed, and within a short timeframe, simple to quantify, and of biological significance and ecological relevance .

MO 382Ecosystem functions vs. functional groups: do they respond in the same way to stress?F . de Laender1, F . Taub2, C .R . Janssen1

1Ghent University, Ghent, Belgium2Washington state university, Washington, United States of AmericaUnderstanding to what extent ecosystem functions respond to chemicals is a major challenge in ecotoxicology . Ecosystem-level experiments (micro- and mesocosms) mostly describe the responses of functional groups to stress . However, it is unsure if these responses are proportional to the responses of associated ecosystem functions . By combining carbon budget modelling with data from a standardized microcosm experiment with a known community composition, we quantified three ecosystem functions (net primary production, NPP; net mesozooplankton production, NZP; net bacterial production, NBP) at three copper concentrations and a control . Changes of these ecosystem functions with increasing chemical concentrations were not always proportional to the copper effects on the densities of the contributing functional groups . For example, copper treatments decreased mesozooplankton density by a factor 100 and increased phytoplankton density 10 to 100 times while increasing NZP and leaving NPP unaltered . In contrast, copper effects on microzooplankton density were comparable to those on the associated function (NBP) . We illustrate that differences in the response of phytoplankton/mesozooplankton densities and the associated ecosystem functions to stress occur because functional rates (e .g . photosynthesis rates/ingestion rates) differ among copper treatments and in time . These variations can be explained by food web ecology but not by direct copper effects, indicating that ecological theory is needed to understand environmental effects of stressors .

MO 383Effects of pesticide toxicity, salinity and other environmental variables on selected ecosystem functions in streams and the relevance for ecosystem servicesR .B . Schaefer1, M . Bundschuh1, E . Szoecs1, P .C . Von der Ohe2, V . Pettigrove3, R . Schulz4, D . Nugegoda5, B .J . Kefford6

1Environmental Sciences, University Landau, Landau, Germany2UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany3Melbourne University, Melbourne, Australia4University of Koblenz-Landau, Landau, Germany5RMIT University, Melbourne, Australia6UTS Sydney, Sydney, Australia Effects of anthropogenic and environmental stressors on freshwater communities can propagate to ecosystem functions and may in turn impede ecosystem services . We investigated potential shifts in ecosystem functions that provide energy for freshwater ecosystems due to pesticides and salinity in 24 sites in streams of southeast Australia . First, effects on allochthonous organic matter (AOM) breakdown using three different substrates (leaves, cotton strips, wood sticks) in coarse and fine bags were investigated . Second, we examined effects on stream metabolism that delivers information on the ecosystem functions of gross primary production and ecosystem respiration . We found up to a fourfold reduction in AOM breakdown due to exposure to pesticides and salinity, where both stressors contributed approximately equally to the reduction . The effect was additive as, no interaction or correlation between the two stressors was found . Leaf breakdown responded strongly and exclusively to exposure to pesticides and salinity, whereas cotton strip breakdown was less sensitive and responded also to other stressors such as nutrients . No functional redundancy for the effects of pesticides and salinity on leaf breakdown was observed . For wood stick breakdown, no relationship to environmental gradients was found, however, the sample size was lower . We did not detect effects of pesticides or salinity on gross primary production or ecosystem respiration . A reduction in AOM breakdown by pesticides and salinity may impair the ecosystem services of food provision and possibly water purification . Hence, future studies should examine the spatial extent of these effects .

MO 384Effects of SSRIs on aquatic predator-prey interactionsM .L . Hedgespeth, J .A . Jönsson, O . BerglundLund University, Lund, SwedenBecause pharmaceuticals and their metabolites have been detected in aquatic environments, there is potential for wildlife exposure and consequent effects on ecosystem structure and function . Not only is it important to examine sublethal effects such as changes in behavior of individuals, but it is also necessary to determine if these result in changes with broader ecological implications . This study focuses on the effects of pharmaceutical exposure on interactions between aquatic predators and prey, i .e . the European perch (Perca fluviatilis) and the small crustacean Daphnia magna . The effects of sertraline, a selective serotonin reuptake inhibitor (SSRI), on predator feeding rates were quantified at different prey densities to examine changes in the predators’ functional response . This research will allow for application of ecological models to predict whether changes in predator-prey interactions caused by pharmaceuticals can alter population structures of aquatic organisms, and potentially community structure in aquatic environments . By taking such potential ecological impacts into account along with traditional methods, researchers and policymakers can improve the assessment of potential environmental risks of pharmaceuticals .

MO 385The ecosystem serrvices triad: linking stakeholder engagement, biophysical models, and ecological production functions to develop indices of ecosystem services for biodiversityN . Booth, S . Law, T . Wickwire, J . Ma, J . SalatasExponent, Bellevue, United States of AmericaThere is tremendous momentum in the regulatory, academic, and business communities to develop approaches for measuring and managing ecosystem services . These efforts are evolving at various landscape levels ranging from site, to watershed, to regions . Most of these approaches rely to some extent on developing linkages between ecological processes and products and their use by humans . These linkages, often termed, ecological production functions, aim to describe and identify ecological inputs and processes that result in production of outputs that are valued by humans in such a way as to render ecologically meaningful measurement endpoints as well as to allow for accurate elicitation of values and preferences among stakeholders . One challenge for ecologists as well as social scientists is to develop analytical frameworks that accurately portray relevant ecological processes in ways that are easy to understand by non-scientists . Another challenge is to identify measurement endpoints that accurately represent ecological processes and functions, are easy and cost-effective to measure, and are good indicators of ecological changes over time . This poster will present results of a field demonstration of a decision support tool to quantify biodiversity-related ecosystem services at a U .S . Department of Defense site in the United States of America .

MO 386Implementation of ecological risk assessment TRIAD for contaminated sites in the Basque Autonomous CommunityL .G . Gutiérrez, P .M . Menger, E .C . CiprianTECNALIA, Derio, SpainThe Ecological Risk Assessment (ERA) methodology currently applied in the Basque Automonous Community (BAC) is based on Pontentially Affected Fraction (PAF %) based on toxicity data from literature and the concentration of contaminants on the site . However ecosystems are complex systems with multiple interactions and PAF may not be considered as a representative measure of ecological impact . .With the aim of improving soil quality assessment methodologies in BAC, BERRILUR RTD project, persues the implementation of TRIAD [1] as a proven ERA approach . Multiple expert groups from the Basque University and Research Centers are commited in BERRILUR gathering expertise from different disciplines . The first phase of BERRILUR has consisted in adapting the existing TRIAD approach to BAC regional conditions such as soil type, soil use and expert´s knowledge . Results and information gathered were combined and three comparative tables were generated, one per LoE . In total, 18 chemical, 35 ecotoxicological and 62 ecological tests have been classified and prioritized . In each table the tests are classified according to the following scheme: (i) name of the test, (ii)organization that carries out the test, (iii) taxonomic group and organism used in the test, (iv) type of contaminant (M: Metal, O: organic and T: all), (v) scoring of each test in each tier and finally, (vi) its corresponding ranking . So far results of the classification process show strong coherence, i .e . cheaper, quicker and highly standardized tests are ahead in tier 1, while the score decreases in tier 2 and 3 . On the contrary the tests in the latter positions of tier 1 are best positioned in tier 3 . Regarding sensitivity of the tests towards nature of pollutant, slight differences could be appreciated only through chemical tests . The species used for testing consisted essentially in: microorganisms, plants, earthworms and slug . It should be noted that colembola, nematodes, arthropods and enchytraedos were not used in any test . So far, this means that available tests are not applicable to every existing soil function . It is therefore that we raise here the need to progress beyond the state of the art in by developing new testing tools and obtaining empirical data to assess specific replicability . In the second stage of the project the above methodology will be tested on a closed landfill in order to assess the ecological risk and define the possible soil uses and/or necessary remediation measures .

MO 387Ecosystem services and environmental decision making: seeking order in complexity

21�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

S .E . Apitz1, S . Deacon2, J . Nicolette3, M .L . Rockel3

1SEA Environmental Decisions, Ltd ., Little hadham, United Kingdom2ENVIRON UK Limited, Box, United Kingdom3ENVIRON International Corporation, Atlanta, United States of AmericaWhile there are still great uncertainties about the links between ecosystem biophysical drivers, biotic community structure and ecosystem functions and services, the concept of ecosystem services (ESS) is being applied in a rapidly expanding number of decision, management and policy frameworks . The majority of these frameworks can be referred to as net environmental benefit analyses (NEBA) or net ecosystem service analyses (NESA) as they seek to understand the net effect that various actions may have on ESS . As emergent properties of ecosystems, ESS are integrators of effects from multiple stressors and biophysical interactions at a range of spatial and temporal scales . ESS thus have the potential to provide a holistic approach using a “common currency”between environmental issues, helping us join up various programmes for a more integrated and sustainable management of the environment . However, how ESS within various frameworks are applied, defined, quantified, modelled and valued ranges widely between applications, potentially hindering their roles as cross-sectoral tools . For example, ESS can be transposed as assessment endpoints for “traditional”EcoRA measurement endpoints; they can be assigned indices such as habitat suitability for comparative assessment; they can be quantified as measurable material or product flows; or they can be mapped based upon landscape uses, each with very different scientific and decision implications . This paper will critically review how these issues are or could be addressed in a broad range of case studies in which ESS play a role, including examples for the assessment of environmental damage, sustainable remediation, International Finance Corporation Performance Standards, dredged material management, Integrated Coastal Zone, fisheries and landscape management, Plant Protection Products Registration, international environmental impact assessment, international due diligence, environmental insurance, and more strategic environmental decision making at catchment and landscape levels . Key assumptions, tools, similarities and differences will be identified, and opportunities for greater scientific relevance, clarity, transparency and read-across will be identified . Finally, the question of how we can ensure that current and evolving scientific understanding of ecosystem function and services in complex systems are relevant to and considered in wide-ranging environmental decision making and policy will be discussed .

MO 388DASEES: a tripartite decision analysis framework to achieve sustainable environment, Economy & society growth and management goalsT .J . Canfield1, T . Stockton2, B . Dyson1, M . ten Brink1, K . Black2, P . Bradley1, S . Yee1, W . Fisher1, J . Carriger1, A . Vega1

1USEPA, Ada, United States of America2Neptune and Company, Los alomos, United States of AmericaMany of Societies management and growth decisions are often made without a balanced consideration of pertinent factors from environmental, economic and societal perspectives . All three of these areas are key players in many of the decisions facing societies as they strive to operate in a more sustainable manner . Typically decisions are made either considering one or two of these perspectives while ignoring the third, or the weighting for one or two of the perspectives vastly outweigh the second or third consideration . Far too frequently the process either ignores or does not adequately factor in consideration of the roles that ecosystem services play in a sustainable system . Most decision-makers do not currently have access to useful or usable methods and approaches when they are presented with economic and social welfare choices that will have significant ecosystem impacts . The goal of the USEPA Ecosystem Services Research Program (ESRP) Decision Support Framework (DSF) Team is to help provide that access by identifying and developing effective and user-friendly decision methods and approaches that empower decision-makers to explicitly, routinely, and substantively incorporate ecosystem services into their decision-making process . The goal of this framework is to provide decision-makers with an understanding of potential outcomes and effects of their planned decisions on economic, social, and ecological systems in order to promote more balanced and sustainable solutions . To this end the ESRP DSF team is developing an open-source, web-based decision analysis framework called DASEES: Decision Analysis for a Sustainable Environment, Economy and Society . DASEES integrates guidance and decision support tools to implement a five step iterative Bayesian decision process designed to: 1 - Understand the decision context; 2 - Define Objectives; 3 - Develop Options; 4 - Evaluate Options; 5 - Take Action . DASEES will be developed with stakeholder and decision-maker input, through case studies, to ensure the guidance, tools, and templates meet user needs and facilitate the incorporation of economic, societal and ecosystem services in the decision-making process .

MO 389Acute exposure of common carp (Cyprinus carpio L.) fry to copper sulfate: a histological studyB .R . Raskovic1, M .M . Markelic2, S .M . Spasic1, Z .M . Markovic1, V .P . Poleksic1

1University of Belgrade, Faculty of Agriculture, Belgrade, Serbia2University of Belgrade, Faculty of Biology, Belgrade, SerbiaOne of the most common chemicals used in carp aquaculture is copper sulfate . It is regularly employed as a bath treatment against parasites as well as a fungicide and a herbicide . When used as a pesticide in plant treatment it can reach streams and rivers by surface runoff . It is known that copper sulfate is toxic, even in small quantities, and its use in fish farming has to be controlled . If used improperly, copper sulfate can cause severe damage to fish vital organs . In this experiment, 2 months old carp fry were exposed to increasing concentrations of copper sulfate (lower doses: 0 .5 mg/L; 1 mg/L; higher doses: 2 mg/L; 4 mg/L) in aquaria for 1, 6, 12 and 24 hours . Fish fry were sampled, formalin-fixed, whole mounted in paraffin moulds and cut longitudinally for microscopic observations . The slides were stained histochemically with PAS (periodic acid-Schiff) or with propidium iodide and observed with light microscope and fluorescent microscope, respectively . Histological analysis showed various alterations in different organs of copper sulfate-exposed fry, especially the eye, the gills, the skin and the kidney . The structure of the eye was altered, it was partly shrunk and its function appeared to be impaired at higher doses . In the gills, complete fusion of secondary lamellae and necrosis of gill tissue were visible after exposure to higher doses of copper sulfate and longer exposure periods (6, 12 and 24 hours), while lower doses induced less severe alterations: hyperemia, hyperplasia, hypetrophy, as well as lifting of the respiratory epithelium (after 12 and 24 hours) . Most common change in the kidney was dilatation and degeneration of glomeruli, which was seen at higher doses and longer exposure periods (12 and 24 hours) . Fragmentation of nuclei in skin cells was increased, indicating elevated level of apoptosis in the skin of copper sulfate-exposed fry . The liver and the intestine did not sustain histopathological changes and appear normal in all treatments . These results are consistent with earlier studies on the toxic effects of copper sulfate on fish, indicating dose-dependent alterations in the vital organs .

MO 390Zebra mussel parasites: potentially useful bioindicators of freshwater quality?L . Minguez1, S . Devin1, D .P . Molloy2, F . Guérold1, L . Giambérini1

1Laboratory LIEBE - CNRS UMR 7146 - Université de Lorraine, Metz, France2Department of Biological Sciences, State University of New York, Albany, United States of AmericaParasites are major components of aquatic ecosystems in terms of diversity, ubiquity and key role in food web structure . This implies that they can be a reliable tool in assessing the effects of ecosystem disturbances, as an alternative or complement to macro-invertebrate approaches . Their value as bioindicators of water quality has been documented with fish hosts, but few studies have focused on bivalves, particularly freshwater species . These hosts have the advantage of being less mobile and therefore provide information on the site-specific quality . In this context, we inventoried and analysed the composition and the structure of parasite communities of zebra mussel populations at twelve sites in France and the United States, characterized by different levels of contamination . Our specific goal was to determine if zebra mussel parasites could be useful as bioindicators of freshwater quality . Each host population was characterized for parasite infection following standard histological methods . We used multivariate analyses and descriptive modelling to study parasite communities in relationship with environmental quality . Our results indicated that sites of different chemical contamination levels exhibited different parasite communities characterized by different trematode species and parasite associations . The contamination promoted infections by microparasites (i .e . bacteria and ciliates), whereas it prevented those by macroparasites (i .e . trematodes) . For example, the occurrence of intracellular bacteria, Rickettsiales-like organisms, was positively correlated with the nickel and chromium concentrations, and sampling indicated the absence of digenean trematode parasites at a highly contaminated site . Moreover, the use of multivariate analyses and modelling are promising tools to study zebra mussel parasite communities related to pollution

MO 391Microcosm studies for evaluating potential of microbial community to degrade pollutants in soil and water ecosystems P . Grenni, A . Barra CaraccioloNational Research Council, Monterotondo, rome, ItalyThe ability of soil and water to recover from chemical contamination is primarily dependent on the presence of a microbial community with the ability to remove it . Furthermore the microbial community characteristics of an ecosystem can indicate changes in resource availability and the presence of pollution . In this way, the microbial community represents an important key to understanding the impacts of environmental and anthropogenic factors on ecosystems . Pollution can influence soil and water quality and productivity, but little is known on the effects on microbial communities, and consequent impacts on its functioning . Soil and water microcosm studies enable the studying, under controlled conditions, the effects of selective pressures, such as the xenobiotic occurrence, on natural microbial communities . We report the overall results of several microcosm degradation studies, using natural soil and water samples, in which the degradation of many chemicals (including the parent compounds of pesticides and pharmaceuticals and their metabolites) in soil and water ecosystems has been evaluated . The natural bioremediation capability was evaluated comparing the microbiologically active microcosms treated with the parent compound (e .g . pesticide or pharmaceutical) with others previously sterilized . The disappearance time of 50% of the parent compound applied (DT50) was evaluated for each chemical in the presence/absence of microbial community . The overall results presented here show the key role of microorganisms in the degradation of the chemicals studied and encourage the use of microcosm approach for assessing more realistic environmental exposure scenarios and establishing the casual relationship between degradation and the role of microbial communities in chemical disappearance form the environment .

ET19P - Veterinary medicines in the environment: Basic research for risk analysis

MO 395Risk assessment of veterinary medicines - lessons learned and research needsS . Hickmann1, J . Bachmann1, A . Hein2, E . Thumm1, C . Schlüter1

1German Federal Environment Agency (UBA), Dessau, Germany2Federal Environment Agency (UBA), Dessau, GermanySince 2005 European legislation and guidelines ensure a harmonised environmental risk assessment for all new veterinary medicinal products . Information on fate and

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effects of veterinary medicines and on their risk to the environement has increased in the past years . The presentation gives an aggregated overview of the results of the enviornmental risk assessments performed at the German Federal Environment Agency since the coming into force of the guidelines . Needs for future basic research are identified . A summary of ongoing guidance development is given .

MO 396Behavior of steroids and veterinary antibiotics in soil: study of transfer and degradation in soil columnsM .V . Salvia1, J . Camilleri2, E . Vulliet1, L . Wiest1, R . Baudot1, C . Cren-Olivé1

1Institut des Sciences Analytiques, Villeurbanne, France2SCA, Solaize, FranceAntibiotics are more and more used for animal treatments and are excreted unchanged . So, these compounds can enter the environment through land application of manure . This phenomenon could induce a resistance to antibiotics in human or animal medicine . Steroids, which are endocrine disruptors are also found in the environment due to animals but also humans wastes . The presence, distribution, fate and impact of veterinary substances and hormones regularly introduced into the soil via land application are far from known and very poorly characterised at the present time . In this context, the project aims at the development of methodologies based on soil columns experiments, in order to characterise the main products of biotic and abiotic transformations and to evaluate the migration and/or retention of selected contaminants and their breakdown products . For this purpose, 45 substances were selected including 11 hormonal steroids, 23 veterinary compounds and 11 other well-known human contaminants . The surface of the columns were first spiked with the different compounds and then regularly watered with artificial rain (0 .01 M CaCl2 in distilled water) . The leachates were regularly collected and analysed using a multi-residue method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) . Several series of columns were set up for periods ranging between 0 and 30 days . After this, the columns were sectioned into 5 horizons that were analysed using an original method based on a sample extraction by QuEChERS followed by a purification by solid phase extraction and an analysis by LC-MS/MS . Then, the presence and distribution of contaminants in the different horizons and/or leachates were established . The influence of various parameters on this transfer was also examined such as the pluviometry and the composition of soil (clay, loam, sand and organic matter contents) .

The degradation of the substances in soil was also studied . The kinetics of disappearance of the target contaminants were evaluated via modelling, by determining their lifetimes and degradation constants . On the other hand, the detection and identification of transformation products were considered by using complementary features of the coupling with two analysers: the time of flight (QqToF) and triple quadrupole (QqQ) .

MO 397Modelling environmental exposure from veterinary medicines with winPRZM-EXAMSJ .M . Cheplick, C .M . Holmes, A .M . Ritter, W .M . Williams, I .K .K . Khanijo, J .J . AmosWaterborne Environmental, Inc ., Leesburg, United States of AmericaThe Pesticide Root Zone Model is used in the FOCUS Surface Water and Groundwater model software (winPRZM) . Outside of these model frameworks, winPRZM linked with the Exposure Analysis Modelling System (EXAMS) may be used to estimate environmental concentrations of veterinary medicines associated with releases from beef cattle manure from pasture, feedlot, and fertilizer sources . This poster will show the enhancements to winPRZM for “applications”to feedlots and pasture . An example watershed scenario (based on the USEPA standard index reservoir scenario) will be shown with predicted exposure concentrations in the receiving water . The watershed scenario incorporated landscape metrics derived from the GIS analysis of watersheds (i .e ., % manure application, % pasture land, and % feedlots in a watershed) . Simulations were conducted for all three modes of manure “application”(pasture, manure, feedlot) and a combined mass (percent crop area adjusted) input into the waterbody to reflect a conservative proportion of each application source within one watershed .

MO 398Temperature dependence of transformation and translocation processes of manure-amended sulfadiazine in soilT . Mueller1, K . Pamplaniyil2, M . Lamshöft2, J . Klasmeier1, M . Matthies1

1University of Osnabrueck, Osnabrueck, Germany2Technical University of Dortmund, Dortmund, GermanySulfadiazine (SDZ) is a widely used veterinary antibiotic . In pigs, it is partly metabolized to N-acetylsulfadiazine (Ac-SDZ) and 4-hydroxysulfadiazine (4OH-SDZ) . Fresh manure applied to agricultural soils contains a mixture of these three compounds . A conceptual kinetic model describing concentration dynamics of SDZ and its metabolites in soil considers hydroxylation of SDZ, de-acetylation of Ac-SDZ, irreversible formation of non-extractable residues (NER) and reversible sequestration into a residual fraction . In field experiments with manure-amended SDZ, a dominant influence of temperature on the environmental fate dynamics was observed . For an application of the model on the field scale, the effect of temperature on the rate constants needs to be quantified . Therefore, fate dynamics were investigated at two different temperatures (10°C and 20°C) . To separately observe all transformation pathways in one experiment, SDZ, Ac-SDZ and 4OH-SDZ were each labelled with different C isotopes and spiked to manure, which was then homogeneously mixed with a silt loam soil . The batches were adjusted to 40% maximum water capacity and incubated in the dark for 150 days . Samples were sequentially extracted with (1) CaCl2, (2) methanol/CaCl2, and with (3) acetonitrile:water via accelerated solvent extraction (ASE) . The extracts were analysed by HPLC-MS/MS . The kinetic model was fitted to the concentration data . Model parameters were optimized by a stochastic optimization algorithm . All considered processes showed higher rate constants in the 20°C experiment . In principal, increasing temperature may exert different effects: (i) chemical and enzyme-catalyzed reactions are accelerated, (ii) microbiological processes (including growth) are enhanced, and (iii) equilibrium sorption coefficients are shifted leading to larger dissolved compound fractions . In accordance, de-acetylation of 13C-Ac-SDZ was several times more rapid at 20°C than at 10°C . The same tendency was observed for the slow hydroxylation of 14C-SDZ, but the effect was not exactly quantifiable . NER formation could only be determined indirectly, but likewise seemed to be enhanced considerably with increasing temperatures . Translocation into the residual fraction and back is assumed to be due to physical processes with less sensitivity to temperature . However, translocation also seemed to be positively correlated with temperature .

MO 399Fate of 14C-sulfadiazine in soil and characterization of its non-extractable residuesT . Junge1, M . Lamshöft2, B . Schmidt1, A . Schäffer1

1RWTH Aachen University, Aachen, Germany2TU Dortmund, Institute of Environmental Research (INFU), Dortmund, GermanyAntibiotics are important therapeutics for the treatment of bacterial diseases in humans and animals . Most antibacterial compounds are metabolized only incompletely by the medicated animals and humans . Therefore biologically active compounds, besides partially active metabolites, are excreted in urine and feces . Manure originating from livestock husbandry is commonly utilized as fertilizers in agriculture . Thus, residues of antibiotics present in manure have been directly released into the environment for decades . [1] Sulfadiazine (N1-[2-pyrimidinyl]-sulfanilamid) belongs to the class of sulfonamides and is one of the most widely-used antibacterial veterinary products . It exhibits a wide spectrum against Gram+ and Gram- bacteria by inhibiting the p-amino benzoic acid in the folic acid metabolism . [2] Non-extractable residues (NER) of xenobiotics in soil are discussed regarding their potential remobilization and bioavailability in the long term . In this study the fate of 14C-labeled sulfadiazine (SDZ) residues was studied in time-course experiments in soil . Silt loam was spiked with 14C-SDZ and amended with pig manure . Balances of 14C are based on mineralization, extractable and non-extractable residues . The main focus of this study was the characterization of the non-extractable residues associated with humic substances . [1] B . Halling-Sorensen et al ., Occurrence, fate and effects of pharmaceutical substances in the environment - A Review . Chemosphere . 1998, Vol . 36, No .2, 357-393 . [2] G .M . Brown, Biosynthesis of folic acid .2 . Inhibition by Sulfonamides . Journal of Biological Chemistry 1962, 237(2): 536 .

MO 400A closer look on the non-extractable residues formed by SDZ and soil organic matterS . Malchow, M . Lamshöft, S . Zühlke, M . SpitellerInstitute of Environmental Research, Faculty of Chemistry, TU Dortmund, Dortmund, GermanyBy application of the 14C-labeled veterinary drug Sulfadiazine (SDZ) to soil and incubation for 200 days the formation of non-extractable and/or bound residues with soil organic matter is studied . The study focuses on the alkaline extractable part of the soil organic matter which is referred to as humic acids . Extracted humic acids from the different soil samples are redissolved and separated utilizing size-exclusion-chromatography . Further analysis is carried out by radio-chromatography and high resolution mass spectrometry (HRMS) . The radioactivity balance study of the storage experiment shows that the organic and acidic extractable radioactivity decreases over time . In contrast to these findings the alkaline extractable radioactivity increases which means that the SDZ gets enriched in the humic acid fraction . The radio-chromatograms of the redissolved humic acids show two separated peaks . The first peak is broad and can be associated to SDZ which is somehow bound or trapped in the humic acids . The second peak is sharp and is identified by mass spectrometry as free SDZ . This shows that a fraction of the non-extractable sulfadiazine is not bound to the humic acids . By quantification of the SDZ in these fractions and the evaluation of the results in context of the storage experiment the nature and dynamics of SDZ in soil can be characterized . This will lead to a further insight into the nature of binding of SDZ and in general xenobiotics to soil organic matter .

MO 401Effects of amoxicillin, difloxacin and sulfadiazine on microorganisms and the nitrogen cycle in soilR .K . Kindler, A .K . Kotzerke, B .M . WilkeTechnische Universität Berlin, Berlin, GermanyVeterinary drugs, e .g . antibiotics, enter the environment during fertilization with manure and may influence the soil organisms and nutrient cycles .

The present work summarizes information on the effects of different antibiotics added with pig manure on soil microorganisms and the nitrogen cycle pathways (potential

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nitrogen mineralization, nitrification, and denitrification activity) . The effects of amoxicillin (AMX; a beta-lactam), difloxacin (DIF; a fluoroquinolone) and sulfadiazine (SDZ; a sulfonamide) were investigated in batch experiments, a mesocosm trial under controlled moisture, temperature and light conditions and a field trial . In the batch experiments, antibiotic spiked manure was added to Ah material of an agricultural soil (Orthic Luvisol) at a ratio of 1:25 manure:soil (w/w) . The antibiotic concentrations amounted to up to a final content of 100 mg kg-1 soil . In the mesocosm and field trials, manures of DIF- and SDZ-treated pigs were used for fertilization and maize were cultivated on the soil during incubation . Analyses of the microbial activities were performed at the beginning and in distinct intervals after manure addition . The microbial biomass amount and all processes of the nitrogen cycle were stimulated by manure addition . The antibiotics inhibited the enhancement but the effect depended strongly on the kind of antibiotic . AMX and DIF showed only insignificant effects at medically caused concentrations . The effects at higher concentrations were less pronounced and only temporary . The reasons are the biological degradation of AMX and the strong and fast sorption of DIF to soil particles . In contrast, SDZ showed pronounced effects at medically caused concentrations over long time periods . Only, the nitrogen mineralization rate was inhibited temporarily . Potential nitrification and denitrification activities showed decreased activities (up to 30% inhibition) during the whole incubation time of the mesocosm and field trials . Considering the world wide applied amounts of veterinary antibiotics, particularly sulfonamides, these results suggest a significant risk by fertilization with antibiotic containing manure for crop production and fundamental soil functions . MO 402Sublethal effects of Ivermectin on dung beetles of the genus Onhtophagus (O. taurus, O. nuchicornis)A . Scheffczyk1, K . Floate2, J .P . Lumaret3, N . Adler4, J . Roembke1

1ECT GmbH, Floersheim, Germany2Lethbridge Research Center, Lethbridge, alberta, Canada3University Paul Valery Montpellier III, Montpellier, France4Federal Environment Agency (UBA), Dessau-Roβlau, GermanyEuropean legislation requires parasiticides to be tested for excreted residues using species of dung flies (OECD 228) and dung beetles (OECD GD 122) . In the latter case, the beetle Aphodius constans, a dweller species has been approved as a test species for insecticidal residues in dung (OECD 2010) . Larvae of dweller species develop in dung on the soil surface while larvae of tunnelers develop in dung buried in the soil by adult beetles . Therefore, results of toxicity tests using A. constans may not be relevant to tunnelers . Recently an international ring test was started using two tunneler species of the genus Onthophagus by the SETAC advisory group DOTTS (Dung Organism Toxicity Test Standardization) . The suitability of two species (O. nuchicornis, O. taurus) was tested using the parasiticide ivermectin as model substance . The new test covers more than one life stage and a sub-lethal endpoint, aiming to increase the sensitivity of the test . It has already been set-up at eight laboratories in five countries . Test concentrations were spiked into fresh cattle dung using acetone as a solvent . All concentrations are given as nominal values in mg a .i ./ kg dung fresh weight (FW) . Despite the fact that different concentrations were used in the tests with the two species, the resulting EC50 values concerning the number of hatched beetles were almost the same . For example, EC50 values of 31 .3 (12 .2 - 186 .9) and 34 .1 (13 .9 - 85 .1) µg a .i ./ kg dung (FW) were determined at ECT GmbH . In comparison, EC50 values of 100 - 176 µg a .i ./ kg dung (FW) were determined in tests with A. constans . Thus, the new method is more sensitive than OECD test 2010 . Future objectives include adoption of the new method as an OECD guideline to facilitate Phase II, Tier B testing when such tests are required

MO 403Behavioural response of soil invertebrates to veterinary ionophore antibioticsS . Žižek, M . Pogacnik, P . ZidarUniversity of Ljubljana, Ljubljana, SloveniaMonensin and lasalocid are polyether ionophore antibiotics used in the poultry industry for treatment and prevention of coccidiosis . They enter the environment by the application of contaminated manure . They are therefore a potential threat to non-target soil invertebrates . In the present study, we were interested in potential behavioural responses of earthworms (Eisenia andrei) and woodlice (Porcellio scaber) to the two ionophores . The standardised earthworm avoidance test was used (ISO 17512-1:2008) and the same protocol was also adopted for use on isopods . Preliminary results indicate that the behavioural assay could be as sensitive as the standard toxicity tests . The environmental implications of avoidance behaviour should therefore be taken into account in risk assessment .

MO 404Development of a guidance for testing transformation of veterinary medicinal products (VMP) in manureD . Hennecke1, M . Herrchen1, U . Merrettig-Bruns2, T . Junker3, S . Jäger4, S . Berkner4

1Fraunhofer IME, Schmallenberg, Germany2Fraunhofer UMSICHT, Oberhausen, Germany3ECT, Flörsheim, Germany4Umweltbundesamt, Dessau, GermanyResults of a project to develop a test method to determine VMP transformation in manure are presented . Project aims are: adaptation of existing methods to study the fate of VMP and biocides under special emphasis on the complex matrix manure; method validation by use of an appropriate statistical evaluation tool and by intra- and inter-laboratory testing; development of Standard Operation Procedures; preliminary outline for an OECD-testguideline to be introduced into the OECD programme . The project is subdivided into three subsequent phases: 1 . Method development using cattle manure of different origin and the VMP salicylic acid . Method development includes an intra-laboratory validation . Fate of salicylic acid is described by dissipation of the parent, formation of characterized transformation products, NER, CO2 and CH4, mass balances . 2 . Method refinement by using two further substances (paracetamol and a biocide) and pig manure . 3 . Inter-laboratory validation . The selected experimental setup is a suitable basis to study transformation of VMP and biocides in manure . The test design gives reliable and reproducible results . Statistical analyses yield minor variations in DT50- and DT90-values of the test substance . Also, quantification and characterization of transformation products as well as determination of formed NER, CO2 and CH4 is achieved reliably . Still crucial points are: the influence of a static versus a flow-through incubation system on the ratio of formed CO2 and CH4, suitability of methods to determine the microbial activity (using 14C-glucose as reference substance in comparison to the fumigation method used for anaerobic transformation studies), and the influence of storage temperature and storage duration on the test manure .

MO 405Co-extraction and -quantification of 16 veterinary antibiotics and the evaluation of their bioavailability in dairy manureT .K . Wings1, F . Platzbecker2, W . Dott1

1RWTH Aachen University, Aachen, Germany2RWTH Aachen University/ Institute for Hygiene and Environmental Medicine, Aachen, GermanyPharmaceutical products, most notably antibiotics are being applied on a large scale in animal husbandry . With close to 800t administered in 2005 they form the most avidly applied group of veterinary pharmaceuticals in Germany . Such quantities arise because antibiotics are being used not only for acute disease treatment, but are also administered prophylactically to prevent disease-expansion to an entire livestock . But the continuous feeding of antibiotics demonstrably leads to the dispersal of antibiotic substances in manure, soil and rural waterbodies thusly creating the selective pressure of constant subtherapeutical levels which are known to promote the spread of resistant and potentially pathogenic microorganisms . Accordingly the intermediate goal of this work was to develop a method for the co-extraction and subsequent quantification of 16 representatives of the most notable veterinary medicinical classes (350t tetracyclines, β-lactams 199 .2t, 97 .5t sulfonamides, 52 .6t macrolides and 3 .7t fluoroquinolones in 2005) in manure . The Extraction of antibiotics from manure was done using a McIlvain buffer . Purification and concentrating of the analytes were accomplished through co-polymer-based solid phase extraction (SPE) . The chromatographic determination, using UPLC-MS/MS, ran over a BEH C 18 column with an acetonitrile gradient . The antibacterial substances were identified from retention times and fragmentation pattern of each analyte using ESI-tandem quadrupole mass spectrometry and detection by MS / MS in MRM mode . The detection limits for the MS / MS in dairy manure were determined at 0 .4 µg/kg . The recoveries between 28% and 158% were determined in dependence of the analyte and its concentration . For the investigation of the antimicrobial activity and bioavailability of the individual compounds an agar diffusion test was applied showing significant inhibition of the antibiotic potency under the influence of manure matrix in contrast to application of the pure substance . Further work will provide an extended method for the use on fatstock manure, due to a dramatically higher antibiotic consumption and correspondingly higher residues that can be expected .

MO 406Determination of the veterinary antibiotic erythromycin’s potential to bioconcentrate in the aquatic invertebrates, Lumbriculus variegatus and Daphnia magna, and evaluation of a surrogate modelJ .R . Coats1, A .M . Jessick1, K .L .D . Carstens1, C .R . Seery2, N .L . Cooper2, J .R . Bidwell2, T .B . Moorman3

1Iowa State University, Ames, United States of America2Oklahoma State University, Stillwater, oklahoma, United States of America3USDA-ARS National Laboratory for Agriculture and the Environment, Ames, iowa, United States of AmericaThe bioavailability of 14C-radiolabeled erythromycin was evaluated in aquatic microcosm systems with and without the addition of pond sediment extraction disks and the aquatic worm, Lumbriculus variegatus, and using C8-Empore[TRADEMARK] disks as surrogate passive sampling devices for comparison . An aquatic microcosm was also examined using the crustacean Daphnia magna to assess the potential of erythromycin to bioconcentrate in non-target aquatic organisms . After 14 days of exposure, the worms, daphnids, and disks were extracted, and uptake of 14C-erythromycin residues in tissues was determined . Comparisons of relative uptake indicated a steady-state bioconcentration factor of 2 .2 for L . variegatus compared to 18 for the disks in aquatic systems (reached between 3 and 7 days) . The disk bioconcentration factor (BCF) was 8 .2 times the worm bioconcentration factor without normalization to lipid and carbon content . In the pond sediment-containing microcosms, worms exposed to 0 .05 and 0 .5 ppm demonstrated a mean BCF of 0 .04, compared to 0 .13 for the 5-ppm treatment . The disks showed a peak “BCF”at day 3 in sediment-containing microcosms with no significant differences seen between concentrations and timepoints examined . The daphnids showed the highest erythromycin BCF at day 3, followed by a decrease in tissue content of the compound through day 14 . These organisms had higher BCF values at 0 .005 ppm with a maximal BCF of 8 for 0 .005 ppm at day 3, compared to the higher concentrations examined (0 .05, 0 .5 and 5) . The higher BCF in daphinds in aquatic microcosms compared to the worms is potentially due to the daphnids being fed throughout the study, system type (renewal vs . static),

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and/or tissue permeability . The comparative data presented could be used to evaluate antibiotic compounds in aquatic environments to better assess their bioavailability to non-target organisms within water and sediment .

MO 407Potential environmental impacts of two veterinary ionophore antibioticsS . Žižek, M . Pogacnik, P . ZidarUniversity of Ljubljana, Ljubljana, SloveniaMonensin and lasalocid are polyether ionophore antibiotics used in the veterinary medicine for treatment and prevention of coccidiosis in broiler chickens and turkeys . They enter the environment by the application of contaminated manure and are therefore a potential threat to non-target soil invertebrates . The goal of our work is to ascertain what potential effects ionophore-contaminated manure could have in terrestrial ecosystems . Although earthworms (Eisenia andrei) are relatively susceptible to monensin and lasalocid and exhibit acute and chronic effects at low levels of ionophores, woodlice (Porcellio scaber) have not shown any effects to monensin or lasalocid even at very high concentrations and regardless of the route of exposure (soil or food) . We were also interested in the effects of ionophore antibiotics on the bioaccumulation of cadmium and lead in woodlice and we examined potential behavioural response to contaminated soil . The acute toxicity of monensin and lasalocid is probably not environmentally relevant because PECs in soil are well below the obtained LC50 values . However, they could have some collateral effects as ionophores in their ability to facilitate cation transport across biological membranes . Mobile animals such as woodlice might be able to evade any harmful effects since they exhibit avoidance behaviour in the presence of ionophores .

MO 408Determination of the antiparasitic pharmaceutical toltrazuril and two metabolites in environmental water, soil and animal manureJ . Olsen, M . Hansen, E . Björklund, K .A . KroghUniversity of Copenhagen, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark Toltrazuril is an organohalogenic endoparasiticide pharmaceutical used in the acute and prophylactic treatment of livestock such as poultry, piglets, sheep and cattle against coccidiosis . Despite that toltrazuril has been used as veterinary pharmaceutical for more than a decade, little is known about its environmental occurrence and fate . Already in 2003 concerns about this compound was expressed and toltrazuril was considered as a high-priority pharmaceutical with respect to especially environmental and toxicological concerns . Toltrazuril is metabolized into two pharmacologically active metabolites; toltrazuril sulfoxide and toltrazuril sulfone . In this work, we present a novel methodology able to determine toltrazuril, toltrazuril sulfoxide and toltrazuril sulfone in environmental water at low ng/L-level, utilizing LC-MS/MS in combination with solid-phase extraction (SPE) . Soil and animal manure matrices were also possible to quantify at trace levels using pressurized liquid extraction (PLE) with an inverse and an integrated clean-up procedure . A field site study, investigating the occurrence of toltrazuril and its metabolites in manure, soil, run-off and drainage water is presented .

MO 409Determination of tetracyclines and sulphonamides in swine wastewaterJ .M . LinInstitute of Environmental Health, Taipei, Taiwan The animal husbandry industry provides a stable source for livestock products . There is a huge demand for veterinary antibiotics in concentrated animal feeding operations (CAFOs) on treating and preventing diseases as well as on promoting growth . This practice may favor a selection of antibiotic-resistant genes . To evaluate the potential impact of emitting antibiotics on the environment, a good analytical method is desired for measuring veterinary antibiotics in environmental matrixes . Tetracyclines (TCs) and sulphonamides (SFs) are broad-spectrum antibacterial agents, and were once heavily used in animal husbandry and as feed additives . This study developed a method to determine three TCs and eight SFs in swine wastewater and river water using deuterium-labeled TC and SFs as internal standards . The samples of influent and effluent were prepared with solid-phase extraction (SPE), and were analyzed using UHPLC-MS/MS at positive electrospray ionization . The recoveries of tetracyclines were 40% - 60% in influent and 50% - 100% in effluent, respectively . The limit of detection (LOD) and the limit of quantification (LOQ) of tetracyclines in influent were 40 - 75 ng/L and 150 - 250 ng/L, respectively, which were both higher than those in effluent (12 ng/L - 28 ng/L and 40 ng/L - 90 ng/L) . Sulfamethazine (SMT) was found to be the highest among the analytes in both the influent (340 μg/L) and the effluent (450 μg/L) from a piggy farm at Tao Yuan, Taiwan . The similar concentrations of SMT in the influent and effluent suggested limited removal from the wastewater treatment processes . The emission of SMT was estimated to be about 5,450 mg per day . The concentrations of all analytes in most river waters were either not detectable or lower than the limits of quantitation, which indicated a good dilution of the emitted effluent . Chlortetracycline and SMT was observed in a downstream sample at 160 ng/L and 37 ng/L, respectively, although they were not detected upstream . This suggested that there might be other emission sources of these antibacterial agents . This study provided a method for determining tetracyclines and sulphonamides simultaneously in swine wastewater and river water . The method could be utilized for investigating the distribution and fate of tetracyclines and sulphonamides

MO 410Degradation products of Oxytetracycline in anaerobic digestion and its effects on biogas production and microbial community structuresG . Turker1, O . Ince2, E . Ertekin1, H . Coban1, B . Ince1

1Bogazici University, Istanbul, Turkey2Istanbul Technical University, Istanbul, TurkeyOxytetracycline (OTC) is one of the important antibiotics used in animal husbandry which is poorly metabolized and excreted in form of liquid and solid manure . Antibiotic presence in manure may inhibit microbial activity thus inhibiting biogas production in manure digesters . In this study, change in microbial community structures due to presence of OTC was investigated along with degradation products of OTC and its inhibition on biogas production in anaerobic digestion . Blank manures were diluted with tap water and established manure slurries were spiked with 50, 100 and 200 mg/l OTC . Blank and spiked manure slurries were digested in 120 ml serum bottles at 37±1°C for 30 days . During digestion, biogas production, biogas and volatile fatty acid compositions were also monitored . After 30 days, degradation products of OTC was analyzed with LC-MS/MS which showed that OTC was mainly degraded to 4-epi-OTC at a ratio of 5-10% of initial concentration . Other degradation products were a-apo-OTC and b-apo-OTC which were present at 1-2% of initial parent concentration . According results, half life of OTC was less than 30 days . 190±3 l/kgVS biogas yield was obtained in the control microcosm where inhibitions of 41%, 57% and 61% were monitored in spiked microcosms, 50, 100 and 200 mg/l OTC, respectively . IC50 concentration of OTC was found to be 68 mg/l . Microbial communities in microcosms were characterized by 16S rDNA targeting clone library formation as changes in community structures were characterized by Denaturing Gradient Gel electrophoresis (DGGE) . Bacterial clone library was made up Firmicutes by 82% (33% Clostridiales and 49% Bacillales) and Proteobacteria by 15% (14% Acinetobacter) . Methanosarcina mazei (52%) and Methanobacterium spp. (46%) were found in archaeal clone library . DGGE analysis showed that similarity changes of bacterial communities were higher than similarity changes of archaeal communities and changes increased with increasing OTC concentration .Statistical analyses of clone libraries with environmental parameters were made by Canonical Correspondence Analysis (CCA) . Analyses showed that Bacillales were affected negatively by increasing OTC concentration while some Clostridiales were affected positively . Inhibitory effect of OTC on Methanosarcinales was larger than on Methanobacteriales.

MO 411Flubenzurons: risks and developmental effects on non-target species, a Norwegian perspectiveA .L . Macken, A .D . Lillicrap, K . LangfordNIVA, Oslo, NorwayChitin Synthesis inhibitors (CSI) are part of a class of insecticides known as Insect Growth Regulators (IGRs) which interfere with the hormonal system of insects . Flubenzurons are CSIs and the two which are most often used in aquaculture in Norway are diflubenzuron (DIF) (market name Releeze) and teflubenzuron (TEF) (market name Ektoban) . In Norway there was a voluntary ban at the end of the 1990s, however, in recent years they have been reintroduced due to the resistance of sea lice to emamectin benzoate, the replacement product for flubenzurons . Throughout the world, the monitoring of new and emerging contaminants is of increasing concern and although the flubenzurons cannot be considered a new contaminant, as they have been around since the 1970s, their re-entry to the market place and their recent increased use has become of concern . Non-target species of ecological importance may be affected by the use of these compounds, especially crustaceans and arthropods . Tisbe battagliai was selected as the test species as it is a crustacean and its life mode is similar to those of the target species, the sea-lice . Acute naupliar (< 3 hr old) single substance and mixture toxicity testing was conducted with TEF and DIF and the copepod T .battalgiai . A further 7 day developmental naupliar experimental design was developed to investigate the ability of the copepod to successfully develop through the 6 naupliar and 5 copepodid stages to adult copepod after exposure to DIF, TEF and a mixture . The exposure concentrations were based on environmentally relevant levels employed and measured in Norwegian sediment and waters near fish farms treated with these insecticides . The results on development rate indicated significant impediment of copepod development at ng/L with the two compounds . Based on these results, recommendations on improvements in study design and ease of use are mead, in addition to recommendations to regulatory authorities regarding the continued use of these pesticides .

MO 412Assessment of tetracyclines toxicity with two Lemna minor assaysJ . Kolasinska1, A . Drobniewska2, B . Adomas3, A . Piotrowicz-Cieslak4, J . Barszczewski5, G . Nalecz-Jawecki6

1Medical University of Warsaw, Warsaw, Poland2Medical University of Warsaw, Dept of Environmental Health Sciences, Warsaw, Poland3Department of Air Protection and Environmental Toxicology, University of Warmia, Olsztyn, Poland4Department of Plant Physiology and Biotechnology,University of Warmia and Mazury, Olsztyn, Poland5Department of Grassland, Institute of Technology and Life Sciences, Raszyn, Poland6Department of Environmental Health Sciences, Medical University of Warsaw, Warsaw, PolandTetracyclines are used as antibacterial agents in veterinary medicine including aquacultures . The duckweed Lemna minor plays an important role in aquatic ecosystems . It is an important food resource for many fish and birds (notably ducks) . L . minor is used as a model organism for higher water plant (ISO 20079:2006) . The sensitivity of L . minor strain taken from fish aquaculture was evaluated with two assays . A microplate assay was conducted according to standard ISO protocol with some modifications .

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The field samples needed for semifield assay were taken using the Kajak sediment core sampler . In the semifield assay special cylinders were used, which contained the 5 cm sediment layer and 10 cm of water column spiked with tested antibiotic . Two growth parameters of L . minor were measured: frond number and area . Concurrently, the concentration of tetracyclines in water column was monitored with HPLC-DAD . The main objective of the study was comparing the phytotoxicity of tetracyclines to Lemna minor in semifield and microplate assays . Three antibiotics were tested tetracycline, oxytetracycline and chlortetracycline . In the semifield assay the concentration of the antibiotics decreased rapidly, much faster than in the microplate assay . However, in both assays the fronds showed the signs of chlorosis and necrosis . The endpoints did not correlate with the actual concentration, but with initial concentration of the tested drug . It means that even the toxic agent (tetracyclines) has relatively short environmental half-lives, it can cause the negative changes in aquatic plants .

MO 413Effects of temperature and florfenicol on Daphnia magna feeding behaviourA .S . Martins1, L .M . Guimarães2, L .M . Guilhermino1

1ICBAS & CIIMAR, University of Porto, Porto, Portugal2CIIMAR, University of Porto, Porto, PortugalThe aim of the present study was to investigate the influence of temperature variation on the toxicity of the veterinary antibiotic florfenicol to the freshwater grazer Daphnia magna . The assays were carried out for 24h at three different temperatures (15, 20 and 25ºC), in the presence and absence of light with juveniles and adult females using feeding inhibition as effect criteria . The results indicate that temperature variation changes the effects of florfenicol on D . magna feeding behaviour highlighting the need of more research on the combined effects of temperature and pharmaceuticals on freshwater grazers . This work was supported by EU-FEDER and national MCTES funds through a PhD grant from the Portuguese Foundation for the Science and Technology (FCT) to Alexandra Martins (SFRH/BD/65436/2009) .

MO 414Histological changes in fish Astyanax altiparanae) exposed to 17-Alpha-Methyltestostone N . Ferreira, S .C .D .E . Sousa, A .C . Borges, C .L .G . Wendt, C .K . GrisoliaUniversidade de Brasília, Brasilia, BrazilThe synthetic androgen 17-alpha-methyltestosterone is excreted almost entirely by the gills, urine and feces . This study aims the histological analysis of the effect of this in the kidney, liver and intestines of a Brazilian native fish Astyanax altiparanae . Two tanks were kept for 28 days, each with 80 specimens fed diets, one without hormone and other with hormone concentration traditionally used in tilapia fish farming . Temperature was controlled (26 ± 1 C) and aeration was abundant . At 15, 20 and 28 days of experiment ten individuals were euthanized and organs was subjected to standard processing procedure for ligth microscopy and stained with hematoxylin and eosin . The control group showed normal histological structure . The group fed with feed to which was added to 17-alpha-methyltestosterone, showed histopathological lesions in the intestine, liver and kidneys . In all organs blood vessels had few blood cells, with reduction of erythrocytes and plasma accumulation . In the intestine, the epithelial lining had groups of hypertrophic cells . In the liver, hepatocytes showed cytoplasmic vacuolation and changes in the architecture of the organ . Kidney ducts had enlarged its lumen, the cuboidal epithelium showed apical vacuoles in the cytoplasm and the space capsule of the glomeruli were dilated . Changes were seen as early as the first sample . The lesions gradually progressed, with worsening of histopathology, culminating in the appearance of necrosis in the epithelium lining the intestinal lumen, the liver parenchyma and renal tubules . During exposure to the hormone, the individuals were fed without much greed, unlike observed in the control group . It was possible to observe the feeding using the light microscope to observe the presence of food in light of the digestive tract . The growth of the exposed group was lower than in the control group, contradicting the anabolic function in the case of lambari . The species was considered ‘non-target species’, since the cultivation of the same is not made use of 17-alpha-methyltestosterone . However, being a native species and genus widely distributed in inland waters in Brazil, it can be used to monitor water quality for the presence of the hormone, because it proved very sensitive to the presence of the same . The data show, therefore, that the hormone 17-alpha-methyltestosterone induces pathological changes in kidney, liver and intestine related to species Astyanax altiparanae . Financial support: FINATEC, CNPq .

MO 415Use of antibiotics in tilapia cage aquaculture: a case study in the Tha Chin river (Thailand)A .R . Rico1, F . Knopperts2, J . Khatikarn3, K . Satapornvanit3, P .J . Van den Brink4

1Wageningen University, Wageningen, Nederland2Department of Animal Ecology, VU University, Amsterdam, Nederland3Department of Fishery Biology, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand4WUR, Alterra, Wageningen, NederlandThe use of antibiotics to prevent and treat bacterial infestations in aquaculture has raised concerns worldwide because of their potential environmental release and the potential effects on the structure and functioning of surrounding aquatic ecosystems . The majority of the available studies addressing this issue have focused on marine aquaculture produced in temperate and cold regions, whereas the great bulk of the global aquaculture production takes place in tropical and sub-tropical regions of Asia . In the present study, the use of antibiotics in tilapia cage production in the Tha Chin river (Suphanburi Province, Thailand) was investigated . Semi-structured questionnaires were performed in 15 farms between the 6th and 22nd of April of 2011 . All the interviewed farmers reported to have used antibiotics during the last year with enrofloxacin (73 .3% of farmers used it), oxytetracycline (60 .0%) and amoxicillin (40 .0%) being the most common ones, followed by sulfadiazine potentiated with trimethoprim (33 .3%) and sulfamonometoxazine (6 .7%) . The 80% of the interviewed farmers reported to apply antibiotics mixed with feed for about 7 days to prevent fish mortalities at stocking and all throughout the culture cycle when mortalities increased . The antibiotic dosages reported to be applied during the stocking period were found to be more than two-fold the recommended amounts in one third of the interviewed farms . The application of antibiotics was reported to be more frequent during the dry season and during the months with adverse climatologic conditions (i .e ., monsoon periods), which corresponded with the periods of lower water quality in the river and higher fish mortalities . The results of this survey suggest that downstream aquatic ecosystems will be chronically exposed (i .e ., multiple-pulse exposure in the water phase and prolonged exposure in sediments) to the antibiotics applied in the tilapia cages since a great proportion of the applied compounds will immediately cross the nylon netting structure of the cages and reach the surrounding tropical waters . In this presentation, detailed results of the antibiotic use survey will be presented together with the results of an antibiotic monitoring campaign performed between April and July of 2011 in the Tha Chin river .

MO 416Tetracycline and Fluoroquinolone concentrations in the Istanbul strait sediments Karacik1, K . Li2, A . Yediler3, O .S . Okay1

1Istanbul Technical University, Istanbul, Turkey2Research Center for Eco-Environmental Sciences, Beijing, China3Helmholtz Zentrum München, Munich, GermanySurface sediments from twelve different locations of the Istanbul Strait and Marmara Sea, Turkey were analysed for five antibiotics belonging to two different groups of widely used pharmaceuticals, tetracyclines (TCs) and fluoroquinolones (FQs), by solid-phase extraction and high performance liquid chromatography . These two groups of antibiotics, mainly used to prevent or treat illness for humans as well as for animals, are frequently detected in the effluent of municipal sewage plants, in the aquatic environments and in soils after being spread by liquid manure . The results of analysis revealed that measured concentrations of individual antibiotics were significantly different depending on sampling location . Chlortetracycline (CTC) was not detected in any of the samples . High concentrations were mainly found in urbanized regions of the Strait . The concentrations of the two tetracyclines ranged from not detectable to 27 .3 µg kg-1 in freeze dried marine sediments . Comparable results were obtained for the two fluoroquinolones with concentration levels from 1 .3 µg kg-1 up 34 .1 µg kg-1 . This study is the first attempt to show the contamination degree of the Istanbul Strait sediments by emerging contaminants . Particular concern should be given concerning their potential side effects caused by the frequent consumption of mussels and fishes captured in the Istanbul Strait .

MO 417On-line SPE-LC-MS for ultra trace analysis of antibiotics in river and ground waterM . Martin1, D . Gode2, V . Remenyi2, F . Steiner1, C .G . Huber3

1Thermo Fisher Scientific, Germering, Germany2Saarland University, Saarbruecken, Germany3University of Salzburg, Salzburg, AustriaEndocrine disrupting compounds (EDCs) represent a broad range of natural or synthetic chemical substances, mostly of anthropogenic origin . Some typical representatives of EDCs are industrial chemicals, cosmetics, pesticides, sexual hormones, medical products and many common pharmaceuticals . Among the latter group, antibiotics are of a special interest as their presence in the - mostly aquatic - environment is a potential cause for antibiotic resistance of many bacteria . Obviously, the concentrations of these compounds in environmental samples are very low, i .e . in the µg/L scale (ppb to ppt range) which is a real challenge for analysis . An analytical method for the analysis of selected antibiotics in aqueous matrices like river water is presented . It comprises a sample clean-up and pre-concentration procedure combined with high-performance liquid chromatography (HPLC) for the separation and mass spectrometry (MS) for detection at highest sensitivity and selectivity . The sample preparation step is accomplished with solid phase extraction (SPE) . The intended system is fully automated and performed on-line with the HPLC-MS step in an integrated set-up with column switching abilities and under single point software control . The feasibility of the system will be demonstrated by analyzing a selection of representative antibiotics, mostly from the tetracycline and macrocyclic type .

RA04P - Established and emerging footprints – striving towards a valid and comprehensive support for decision-making processes

MO 421Does ‘green power’ purchase affect Carbon footprint?H .L . Raadal, C . Askham, E . Svanes, I .S . Modahl, O .J . HanssenOstfold Research, Fredrikstad, Norway

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Which electricity models to be used for Carbon footprint is often debated; e .g . how is “national grid”understood and defined? However, when introducing Guarantee of Origin (GO) and Electricity Disclosure the according to the European Renewable Energy Directive (2009/26/EC) and Electricity Market Directive (2009/72/EC), respectively, as well as US Renewable Energy Certificates, the issue gets more complicated . A GO is defined as a means of proving the origin of electricity, while the objective of the Electricity Disclosure is to provide consumers with relevant information about power generation and to allow for informed consumer choice, not only based on electricity prices . The suppliers of electricity are required to disclose their electricity portfolio with regards to energy source and environmental impacts, specifying the emissions of CO2 and the amount of radioactive waste . Consumers can make an active choice regarding the environmental performance of the purchased electricity by voluntarily purchase of GO . In US Renewable energy certificates (RECs), represent a similar voluntary product . RECs represent the environmental attributes of the power produced from renewable energy projects and are sold separate from commodity electricity . Several standards and guidelines (e .g . ISO14067 Carbon footprint of products, GHG Protocol) are working on general rules for how such products should be interpreted in Carbon footprint calculations . The aim of this presentation is to show how different ways of dealing with this issue affect the Carbon footprint calculations . There seems to be a need for common guidelines for the interpretation of GOs/RECs in Carbon footprint calculations are needed to be able to make fair comparisons between products and services .

MO 422Climate impacts of transitioning from coal to natural gas fired electricity generation over timeS . Schwietzke, W .M . Griffin, H .S . MatthewsCarnegie Mellon University, Pittsburgh, United States of AmericaTransitioning from coal fired electricity generation towards a greater share of natural gas (NG) is increasingly perceived to substantially mitigate climate change due to lower life cycle greenhouse gas (GHG) emissions . Life cycle assessment (LCA) is an accounting tool that compares technologies based on the GHG emissions over their life cycle . The life cycle GHG balance of a specific technology does not represent actual climate impacts because (1) it is based on a per energy basis, and (2) using GHG emissions as a metric masks the physical processes leading from GHG emissions to climate change and climate impacts . The physical processes include the generation of radiative forcing (RF) and temperature change (ΔT), which are sensitive to the timing and the type of the GHG emissions . We apply life cycle emissions distributions of coal and NG fired electricity generation to a stochastic climate model to quantify the absolute climate impacts of different transitioning scenarios . We extend current models by explicitly modeling two major sources of uncertainty . First, we bound bottom-up life cycle CH4 emissions from leakage during NG production using top-down atmospheric measurements . Bounding is important given the current wide distributions of CH4 leakage in the literature and the resulting controversy regarding the “correct”leakage rate . Second, transient climate sensitivity, which accounts for the magnitude and temporal distribution of the climate response to GHG emissions, is represented stochastically in the climate impact module . Quantifying uncertainty in the climate response is vital for drafting policy recommendations that go beyond GHG emissions by addressing climate change directly . Results show that substituting coal with NG provides little if any climate benefits despite significantly correcting the CH4 leakage to lower levels . Assuming that U .S . power generation from NG increases by 25% beyond current projections until 2035, thereby reducing coal power by the same absolute amount, RF will be reduced by only about 1-3% over 25-100 years (0 .0005-0 .0007 W/m2) . Moreover, if reduced coal power generation in the U .S . is used to meet coal demand elsewhere, the coal-NG substitution may actually increase total RF . Current work aims to quantify the influence of climate model uncertainty on these results .

MO 423Reducing energy and GHG footprints of microalgae biodiesel by the use of co-products from the sugarcane ethanol industryJ .A . de Almeida Neto1, H .L . Marandua1, S . Robra1, I . Nascimento2

1Universidade Estadual de Santa Cruz, Ilhéus, Brazil2Federal University of Bahia, Salvador, BrazilGreenhouse gases (GHG) and energy assessments are useful techniques to assess environmental impacts resulting from the distinct stages of an industrial practice . Such stages may include upstream and downstream processes in addition to the main activities carried out during the manufacturing of a specific product . In this study, Life Cycle Energy and GHG Assessment (8, 9, 10) were carried out in order to compare the environmental impact of biodiesel production from microalgae cultivated in open ponds . Biodiesel from microalgae is being considered today as a superior alternative to land-crops for the production of biofuels . Comparatively, cultivation of microalgae offers a better potential for increasing biodiesel productivity and improving the capacity for CO2 sequestration (4, 5) . This study considered two distinct scenarios in a pilot-scale process using microalgae cultivation for biodiesel production . Brazil is an important worldwide player in the biodiesel industry with an installed productivity capacity of 5,5 million liters per year (3) . The use of biodiesel in Brazil has been favored due to the availability of agricultural land . However, the expansion of oil-seed crops may lead to land use changes which in turn favor the release of CO2 and nitrogen gases stored in soil (1) . The production of biodiesel from microalgae does not show such disadvantages . The default scenario: (i) biodiesel production based on microalgae biomass outputs, in a system using commercial energy, CO2 and chemical fertilizers as nutrient sources . In this scenario, the algal cake is also submitted to pyrolysis for the production of oil, gas and biochar; was compared with: (ii) the same conditions as described in the former item (i), but now using CO2 and biodigested vinasse from the sugar cane industry to supply part of the fertilizer demand of the algae, the other part being supplied by the digestate of the anaerobic digestion of algal cake . Life Cycle Energy and Carbon Footprint assessments allow the identification of possible synergistic aspects of algal biodiesel production and the sugarcane ethanol industry, considering a future integration of the two production system . The preliminary results indicate significant positive effects on the energy and carbon footprints of biodiesel from microalgae .

MO 424Including urgency in global warming assessment in LCAS .V . Jørgensen1, M .Z . Hauschild1, P .H . Nielsen2, J .H . Kløverpris2

1Technical University of Denmark, Lyngby, Denmark2Novozymes A/S, Bagsværd, DenmarkClimate change mitigation is more relevant today than ever, as clearly illustrated by the conclusions in the World Energy Outlook 2011 just published by the IEA, giving us only until 2017 to take action in order to stay below a global warming of 2 degrees Celsius . However, the issue of assessing climate change impacts of greenhouse gases is complex and still widely discussed . At midpoint level the use of the global warming potential (GWP) is completely dominating, but a major short coming of the GWP is the lack of consideration of short term issues, such as important climatic target times - the points in time where climatic tipping points are expected to be crossed . Several alternative metrics to the GWP have been suggested in recent literature . Some include considerations of short term targets but have problems describing more long term impacts occurring beyond such target times . Addressing both of these issues are essential in the pursuit of mitigating climate change, however they should be addressed separately, as they represent two different aspects of the climate change challenge . This paper introduces an additional impact category in life cycle assessment (LCA), called the climatic tipping point impact category, addressing the urgency related to prevent the crossing of climatic tipping points . The new suggested impact category will take into account both atmospheric lifetimes of different greenhouse gasses and increasing urgency when approaching tipping points . Furthermore, it is illustrated how this addition to existing LCA methodology complements the existing impact category on global warming, ensuring easily interpretable results in both the urgency and the long term category . Solving the issue of urgency with respect to climate change in LCA further deals with the yet unsolved issue of assessing the climate change mitigation value of temporary carbon storage .

MO 425Study on the community CO2 emission based on LCA in BeijingC .S . Chen, Y .P . Li, X .G . Yang, L .P . LipingBeijing University of Technology, Beijing, ChinaThis research assessed the size and major components of a community carbon footprint based on the life cycle assessment approach combined with IPCC methods . Data related to CO2 carbon footprint were collected, which included household energy consumption, public and car transportation, waste production, etc . The result showed that energy consumption including electricity consumption, natural gas and heating accounted for more than 60% of CO2 emission, with transportation and waste production resulting in a further 24% and 15% of total emissions, respectively . The carbon absorbing effect of public green place was analyzed . As the result showed, the public green place in the community reduced about 25,262kg CO2 emission every year . According to the status of Beijing, the proposals for reducing the CO2 emission and the low-carbon community building were put forward . Acknowledgement: The research was funded by the Key Projects in the National Science & Technology Pillar Program during the 12th Five-year Plan Period “Research on carbon footprint of typical products of electronic information, paper making and printing”and “Research on the key technique for certification of carbon emission and carbon reduction

MO 426How valid and comprehensive is our CED practice today?I .S . Modahl, K .A . Lyng, H .L . Raadal, C . AskhamOstfold Research, Kraakeroy, NorwaySeveral indicators are under development, while others, such as carbon footprint and cumulative energy demand (CED), are more or less regarded as fully developed and integrated into conventional LCA studies . This paper will, however, focus on methodological gaps and challenges such as double counting and missing characterisation factors used for this ‘fully developed’ environmental impact indicator CED . These challenges are connected to the following aspects: - Use of specific data (for example for energy carriers with a heat value different from the pre-defined energy carriers in the database and characterisation methods used) . - Feedstock issues: - How do you define the raw material used for building a wooden shed? - Should this definition change somewhere in the life cycle, if you extend the analysis from cradle-to-gate to cradle-to-grave, when using the wooden material as an energy source after dismantling?

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The methods for calculating cumulative energy demand depend on the correct input, in the correct form, for calculating comparable indicators . In contrast to calculating carbon footprint, the ‘correct’ input for calculating CED is heavily dependent on the LCA practitioner being aware of how the LCI indicators are calculated, and the practitioner’s knowledge of how to manipulate the pre-defined CED method . In addition, the different CED methods available in LCA software tools are not always implemented with awareness of the challenges connected to feedstock issues . It is also interesting that older CED methods seem to be more accurate than newer ones, due to the inclusion of more substances . The paper will illustrate these challenges by describing quantitatively the inconsistencies introduced by using specific energy carriers and raw materials which can both be used as feedstock and energy sources . The aim of the paper is to raise awareness of the CED challenges and to improve current practice in order to increase the accuracy of the CED results .

MO 427Development of water inventory database considering water type and form of water useY . Ono, K . Horiguchi, N . ItsuboTokyo city university, Yokohama, JapanWaterfootprint (WF) is paid attention by water resource depletion and eccentric water . WF can be expressed as total amount of water used at entire life cycle of products .WF is expected to be effective the following four . First WF can understand the magnitude of the impact of water use . Second risk management strategies for water use . Third products, processes, promote the optimization of water efficiency and water management at the organizational level . Finally Communication and marketing strategies .WF is discussed for standardization in ISO .According to ISOWD, they require quantity of water used, type of water used, form of water use, geographical location of water withdrawal and return in water inventory . Though these database are not exhaustive, this study developed about 3700 sector water inventory database considering water resource, water type and form of water use with input-output table in Japan . As characteristics of database, in the input aspects water is classified into rain, river, ground and reused water, and consumption aspects it has three types: rain, river and ground water . Based on the database, annual water use by one average family in Japan and import and export water have been calculated .

MO 428Conceptual framework for grey water footprint of copper mining productionC .P . Pena1, V . Carmona2

1Research centre for mining and metallurgy, Santiago, Chile2University of Chile, Santiago, ChileGiven the concerns about global water resources and the particular situation of aridity and water scarcity in the main mining areas of Chile, a priority for mining industry is to advance towards an effective sustainable resource management of water . The grey water footprint is defined as the volume of freshwater that is required in order to assimilate the load of pollutants discarded a water body taking into consideration the natural concentrations and the existing ambient water quality standards . This work aims to develop a conceptual framework to adequate measure a relevant sustainability impact indicator for the copper mining production through the water footprint method, which is one of the most widely used methods for assessing the consumptive use of water . Therefore, the study focuses on developing a framework for accounting the grey water footprint and establishing the most critical pollutants in the effluents of the copper cathodes production, of both the sulfide and oxide production lines, according to the water footprint method . The methodology of life cycle assessment was applied to establish the system boundaries, the calculation basis and the allocation criteria .

MO 429Closing methodological gaps of water footprint: scope and aggregation issuesS . Pfister1, S . Suh2

1UC Santa Barbara / ETH Zurich, Zurich, Switzerland2UC Santa Barbara, Santa barbara, United States of AmericaWhile for many impact categories such as GHG emissions and fossil resource consumption indicators with long experience exist, for water resources impact assessment the knowledge is still limited and bears the additional problem of high spatial variation . The integration of the different aspects of water impacts into a single water footprint metric is a required for proper and robust communication, also addressing the different levels of uncertainties . Also there is a relevant discussion about what impacts should be included in a water footprint in order to avoid double counting and to have a meaningful footprint . In this project, all impacts related to emissions to or consumption of freshwater water are included . While for substance emissions to water many sophisticated model exist, there is still a lack of proper assessment of heat releases and anti-fouling detergents that are crucial for thermal power production . These impacts have been addressed and integrated . In a second step, we developed a scheme to combine the various existing LCIA methods related to water resources on midpoint level of the LCIA factors as well as on endpoint level for ecosystem quality impacts . For a illustrating the issues related to aggregation and communication of such a water footprint, we developed specific inventories for cooling water technologies and state of the art coal and gas power plants, including regional differentiation of the supply chain and corresponding geographies for the impact assessment ., creating maps of aggregated water footprints . More detailed analysis of the results revealed that the aggregation step and hence the final metric is very sensitive to underlying assumptions, aggregation procedures and the coverage of included methods . Although in the ISO 14040 series full aggregation of impacts is not allowed for public reporting, the demand for water footprint labels requires a single number and therefore our results are accounting for and reporting the different uncertainties involved .

MO 430Evaluation of a range of impact methods for assessing water quantity and water quality for a liquid laundry productA . Kounina1, H . Franceschini2, I . Munoz2, S . Humbert1, S . Vionnet1

1Quantis, Ecublens, Switzerland2Unilever, London, United KingdomA large number of impact assessment methods have been proposed for measuring the water quality and water use aspects of products . Twenty methods including USEtox, grey water from the Water Footprint network and midpoint indicators based on Water Stress Index (WSI) were applied to a case study of a European liquid laundry product . The objectives of the study were to: 1) understand the strengths and limitations of each method ; 2) evaluate their informational requirements and identify gaps in the data available for the life cycle of a detergent product ; 3) contrast potential impacts related to water quantity and water quality at the midpoint and endpoint level and 4) provide some initial insights in understanding the water flows of the product system through a mass balance at the inventory level . The case study was based on a concentrated liquid detergent used in France . The functional unit was one wash at 40°C . Life cycle inventory data was obtained from ecoinvent and the Quantis Water Database . The life cycle stages taken into consideration were chemical supply, manufacturing, consumer use, and end of life (wastewater treatment and packaging disposal) . The quality of the results is directly linked to the quality of the data used in this evaluation . For example there were data gaps in the primary data collected e .g . country of origin of the raw materials and therefore assumptions were made . Typically the consumer use stage dominates the impact scores for 15 out of 20 impact assessment methods applied .

MO 431Water assessment of a hand dishwashing product using an LCA approach: opportunities and limitationsG . van Hoof1, A . Kounina2, B . Buyle1, S . Humbert2

1Procter & Gamble, Strombeek-bever, Belgium2Quantis Switzerland, Lausanne, SwitzerlandOnly since a few years, LCA studies are including metrics to evaluate products from a water use impact perspective . Few methods are ready to use, some are under development . However, the applicability of the existing methods has been primarily demonstrated on agricultural materials or products . For example, water irrigation requirements in food production can be water intensive . In view of an increasing interest on water impact from different products, we ran a study on the water impact a hand dishwashing product . A number of water assessment methods were applied with the purpose of identifying both product improvement opportunities as well as understanding the state of the art both from a database and a method perspective . The study focused on environmental issues related to water use, looking in depth at inventory methods, midpoint and endpoint methods (14 in total) and covered the entire life cycle of the product . The use of a single product with the same global supply chain, sourced from one location was evaluated in two countries with a different water scarcity conditions . The study suggests that the consumer use stage is the most important one (99% using the Ridoutt and Pfister method), driven by both direct water use (dishwashing process) as well as indirect water use (electricity to heat the water) . Therefore, the largest improvement opportunity is to build consumer awareness on habits and practices in water scarce regions . The study also showed a very different profile if spatially explicit methods are applied on one product used with the same use conditions . From a methodological approach, a lot more work is necessary to develop good inventory databases (in terms of flows covered and accurate data), which is key for all methods . This will be necessary for the application of water assessment methods in a business context . A number of the applied methods are not regionalized, which was demonstrated to be a critical aspect in comparing the overall water profile of the product . Finally, improvements to some methods are suggested .

MO 432Science behind biodegradable claims on down the drain consumer productsS . Gimeno1, D .J . Versteeg2, T .W . Federle2

1Procter & Gamble, Strombeek bever, Belgium2Procter & Gamble Environmental Stewardship Organisation, Cincinnati oh 45252, United States of AmericaThis poster provides an overview of various approaches that could be used to evaluate the biodegradability of consumer products . The biodegradability of formulations is increasingly perceived as a key attribute for modern “sustainable”products . Despite biodegradability becoming a common environmental claim on the packages of consumer goods products, there is no standard technical approach to assess the biodegradability potential of formulations (i .e . complex mixtures, preparations, finished product, whole-product formulations) . This creates the potential for misleading or even incorrect claims . For example, biodegradation is only relevant for organic constituents and

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not for inorganic ingredients (e .g . water) . Biodegradability assessments are typically required by law for the hazard and risk environmental assessments of individual organic substances . The existing standard procedures (eg OECD 301 Testing Guidelines) for ready biodegradability are only appropriate for individual organic chemicals or mixtures of similar chemicals (i .e . closely related homologues) . The OECD guidance specifically states that these tests are inappropriate for mixtures of dissimilar substances as would exist in a product formulation . Hence, ready tests of whole products are insufficient for establishing the full biodegradability of a product formula . Furthermore, even when one has appropriate biodegradation data on the individual ingredients in a formula, different rules and calculation methods can yield quite different results . For example, the percent biodegradability for a single formulation can vary from <70% to > 90%, depending on whether the percentage is based upon the total number or mass of ingredients as well as if all constituents or only those constituent for which biodegradability is a relevant attribute are included in the calculation . We evaluated the pros and cons of each approach and we conclude that the most scientifically rigorous approach is to calculate percent biodegradability of a formula based upon the mass of only those ingredients for which biodegradation is relevant . Going forward, there is a need for a common scientifically based approach and internationally accepted calculation method to assess the biodegradability of whole product formulations to safeguard the credibility of environmental claims among the general public and consumers organizations .

MO 433Development of impact assessment for chemical substances covering PRTR substances S .A . Arai1, T .K . Kubo2, N .I . Itsubo3

1Tokyo City University, Yokohama, Japan2MIzuho Information&Research Institute, Chiyoda-ku, Japan3Tokyo city university, Yokohama, JapanThe problems of existing assessment methods for chemical substances in LCIA are the lack of covering chemical substances and that of reliability in model . The purpose of this research is to develop the evaluation method and characterization factors for chemical substances improving the above problems . Concretely, the approach of this research are to develop Characterization factors on ecosystem for chemical substances prescribed by PRTR(Pollutant Release and Transfer Register)in Japan and to develop those on ecosystem and human health using USEtox that is consensus model for LCIA developed by UNEP / SETAC Life Cycle Initiative . This model was developed based on the knowledge gained from comparative verification by developers of existing evaluation method . Furthermore, this research will consider developing those corresponds to the evaluation in Asian countries have recently been spreading LCA method . The brief process of this research can be described as follows . 1 . Fate model which relates inventory with concentration in each environmental media will be developed . USEtox, one of the most advanced methods will be applied in this research . 2 . Exposure analysis will be adopted to estimate the amount of exposure caused by the emission of amount of chemical . 3 . Effect analysis will be applied to evaluate impacts on ecosystem and human health . Characterization factors reflecting environmental conditions of Japan and Asian countries will be developed through these analyses .

MO 434LCIA and fate of metal-oxide engineered nanomaterials in freshwaterM .M . Miseljic1, B . Salieri2, S .I . Olsen1

1Technical University of Denmark, Lyngby, Denmark2Laboratory of Environmental Sciences „R . Sartori„, University of Bologna, Bologna, ItalyLife cycle assessment (LCA) have in past years proven to be a valuable tool for environmental impact assessment of products and systems, but with the introduction of more new products that contain engineered nanomaterials (ENMs) the tool has experienced an obstacle in terms of performing a holistic life cycle impact assessment (LCIA) of ENMs and the engineered nanoparticles (ENPs) that these materials can release to the ecosystem . Metal-oxide ENMs are often used in consumer nanoproducts where TiO2 and ZnO are considered industry-popular within this group, along with Ag being a widely used ENM metal in products . Today there is a limited amount of LCA case studies on ENMs published as scientific articles . To be exact these count around 13 studies and in most of those there is a focus on non-toxic impact assessment categories that mainly are related to the raw material and production stages of an ENM product life cycle . Performing a conventional LCIA of an ENM product it can be also concluded that the modelling is rather difficult due to missing inventory data and many assumptions that in the end result in cradle-to-gate processes (usually associated to bulk metal production) dominating the impact . The metal-oxide and metal ENMs are incorporated into a polymer matrix in the performed LCA study . However, release of ENPs to the environment may still occur through e .g . aging and wear . The fate, exposure and effect of ENM has received much focus but this scientific domain still struggles with a low understanding of many of the processes affecting the fate, exposure and effect . Fate can be considered a central research area that needs better understanding and is the focus of this study . Fate modelling is performed on metal-oxide ENMs with a focus on the two important processes of aggregation and dissolution . By quantifying fate in freshwater through these two processes there is a possibility to apply a simplified model to predict the removal of metal-oxide ENPs due to aggregation followed by sedimentation, and the removal of ENPs through dissolution . Thereby concentration in the freshwater compartment can be estimated and used for improving LCIA in the assessment of particle release by linking the metal ENP concentration in freshwater to potential exposure and effects .

MO 435Moving forward: sustainability and the U.S. cleaning products industryK .L . Stanton, B .T . Sansoni, R .I . SedlakAmerican Cleaning Institute, Washington, United States of AmericaSince the Brundtland Commission of the United Nations in 1987 defined sustainable development, the pursuit of sustainability has been a growing movement for all of society, including industries . While the call for sustainable services, packaging, products and processes has been heard, the answer to how these should be assessed and reported has not been determined . Over recent years, the American Cleaning Institute® (ACI) has worked to define and describe how the Institute and its members are committed to sustainable environmental, health and socio-economic practices . From a foundation of adopted principles on sustainability, ACI has initiated a program to substantiate actions taken by industry to be more sustainable . In order to be as comprehensive as possible, and to include both ACI’s ingredient supplier and formulator members, an initial evaluation of sustainability metrics across the membership was conducted . From there, attributes including environmental characteristics were chosen for which member companies reported three consecutive years of data . This information is being used for benchmarking and trend assessment purposes . Cleaning product manufacturers and their ingredient suppliers are being faced with a multitude of different demands and requests for data and information that could support development of sustainability reports up and down the supply chain . ACI’s work is creating a program that demonstrates the contributions of the industry to the goals of sustainable development, while engaging in the development of numerous sustainability programs . Engagement in the sustainability programs of other organizations provides the opportunity to have the industry’s approaches incorporated into their processes . The United States cleaning products industry is working towards sustainability and the development and refinement of tools to assess the industry’s achievements . While the overall picture of approaches for reporting sustainability is still developing, the U .S . cleaning products industry is making substantial progress in reporting major industry-wide trends .

MO 436Comparing the chemical footprint of hydraulic fracturing fluids used in natural gas extractionK . Hitchcock1, P .K . Scott1, K .M . Unice2, J .M . Panko1

1ChemRisk, Pittsburgh, United States of America2ChemRisk, LLC, Pittsburgh, United States of AmericaHydraulic fracturing, commonly referred to as fracking, is a process by which high pressure fluids are injected into a well to propagate fractures in the rock formation to enable extraction of natural gas and oil from previously untappable sources such as deep shale formations . Although fracking is sometimes used in reference to drilling and extraction practices, it represents only one portion of the life cycle of a well . A variety of chemicals are used in fracking fluid including biocides, breakers, surfactants, scale inhibitors, clay stabilizers, and friction reducers . Concern from the public and the regulatory community regarding the potential contamination of groundwater and surface water bodies by the fracking fluid chemicals has risen to a fever pitch . Recently, some companies have begun to voluntarily disclose the chemicals that they use in their fluids and some regulatory agencies in the United States are now requiring disclosure . Using composition information available through the public database, FracFocus, we have evaluated the chemical footprint of several fluids to describe the overall potential risk posed by each should the chemicals reach human or ecological receptors . The chemical footprint provides an assessment of the environmental performance associated with each fluid and a means for selecting between various ingredients .

MO 437Application of ERICA index to evaluation of soil ecosystem health according to sustainability threshold for industry impact E .B . Boriani1, D . Baderna2, A .L . Lombardo2, E .B . Benfenati2, M .T . Thomsen1

1Aarhus University, Roskilde, Denmark2Mario Negri Pharmacological Research Institute, Milan, ItalyThe improved ERICA methodology can be able to show how much an additional stressor can cause or not further risk to the target environment compartment . The idea behind ERICA, Boriani et al . 2010, was to build a modular and upgradable index . The aim of the improved ERICA is to give an instrument that can be used to measure the effect of new engineered substances (e .g . waste from new technological production, pharmaceuticals) introduced into the environment . This will be of great help for “green”processes and sustainable industries and may help to advertise their product as safe for the environment following impact assessment not derived only from political and economical chosen limit thresholds values . In this work we have added new theoretical indicators to be used for the soil compartment in assessing sustainable use of new chemicals in the environment . The fact that a certain new compound added to an existing scenario will not pose additional risk is currently not considered by any legislation . Thus, industries able to show this will get an extra value for their products . Furthermore this will lead to a awareness by industries of how much their product are impacting the environment in a realistic way as most as possible . For example degradation is of extreme importance in the soil compartment to understand how the chemical will be transformed and how properties like e .g . persistency and mobility will be different in case of transformation products of the parent compounds . In a concise methodology ERICA can be used as a prioritization system, while in case a detailed risk assessment is needed, it is possible to check every step of ERICA procedure and obtain the partial results concerning for example different compartment impact or human health risk assessment or environmental specific target risk assessment .

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ERICA innovated tool may be used to monitor and document environmental performance and to quantify the sustainability of actual and potential land use . Its main objective will be to develop a new methodology intended to help industry and regulatory bodies to evaluate the positive effects resulting from mitigation actions and to promote eco-efficiency . 1) Boriani E ., Mariani A ., Baderna D ., Moretti C ., Lodi M ., Benfenati E . ERICA: a multiparametric toxicological risk index for the assessment of environmental healthiness . Environment International 2010 36 : 665-674

MO 438Impacts on carbon pools and biodiversity due to land use and land use changes, exemplified with a case study on Norwegian forestryO .M . MichelsenNorwegian University of Science and Technology, Trondheim, NorwayThere is a strong need for methods within life cycle assessment (LCA) that enable the inclusion of all complex aspects related to land use and land use change (LULUC) . Here a proposal on combining impacts on biodiversity and carbon pools is presented and exemplified with a Norwegian case study on forestry . In the case study the use of one hectare (ha) of forest managed for production of wood for bioenergy production is presented . Impacts from LULUC are measured as a product of time and area requirements, as well as changes in quality . At present, there is no consensus on how to measure quality and quality changes due to LULUC in LCA . A range of methodologies are proposed, but these will often give diverging results and might thus motivate different actions . Here both permanent and temporary changes in biodiversity and carbon pools, limited to above-ground biomass, are included . In the case study different management options and their impact on biodiversity and carbon pools are assessed; transformation from unmanaged to a managed forest, transformation from fallow land to a managed forest, consecutive rotations with Norwegian spruce, and consecutive rotations where Sitka spruce is introduces to the area . The results from the case study show that the impact on climate change from LULUC is more important than the impact from forestry operations . This clearly demonstrates the need to include LULUC in LCA of forestry products . However, LULUC causes a wide spectrum of impacts . Here this is exemplified by biodiversity and carbon pools, presumably the two areas of protection most affected by LULUC . Still, by only including these two, the case studies show large variability based on what assumptions are made and the results are sensitive to both selection of time horizon and allocation of transformation impacts . Impacts from LULUC can be both positive and negative and the results show that there in some situations will be a trade-off between impacts on biodiversity and carbon pools . This underlines the fact that a mere measure of land used does not provide any meaning in LCAs since it is not possible to know whether the impacts are positive or negative .

MO 439Impacts of land occupation and water consumption through life cycle of bio-ethanol from rice strawM . Motoshita1, C . Yang1, K . Tahara1, A . Inaba2

1National Institute of Advanced Science and Technology, Tsukuba, Japan2Kogakuin University, Tokyo, JapanBio-ethanol may contribute to reduce carbon footprint of fuel use and supply renewable energy . In addition, second-generation bio-ethanol made from agricultural residues and wastes is expected to avoid the competition for food and energy supply . However, biomass production as a raw material for bio-ethanol seems to be strongly related to land occupation and water consumption . In this study, the significance of land occupation and water consumption related to bio-ethanol from rice straw was analysed based on life cycle impact assessment . Carbon footprint of bio-ethanol is certainly smaller than that of gasoline . On the other hand, weighted impact assessment result indicates that the impact of land occupation (Net Primary Production loss) for rice straw production is dominant in the case of several countries (27~68% of total impact) . The impact of water consumption (health damage due to the increase of infectious diseases and undernourishment) will vary from countries but significant effect can be found in the case of Thailand (58%) . Footprint of single issue is very clear but results in misleading . Particularly, bio-products have close connection with land and water use . The result of this analysis shows the significance of both impact categories for assessing the availability of bio-ethanol and implies site-dependent conclusion of bio-ethanol availability .

MO 440Environmental indicators for sustainable forestry and forest industry (EVERGREEN)T . Helin1, E . Saarivuori1, H . Wessman1, M . Ovaskainen1, L . Sokka1, S . Soimakallio1, T . Pajula1, K . Antikainen2, J . Hynynen3, S . Launiainen3

1VTT Technical Research Centre of Finland, Vtt, espoo, Finland2Finnish Environment Institute, Helsinki, Finland3Finnish Forest Research Institute METLA, Vantaa, FinlandThe aim of the EVERGREEN project (2010-2013) is to increase environmental competitiveness of forest industry products and fibre-based raw material . Scientific-grounded solutions will be developed to evaluate and communicate the most relevant environmental indicators: biogenic carbon balance, water footprint, land use and biodiversity . Carbon footprint methodology is the most established indicator in the forest industry but still lacks the inclusion of biogenic carbon . The aim is to develop and test methods to attribute the forest management related impacts on biogenic carbon stocks for forest products with various life times by using LCA as a tool . Water footprint is under rapid development, but the existing methodologies do not meet the requirements for product specific assessment . This project will evaluate the existing methodologies and participate in method development in ISO 14046 . The aim is also to develop a hydrologic model for the ‘green water’ in Boreal forests and discuss its relevancy . Land use and biodiversity are gaining more interest but are to be defined more precisely from the forestry basis . The aim is to produce biodiversity indicators that are applicable throughout the supply chains of forest based industry . Focus is on indicators on species diversity and habitat diversity . Land use aspects related to forest biomass use are currently gaining more interest . The methodology is still open and up-to-date scientific views are needed to identify the land use related environmental aspects and to quantify the impacts . Focus is in resource depletion and soil quality impacts of forestry .

MO 441Life cycle impact assessment (LCIA) method selection: an LCA practitioner’s perspectiveN . Vipparla, S .S . GhongeEaton, Pune, IndiaLCIA phase in an LCA study aims at assessing the potential environmental impacts based on the life cycle inventory (LCI) and communicating them through a common commensurate for each impact category . There are different LCIA methodologies developed by various researchers leading to difficulty in selection during an LCA study . Moreover, for certain impact categories, these methods use different approaches to derive the characterization factors leading to inconsistency . Selection of an appropriate LCIA method by the LCA practitioner is thus, dependent on multiple factors . The major factors that influence this decision can be - 1 . The goal and scope of the LCA Study 2 . Type of intended communication - internal communication, business to business communication or business to consumer communication 3 . Impact categories covered in the methodology 4 . Pollutant emissions obtained from LCI versus the characterization factors available in the LCIA methodology The paper tries to explore, with examples, the influence of these on decision making of an LCA practitioner . The paper also tries to explore some of the gaps that are usually overlooked in the process .

MO 442Comprehensive life cycle assessment for cheese and whey products in U.S.G .J . Thoma1, D . Nutter1, R . Ulrich1, D . Kim1, G . Norris1, F . Milani2

1University of Arkansas, Fayetteville, United States of America2University of Wisconsin, Madison, United States of AmericaA comprehensive life cycle assessment (LCA) has been carried out to account for the entire supply chain of cheddar and mozzarella cheese consumption in the United States . This includes specifically product loss at various stages of the supply chain, as well as consumer transport and storage of products . SimaPro 7 .3 (Pre Consultants, The Netherlands) was used as the primary modeling software . The EcoInvent database provided information on the “upstream”burdens associated with materials like fuels and refrigerants . Data from the surveys and other U .S . specific information was incorporated into the model to the extent that it was available . Greenhouse gas (GHG) emissions are of significant interest, and on a milk solids (dry) basis the carbon footprint of cheddar and mozzarella are approximately 12 .3 and 11 .8 metric tons CO2e per metric ton of cheese consumed, respectively . The 95% confidence band ranges 8 .1 to 18 .2 metric tons CO2e per metric ton of cheddar consumed, and 7 .9 to 17 .2 metric tons of CO2e per metric ton of mozzarella cheese consumed . For an average moisture content of 35 .0% for cheddar as sold at retail, the carbon footprint is 8 .0 kg CO2e per kg cheddar cheese consumed with a confidence band of 5 .3 to 11 .8 kg CO2e per kilogram cheddar consumed . On average moisture content of 42 .5% for mozzarella as sold at retail, the carbon footprint is 6 .8 kg CO2e per kg mozzarella consumed, with a 95% confidence band of 4 .5 to 9 .9 kg CO2e per kg mozzarella consumed .

MO 443ReCiPe, USEtox, IMPACT 2002+, and TRACI 2 impact assessment methodologies in the context of fluid milk delivery systems LCAJ . Burek, G . Thoma, D .S . KimUniversity of Arkansas, Fayetteville, United States of AmericaThe purpose of the fluid milk delivery systems Life cycle assessment (LCA) was to provide results and interpretation of the life cycle impact assessment (LCIA) for the U .S . dairy industries that would allow them to engage in more sustainable approaches and reduce their environmental impacts . In order to be able to interpret and communicate results in a transparent way LCIA method sensitivity analysis was performed . The selected methodologies were two midpoint USEtox, and TRACI 2 and two endpoint ReCiPe and IMPACT 2002+ methodologies . The impact category chosen was ecotoxicity or ecosystem . The results have shown differences in cradle-to-grave vs . farm gate-to-grave assessment . For example, in ecotoxicity USEtox midpoint category the largest impact was attributed to raw milk production (almost 100%), but in TRACI 2 only 50% was attributed to raw milk production and 40% to consumption LCA stage . Significant differences were also visible within farm gate-to-grave assessment where LCA stages show different relative contribution in total impact for example, in IMPACT 2002+ ecosystem quality category, 35% of impact is attributed to processing plant, 30% to consumption, 18% to raw milk transportation, 8% to distribution center, 6% to retail and 2% to container construction . Harmonizing LCIA methods will take some time . Meanwhile, sensitivity analysis on LCIA methods and ranking of methodologies by result and relevancy of that information for a studied product targeted audience appeared to be a simple technique in selecting the most appropriate LCIA method, without completely excluding the others . Although all the methodologies provided valuable results, in the context of this project and targeted audience, ReCiPe method has shown advantage by allowing direct identification of the “hotspots”on the level of dairy industry

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processes e .g . pasteurization, refrigeration, container material construction etc . Nevertheless, other methods were kept in the framework of the sensitivity analysis . They gave a valuable insight of the drivers that might appear in the results and help in further refining of the interpretation of the result .

MO 444IMPACT World +: a new global regionalized life cycle impact assessment methodS .M . Bulle1, O . Jolliet2, S . Humbert3, R . Rosenbaum4, M . Margni1

1CIRAIG - Departement de Genie Chimique - Ecole polytechnique de Montreal, Montreal, Canada2Dept . of Env . Health Sciences, School of Public Health, Univ . of Michigan, Ann harbor, United States of America3Quantis, Lausanne, Switzerland4QSA, Department of Management Engineering, Technical University of Denmark, Lyngby, DenmarkMost of the impacts modelled in life cycle impact assessment (LCIA) are regional or local . However, LCIA methodologies currently offer generic characterization factors (CFs) that do not account for the spatial variability of impacts . Some LCIA methodologies have partially addressed the issue of regionalization (IMPACT 2002+, ReCiPe, LIME, or LUCAS), but they only cover a specific region of the world . Characterizing life cycle inventories collected within a global economy using for example European CFs implies an underlying assumption that all the emissions occur in Europe or at least under European conditions, which is not necessarily a better assumption than applying global or generic CFs . IMPACT World+ was developed out of the need to offer a regionalized methodology at a global scale, implementing state-of-the art characterization modelling approaches developed since the publication of IMPACT 2002+ and LUCAS, and including uncertainty information encompassing both spatial variability and model uncertainty . This not only allows applying more environmentally relevant CFs, but also to regionally assess any geo-referenced emission, and ultimately to determine the uncertainty related to an unknown emission location by associating the corresponding geographical variability to each CF at a given geographical scale . The novelties introduced in IMPACT World + are numerous: USEtox model for toxic impacts and water use impacts are included for the first time in an LCIA method with consistent spatialized alternatives, major modelling improvements have been conducted in regards to land use with the inclusion of ecosystem services, acidification with an improved modelling of the atmospheric fate, resource use with the introduction of an extraction-consumption-competition approach, respiratory effects with new epidemiologically derived factors and finally, for eutrophication with a world model at 0 .5 x 0 .5 degrees . IMPACT World+ is a new LCIA method which will increase both the relevance and the dicriminating power of LCA by allowing to account for uncertainties and spatial variability . Many other improvements have been integrated into the methodology making it more environmentally relevant and scientifically robust .

MO 445An open source software for Emergy calculation with life cycle inventory databasesA . Marvuglia1, B . Rugani1, G . Rios2, J . Muller3, E . Benetto1, L . Tiruta-Barna4

1Public Research Centre Henri Tudor, Esch-sur-alzette, Luxembourg2Cork Constraint Computation Centre, Cork, Ireland3Université de Toulouse, Toulouse, France4INSA Toulouse, LISBP, Toulouse, FranceEmergy evaluation is a relatively novel environmental accounting method which has gained credit during the last decade, but is still affected by several drawbacks in its calculation procedures and in its general methodological background, which prevent it from being accepted by a wider community . Emergy accounting could benefit from the use of existing Life Cycle Inventory (LCI) databases, which comprise hundreds of environmental interventions in thousands of common industrial processes . However, it requires the compliance with a set of algebraic rules that are completely different than those applied in Life Cycle Assessment (LCA) and this has hampered the implementation of emergy calculation through automatic routines in specific software tools . This work presents an open source software specifically created for allowing the rigorous calculation (respecting all the algebra rules emergy accounting has to comply with) of the Emergy associated to each of the commodities involved in a given product’s life cycle along with its related inventoried resources . We formalized the problem in a matrix based structure which comes directly from the LCA framework and developed a variant of the well known track summing algorithm . Two versions of the calculation routine were implemented: one in Scala (a general purpose programming language integrating features of object oriented and functional languages) and one in C++ . The former is a multi-threaded breadth first search (BFS), the latter follows a depth first search (DFS) and is more efficient in terms of memory usage . The beta version of the software has been tested on different case studies, the most complex of which involved a square matrix of 2154 rows . Besides the emergy of the studied product, the software also allows the calculation of some statistics related to the complexity of the whole network of involved processes . For a toy problem involving only a 7 by 7 matrix the results were also validated using the software Emsim, a free share Emergy simulator able to work with lifecycle systems using a graph instead of a matrix . Differently than our software, Emsim does not allow a direct link to automatic calculation routines, since it requires the system’s diagram to be drawn by the operator . The software could be potentially used for a consistent emergy calculation of any product in a LCI database and therefore for a complete combination of LCA and Emergy perspectives in environmental assessment .

MO 446Complementing ecological footprint with risk analysis for the ecodesign of footwearM . Herva1, A . Alvarez2, E . Roca1

1University of Santiago de Compostela, Santiago de compostela, Spain2Inditex, A coruña, SpainThe ecological footprint (EF) was created in the early nineties and has been gaining in popularity ever since . As a pioneering footprint, its applications has expanded and now they are not limited to the regional or national level but include the evaluation of production processes and products . Nevertheless, as a land-based indicator it can hardly account for all of the environmental burdens that may occur during an industrial process or the development of a business activity . Therefore, it is advisable its combined application with complementary environmental evaluation tools . In this respect, this work proposes the joint application of EF and environmental risk assessment (ERA) to evaluate footwear design proposals from an environmental point of view . On the one hand, the EF component approach based on life cycle inventory was employed to appraise the environmental impact of the shoe-making process . Thus, individual EFs were calculated for each input/output material and energy flow in the inventory data and then aggregated to estimate the total EF of the pair of shoes . On the other hand, the ERA is a standardized process for the estimation of the magnitude, probability and uncertainty of adverse effects on health derived from the exposure to substances present in the environment . It was used to estimate the risk derived from the exposure to certain hazardous substances that raw materials may contain (as some organic compounds or heavy metals) and that would affect the final consumers of the product . Hence, the hazard quotient (HQ) and cancer risk (CR) were determined as additional indicators to EF . Four models of children shoes were evaluated under the viewpoint of EF and ERA and compared . The synthetic shoes obtained a smaller EF (6 .5 gm2) when compared to the leather shoes (11 .1 gm2) . However, high concentrations of hazardous substances were detected in the former, even making the HQ and CR exceed the recommended safety limits for one of the synthetic models analysed . Risk criteria were prioritized in this case and, consequently, the design proposal was discarded . For the other cases, the perspective provided by the indicators of different nature was balanced to accomplish a fairest evaluation . The selection of fibres produced under sustainable criteria and the reduction of the materials consumption was recommended, since the area requirements would be minimized and the absence of hazardous compounds would ensure safety conditions during the use stage .

RA11P - Guidance documents and guidelines for environmental risk assessment (ERA): needs, developments and progress

MO 449Mollusc reproductive toxicity tests - Development and validation of test guidelinesV .P . Ducrot1, A .J . Sieratowicz2, J . Oehlmann3, L . Lagadic1, D . Azam1, R . Brown4, M . Collinet1, M . Coke1, J . Dobrick5, P . Egeler5, H . Holbech6, T .H . Hutchinson7, A . Jach8, K .L . Kinnberg6, G . Le Page4, P . Lorentz5, I . Planojevic9, K . Ruppert3, U . Schulte-Oehlmann3, C . Schmitt9, A .J . Smith10, L . Weltje8, P . Matthiessen11

1INRA, Rennes, France2Johann Wolfgang Goethe University, Frankfurt, Germany3Johann Wolfgang Goethe University Frankfurt am Main, Frankfurt, Germany4AstraZeneca, Brixham, United Kingdom5ECT Oekotoxikologie GmbH, Flörsheim/main, Germany6Institute of Biology, University of Southern Denmark, Odense, Denmark7Centre for Environment, Fisheries and Aquaculture Science, Weymouth, United Kingdom8BASF SE, Limburgerhof, Germany9Universiteit Antwerpen, Wilrijk, Belgium10CEFAS, Weymouth, United Kingdom11Consultant Ecotoxicologist, Cumbria, United KingdomThe Organisation for Economic Cooperation and Development is promoting the development and validation of mollusc toxicity tests within its test guidelines programme, eventually aiming for the standardization of mollusc apical toxicity tests . Through collaborative work between academia, industry and stakeholders, this study aims to develop innovative partial life-cycle tests on the reproduction of the freshwater gastropods Potamopyrgus antipodarum and Lymnaea stagnalis, which are relevant candidate species for the standardization of mollusc apical toxicity tests assessing reprotoxic effects of chemicals . Draft standard operating procedures (SOPs) have been designed based upon literature and expert knowledge from project partners . Pre-validation studies have been implemented to validate the proposed test conditions and identify issues in performing the SOPs and analyzing test results . Pre-validation work for P. antipodarum consisted in a comparative 28-day study between four laboratories and with two test substances: cadmium (Cd) and bisphenol A (BPA) . For L. stagnalis, pre-validation consisted in a comparative 56-day study between six laboratories and with two test substances: Cd and tributyltin (TBT) . Cumulated fecundity per individual over the test period was

22�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

the main test endpoint . Tested animals came from a single laboratory culture (i .e . the University of Frankfurt for P. antipodarum and INRA for L. stagnalis) . Snails were acclimated in partner laboratories and exhibited homogenous age and size at the beginning of the tests . All laboratories found a concentration-dependent decrease of fecundity in both species under Cd exposure . Values of NOECs and LOECs were generally homogenous among laboratories . Results of the pre-validation studies with P. antipodarum and L. stagnalis exposed to Cd are thus very encouraging . Results for BPA highlighted issues in controlling exposure concentrations, and possibly BPA leaching from test equipment . This study also highlighted that factors, such as e .g ., snail origin, temperature and test chemical stability in water, must be controlled in mollusc toxicity tests to avoid data dispersion which may impede the interpretation of effects . Applicability and limitations of the SOP proposed for L. stagnalis will be assessed after completion of the on-going pre-validation work . In both cases, optimization of experimental conditions and test procedures is required before the SOPs are ready to be proposed as OECD test guidelines .

MO 450Temperature effects on reproduction in Potamopyrgus antipodarum - optimizing a standard test protocol with molluscsK . Ruppert, K . Bender, A .J . Sieratowicz, U . Schulte-Oehlmann, J . OehlmannJohann Wolfgang Goethe University, Frankfurt am main, GermanyAquatic ecosystems are exposed to a multitude of chemicals via surface runoff, landfill sites or sewage treatment plants . Many of these substances may affect growth, development and/or reproduction of aquatic species and therefore endanger the survival of populations . Standardized and internationally accepted OECD guidelines for the testing of chemicals with invertebrates currently include tests with arthropods and annelids but exclude molluscs, which represent the second largest clade . The German Federal Environment Agency (UBA) and the Department for Environment, Food and Rural Affairs of the United Kingdom have supported the preparation of a Detailed Review Paper on Molluscs Life-cycle toxicity testing [1] and a pre-validation round-robin with the parthenogenetic mudsnail Potamopyrgus antipodarum as standard test organism . Currently, the project aims to optimize and define test and breeding conditions to minimize variations of test results in preparation of an international validation exercise . In this study we investigate reproduction variations at the proposed standard temperature (16°C) and two lower temperatures throughout the year . Therefore, we acclimatized P. antipodarum at 10, 13 and 16°C and compared reproduction (embryo numbers in the brood pouch) . Because test temperature and natural seasonal reproduction patterns can modulate reproductive responses towards a chemical and even mask effects, we assessed sensitivity variations caused by these factors in reproduction tests with the endocrine disruptor octylphenol . To this end P. antipodarum was exposed to 1, 3, 10, 30 and 100 µg/L octylphenol at each temperature scenario and at different times of the year and the resulting effect concentrations were compared . [1] OECD . 2010 . Detailed review paper on molluscs life-cycle toxicity testing . ENV/JM/MONO(2010)9, Paris, France . Acknowledgement - The authors thank the UBA for funding (UBA projects 370861402 and 371165417)

MO 451How breeding conditions influence substance effects - the need for a standard test protocol with molluscsA .J . Sieratowicz1, U . Schulte-Oehlmann2, A . Wigh2, J . Oehlmann2

1Johann Wolfgang Goethe University, Frankfurt, Germany2Johann Wolfgang Goethe-University, Frankfurt, GermanyUp to now effect studies with molluscs play a minor role in regulatory risk assessment of substances because OECD guidelines for the testing of chemicals have not considered this phylum . The German Federal Environment Agency and the Department for Environment, Food and Rural Affairs of the United Kingdom supported the preparation of a Detailed Review Paper (DRP) on Molluscs Life-cycle Toxicity Testing proposing inter alia the parthenogenetic mud snail Potamopyrgus antipodarum as standard test organism[1] . To investigate the impact of breeding and test conditions on the outcome of reproduction tests with P . antipodarum we bred specimens in different media and at vaying densities or temperatures . In the “medium”experiment, snails were kept in 800 mL DRP-[1], ISO- or Elendt M4-[2] medium for 12 weeks . Reproduction was assessed after 4, 8 and 12 weeks by the number of embryos in the brood pouch . For the density experiment, reproduction of snails kept in groups of 5, 10, 20, 40, 80 or 160 specimens per 200 mL DRP medium was investigated after 4 weeks . Subsequently, snails from each density group were exposed to 12 µg cadmium/L (CAS: 67-68-5) without further density stress for 4 weeks . In the temperature experiment, snails were acclimated at 7 and 25°C for 4 weeks . Then they were additionally exposed to 5, 10, 20 or 40 µg bisphenol A/L (CAS: 80-5-7) at 7, 16 and 25°C . Reproduction was assessed after 4 weeks . The use of different breeding media resulted in significantly varying embryo numbers of P . antipodarum between all test media after 4 and 8 but not after 12 weeks . Varying snail densities affected reproduction significantly . Furthermore, differing snail densities before the start of the cadmium exposure had a significant impact on the sensitivity towards the heavy metal: the sensitivity increased with density stress in the pre-exposure period . At 7°C and 25°C, a significant increase of reproduction was already observed for BPA exposed snails at 10 µg/L and thus at lower concentrations compared to the standard temperature (16°C)[1] where a significant effect occurred only at 40 µg/L . This confirms temperature as a trigger for reproductive output . In summary, different breeding and test conditions like snail density or temperature affect the sensitivity towards chemical exposure of P . antipodarum in subsequent reproduction tests . To gain reproducible test results with low variability among laboratories, the definition of standard test conditions is essential .

MO 452A partial life-cycle toxicity test to assess the effects of endocrine disruptors in the great pond snail Lymnaea stagnalisA .E . Barsi1, V . Ducrot1, T . Jager2, L . Lagadic1

1INRA, Rennes, France2Vrije Universiteit, Amsterdam, NederlandMolluscs are highly sensitive to endocrine disrupting compounds (EDCs) and therefore are of great interest for environmental hazard and risk assessment . The Organisation for Economic Co-operation and Development (OECD) is thus promoting optimization and validation of mollusc-based toxicity tests . Standardised test methods for assessing effects of reprotoxicants, and hence a number of EDCs, are currently under development in various mollusc species, including the great pond snail Lymnaea stagnalis . Based upon this on-going work, we designed and performed a partial life-cycle toxicity test in which adults of L. stagnalis were exposed to two androgenic compounds (triphenyltin and 17α-methyltestosterone), and two estrogenic compounds (nonylphenol and 17α-ethinylestradiol) . This test consisted in two parts . In the first part, snails were exposed to the toxicants during 56 days in semi-static conditions, as proposed for the OECD test under development . Survival, growth, and fecundity were regularly monitored . Egg-clutches produced in this part of the test were collected and exposed to clean test water during 35 days, which represented the second part of the test . The biological quality of offspring was assessed through the estimation of the frequency of abnormal eggs in clutches, their mean hatching rates, and the mean shell length and dry weight of hatchlings . Data were analyzed using standard statistical approaches, as recommended by OECD for the analysis of toxicity test data . Preliminary results generated over the first week indicated that exposure of adult snails to the higher tested concentrations of triphenyltin (nominal concentrations of 0 .75 and 2 .60 µg/L) and nonylphenol (500 µg/L) led to a decrease in food consumption and also to a decrease in growth and reproductive output . This study will allow to further examine whether or not a 56-day partial life-cycle test in adults of L. stagnalis is sensitive enough to highlight the reproductive effects of the selected EDCs, or if the monitoring of offspring performances is also required to significantly detect those effects . Obtained results will thus contribute to the process of optimizing the sensitivity of toxicity tests based upon L. stagnalis for use in environmental hazard and risk assessment .

MO 453Environmental risk assessment of a disinfectant for milk extraction systems: comparison of 2 emission scenario documents and EUSESH .M . Dosogne1, A . Ghekiere1, F .A .M . Verdonck1, L . Uytterhaegen2

1ARCHE, Gent, Belgium2DeLaval, Ghent, BelgiumThe environmental risk assessment of a disinfectant for milk extraction systems was performed with 1) EUSES 2 .1 based on local consumption estimates extrapolated from EU/regional tonnage and 2) two Emission Scenario Documents for Biocides (ESD), published in 2002 and 2011 , based on local consumption estimates from a dairy farm . The ESD of 2002 describes only emission to manure followed by application to agricultural soil, whereas the ESD of 2011 only describes emission to sewage . Moreover in the ESD of 2011, degradation is not taken into account in Tier 1, whereas in the ESD of 2002 a formula for degradation was already included . Calculations were made for 100% emission to sewage as a worst-case scenario . Scenario 2 .5 .1 of EUSES (Medical, disinfectants in accomodations) was selected from the exposure scenarios of biocides on a local scale, because there is currently no scenario for PT3-4 biocides evaluation included in EUSES and because scenario 2 .5 .1 resembled most the PT3-4 use . With the ESD of 2002, the RCRs were all below 1, whereas with the ESD of 2011, the RCR was < 1 for one active substance, but > 1 for the other substance and a refinement of the calculation was required . PEC values obtained with the Tier 1 ESD 2011 calculations were about 10 times higher than those obtained with the ESD of 2002 and about 100 times higher than with EUSES . This can be explained by the fact that the ESD of 2011 does not take into account degradation . An advantage of the ESD of 2011 is that the scenario is more consistent with current practices and that EUSES could be applied in replacement of the ESD . A more accurate environmental risk assessment would be possible with EUSES when a PT3-4 scenario becomes available .

MO 454Can we assess environmental risks of antifouling paint usage with the prevailing BPD?P . Ytreberg, L . Johansson, B . EklundStockholm University, Stockholm, SwedenSince many antifouling paints are designed to leach toxic biocides, it is essential to evaluate, prior to use, their effects on aquatic ecosystems, including toxicity to aquatic organisms . This evaluation process, known as environmental risk assessment (ERA), is used during the authorization of anti-fouling coatings and is regulated in the European Union (EU) under the biocidal product directive (BPD) . In the ERA, the predicted environmental concentration (PEC) of biocides, due to leaching from the coating, is determined using the modeling tool MAMPEC . In this model, default scenarios representing an EU harbor and an EU marina is incorporated in the software and used in the process of deriving a PEC-value of biocides . However, there may be other sources of biocides to these marine systems, sources that have been largely overlooked in ERA of antifouling formulations . Removal of fouling by pressure hosing usually occurs close to the marina, e .g . at a slipway, when the boat is lifted out of the water after the season . The fouling that is still present on the hull after pressure hosing is typically being scraped off at the boat yard . In this process also antifouling paint particles (APP) will be removed and deposited on the ground . These cleaning activities are not regulated to any great extent, i .e . leisure boat owners are not obligated to collect the waste and the removed APP . Hence, the boat yard may act as a substantial source of contaminants to the recipient (i .e . the marina) as precipitation infiltrates the soil resulting in leaching

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and transportation of biocides . The aim of the current study was to determine the leaching of biocides from a boat yard allocated in Stockholm archipelago . We designed the experimental part to quantify the annual leaching of biocides from the contaminated soil to the marina . The data were subsequently used in a full-scale ERA of antifouling paint usage by determining the contribution of different biocidal sources to a marina i .e ., from boat hulls, pressure hosing and from the soil at the boat yard . The results showed the biocidal release from pressure hosing and soil to be a substantial source of biocides to the marina . These findings suggests that a PEC-value based on only the biocidal release from boat hulls to be inadequate in predicting environmental risks of antifouling paint usage .

MO 455Ready biodegradability testing for Insoluble and volatile substancesS . Fiebig, L . Geisler, N . Mendel, U . NoackDr .U .Noack-Laboratorien, Sarstedt, GermanyUnder REACH a PBT/vPvB assessment is required for substances that are manufactured or imported in amounts of > 10 tons per year . The first step of persistency assessments is a screening like the tests on ready and inherent biodegradation provide . Often substances fail the criterion for ready biodegradability or an unequivocal assessment is not possible because the substance is not bioavailable for the degrading bacteria due to its low water solubility and/or volatility . Careful consideration of the physic-chemical properties of a substance is therefore necessary to choose the appropriate test design . As the biodegradation of a substance strongly depends on its mass transfer and the bioavailability for the degrading bacteria, there are several technical issues which need to be addressed while testing the biodegradability of poorly water soluble and/or volatile substances . The OECD guidelines for testing ready biodegradation comprise seven different tests (OECD301A-F, OECD310), which differ mainly in the vessel size, the ratio test solution/headspace, the agitation and aeration during testing and the type of analysis . Based on the substance properties the test type has to be chosen carefully e .g . the use of the aeration or agitation method can result in losses of the substance and underestimated biodegradation . The influence of the test type on the biodegradation of insoluble and volatile substances was investigated and the results will be presented .

MO 456OECD Guideline 209: investigation of validity criteriaS . Fiebig, K . Klee, L . Geisler, K . Sander, N . Mendel, U . NoackDr .U .Noack-Laboratorien, Sarstedt, GermanyThe revision of the OECD guideline for the Activated Sludge Respiration Inhibition Test (ASRIT) was finalized in 2010 . Within the revision the validity criteria were changed significantly and a new criterion was included . The specific oxygen uptake rate of the activated sludge should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour . As the oxygen uptake rate of active sludge from many municipal treatment plants shows fluctuations and periodically decreases below 20 mg O2/(g[GREEKX]h), the significance of the validity criterion was examined . It is assumed, that parameter like season, sewage flow and weather conditions influence the oxygen uptake rate . To confirm the assumption, reference tests were conducted over several months and the activated sludge was collected at different days of the week . The dependence of the oxygen uptake rate on the assumed parameter was investigated . Furthermore the correlation between the oxygen uptake rate and the sensitivity of the activated sludge was examined . The results of these investigations will be presented .

MO 457Development of interim guidance for the inclusion of non-extractable residues (NER) in the risk assessment of chemicalsC . Roberts1, C . Finnegan2, G . Sanders3, J .O .Y . Worden4, M .I .C .H .A Spiteller5, M . Galay Burgos6, M .S . Holt6

1AstraZeneca, Brixham, United Kingdom2Unilever, Bedford, United Kingdom3Givaudan Schweiz AG, Vernier, Switzerland4Shell Technology Centre, Thornton, United Kingdom5Dortmund University of Technology, Dortmund, Germany6ECETOC, Brussels, BelgiumBound residues (BR), including non-extractable residues (NER), are an important factor in PBT (Persistence, bioaccumulation and toxicity) assessment and risk assessment of chemicals . Precautionary risk assessments usually assume 100% bioavailability, i .e . all of the chemical present, is available for degradation or to have potential toxic effects on the biota . This precautionary approach generally overestimates the exposure concentration by the amount that is not available and therefore overestimates the level of risk to biota in the environment . It is also well documented that chemicals that are irreversibly bound to solids are less degradable and less toxic than the total residue would predict . Currently, there is no agreed guidance on how to determine what is available or not, and how it should be considered in the risk assessment . An ECETOC workshop ‘Significance of Bound Residues in Environmental Risk Assessment”was held on 14-15 October 2009 in Brussels, and produced a framework outlining a possible decision tree for improving the risk assessment of NER, together with the identification of key research needs to address gaps in the current knowledge base . Subsequently, an ECETOC Taskforce was set up to i) critically evaluate the proposed risk assessment framework developed following the ECETOC workshop and assess its utility as an interim approach for regulatory assessment of chemicals; ii) develop suitable guidance and trigger values to enable the decision tree to be used and test the utility of the scheme using suitable case studies, and; iii) Provide guidance on study design to provide the appropriate quality of data needed for the risk assessment framework to function within a regulatory decision making system . The progress of this taskforce will be presented, along with recommendations for addressing Bound/non-extractable residues . An example of a risk assessment using this approach will be presented .

MO 458Granivorous birds - exposure reduction by de-husking behaviour J . Gerlach, J . Schabacker, J .D . LudwigsRIFCON GmbH, Hirschberg, GermanyWith the publication of the current EFSA Guidance document on bird and mammal risk assessment, exposure of granivorous birds to treated seeds has received increased attention . Seed-eaters are considered no longer as indicator species only for seed treatment uses, but also regarding foliage spray applications resulting in exposure through contaminated weed seeds - here small granivorous bird species need to be evaluated as well . For granivorous mammals several studies have been conducted and data is published demonstrating that de-husking i .e . removing of the husk (including large portions of residues) prior to ingestion, can significantly reduce the exposure in crop seeds (e .g . Brühl et al . 2010, DEFRA research report 2010) . For birds it is long and well known that several behavioral skills and traits are common across different bird families to remove the husk from seeds (e .g . Ziswiler 1965) . One method to quantify the reduction of such exposure routes is to conduct pen studies, i .e . offering treated seeds to caged individuals and measuring the amount of residues (or marker substance) in the remaining seed husks . Such approaches and test designs are feasible for crop and weed seeds . Since small granivorous birds are often considered as worst-case indicator species (due to their low body weight) their theoretical exposure is relatively high in default risk assessments . Based on the de-husking behaviour of birds (e .g . finches, sparrows and buntings) we will present data and study design proposals on how to use this behavioural trait as refinement tool for de-husking species in higher tier wildlife risk assessments .

MO 459ECETOC Guidance on identifying endocrine disrupting effects: specificity for environmental speciesA . Weyers1, L . Weltje2, J .R . Wheeler3

1Bayer CropScience, Monheim am rhein, Germany2BASF SE, Crop Protection - Ecotoxicology, D-67117 limburgerhof, Germany3Syngenta Product Safety, Jealott’s Hill International Research Centre, Bracknell, United KingdomThe first ECETOC technical report and associated workshop presented a science-based concept on how to identify endocrine disrupting properties of chemicals for both human health and the environment . To be able to discriminate chemicals with endocrine disrupting properties of low concern from those of high concern (for regulatory purposes), it was recognized that the concept needed further refinement, including specificity . For environmental assessments the lead toxic effect can be considered within a study/species or between different studies/species within an environmental compartment . Within a study an endocrine mediated endpoint can be affected below, at the same, or above the threshold of general toxicity . This should be considered in the first part of the original ECETOC assessment, when linking mechanistic and apical in vivo studies to decide whether a substance is a potential endocrine concern or not . A substance should only be considered of high concern when the endocrine mediated effect occurs at concentrations lower than those that cause other significant toxicity . In the case where the endocrine mediated effect is not the lead effect, i .e . the endocrine effect is observed at a higher concentration than that causing other toxicity, then the substance should then proceed with a risk assessment based on the most sensitive (non-endocrine) lead effect . Once a population-relevant endocrine effect has been confirmed, the next step in the environmental evaluation is to consider the specificity across taxa . Although endocrine effects may be observed as the most sensitive (=lead) effect within one study or organism, this effect may be accounted for in a risk assessment by more sensitive non-endocrine endpoints observed in other taxonomic groups . Risk assessment then allows for a margin of safety that sufficiently covers endocrine specific effects . Any risk management measures based on the lead toxic effect in one taxon will also be protective of other toxic effects (including endocrine effects) in other taxa . Such substances should not be considered as endocrine disruptors, since they do not cause adverse endocrine effects in non target species under realistic conditions of use .

MO 460Concentration-response relationship for teratogenic effect of 17β-oestradiol in eelpout Zoarces viviparusN . Brande-Lavridsen, J . Ebsen Morthorst, B . Korsgaard, P . BjerregaardUniversity of Southern Denmark, Odense, DenmarkIn recent years increased frequencies of malformations among eelpout embryos have been detected in monitoring programs in several Baltic countries (1,2,3) . Malformations can be induced in oviparous fish species by exposure to chemicals, including endocrine disrupting substances, in the laboratory . Eelpouts are the only viviparous fish in Northern Europe, which makes them very suitable for investigation of mother-offspring interactions and effects in the offspring upon maternal exposure to various chemicals . The causative agent or agents in underlying the malformations observed in the monitoring programmes are not known, but malformations upon exposure of pregnant eelpout

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to octylphenol and 17β-oestradiol (E2) have been observed (4) . To investigate the concentration-response relationship for malformations in eelpout embryos induced by E2, we investigated the embryonic development upon exposure of pregnant eelpouts to (E2) at nominal concentrations between 12 .5 and 500 ng E2/l . The experiment was terminated after 40-44 days of exposure . The various types of abnormal development among the embryos (early death, malformations of eyes and spinal cord) were evaluated and the frequencies were recorded for each experimental group . Exposure of female eelpouts to environmentally realistic concentrations, 132,7 ng/l, of 17β-oestradiol early in the pregnancy increases the abundance of malformations in the embryos . The study further showed that delaying the onset of exposure to the highest dose of E2 by approximately three weeks reduced the frequency of abnormal larvae, suggesting that timing of the exposure is crucial . (1) Gercken J, Forlin L, Andersson J . 2006 . Developmental disorders in larvae of eelpout (Zoarces viviparus) from German and Swedish Baltic coastal waters . Mar . Pollut . Bull . 53, 497-507 . (2) Strand J, Bossi R, Dahllof I, Jensen CA, Simonsen V, Tairova Z, Tomkiewicz J . 2009 . Dioxin og biologisk effektmonitering i ålekvabbe i kystnære danske farvande . Report from the National Environmental Reseach laboratory . (3) Hedman JE, Rüdel H, Gercken J, Gergek S, Strand J, Quack M, Appelberg M, Förlin L, Tuvikene A, Bignert A . 2011 . Eeelpout (Zoarces viviparus) in marine environmental monitoring . Mar .Pollut .Bull . 62: 2015-2029 . (4) Brande-Lavridsen N, Nielsen RV, Rasmussen TH, Korsgaard B, Bjerregaard P . In prep . A novel type of endocrine disrupting effect: Octylphenol and 17β-estradiol cause abnormal development in embryos of the viviparous eelpout Zoarces viviparus

MO 461Use of field studies in different regulatory frameworksF .M .W . de JongRIVM, Bilthoven, NederlandThe poster discusses the different roles of field studies in the different regulatory frameworks . In the tiered approach followed in the registration procedure of plant protection products (PPP’s), field studies are frequently used in the higher tier . These higher tier studies are used to generate additional information, in order to lower the safety factors of the lower tiers, and to show recovery after an initial effect . For a number of field study types (e .g . mesocosm studies) detailed guidance is available, as well as guidance for the assessment of the studies . In most other regulatory frameworks (e .g . biocides, pharmaceuticals, GMO’s, REACH) the role of field studies is less clear . At the most limited guidance is available, that is not very specific . The poster discusses the possibilities and the impossibilities to use the guidance for field studies from one framework for another framework . In some cases the same active substance is assessed in different frameworks (e .g . PPP’s and biocides) . In this perspective a number of initiatives have been taken to study whether and how field studies, designed and conducted for PPP registration can be used for biocide registration . As an example the use and assessment of mesocosm studies in PPP and biocide registration is elaborated .

MO 462Experimental design and statistical analysis of ecotoxicological community studiesA .J . Lawrence1, K .C . Brown2, G .K . Frampton3, P .J . Van den Brink4

1Cambridge Environmental Assessments, Cambridge, United Kingdom2Independent Consultant, Devon, United Kingdom3University of Southampton, Southampton, United Kingdom4WUR, Alterra, Wageningen, NederlandEcotoxicological field studies may be conceived by industry notifiers in association with external consultants and contract research organisations (CROs) . The results may then be interpreted and summarised before inclusion in a regulatory submission in support of a product registration . The submission will be reviewed by representatives from Member State (MS) Competent Authorities, in terms of both study quality and meaning . Not all involved parties may be experts in the conduct and interpretation of such studies . There is a need, therefore, for guidance on both ecotoxicological field study experimental set-up and statistical analysis of ecotoxicological community data - from aquatic mesocosms (invertebrates, algae and/or higher plants), surface/foliar-dwelling non-target arthropod (NTA) field studies and full-field soil mesofauna studies . Firstly, this project seeks to provide guidance on experimental set up of ecotoxicological community studies from a statistical point of view, such as replication, plot size, study duration, randomisation . Secondly, the project will provide guidance on the statistical analysis of ecotoxicological community effect data . This will include use and interpretation of both univariate and multivariate methods . The project will summarise existing guidance and use examples available in the literature to illustrate key points . This work is funded by the UK Chemicals Regulation Directorate .

MO 463Development of a framework for toxicological and ecotoxicological risk assessment of fertilisers, soil improvers and related productsG .J . Heijerick1, K . Oorts1, F .A .M . Verdonck1, E . Smolders2

1ARCHE, Gent, Belgium2K .U . Leuven, Leuven, BelgiumThe use of fertilisers, soil improvers and related products may entail toxicological end ecotoxicological risks through the presence of specific chemical substances and/or pathogens . In Belgium, there is currently no real technical guidance on how to conduct a toxicological and ecotoxicological risk assessment of fertilisers, soil improvers and related products . Therefore, an evaluation is made of existing EU methods for toxicological and ecotoxicological risk assessment in similar domains (REACH, plant protection products, biocides, etc[3DOTS]) and of existing national approaches of risk assessment of fertilisers, soil improvers and related products in other EU member states . The application and relevancy of the identified relevant methods is further evaluated and tested in case studies for specific products . The results demonstrate that screening methods, identifying the most critical human health and environmental endpoints based on the hazards of the constituents, improve the usability and applicability of the selected risk assessment methods .

MO 464Chemical risk assessment under chemical substances control law in JapanW .N . Naito1, Y .H . Hirai2, M .M . Murata3, N .M . Miyasaka3

1AIST, Tsukuba, Japan2National Institute of Technology and Evaluation (NITE), Tokyo, Japan3NITE, Tokyo, Japan The purpose of Chemical Substances Control Law (CSCL) is to evaluate, before manufacture or import, whether or not new chemical substances have properties such as persistence, and to implement necessary regulations, in order to prevent environmental pollution caused by chemical substances that pose a risk of impairing human health or interfering with the inhabitation and/or growth of flora and fauna . To minimize the significant adverse effect of chemical substances on human health and environment by 2020 (Agreement in the Environmental Summit in 2002), CSCL has been amended in 2009 and implemented in 2011 . Under amended CSCL, risk assessment will be conducted in step-wise manner for all chemicals including existing chemicals . In this presentation, we will explain chemical risk assessment scheme and approach under amended CSCL and comparing it with other regulatory schemes .

MO 465Status quo and challenges in the EU scheme of environmental risk assessment for nanomaterialsJ .W . Kim, B .H . Ryu, H .P . Jeon, J .S . Ra, S .H . Kim, J .W . HwangKorea Institute of Science and Technology Europe, Saarbruecken, GermanyAlthough high potential benefits of engineered nanomaterials (NMs) for overcoming the technological limitations of conventional materials, the unique properties of NMs raise new emergent challenges in understanding, predicting, and managing potential risk to environment . For establishing a standard risk assessment of NMs, different regulatory frameworks have been suggested in recent years in various fields of chemicals, cosmetics, foods, environmental safety and waste in the EU . As a representative regulation for controlling the potential risk of NMs manufactured or imported in the EU, REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) regulation controls the use of NMs . In order to address the specific properties, hazards and risks associated with NMs, additional testing or information is required . Nevertheless, specific REACH guidance for NMs is not readily available . For develop the draft technical guidelines for risk assessment of NMs, the REACH implementation Projects on NMs (RIP-oNs) have recently been conducted by the EU Commission . However, the draft guidelines are still placed on the unsatisfactory level to reach a consensus on the risk assessment scheme of NMs in accordance with regulatory purposes . The objectives of this study are to analyse and map the status quo and future research challenges in the EU Nano risk assessment scheme based on the RIP-oNs and related recent studies with focusing environmental risk assessment (ERA) . The major challenges in regulating NMs are identified as the wide variety of NMs and applications, the limited knowledge on the toxicity of NMs in living system and their transport in living and environmental systems, the lack of harmonized standards or guidance, and the issues related to classification of NMs .

MO 466Is European chemical legislation suitable for regulating engineered nanoparticles?K . Syberg1, G . Banta1, V .E . Forbes2, F . Khan3, S .N . Luoma4, P . Reip5, H . Selck1, E . Valsami-Jones6

1Roskilde University, Roskilde, Denmark2University of Nebraska-Lincoln, Lincoln, United States of America3Department of Mineralogy, Natural History Museum, London, United Kingdom4John Muir Institute of the Environment, University of California at Davis, Davis, United States of America5Intrinsiq Materials Ltd, Farnborough, United Kingdom6School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, United Kingdom

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The nanotechnology industry is rapidly growing, and the production and use of engineered nanoparticles (ENP) is already a billion euro industry . ENPs are used in a wide range of applications (e .g . consumer goods and plant protection products) which will inevitably result in exposure to the environment . The current study reviews three major European chemical legislative instruments (REACH, the Water Framework Directive and the Cosmetics Regulation) in order evaluate whether the risk assessments under these instruments are suitable for ENPs or whether nano-specific adaptations to these frameworks should be recommended . REACH and the Cosmetic Regulation were chosen since they regulate some of the most important uses of ENPs (e .g . cosmetics and industrial applications) . The Water Framework Directive aims at protecting aquatic environments in the European Union, and is thus essential in regard to environmental risk assessments . The criteria for this evaluation are based on the substantial amount of experimental data that have been generated in the EU funded NanoReTox project (insert project number here) . Within the project we have studied the fate and toxicity, to both humans and the environment, of selected metal ENPs . The empirical approach allows us to evaluate the risk assessment frameworks with regard to very specific criteria and thus gives the analysis another dimension compared to an analysis that would have been based soley on theoretical considerations .

MO 467PBT-Assessment: the need for an agreed guidance across regulations - assessment of persistence as an exampleC .A . Rauert, U . Jöhncke, C . Hufenbach, M . Neumann, A . Wiemann, G . Hermann, I . Prutz, A . Friesen, J . Schönfeld, K . Willhaus, A . Kehrer, S . DuquesneUmweltbundesamt, Dessau-roβlau, GermanyThe PBT-assessment has been established across the various European substance regulations i .e . plant protection products, biocides, pharmaceuticals and REACh but references between regulations are sometimes lacking . For the three criteria P (persistence), B (bioaccumulation) and T (toxicity), the trigger values are consistent among the mentioned regulations, but the accompanying guidance differs and does not always give sufficiently detailed information . Also, data availability and risk assessment procedures differ across regulations which will influence the outcome of a PBT-assessment . This may be especially problematic for substances registered under more than one regulation, e .g . an active substance that is used both as a plant protection product and a biocide or a veterinary pharmaceutical . Therefore a consolidated guidance across regulations is needed to ensure the identification of PBT-substances on a common basis . In our experience at the German Federal Environment Agency persistence is the criterion where results of a PBT-Assessment potentially differ most across the different substance regulations, and hence, experts from these regulations across the EU should consult most urgently . In this poster, we present the current situation and introduce our proposal for a harmonized persistence assessment to overcome this situation . MO 468Stakeholders’ view on ecological risk assessment in the EU: what are we trying to protect and why might we be failing?A .D . Hunka1, M . Meli1, A . Palmqvist1, P . Pernille2, V .E . Forbes3

1Roskilde University, Roskilde, Denmark2Syngenta Jealott’s Hill International Research Centre, Bracknell, United Kingdom3University of Nebraska-Lincoln, Lincoln (ne), United States of AmericaThe EC Regulation 1107/2009 states that “A plant protection product ([3DOTS]) shall have no unacceptable effects on the environment, having particular regard to ([3DOTS]) its impact on non-target species, including on the ongoing behaviour of those species” . General goals for protection are thus well established in the legislation, whereas specific protection goals are less well defined . Still, accurate protection goals are vital in scientific risk assessment process . Therefore, we asked stakeholders (both practitioners and theorists) involved with ecological risk assessment (ERA) about the challenges they meet in the current ERA scheme and the way they perceive recent changes in guidance documents and legislation . We conducted 43, semi-structured, confidential interviews with representatives of plant protection product industry, regulatory bodies and academic ecotoxicologists and ecologists . The topics were related to the current protection goals (including introduction of the ecosystem services concept), the strengths and weaknesses of the current ERA and the changes in regulations . We employed the key informant approach in recruiting our participants . They were first identified as key stakeholders in ecological risk assessment of pesticides and then sampled by means of a snowball sampling . Our preliminary results show that participants, in general, find the current regulations (EC 1107/2009) too vague and the protection goals unclear . They put much hope in the ecosystem services concept, while on the other hand, many participants were concerned that the framework is overcomplicated and adds extra bureaucracy to the procedure instead of making it more practical . There is also much concern about the ecosystem services which cannot be directly quantified, especially from risk managers . It seems promising that all three stakeholder groups have a similar view on the ERA scheme, although there were some differences with regard to the level of complexity and protection provided by the current ERA . Stakeholders were concerned whether the increasing number of procedures directly translates into a better assessment of risk . Main hopes associated with changes in regulations were to address issues that are not clear at the moment, such as the mixture toxicity problem or implementation of ecological models .

MO 469Environmental assessment regulations for active pharmaceutical ingredients: a Canadian contrastA .G .M . Rattray, A . Beck, R .G . StringerHealth Canada, Ottawa, CanadaIn Canada, the Canadian Environmental Protection Act (CEPA) 1999 requires that all new substances for use in Canada be evaluated for their potential risks to the Canadian environment and human health while the Food and Drugs Act regulates the sale of human and veterinary drugs . Health Canada, in consultation with representatives from industry, non-governmental organizations and consumer groups has developed environmental assessment frameworks for new substances in products regulated by the Food and Drugs Act . One framework is specific for active pharmaceutical ingredients (APIs) in human and veterinary drugs and has been designed to align with the drug development/approval process . When developing this framework, the starting premise was that the regulatory requirements must be science-based and in proportion to potential for risk . Furthermore the approach taken was that any new framework should leverage the work done for environmental assessment guidelines in other jurisdictions internationally and harmonize where feasible . This talk will explore similarities and differences between the proposed Canadian Environmental Assessment Regulations and requirements in other jurisdictions with an emphasis on issues that are distinct to the Canadian context .

MO 470Mitigation measures for surface and groundwater based on risk assessment of Plant Protection Products in France: from the risk assessment to the stakeholders A .F . Boivin1, H .L . Larue1, N .D . Domange2, V . Poulsen1

1ANSES, Maisons alfort, France2ONEMA, Vincennes, FranceANSES provides opinions prior to Plant Protection Products (PPP) registration . All opinions are amended with the specific Standard Phrases for safety precautions related to the environment (SPe) in accordance with regulation (EC) No 1107/2009 . SPe are based on the risk assessment for the active substance(s) and their metabolite(s) . This work focuses on Standard Phrases for safety precautions related to the environment (SPe 1 to 3), that were purposely designed to protect groundwater; and/or aquatic organisms . For the different uses assess all PPP may then be labeled with the following phrases, which should be supplemented with specific recommendation regarding the time period or frequency of application (SPe1); and/or specific soil type or situation (SPe2) and /or specific unsprayed buffer zone (SPe3) . Since these Standard Phrases for safety precautions do focus on potential risks accounting for both the exposure and the toxicity for various uses of the PPP they are supposed to be suitable indicators since they summarize the outcome of the environmental fate and/or ecotoxicological risk assessments and thus the main outcome for PPP . The likening between PPP (active substance and metabolites) and specific SPe allow to identify if correlation between a specific waterbody (ie surface water and/or groundwater) and a PPP type (e .g . herbicide, fungicide insecticide) may be set up or not . In addition, SPe data collection ant following connections between PPP properties will be used to create a “comprehensive environmental package”reflecting the main conclusions of the risk assessment conclusions to stakeholders like national water agencies which would like to implement specific groundwater and/or surface water monitoring; or national agencies in charge of aquatic environment protection by inspecting use and enforcing regulations for instance .

MO 471At which level is an additional factor for EPM within the risk assessment of the benthic environment justified?S . Hahn, T . Soyka, J . Regelmann, G . Könnecker, O . Licht, A . BitschFraunhofer ITEM, Hannover, GermanyFor the environmental risk assessment data for aquatic organisms are in most cases available; data for sediment organisms are often lacking . In this situation the PNECsed can be provisionally calculated using the equilibrium partitioning method (EPM), as described in the REACh guidance . The EPM is based on the assumption that both sediment and aquatic organisms are equally sensitive . Only the uptake via the water phase is considered, assuming that substance concentrations in sediment and interstitial water are in a thermodynamic equilibrium . However, notable uptake may also occur via ingestion of sediment or direct contact to sediment . Thus, according to the REACh guidance, the EPM method has to be applied in a modified way for compounds with an octanol/water partitioning coefficient (log Kow) > 5 (or with a corresponding binding behavior, e .g . ionisable substances) . In these cases the uptake via ingestion of sediment should be taken into account by increasing the PECsed/PNECsed ratio by a factor of 10 . In this study we gathered information for data on sediment organisms as well as aquatic data and adsorption values from different sources, i .e . EU-RAR, BPD and REACh . From aquatic data and the distribution coefficient, sediment effect values were modeled using the EPM . Hereby we preferred the suspended matter-water partition coefficient Kpsusp as the log Kow is only an indirect descriptor for the adsorption on sediments . The comparison of the two values revealed that the EPM will not necessarily underestimate the risk, even in cases when no additional factor is used . In most cases NOECsed und NOECsed-EPM are within the general variability of ecotoxicity test results and below the variability between different species . This is also true for surface active compounds with a high adsorption potential . The results clearly demonstrate that for the risk by oral ingestion, the adsorption potential on sediment, the uptake efficiency in the GI tract has to be taken into account as well . For hydrophobic substances such as PCBs the log Kow > 5 is assumed to be sufficient as trigger, due to the fact that adsorption and uptake efficiency are both correlated to the log Kow . This log Kow > 5 is equivalent to a Kpsusp of > 1410 L/kg . For highly adsorptive, especially ionisable substances, however, a decision based exclusively on the adsorption is questionable .

MO 472Implications of differences in higher tier studies for deriving threshold concentrations

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S . Duquesne, S . Matezki, J . WogramFederal Environment Agency, UBA, Dessau, GermanyWithin prospective risk assessment of PPPs, uncertainties are associated to a number of extrapolations needed from test-systems towards the ecosystem . These uncertainties are considered by applying assessment factors to derive a threshold concentration, below which unacceptable effects on natural communities are avoided . The respective procedures are standardized for a lower tier risk assessment, but they are not at the higher tier level because tools and techniques are diverse and substance-specific information need to be included . This can thus lead to differences in terms of endpoints delivered and data interpretation . To identify magnitude and reasons for variability in endpoints and data interpretation, we review datasets from higher tier aquatic studies to assess if their analyses and interpretation in terms of uncertainties and extrapolations are consistent across the type of study (ex: SSD, micro- mesocosms) and could thus lead to similar acceptable threshold concentration in the field . This analysis will account for the variability between different classes of pesticides (insecticides, herbicides, fungicides), with various modes of action (ex: pyrethroids, neonicotinoids) . It will be based on existing datasets and information from the scientific literature and regulatory world . The analysis aims at contributing to the further development of the EU Guidance Document of Aquatic Ecotoxicology .

MO 473The adoption or adaptation of EU predicted No Effect concentrations for use as Canadian water quality guidelinesD .H . Leverett1, S . Roe2, T . Fletcher2, C . Crane2

1Wca-environment, Faringdon, United Kingdom2Environment Canada, Gatineau, CanadaThe Canadian Council of Ministers of the Environment (CCME) is a major intergovernmental forum in Canada for discussion and joint action on environmental issues of national concern . The 14 member governments work as partners in developing nationally consistent environmental standards and practices . The CCME Water Quality Task Group (WQTG) develops Canadian Water Quality Guidelines (CWQGs), which are derived according to nationally approved scientific protocols that describe data requirements and evaluation procedures . CCME WQTG is considering adopting or adapting Predicted No-Effect Concentrations (PNECs) and Environmental Quality Standards (EQS) for freshwater and marine aquatic life that have been developed in the EU for use as CWQGs as a means of: - benefiting from work conducted in the EU and avoiding duplication of effort, - rapidly augmenting the existing suite of CWQGs; and, - harmonizing CWQGs with international environmental quality benchmarks . Work was undertaken to develop procedures and guidance for the evaluation of EU PNECs/EQS values as potential candidates for adoption/ adaption as CWQGs, and specifically included: i) identification of a candidate list of substances for which EU Risk Assessments under the Exiting Substance Regulations and REACH or Water Framework Directive chronic aquatic PNECs or EQS had been derived and for which the detail of the derivation had been reported and was readily available, ii) evaluation of the test species used to derive the EU PNEC/EQS and a comparison with the species used to derive CWQGs, including the development of criteria for assessing the suitability of non-Canadian test species as potential surrogates for native Canadian species, iii) evaluation of the criteria used to assess the quality and reliability of study data used to derive EU PNEC/EQS, and a comparison with the criteria applied to study data used to derive CWQGs, iv) development of a procedure and guidance for evaluating EU PNECs as candidates for adoption as CWQGs . This presentation will detail the outcomes of the work and describe the guidance and recommendations to CCME WQTG with respect to the evaluation of EU PNEC/EQS for their potential to be adopted as CWQGs . This work was funded by, and conducted under the auspices of, the CCME WQTG, however, this presentation and the draft guidance report do not necessarily reflect the views of the CCME Water Quality Task Group .

MO 474Information systems on environmental quality standards (EQS) and quality criteria (QC)D .S . Schudoma1, C .H . Heiβ1, R .K . Kase2, J .M . Janssen3

1Federal Environment Agency (UBA), Berlin, Germany2Swiss Centre for Applied Ecotoxicology, Dübendorf, Switzerland3Expert Centre for Substances , RIVM, Bilthoven, NederlandEnvironmental Quality Standards for priority substances and national EQS need to be updated on a regular basis . Differences in data, methods and expert judgment have been a major source for a large variety of national EQS-values . Differences in data might be minimized by a targeted data exchange and evaluation . But this needs a targeted initiative by national authorities . For pragmatic reasons, one first step in evaluation of existing EQS or the prioritization of monitoring data is the IT-search of existing EQS and studies published by other national authorities . Existing web pages from RIVM, risicos van stoffen (http://www .rivm .nl/rvs/), INERIS Chemical Substance Portal (http://chimie .ineris .fr/en/index .php , Swiss Centre for Applied Ecotoxicology, Qualitätskriterien (http://www .oekotoxzentrum .ch/qualitaetskriterien) or the UBA ETOX database (http://webetox .uba .de/webETOX ), and others have many similarities, but there are also a lot of differences in handling and content . While OECD has harmonized templates for reporting chemical test summaries (OHT), similar formats does not exist for QC or QS to make data exchange and comparison easier . Quality Criteria (QC) could be derived under different regulations, for different protected assets, matrixes, monitoring aspects, etc . therefore the values are better to analyse, if this background information is easily available . EQS set under (2008/105/EC) or national EQS normally don’t provide this essential background information, which is necessary for a comparison, how the value is derived . A Comparison of EQS of Catchment Basin Specific Pollutants (CBSP) set for the demands of WFD (Arle 2011) showed an extreme difference between EQS for the same substance in a lot of cases . In future differences of EQS for CBSP might be minimized, because the derivation should follow TG-EQS (EC 2010) . But reasons for the discrepancy in values can only be explained, if background documents exist . The aim of this study is to identify the necessary information which is needed for a better comparability of QS and QC . Data bases are run by national authorities for their needs and users, the poster will give an overview of existing systems, and will make a proposal, which basic information’s are needed to make values of QC (non-legal binding) and QS (legal binding) derived by different institutions better comparable . A first outline for minimum criteria for reporting and harmonization of exchange formats will be offered .

MO 475Variability in Environmental Quality Standards - how much is there and what are the causes?P . Whitehouse1, M . Junghans2, S . Von Arb2, I . Werner2

1Environment Agency, Wallingford, United Kingdom2Swiss Centre for Applied Ecotoxicology, Duebendorf, SwitzerlandEnvironmental Quality Standards (EQSs) define an acceptable concentration of chemical contaminants in waterbodies . Under the Water Framework Direcive, EQSs for the most hazardous substances (Priority and Priority Hazardous Substances) are developed at EU level and Member States (MS) must put in place steps to ensure they are not breached . In addition, MS must identify Specific Pollutants that are discharged in significant quantities and set their own national EQSs for these substances . Many of the Specific Pollutants are common to several MS . Given similar data and protection goals, we would expect different MS to reach similar conclusions about the EQS for the same substance . However, the resulting threshold can vary between MS . In cases where different MS share a river basin, differences in the EQS for the same substance could result in compliance with the EQS in one part of the catchment but failure in another, even if the level of environmental exposure has not changed . This could lead to bias in chemical regulation . Clearly, derivation of EQSs should be harmonised as much as possible . The completion of detailed Technical Guidance for deriving EQSs in 2011 should help promote consistency in the way EQSs are derived . However, programmes of EQS development for Specific Pollutants are already well advanced in several MS . It is therefore timely to assess the degree of variability between EQSs, the causes of that variability, and to understand whether the new EQS Technical Guidance will help reduce between-MS variability for new EQSs, or revisions of existing EQSs . Our poster compares EQSs developed independently for the same substances . It describes the variability in EQS, and an analysis of the major reasons for variability, focussing on: - Underlying assumptions such as the form in which the EQS is expressed (e .g . dissolved vs bioavailable concentrations of metals, free acid vs neutral organic acids) - Comprehensiveness of the ecotoxicity data on which the EQS is based - Quality assessment of ‘critical’ data, including treatment of ‘outliers’ - Models used to undertake extrapolation (e .g . deterministic vs probabilistic methods) - Allowance for uncertainty (size of assessment factors) We conclude by offering a view on the extent to which the new EQS Technical Guidance will help reduce variability in future, and whether any particular aspects need to be emphasised to promote consistency .

MO 476Aquatic effect assessment for plant protection products. I. A Dutch proposal addressing the requirements of the Plant Protection Product Regulation and the Water Framework Directive C .E . Smit1, T .C .M . Brock2, G .H .P . Arts3, T .E .M . ten Hulscher4, F .M .W . de Jong1, R . Luttik1, E .W .M . Roex5, P .J .M . van Vliet6

1RIVM, Bilthoven, Nederland2Alterra, Wageningen, Nederland3Alterra WUR, Wageningen, Nederland4RWS-Waterdienst, Lelystad, Nederland5Deltares, Utrecht, Nederland6Ctgb, Wageningen, NederlandChemical monitoring programs revealed that in a large number of surface waters in the Netherlands, measured concentrations of certain plant protection products (PPP) exceed Dutch quality standards (QS) for water . One reason could be that there have been flaws in the registration procedure or in the derivation of the QS . Another cause

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might be that different effect assessment methods are used for registration or standard setting . The responsible ministries decided to update the aquatic effect assessment procedures for plant protection products (PPPs) in the Netherlands, taking into account the requirements laid down in European legislation with respect to PPP authorisation (Regulation 1107/2009/EC) and the derivation of QS under the Water Framework Directive (2000/60/EC) . In this project, decision trees have been developed for the aquatic risk assessment that can be used in the pre-registration authorisation and post-registration monitoring and evaluation . The core of the approach is that risk assessments are performed at two places in the water system, viz . (1) in edge-of-field surface water and (2) further downstream in surface water that has officially been assigned as WFD water bodies . In smaller edge-of-field surface waters (e .g . drainage ditches) the pre-registration criteria of the PPP Regulation apply, whilst in the larger WFD water bodies, the standards derived according to the WFD methodology apply . Post-registration verification of the exposure concentrations in the WFD water bodies against WFD water quality standards will take place using measurements . If results of chemical monitoring programs indicate exceeding of QS for a specific compound which can be attributed to the current “GAP”(good agricultural practice), this may have consequences for its authorisation (post-registration risk assessment procedure) and/or adequate mitigation measures have to be implemented . In this poster, we summarise the methodology for the edge-of-field assessment and the derivation of QS, taking the information in a regular pesticide dossier as a basis . This includes guidance on first tier and higher tier risk assessment, linking of exposure and effects and the incorporation of information on the specific mode of action of PPP in the methodology of QS derivation . In a second poster we present the results of case studies used for the verification of the proposed methodology .

MO 477Aquatic effect assessment for plant protection products II. Case studies for the verification of a new procedure in the Netherlands C .E . Smit1, T .C .M . Brock2, G .H .P . Arts3, T .E .M . ten Hulscher4, R . Luttik1, P .J .M . van Vliet5

1RIVM, Bilthoven, Nederland2Alterra, Wageningen, Nederland3Alterra WUR, Wageningen, Nederland4RWS-Waterdienst, Lelystad, Nederland5Ctgb, Wageningen, NederlandA proposal has been made to update the aquatic effect assessment procedures for plant protection products (PPPs) in the Netherlands, taking into account the requirements laid down in European legislation with respect to PPP authorisation (Regulation 1107/2009/EC) and the derivation of QS under the Water Framework Directive (2000/60/EC) . The core of the approach is that risk assessments are performed at two sites in the water system, viz . (1) in edge-of-field surface water and (2) further downstream in surface water that has officially been assigned as WFD water bodies . In our first poster1, we summarise the methodology for the edge-of-field assessment and the derivation of QS, taking the information in a regular pesticide dossier as a basis . In this second poster, we present the results of case studies that we performed to verify the proposed procedures . For different types of PPP (two insecticides, a herbicide and a fungicide), we performed first and higher tier risk assessments for the edge-of-field on the basis of dossier data and estimated concentrations in water from model calculations for drainage ditches . In addition, we derived QS according to the methodology of the WFD and compared those with measured concentrations on WFD-monitoring sites . On the basis of the results, recommendations for refining the proposed procedures are made . 1: Aquatic effect assessment for plant protection products . I . A Dutch proposal addressing the requirements of the Plant Protection Product Regulation and the Water Framework Directive

MOPC1 - Soil ecotoxicology

MOPC1-1Linking environmental availability and bioavailability of metals to snails: holistic approaches using single chemical extractions and DGT applied to field contaminated soilsF . Gimbert1, V . Sappin-Didier2, B . Pauget1, R . Scheifler1, A . de Vaufleury1

1University of Franche-Comté, Besançon, France2INRA, UMR TCEM, Bordeaux, FranceRisks of trace metals in soils are currently evaluated on the basis of total concentration although it appears often inappropriate for bioaccumulation or toxicity assessments . Chemical extractions and measurements of metal in the soil solution can provide alternative approaches . As dynamic chemical interactions also matter for bioavailability assessment, the DGT (Diffusive Gradient in Thin films) was proposed as a promising tool in this purpose . Although it has been successfully used to predict metal concentration in plants, its application to soil fauna is still scarce . The objective of the present research was to determine the ability of various chemical methods (conventional extractions and DGT) to predict Cd, Pb and Zn bioavailability to terrestrial snails, focusing on the influence of soil properties and the comparison of flux or concentration to predict bioavailability . Subadult Cantareus aspersus snails were therefore exposed for 28 days to fifteen field contaminated soils . We conducted a toxicokinetic experiment allowing to estimate metal bioavailability by mean of assimilation fluxes obtained from one-compartment modelling . Environmental availability was assessed using seven single extraction methods and the deployment of DGT devices . The latter allows to calculate both concentrations and fluxes in the resin that we compared to internal concentrations reached at the end of exposure and assimilation fluxes in snails . For non excreted metals such as Cd, concentrations in snails and DGT were quite well correlated and the addition of soil parameters in regressions did not improve the fit, testifying to the ability of the DGT to properly predict Cd transfer . Considering Cd fluxes, accounting for the influence of CEC allowed to improve the prediction ability of DGT (r²=0 .59) . For Zn, the consideration of soil pH and Alox allowed to better predict bioavailability to snails (r²=0 .80) . For Pb, the addition of soil characteristics in regression allowed to greatly improve the prediction (r² up to 0 .72) although they were not significant . To be a good predictor of bioavailability, a chemical method has to implicitely integrate the physico-chemical and physiolgical processes underlying bioavailability . In this sense, DGT did not bring a clear added value compared to conventional extraction methods, but, by considering fluxes and modulating effets of soil characteristics, allows to properly predict Cd, Pb and Zn bioavailability to C . aspersus snails .

MOPC1-2Impact of biosolids on soil fauna over a period of several yearsA . Coors1, J . Römbke1, T . Moser1, R . Schmelz2, E . Topp3, D .L . Lapen3

1ECT Oekotoxikologie GmbH, Floersheim, Germany2University of A Coruna, Coruna, Spain3Agriculture and Agri-Food Canada, London, CanadaBiosolids derived from municipal sewage treatment plants are applied to agricultural land throughout the world with regulations differing regarding amount and frequency of application as well as regarding limits set to contaminants contained in the applied biosolids and the receiving soil . In the present study, the fate of pharmaceuticals and other substances contained in biosolids was studied after farm-scale application of biosolids from Ottawa city to agricultural land . Together with the fate of conatminants, we followed structural and functional endpoints of soil fauna over a period of several years . Abundance and diversity of enchytraeids and nematodes were selected as structural parameters, while the utilization of carbon by the soil organism community served as a functional parameter and was investigated by means of bait lamina tests . The results of the present study clearly demonstrated an enrichment effect of the application of dewatered biosolids . This effect vanished after two years with regard to structural parameters, but not with regard to functional parameters . No apparent impact on pollutant-sensitive nematode species such as predators and omnivores was detected during the study period . On-going research will verify if long-term effects of biosolids-associated contaminants can also be excluded for the studied parameters and for another relevant organism group, earthworms .

MOPC1-3Microbial degradation of organic compounds (natural, xenobiotics, and pesticides) and the formation of soil organic matter or biogenic non-extractable residuesM . Kästner, A . MiltnerUFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyCarbon from any biodegradable organic compound in soil partitions into parent compound, metabolites, non-extractable residues (NER), CO2, and microbial biomass . This distribution must be known to assess the fate of the compound in soil . For example, NER from pesticides are considered to consist of adsorbed and sequestered parent compounds or metabolites and thus as hazardous residues . However, they may also partly derive from bacterial biomass, resulting in harmless biogenic residues . In addition, the formation of soil organic matter (SOM) or humic compounds has long been a dominating topic in soil science because the amount and composition of SOM determines soil quality but the processes are still not yet really understood . Microbial biomass residues could be identified as a significant source for SOM . The results indicate that a high percentage of the biomass-derived carbon remains in soil, mainly in the non-living part of SOM after extended incubation . The results provide a simple explanation for the development of the small, nano-scale patchy organic materials observed in soil electron micrographs . These conclusions were confirmed by studies on the biodegradation of isotope labeled pesticides in soil which quantified the contribution of microbial residues to the NER in soil . The amount of label found in biomolecules indicated that virtually all of the NER of the compounds are derived from microbial biomass .

MOPC1-4Trophic transfer of gold nanoparticles in a soil food chainY .J . An, S .W . KimKonkuk University, Seoul, South-Korea (Rep)Nanoparticles can be transferred from low trophic level organisms to high trophic level organisms . The present study investigated adverse effects caused by trophic transfer of gold nanoparticles . The low and high tropic level organisms of this study were Escherichia coli and soil nematode Caenorhabditis elegans . E. coli was initially exposed to gold nanoparticles, and C. elegans were fed by the exposed cells . The results showed that gold nanoparticles can transfer from E. coli to C. elegans via food exposure . We observed that the reproduction of C. elegans was decreased with increasing concentration of gold nanoparticles exposed to E. coli . This study reports the first observation of reproduction inhibition of C. elegans by dietary uptake of gold nanoparticles . This work was supported by the National Research Foundation Grant funded by the Korean Government (NRF 2011- 0015985).

MOPC1-5DNA barcoding tackles the identity crisis in earthworm populations used in ecotoxicology: The case study of South AfricaP . Voua Otomo, M .S . Maboeta, C .C . Bezuidenhout

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North-West University, Potchefstroom, South AfricaDNA barcoding was used to investigate the phylogenetic delimitations of the major earthworm populations used in ecotoxicological research in South Africa . A total of four focal populations (used in published works) and two non-focal populations were assessed . Three of the focal populations (two laboratory based and one field) are referred to as Eisenia fetida in the literature . The last focal population (a field based) has been occasionally used as a source of Dendrodrilus rubidus specimens . Phylogenetic analyses of the COI gene revealed that all the Eisenia populations had been misidentified as all the generated sequences grouped unequivocally with published sequences of E . andrei . Moreover, very high sequence divergence (> 25% K2P) found within E . andrei could signal the occurrence of hitherto non-described cryptic species . The last focal population was found to be a mix colony harbouring D . rubidus, Microscolex dubius and at least two other unidentified species . These findings are discussed with an emphasis on their inevitable implications on the current use of earthworms in ecotoxicological bioassays . Left unaddressed, this situation could globally become a considerable setback to ecotoxicological testing as the present study might not be an isolated case .

MOPC1-6What is the most relevant earthworm species for testing pesticide effects? A meta-analysisC . Pelosi, S . Joimel, D . MakowskiINRA, Versailles cedex, FranceHuman land use, through intensification of agricultural practices and particularly soil pollution by chemical inputs, has often resulted in loss of biodiversity . Earthworms represent the essential of living biomass in soils . They perform important agro-ecological functions since they influence organic matter dynamics and soil structure . They are considered as indicators of soil biodiversity and quality . Pesticide impacts on earthworms are difficult to measure under field conditions due to confounding effects . Ecotoxicological risks are assessed mainly under laboratory conditions before marketing authorization . The species Eisenia fetida, which is not present in cultivated fields, is used as biological models for these tests . It is also often used in laboratory studies thanks to its short generation time and to its easy culturing . However, there is a controversy about the sensitivity of this species towards pesticides, when compared to other species naturally present in agrosystems where chemicals are used . Using a meta-analysis, we address the following questions: Is E . fetida less sensitive to pesticides than other species? Are earthworms belonging to different genus and ecological groups differently influenced by pesticides due to their different behavior? What is the relevance of using only mortality as indicator in such studies and tests? Results will allow us giving recommendations for testing effect of pesticides on earthworms in a relevant way . We will also pinpoint the gaps we have to fill in order to understand how pesticides influence earthworm population dynamics in agricultural fields .

MOPC1-7Soil threats in the NetherlandsM .J .D . Hack1, J .J .H . Van den Akker2, T . Hoogland1, F . de Vries1

1Alterra, Wageningen, Nederland2WUR, Alterra, Wageningen, NederlandConsidering the EU Soil Thematic Strategy and the proposal for a framework Directive the Dutch government wanted to know what this could mean for the Netherlands in terms of policy and measures or in terms of impact on for instance agriculture and environment . The proposal for the Framework Directive to protect soil mentions the following soil threats: soil sealing, erosion, organic matter decline, salinisation, compaction, landslides and contamination . Focusing on the soil threats for which risk areas (also called priority areas) should be identified we were asked to try and locate where such areas would possibly be located within the country . For some threats we adopted methods from the ENVASSO-project and for other threats we used traditional land evaluation methods . Our research resulted in maps showing potential risk areas in the Netherlands (Hack-ten Broeke et al ., 2009) . These maps do not indicate where risks are considered important enough to call for policy measures .

MOPC1-8Monitoring of the state and development of soil parameters in the Czech RepublicŠ . PolákováCentral Institute for Supervising and Testing in Agriculture (ÚKZÚZ), Brno, Czech RepublicThe quality of agricultural soils in the Czech Republic is monitored by the Central Institute for Supervising and Testing in Agriculture (ÚKZÚZ) via three programmes . These are “Monitoring of the agricultural soils”, “Monitoring of inputs into the soil”and an adminitration of the database “Registry of contaminated sites” . Monitoring of agricultural soils is focused on characterization of the status and the changes of main soil parameters (physical, agrochemical, chemical) at a stable monitoring network . Protection of food chain and preservation of the soil quality is the main reason for monitoring of materials entering the soil . Within the frame of monitoring of inputs into the soils aims at the quality of sewage sludge, lake sediment and fertilizers . The Registry of contaminated sites give information on level of risk elements soil contamination in the Czech Republic . These programmes and their data compilation enable to obtain information concerning soil contamination effect on agricultural plants, quality of soil after sewage sludge application, etc . Data can be used by other research institutes or universities

MOPC2 - Endocrine disrupting chemicals: recent developments

MOPC2-1Combined effects of estrogen receptor antagonists on in vitro vitellogenesisK .E . Tollefsen1, K . Hylland2, K . Petersen1

1NIVA, Oslo, Norway2University of Oslo, Oslo, NorwaySome environmental compounds are known to have anti-estrogenic activity and their modes of action (MoA) are believed to include competitive inhibition of 17β-estradiol (E2) binding to the estrogen receptor (ER) or interference with ER-dependent processes through different pathways including increased metabolism of E2 and receptor crosstalk . Concentrations of single substances in the environment are generally low and effects rarely observed . Presence of multiple compounds having the same MoA may cause concern, however, as their effects may be additive . Additive effects of mixtures can be modelled by the two prediction models; concentration addition (CA) and independent action (IA) . The objective of the present study was to determine if the combined effects of mixtures of ER antagonists on the ER-mediated production of the estrogenic biomarker vitellogenin (Vtg) in rainbow trout (Oncorhynchus mykiss) hepatocytes could be predicted by the CA and IA prediction models . Model anti-estrogens (4-hydroxy tamoxifen and ZM 189 .154) and environmentally relevant compounds (PCBs and PAHs) were tested to ensure inclusion of compounds from different chemical classes with more or less well known mechanisms of anti-estrogenicity . All eleven tested compounds had the ability to inhibit E2-induced production of Vtg . The potency of the tested compounds differed by four orders of magnitude based on the concentrations for 50% inhibition (IC50) . The observed order of potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin > 4-hydroxy tamoxifen > 3,3’,4,4’,5-Pentachlorobiphenyl > benzo(k)fluoranthene > 3,3´,4,4´-tetrachlorobiphenyl > С-naphthoflavone > ZM 189 .154 > indeno[1,2,3-cd]pyrene > benzo(b)fluoranthene > benzo(a)pyrene > benzo(a)anthracene . All tested mixtures inhibited E2-induced production of Vtg in a concentration-dependent manner . The mixtures consisting of compounds with similar MoA were well predicted by the models . The mixture consisting of compounds with different MoAs caused deviation from the prediction models consistent with more than additive effects . The results from this study show that CA and IA prediction models can be used to assess combined effects of mixtures of estrogen antagonists with similar MoA . The rationale for the deviations observed with the mixture of compounds with different MoAs warrants additional studies to determine the MoA of the interactions occurring and to assess potential impacts on the health of organisms . MOPC2-2Dependence between models and tests in the assessment of the endocrine disruption potential of chemical substancesT . Aldenberg1, M . Nendza2, A . Wenzel3

1RIVM, Bilthoven, Nederland2Analytisches Laboratorium, Luhnstedt, Germany3Fraunhofer IME, Schmallenberg, Germany

MOPC2-3Testing thyroid disruption: which data are available in different regulations to support an initial suspicion for the environment?T .J . Juffernholz, A .K . Klein, C .N . NeubertUmweltbundesamt (UBA), Dessau-roβlau, GermanyIdentification, assessment and regulation of substances having an impact on the endocrine system of ecotoxicological relevant organisms are of special interest in several legislations in the EU (Chemicals: REACH (EC) No . 1907/2006, Plant protection products: 1107/2009 EEC, Pharmaceuticals: 2001/83/EG (2009) and 2001/82/EG (2009), Biocides: 2009/0076 ) . Especially, since for this group a hazard-based approach is proposed by all of the legislations . However, identifying potential endocrine disruptors (EDs) might be pretty difficult as special tests systems for EDs are not well established in ecotoxicology and still under development . Therefore, the currently requested test systems are not prepared to identify endocrine disrupting properties alone and correlate them to adverse effects . During the last years research effort was mainly focused on sexual endocrine-disrupting chemicals, inter alia identifying effects on fish . Nevertheless, effects on the thyroid system were also investigated but are not part of standard procedures in ecotoxicology, yet . Thyroid hormones play an essential role regarding development processes in mammals, birds, amphibians or fish . Therefore, influences on the thyroid system might result in adverse effects comparable to effects on the sexual endocrine system given that an initial suspicion for effects on the thyroid system can be derived from standard toxicity test data, more advanced and specialized test systems could be requested (special concern has to be proved in most regulations) . But when is a substance expected to have an effect on the thyroid system? And based on which data? And what to do if ecotoxicological studies are lacking completely? Do available non-clinical toxicological data and results of in-vitro test help to decide whether a compound affects the thyroid system? This evaluation aimed at summarizing existing data and investigating the suitability of these data to identify substances which affect the thyroid system . Exemplified data sets of different regulations were compared . Studies and endpoints were investigated and (if available) compared with results of tests on the thyroid system . Additionally,

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toxicological data were screened to look for results which indicate an initial suspicion .

MOPC2-4Non-aromatizable androgen exposure to mosquitofish (Gambusia spp): links between molecular, physiological and reproductive system changesK . Anderson1, Y . Ogino2, D .S . Barber1, I . Taisen2

1University of Florida, Gainesville, United States of America2Okazaki Institute for Integrative Bioscience, Okazaki, JapanMosquitofish (Gambusia spp) have the potential to become a bioindicator for EDCs . Male mosquitofish have an extension of the 3rd-5th anal fin rays, and this growth is inducible in females and fry by androgen exposure . Issues with using mosquitofish as a bioindicator include the lack of knowledge of the mechanism of anal fin elongation . Previous studies indicated three genes (sonic hedgehog, muscle segment homeobox C, and fibroblast growth factor 1) as upregulated in juvenile mosquitofish or swordtail anal fins by androgen treatment . The relationship between anal fin growth and reproductive effects also needs to be understood . To address these issues, the objective of this project is to evaluate the effects of androgen exposure on mosquitofish . Two specific aims were developed: 1) Determine the expression patterns of Shh, MsxC, and Fgfr1 in the anal fin during androgen exposure, and 2) Evaluate the relationship between anal fin growth and the expression of the egg yolk precursor protein vitellogenin (vtg) . Female mosquitofish were exposed to a vehicle control or one of three doses of the androgen 17β-trenbolone (TB) for 3, 7, 14, or 21 days . Body morphometrics were assessed and livers and anal fins were removed for quantitative polymerase chain reaction (qPCR) . In situ hybridization (ISH) was used to evaluate Shh gene expression in the anal fin at days 7, 14, and 21 . Results demonstrate that 10 ug/L TB can induce anal fin growth at early time points while 1 ug/L TB is significant from the controls at later time points (one-way ANOVA, p<0 .05) . This may be due to the ability of a higher dose of androgen to stop elongation and promote the formation of advanced structures (hooks and serrae) . MsxC was increased in all TB doses at day 7 and Fgfr1 was increased at days 3 and 7 by 10 ug/L TB (one-way ANOVA, p<0 .05) . Shh was strongly expressed in the distal tip of the anal fin at days 7 and 14 . Mosquitofish exposed to a 10 µg/L TB had reduced hepatic vtg expression (one-way ANOVA, p<0 .05) . Future experiments will determining how TB dose affects anal fin elongation versus differentiation, evaluating MsxC and Fgfr1 expression at additional early time points, and developing an assay to measure fry output during TB exposure to compare these data with vtg gene expression levels . Knowledge gained from these and future experiments will be a starting point for future research towards establishing relationships between biomarkers of androgen exposure and reproductive health .

MOPC2-5Assessment of Nonylphenol for the environment: do the regulatory conclusions differ if we look at endocrine disruption or ‘classical’ endpoints? T .V . Juffernholz, F .K . KaβnerGerman Federal Environment Agency (UBA), Dessau-roβlau, GermanyNonylphenol is one of the gobal high production volume chemicals and the debate of the risk posed by it -especially for surface waters - is a global on too . Levels of nonylphenol in surface waters exceeding the environmental quality standards (EQS) have been found in several waters in the EU although a restriction on the uses of nonylphenol and its ethoxylates under the limitations directive 76/769/EEC (now in Reach Annex XVII, entry 46) have been in force since January 2005 . Moreover, its endocrine disruption properties were assessed in the EU by different organizations . An in depth analysis of its endocrine disrupting properties was performed in 2002 (European Commission, 2002) . It substantiated the endocrine disrupting properties of nonylphenol with respect to aquatic organisms and concluded that it may present a risk to aquatic organisms due to adverse effects that may be endocrine mediated . Nevertheless, there is an ongoing discussion in the scientific and regulatory communities about the severity of the endocrine disrupting properties in comparison with the systemic ecotoxicity of nonylphenol . For the EU risk assessment (RAR 2002) no endocrine disrupting properties were taken into account . The PNECwater was derived by using the algae biomass NOEC of 0 .33µg/L . Even if we would not consider endocrine disrupting properties in our assessment under REACH algal biomass is no valid endpoint (see ECHA Guidance document: R7b R7 .8 .5 .3) and therefore PNEC derivation would have to change . A different lowest NOEC would have to be used: Mysidopsis bahia NOEC 3,9 µg/L . A consideration of the endocrine disrupting properties could lead to NOEC values as low as 1µg/L . For example with the following species:Crassostrea gigas NOECsperm motility <1µg/L sowie Arbacia lixula NOECdev anormalities < 0 .937 µg/L This presentation aims at comparing relevant systemic toxicity endpoint with endocrine mediated endpoints and pointing out the regulatory important conclusions .

MOPC2-6Detection of endocrine effects: changes in the steroid synthesis and reproduction toxicityJ . Kuckelkorn, H . Hollert, S . Maletz, T .B . SeilerRWTH Aachen University, Aachen, GermanyThe joint project “Tox-Box - Risk assessment of anthropogenic micropollutants to assure the drinking water supply”aims for a harmonized, hierarchic test strategy to assess the toxicity of drinking water . As one part of this project the Institute for Environmental Research at RWTH Aachen University will analyze and establish endocrine activity as an important toxicological endpoint within the risk assessment of drinking water by means of the GOW-concept developed by the German Federal Environment Agency (UBA) . The first step will be to screen drinking water samples and optimize biotest protocols . The samples will be provided by Rheinenergie AG as a water supplier and the Helmholtz Centre for Environmental Research (UFZ), and they will be subjected to a set of different biotests . The ER/AR-Calux®-Test (Estrogen-/Androgen-Responsive Chemical-Activated Luciferase gene eXpression) will detect receptor-mediated endocrine activity in the T47DLuc human cell line and the H295r-Assay will identify alterations in the steroidogenesis of the human adrenocortical carcinoma cell line (H295r) . The third biotest will provide data on the reproduction toxicity of the water samples using the snail Potamopyrgus antipodarum . In the second step of this module endocrine active single substances will be used in the H295r-Assay to investigate the mode of action of the disruptors . The data will be used to assess sublethal/chronic toxicity and to transfer the mechanisms to risks for human health . The third step will include research on drinking water samples by effect-directed analysis (EDA) to identify endocrine active single substances which are currently not being measured . The results from the whole Tox-Box project will be used to establish a new guideline regarding the risk assessment of anthropogenic micropollutants in drinking water .

MOPC2-7The development of quantitative weighting factors for use in a weight of evidence framework for the evaluation of endocrine activityE .M . Mihaich1, C .J . Borgert2, L .S . Ortego3, K .S . Bentley4, C .M . Holmes5, S .L . Levine6, M .S . Marty7, B .H . Neal8, R .A . Becker9

1Environmental & Regulatory Resources, Durham, nc, United States of America2Applied Pharmacology & Toxicology Inc ., Gainesville, fl, United States of America3Bayer CropScience, Research triangle park, nc, United States of America4DuPont Crop Protection, Newark, de, United States of America5BASF Corporation, Research triangle park, nc, United States of America6Monsanto Company, St . louis, mo, United States of America7The Dow Chemical Company, Midland, mi, United States of America8Exponent, Alexandria, va, United States of America9American Chemistry Council, Washington, dc, United States of AmericaWe previously developed a conceptual framework for conducting weight of evidence (WoE) evaluations of endocrine screening and testing data (Borgert et al ., 2011) . The framework was designed to be broadly applicable, but with an emphasis on data emerging from the US EPA’s Endocrine Disruptor Screening Program (EDSP), and specifically tailored to accommodate endpoints measured in the eleven Tier 1 Endocrine Screening Battery (ESB) assays . The framework calls for a transparent process whereby specific hypotheses are formulated and data quality is evaluated with respect to overarching scientific principles, including primary validity of the measurements, reliability of data reporting, and probative capability of the study design to evaluate causation . The framework also calls for assigning quantitative weighting factors to assay endpoints (WREL) that reflect their relevance for deciding specific hypotheses . This is an important step in the WoE framework as it explicitly recognizes the fact that some assays are very specific and sensitive for evaluating certain hypotheses related to endocrine activity but may be irrelevant for evaluating others . For example, an increase in thyroid follicular hyperplasia as assessed in the amphibian metamorphosis assay would be expected to have a very high WREL for evaluating the hypothesis that a chemical has potential thyroid antagonist activity, but would have a very low WREL for evaluating the hypothesis that a substance has potential estrogen or androgen agonist or antagonist activity . The framework recommends that WREL values be derived as numerical weighting factors based on the predictive value of the endpoint for actual endocrine activity in vivo, which would typically be determined in chronic reproductive and developmental toxicity tests, i .e ., EDSP Tier 2 tests . This predictive value, however, may be difficult to evaluate until results for a test set of chemicals are available from the Tier 1 ESB and can be compared to results of Tier 2 tests . As an interim step, however, provisional WREL values can be derived based on a rank ordering of relevance for each hypothesis . This rank ordering should also be based on data, but will necessarily involve considerable scientific judgment . An example derivation of rank ordered WREL values for evaluating Tier 1 EDSP hypotheses is presented, with particular emphasis on endpoints measured in the fish screening and frog metamorphosis assays .

MOPC2-8Modeling mechanisms of nanotoxicity at multiple levels of biological organization through dynamic energy budgetsR .M . Nisbet1, S .H .A .N .N Hanna2, P .A .T .R .I Holden2, K . Tin2, H .U .N .T .E Lenihan2, E .D .W .A .R Mccauley2, R .O .B .E .R Miller2, E .R .I .K .B Muller1, J . Priester2, L .O .U .I .S Stevenson2

1University of California Santa Barbara, Santa barbara, United States of America2UCSB, Santa barbara, United States of AmericaEngineered nanomaterials (ENMs) entering the natural environment may have ecological effects by influencing the fluxes of energy and material that regulate the abundance, distribution, and dynamics of organisms . Empirical data alone cannot provide the understanding to guide policy or management action, so theory is essential . Dynamic Energy Budget (DEB) theory offers a conceptual framework for describing the interplay of physico-chemical processes in the environment with suborganismal and organismal processes in living organisms, and thereby linking to population, communities and ecosystems . This has been long recognized in ecotoxicology; indeed some of the earliest work on DEB theory was motivated by the recognition of the limited ecological insight that follows from standardized toxicity tests . DEB theory has the potential to model mechanistically the effects of ENMs on single cells, entire organisms and on populations . The sophistication of the theory that is required varies greatly among applications, and will be illustrated using case studies from the University of California Center for Environmental Implications of Nanotechnology . These include: (i) estimation of toxicity parameters for metal oxide effects on phytoplankton; (ii) detailed modeling of short-term studies of the physiological responses of a marine invertebrate exposed to metal oxide nanoparticles models, and projection of implications for lifetime reproductive output; (iii) modeling the impact of multiple stressors, based on the same data as (ii);

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(iv) taking account of sub-organismal information and environmental physico-chemical processes in modeling the influence of CdSe quantum dots on bacterial population dynamics; (v) models to predict the results of experiments on the effects of citrate coated silver nanoparticles on phytoplankton-zooplankton interactions . Our approach complements much recent work on adverse outcome pathways (AOPs) by extending the pathways to include ecological interactions without compromising the critical connections with suborganismal biology .

MOPC3 - Non-target analysis and identification of toxicologically significant emerging pollutants

MOPC3-1How to evaluate pollution, degradation and metabolisation of multi-families of pharmaceuticals and endocrine disruptors in multiple compartments of the surface water media?J . Camilleri1, M . Tournier1, A . Kiss1, L . Wiest1, R . Baudot1, A . Buleté1, E . Vulliet1, J . Garric2, C . Cren-Olivé1

1Institut des Sciences Analytiques, Villeurbanne, France2CEMAGREF - UR MALY, Lyon, FranceEvaluations like risks or effects assessments of the presence of various xenobiotics in the environment show a continuously growing interest since the past decades . To establish models for degradation of a family of pharmaceuticals and thus evaluate its impact on the environment, scientists need good analytical methods to reinforce databases . While literature focuses on single matrices or single family, this work aims to develop an integrative approach of the surface water media: water, sediments and biota . Those methods are combined to relatively simple and short time-consuming extraction techniques investigated for various matrices for the 20 various pharmaceuticals and 28 Endocrine Disruptor . NanoLC-MS/MS method has already been applied to a benthic model exposed for eco-toxicological study [1] . All presented LC-MS/MS, automated online-SPE-LC-MS/MS and nanoLC-MS/MS methods have been validated following ICH recommendations . They allow rapid quantification of multiple pharmaceuticals and endocrine disruptors at environmental levels and have been for the study of real ecosystems in French rivers . In parallel thanks to passive samplers calibrated in the laboratory and validated in situ with PRCs, real time weighted average concentration can be determined in surface water . Exposing the benthic model to those concentrations should allow not only the quantification of accumulated and metabolised compounds in biota but also degradation products and coefficient partitioning between water and sediments . Those studies even could correlate fate to effects observed on benthic models . Further investigations with TOF spectrometers should allow non-targeted analysis for identification of new by-products, degradation products or metabolites . Although this analytical challenge is needed for risk and effects assessments on environment and populations, it is really time-consuming for the analyst . Prioritisation of substances is crucial to select representative pollutants to help scientists building models . This project opens the discussion for assessments of multi-families of various ubiquitous pollutants with a relatively large panel of analysis for different matrices of the same water surface media . References [1] Cren-Olivé C, Bulete A, Baudot R, Wiest L, Gust M, Garric J . 2010 . Study of bioaccumulation and metabolization of fluoxetine in benthic invertebrates by Micro-QuEChERS-NanoLC-ESI-MS/MS . Presented during SETAC 21st Annual Meeting . 15-19 May 2011; Milan, IT

MOPC3-2Non-target analysis of emerging contaminants - harnessing antibody selectivityJ . Schneider, A . Bahlmann, J .J . Carvalho, A . LehmannBAM Bundesanstalt für Materialforschung und -prüfung, Berlin, GermanyThe coupling of immunoassays to liquid chromatography is described . Fractions of complex environmental samples obtained by RP-HPLC fractionation have been analyzed with immunoassays for carbamazepine and sulfamethoxazole . Two metabolites, one conjugate and two structurally not so closely related compounds have been identied by this method . Procedures and limitations are discussed in the presentation .

MOPC3-3Development of a water toxicity sensor based on genetically modified bacteriaM . Woutersen1, A . Brouwer2, J . Mink3, A . van der Gaag1, M . Heringa1

1KWR Watercycle Research Institute, Nieuwegein, Nederland2BioDetection Systems, Amsterdam, Nederland3M Sensors, Eindhoven, Nederland The detection and monitoring of pollutants in surface water is routinely done by chemical analysis, biomonitors using aquatic organisms, or in vitro toxicity assays . Chemical analysis is sensitive and can be fast, but only compounds searched for are detected . Biomonitors with algae, Daphnia and mussels are excellent for the detection of the total toxicity of for example heavy metals or pesticides . However, not all toxicants with relevant effects can be measured, for example genotoxicants or endocrine disruptors . Laboratory based in vitro assays on the other hand, do detect these effects, but take a long time to perform and can not be used on-line in the field . To be able to fill this gap, a toxicity sensor has been developed, which employs genetically modified Escherichia coli that emit luminescence when they are exposed to compounds with a certain effect . The effect that can be measured depends on the modification of the bacteria . The strain used in the presented sensor at the moment responds to DNA damage . The bacteria are immobilized in a sol-gel on a glass slide and an optical fiber which were continously exposed to a water flow . The goal is to develop a sensor that can be used on-line in the field with minimal maintenance . The first experiments with the sensor were performed with Dutch tap water, which does not contain added chlorine . The immobilization in sol-gel did not affect the response of the bacteria . The luminescence of the bacteria increased after exposure to nalidixic acid and mitomycin C . The response started approximately one hour after the start of the addition of the positive control . The concentration of added growth medium had a strong influence on the response and was thereby the most important parameter . A concentration of 2% Lysogeny broth (LB) was enough to sustain the signal without causing strong fluctuations . Both the background signal and the response of the bacteria declined with time . One possible reason for this might be competition with other microorganisms in the water, but this is still under investigation . As expected, the fiber yielded a signal that was 100-200 times lower than the signal of the plate . This was the result of the much smaller surface area of the fiber tip .

MOPC3-4Can solar irradiation transform human metabolites of pharmaceuticals back to their parent compound? Insights on the direct photolysis products and pathways of sulfamethoxazole human metabolitesF . Bonvin1, T . Kohn2, K .P . Mcneill3

1Environmental Chemistry Laboratory, Lausanne, Switzerland2Environmental Chemistry Laboratory, EPFL, Lausanne, Switzerland3Environmental Chemistry Laboratory, ETH Zurich, Zurich, SwitzerlandSulfamethoxazole (SMX) is a widely used antibiotic, only partially removed by wastewater treatment and therefore often detected in wastewater effluent and in surface waters . However, after human consumption, SMX is partially metabolized in the human body into various metabolites, some of which retain certain biological activity, and which have also been observed in the aquatic environment . Moreover the back transformation of certain metabolites to the parent compound has also been reported in certain environments . Both direct and indirect photodegradation processes have been reported to be important removal mechanisms of sulfamethoxazole in the environment; however no research has been conducted on the photolysis of SMX metabolites .

This study investigates the direct photolysis kinetics, quantum yields and products of a selection of SMX metabolites, namely: acetyl SMX, nitro-SMX, nitroso-SMX and hydroxy-SMX . Photo-experiments were conducted in a merry-go-round photoreactor equipped with 300 nm mercury lamps with variable concentrations (10-100uM) of SMX metabolites, and analysed via HPLC-UV vis and high-resolution mass spectrometry . Results revealed slower direct photolysis kinetics and quantum yields that were 10 to 100-fold smaller than those of the parent compound SMX . The photoproducts generated from direct photolysis of SMX and metabolites were identified . The structural similarity of SMX and acetyl-SMX photoproducts point towards the same photolysis cleavage sites . Moreover, the back-transformation of nitroso-SMX to the parent compound was observed . These results show that pharmaceutical metabolites can be more recalcitrant to photodegradation and even be a source of pharmaceuticals to the environment .

MOPC3-5Using mutagenic fingerprints for a compound identificationS . Buchinger, H . Pollmann, M . Klumb, G . ReifferscheidGerman Federal Institute of Hydrology, Koblenz, GermanyOne of the major challenges in the field of environmental chemistry and ecotoxicology is the identification of compounds or compound mixtures which are responsible for adverse biological effects . One major tool for the elucidation of cause-effect relationships is the effect directed analysis (EDA) which combines the fractionation of a complex sample with biological assays and a subsequent chemical analysis . One drawback of this highly successful approach is its complexity and time demand . A biological test strategy which directly provides information about the chemical nature of bioactive compounds or compound classes would complement the classical EDA because it facilitates a more target-oriented and thus efficient sample fractionation and/or chemical analysis . In the present study such a methodology is developed for the identification of mutagenic compound classes in environmental samples by the Ames-fluctuation assay . It is well known that many mutagenic compounds are bio-activated by the metabolism of xenobiotics . In recent years different bacterial strains were generated which overexpress one ore more enzymes which are involved in the metabolic toxification and/or detoxification of genotoxicants . If a potentially mutagenic compound is bioactive in a given test strain depends on the metabolic competence of the bacteria . Consequently, compound specific mutagenic fingerprints can be generated by making use of a set of genetically modified bacterial strains with different enzymatic equipments . In order to challenge the working hypothesis that such fingerprints allow the identification of compounds or at least compound classes, dose-response-relationships for model compounds were aggregated in a single value, the concentration dependent number of revertants (cdr) . The combination of all strain-dependent cdr-values for a model compound which represents a chemical class can be treated as a vector which describes the metabolic activability of a compound class . It is tested, if compound classes can be identified by a comparison of these vectors which is based on the calculation of the cosine similarity .

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MOPC3-6Innovative sample preparation steps for the analysis of pharmaceuticals in soil M .V . Salvia1, J . Camilleri2, E . Vulliet1, L . Wiest1, R . Baudot1, C . Cren-Olivé1

1Institut des Sciences Analytiques, Villeurbanne, France2SCA, Solaize, FranceChemical products are more and more used for agriculture and domestic activities and are responsible for the spread of several substances in the environment, which can be harmful for animals or humans . Among these products, hormonal steroids and pharmaceutical compounds are of growing concern . If several analytical methods are available to determine the content of these compounds in aquatic environment, few methods were described to allow their analysis in solid matrices . However, the few available data concerning the content of hormonal steroids in soil reveal contaminations which can reach hundreds of ng/kg . Consequently, the aim of this study was to develop an analytical method for the analysis of traces of 11 hormonal steroids, 16 veterinary or human drugs and 11 other well-known human contaminants, used in this study as pollution tag, in soil . An analytical method both selective and sensitive based on liquid chromatography-tandem mass spectrometry was developed . The optimization allowed the determination of the best conditions for the separation by chromatography and for the detection by mass spectrometry . The analysis of complex matrix such as soil needed a rigorous sample preparation to obtain a repeatable and enough sensitive analysis to achieve the detection limits required . For this purpose, an extraction step using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method was set up . This is a relatively new technique, simple, quick and cheap . The use of this method for environmental matrices such as soil is particularly innovative . This technique is based on a liquid-liquid extraction with an organic solvent in presence of salts and buffers . Several parameters were optimized (sample weight, solvent nature, water and solvent volumes, buffer type) . A further purification step was necessary to reduce the matrix effects, therefore the SAX (Strong Anion Exchange) cartridge was used in tandem with the Strata-X cartridge for the clean-up . This analytical procedure allows the determination of the target analytes in the lower ng/g range . After a validation based on the ICH/2005 norm, this analytical methodology was successfully applied to real samples of soil collected in several areas of France that were treated differently . Some of them were contaminated with manure spreading, others with sludge spreading and others with atmospheric fallouts . Results indicate that all the samples contained several contaminants at level lower than the ng/g .

MOPC3-7NORMAN MassBank - a community driven open access mass spectral database for the identification of organic environmental contaminantsT . Schulze1, E .L . Schymanski2, M . Krauss1, S . Neumann3, C . Hug1, C . Gallampois4, J . Slobodnik5, W . Brack1

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2Eawag - Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland3Leibniz Institute of Plant Biochemistry - IPB, Halle, Germany4Linköping University, Linköping, Sweden5Environmental Institute, Koš, Slovak RepublicThe nontarget screening and identification of unknown compounds such as industrial by-products and/or transformation products in complex environmental samples is of emerging concern . Many emerging pollutants are polar in nature and require the use of liquid chromatography coupled to soft ionisation mass spectrometry [1] . Improvements in mass spectrometry, such as accurate mass and high resolution mass analysers, for example time-of-flight (e .g ., ToF-MS, QToF-MS) and Orbitrap MS, has been demonstrated to be valuable tools for the identification of new compounds (e .g ., [2]) . However, a major drawback in the identification of unknowns is the lack of suitable accurate mass and MSn spectral databases as for GC-EI-MS spectra . Existing LC-MSn databases contain relatively few spectra, especially even less of environmental relevance . The web-based MassBank mass spectral database [3] (http://www .massbank .jp) allows storage and sophisticated search (i .e ., spectra, substructure and peak search) of any kind of high and low resolution mass spectra including EI-MS, ESI-QToF-MSMS and ESI-FT-MS . The main objective of our work is the establishment of a community driven MSn database using the MassBank server software for the NORMAN network (http://www .norman-network .net) to improve identification of unknowns in environmental samples and to support the priorisation of emerging compounds . A database containing such data is the starting point for the further development of spectral interpretation algorithms tailored to environmental data for the identification of specific substructures in MSn spectra to assist in candidate selection . Thus, contributions to a community mass spectra database within the NORMAN network (NORMAN MassBank) containing all types of LC-MSn data will help to address the lack of data and improve identification of environmental unknowns . Furthermore, it facilitates the possibility to check instrument comparability and to spend more time on unknowns identification rather than on building of home-made MSn libraries . The database is available at http://massbank .ufz .de/MassBank and at the NORMAN website . References: [1] M . Krauss, H . Singer, J . Hollender, Anal Bioana Chem 397 (2010) 943; [2] A .C . Hogenboom, J .A . van Leerdam, P . de Voogt, J Chroma A 1216 (2009) 510; [3] H . Horai, M . et al ., J Mass Spec 45 (2010) 703 .

MOPC4 - Development in life cycle inventory analysis and modelling

MOPC4-1Dissemination of LCI data in the context of multiple data platforms: case of the aluminium industryS . Jones1, P . Lesage2, K . Buxmann1, C .M . Bayliss1

1International Aluminium Institute, London, United Kingdom2CIRAIG, École Polytechnique de Montréal, Montréal, CanadaAs a leading industry association, with a wide global coverage, the International Aluminium Institute (IAI) has collected and published representative Life Cycle Inventory (LCI) data since 1998 . However, despite the work expended by the aluminium industry in collecting, analysing and reporting the LCI data, the Life Cycle Assessment (LCA) community as a whole has until now relied on a variety of data sources to conduct LCAs, some of which are outdated and/or reflect a limited part of the global industry . The LCI datasets that IAI publish are built from data collected from both its members and non-member companies, through annual energy and emissions surveys and five yearly life cycle data surveys . Traditionally, the resulting data were assembled in a publically available report . LCA practitioners were hence expected to lift the LCI datasets from the reports for use in their studies . Since the datasets were gate-to-gate, the practitioners were also expected to link the various inputs to compute cradle-to-gate life cycle inventories . Despite its effort to disseminate the data, the IAI in 2008 realized that many LCA practitioners were not using the most up-to-date data on aluminium production in their studies . It therefore sought a solution that would ensure that the data collected were relevant to the different major LCI databases being used, ensuring widespread use of their data, and also ensuring datasets were disseminated in a way that all databases could integrate the data with minimal effort . The approach the IAI settled on is to use the ecoinvent database and its new ecoSpold 2 data format as a launching pad for data dissemination . The approach developed as part of this project aims at facilitating the dissemination of LCI data to as wide an LCA practitioner base as possible and at making data updates, when the IAI acquires relevant data through its various member surveys, an easy and in-house process . The wide dissemination is ensured by the fact that (1) the chosen seed database (ecoinvent) is widely used and integrated in most LCA software and (2) the data can be translated into other existing formats through pre-existing data translation tools . The combination of training on the ecoEditor for version 3 software by IAI employees, the possibility to directly submit data to ecoinvent, and the capability to create updated datasets by changing only relevant flows will enable the IAI to create updated versions of the datasets as often as it sees fit .<br type=’_moz’ />

MOPC4-2BioEnergieDat: Life Cycle Inventories and IT infrastructure for Bioenergy in GermanyL . Schebek1, A . Ciroth2, C . Düpmeier1, L . Eltrop3, S . Simon4, P . Viebahn5, H .J . Wagner, H .-J .6, W .I .E .B .K Biemann1, T . Zschunke7

1Karlsruhe Institute for Technology (KIT), Eggenstein-leopoldshafen, Germany2GreenDeltaTC GmbH, Berlin, Germany3Universität Stuttgart, IER, Stuttgart, Germany4Deutsches Zentrum für Luft- und Raumfahrt e .V ., Stuttgart, Germany5Wuppertal Institut für Klima, Umwelt, Energie GmbH, Wuppertal, Germany6Ruhr-Universität Bochum, Lehrstuhl Energiesysteme und Energiewirtschaft, Bochum, Germany7Hochschule Zittau/Görlitz, Fachbereich Maschinenwesen, Zittau, GermanyLCA studies on bioenergy are based on diverse methodological choices and technology information, resulting in discrepancies in data sets of identical technologies or products . The assessment of technologies for decision support calls for further harmonization of methodological procedures, as well as of validation of input data and assumptions on specific processes . In the context of the German energy and climate policy, the need of an adequate database on bioenergy reflecting German framework conditions has been identified . A two year’s project for a database on energetic use of biomass for Germany has been launched named ‘BioEnergieDat’ . BioEnergieDat is aimed at delivering data sets for selected bioenergy technologies and energy carriers based on German framework conditions for today’s state of technology as well as for the projected state of 2020/2030 . Data sets shall be usable for different stakeholder needs in the field of bioenergy and for a broad range of applications of process-chain based modelling . To enable this, the common ground of work is the concept of a modular data supply: data sets will be generated and validated for unit modules of the process chain; based on the project’s methodological specifications, these unit modules may be combined in a flexible way within different process chains and applications . To provide the technical possibilities for this concept, a web-based Open source IT infrastructure is developed, including data storage for the unit modules and a calculation tool for LCA modelling and for conducting case studies . The presentation will outline projects outcomes which have been achieved so far: Concerning data sets 39 process chains for bioenergy were elaborated by combining biomass feedstocks and technologies presently encountered in Germany . The work on methodology is based on considering ‘perspectives’ in inventory modeling . The work on IT infrastructure includes data storage for the unit modules and a calculation tool for LCA modelling . Due to its modular approach and the related development of IT, BioEnergieDat implies a broader outreach: it shall support methodological development as to specific questions of bioenergy like substitution effects or land use, it shall support data quality procedures, and it shall provide novel IT solutions for efficient, reliable and fast exchange of data and information between different tools, methods and user environments, supporting not only national but also international networking .

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MOPC4-3Improving regional specific life cycle assessment of energy crops in GermanyC . Lingoth-Becerra1, H . Stichnothe1, M . Drösler2, S . Fiedler3, S . Glatzel3, U . Hagemann4, G . Heintze2, H . Kage5, K .H . Mühling5, A . Pacholski5, M . Pohl4, M . Rohwer5, A . Seidel5, A . Werner4, J . Augustin4

1Johann Heinrich von Thünen-Institut (vTI), Braunschweig, Germany2University of Applied Sciences Weihenstephan-Triesdorf, Freising, Germany3University of Rostock, Rostock, Germany4Leibniz-Centre for Agricultural Landscape Research (ZALF), Müncheberg, Germany5Christian-Albrechts- University of Kiel, Kiel, GermanyAmbitious goals have been set to reduce by 40% the German greenhouse gas (GHG) emissions by the year 2020 (compare to 1990 levels) . One of the strategies followed by Germany to achieve this target has been the development, use and promotion of renewable energies . Biogas production is a type of renewable energy that has been growing significantly in Germany over the past decades, being the country with the major biogas production in the world [1] . The contributions of agriculture to GHG- emissions and ammonia volatilization are currently under controversial discussion [2] .Frequently, the estimations of agricultural GHG- emissions within a Life Cycle Assessment (LCA) are based on generic emission factors (e .g . IPCC factors) only, leaving behind all other site specific conditions [3-4] . Another ongoing discussion refers to the potential reduction of such emissions if digestate (residue after biogas production) is returned to the field . To address these questions and support decision makers, our project aims to educe specific emissions factors for ammonia, nitrous oxide and methane that can be included in regional-specific LCA studies for energy crops . These emission factors will be derived from direct field measurements of these gases after the application of digestate as well as mineral fertiliser . Furthermore, the project strives to develop precise but also generalisable statements about ammonia volatilisation and alteration of the soil carbon content due to returning digestate from biogas plants to the field . The outcomes of the joint research project should lead to an improved environmental assessment of energy crop production including additional aspects such as fertiliser use efficiency and soil fertility . The project results should provide approaches - not just - for the sustainable production of energy crops but also for a more efficient cultivation of other renewables . References 1 . Weiland, P . (2010) . Applied Microbiology and Biotechnology 85: 849-860 . 2 . Cherubini, F ., et al . (2009) . Resources, Conservation and Recycling 53: 434-447 . 3 . Heller, M . C ., et al . (2003) . Biomas and Bioenergy 25: 147-165 . 4 . Iriarte, A ., et al . (2010) . Journal of Cleaner Production 18: 336-345 .

MOPC4-4Validation of PestLCI 2.0, an updated and expanded model to estimate pesticide emissions for use in LCIT .J . Dijkman, M . Birkved, M .Z . HauschildTechnical University of Denmark, Kgs . lyngby, DenmarkAs the effects that pesticides have on non-target organisms are directly proportional to the amount of pesticide emitted to the environment, it is important to accurately quantify pesticide emissions to the environment in the Life Cycle Inventory (LCI) phase of Life Cycle Assessment . PestLCI 2 .0 is an LCI model to calculate pesticide emissions from agricultural fields to 3 environmental compartments (air, surface water and ground water) . After calculation of the primary distribution of pesticides, i .e . the distribution between crop and soil, different fate modules are used to model the fate of the pesticides after deposition on the crops and in the soil, thereby determining the compartment specific emission pattern of the pesticide . PestLCI 2 .0 is an updated and expanded version of the PestLCI model (Birkved&Hauschild, 2006) . The model has been expanded with a fate module for macropore flow, the effect of tillage on macropore flow has been added while the algorithm sets of other modules have been updated . The applicability of the model is no longer limited to Denmark: the climate database has been expanded to include sets of climate data for 25 different locations in Europe, and the soil database now contains 7 European soils . The database of pesticides has been expanded to 90 active ingredients . To validate PestLCI 2 .0, pesticide emissions calculated by PestLCI 2 .0 were compared to the emissions calculated by risk assessment (RA) models used in the FOCUS project . For surface water emissions, the MACRO model was used for drainage emissions; PRZM was used to determine the emissions caused by runoff . Emissions to ground water were calculated with PEARL 4 .4 .During validation the input parameters of PestLCI 2 .0 were set to resemble the scenarios of the RA models as closely as possible . The results indicate that the RA models and the LCI model generally are in good agreement . Surface water emissions calculated by PestLCI 2 .0 are less than 2 orders of magnitude lower than those calculated by MACRO and PRZM, reflecting the difference between the realistic worst case approach of RA, as opposed to the average scenario approach of LCA . The emissions to ground water calculated by PestLCI 2 .0 are higher than those calculated by PEARL 4 .4 . This is a consequence of the inclusion of macropore flow into PestLCI 2 .0 . Overall, the results show that PestLCI 2 .0 is considered well-suited to estimate pesticide emissions for use in LCI .

MOPC4-5SewageLCI 1.0, an inventory model to estimate chemical specific emissions via sewage systemM . BirkvedTechnical University of Denmark, Kgs . lyngby, DenmarkWide Dispersive Use (WDU) of chemicals results in considerable quantities of chemicals being emitted to the environment directly or via Sewage System (SS) effluents . Emissions to SSs are in product system modeling handled by inclusion of Sewage Treatment (ST), assuming emission of an average ST effluent with a ST plant class specific effluent composition . It is thus assumed that the specific chemical is 100% removed and that an effluent with an average (chemical) composition is emitted . Performing Life Cycle Assessment (LCA) on unique chemicals with WDU , among other issues, necessitates correct representation of chemical-specific emission patterns via SSs as well as quantification of chemical-specific potential impacts instead of the potential impacts of an average effluent . This poster presents a novel approach for quantification of chemical-specific emissions via SSs . SS configurations and the fraction of a chemical with WDU being emitted via SS effluents are nation specific . In order to reflect the national specificy of SSs configurations, statistical data on the SS configurations in all EU countries were compiled from Eurostat . Combining these data with chemical removal efficiencies of biological treatment from the STPWIN model allows for estimation of removal fractions in the most common sewage treatment systems (primary, secondary and tertiary treatment as well as direct emissions and independent waste water treatment) . Emissions from sewage systems not only occur to aquatic recipients . Besides estimation of the fractions emitted to aquatic recipients, SewageLCI therefore also allows for estimation of the fractions of a chemical emitted to air and the fraction emitted to soil in the case of sludge amendment to soil . The approach of the model is tested on two common chemical groups used in large volumes in detergents; an enzyme (a protease) and an alcohol ethoxylate . Both chemical groups are modeled with and without sludge amendment to soil . In the scenarios with sludge amendment to soil, the fractions emitted to the environment from the fields receiving the sludge are quantified applying PestLCI 2 .0 . The results obtained indicate the fractions of the chemicals emitted to the aquatic recipients varies more than an order of magnitude between different countries due to large differences in the national SS configurations . Further the approach reveals the benefits of a modular inventory modeling approach through the SewageLCI and PestLCI combination .

MOPC4-6Interpretation phase in life cycle assessment software programs: a case study for Brazilian biodieselR .R . Sallaberry1, K .R .A . Nunes1, L . Schebek2

1TU Darmstadt, Darmstadt, Germany2Institute for Technology Assessment and Systems Analysis, Karlsruhe, Germany Software programs to perform Life Cycle Assessment (LCA) have the function of reducing the processing time for inventory and impact assessment . What is not clear is whether LCA software programs are able of generating enough outcome to elaborate reports based on interpretation phase requirements . A set of check tools in interpretation phaseis defined by ISO 14040/44 and the European Platform on Life-Cycle Assessment’s International Reference Life Cycle Data System (ILCD) handbook . According to these references, mainly when a report - conclusions and recommendations to a third-party for comparative studies - is needed, three quality checks have to be performed: (a) completeness, (b) sensitivity, and (c) consistency check . A case study was reproduced in three available LCA software programs (OpenLCA, Umberto, GaBi) . The first three LCA phases were identical in each program . The outcome provided, which differentiates in availability of data to perform the three quality checks . As result, the expectation of the evaluation of three checks inside the interpretation phase was not completely achieved . The completeness check was the only verification tool, which could be performed with the support from the three LCA software programs, although restrictions were reported . The sensitivity analysis by scenario variations could be carried out in general, since new propositions had been manually included . The OpenLCA did not have however a sensitivity analysis . The consistency check was not possible to be executed entirely inside any software program . Arguing that the iterative LCA approach and limitations of the study are attributed by user and not by limitations of program leads to a direct relation of expert judgement and report quality . This means that the information reported and the quality checks are only influenced by the practitioner and all informations needed can be extracted from the data provided . However, to perform a consistency check, for example, information about the differences in data sources, data accuracy and data coverage are necessary and they cannot be obtained by generating data in any LCA software program . The lack of information given by LCA software programs leads to a weaker third-party report . In spite of the fact that is possible to access and view most of the data needed in order to carry out the checks, the use of software program do not exclude further data process and the critical review by specialists .

MOPC4-7Methodological approach for validation of LCI modules of Italian life cycle inventory database (I-LCA v. 3.0)L . Maiorino1, S . Minestrini1, M . Pirilli2

1ISPRA - The Italian Institute for Environmental Protection and Research, Roma, Italy2Stager - ISPRA, Roma, ItalyISPRA is involved in research activities to build and to implement a national, on-line and public database for LCA information (I-LCA), as priority action to support the development of competitiveness for enterprises according sustainable paradigms . The starting point was the database I-LCA version 2 .0 developed by ANPA in 2000 . Now

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ISPRA intends to update and develop that project in a way to be in compliance with the European Platform on LCA (EPLCA) . In this context, the objective of this paper is to illustrate the appropriate methodological approach on the conformity of the data set through criteria (quality, method, nomenclature, etc . . .) in order to be in compliance with the format definted by European Platform on LCA . Furthermore, ISPRA is now planning out a research design aimed at developing of new specific-site LCI (Life Cycle Inventory) modules concerning the national transport sector based on primary data already available . Through the acquisition of a methodological approach has been possible to identify the criteria that give the data inventory characteristics of validity and conformity to standards set by the European Platform of LCA, namely quality, consistency and robustness of data . This methodological approach was applied to the modules contained in the database I-LCA v 2 .0 . This LCI database, currently under revision and updated enclosing about 400 manufacturing processes and services, is divided into four main areas (materials and processes, energy, transport and end of life) . The verification of conformity was carried out on some modules (related to the energy sector) that previous were been transcoded for I-LCA v .3 .0 according the format developed by the European Platform on LCA . This first internal review has showed that some of the modules are in compliance with the reference standard, while other modules should be implemented in order to achieve the same degree of completeness . The creation of a public database LCA, with quality and consistent data, methods and assessments, it is extremely important not only for the crucial help in implementing LCA studies, but also in order to promote a different orientation patterns in consumption and production . In this context, ISPRA intends to plan out a research design aimed at developing experimental application to a specific transport sector based on primary data already available .

MOPC4-8Setting up collaborative research on LCA and related topicsP .C .F . Osset1, B . Couffignal2, D .E .N .I .S Le Boulch3, C . Hugrel4

1Solinnen, Paris, France2RECORD, Villeurbanne, France3EDF, Moret sur loing, France4Bleu Safran, Lyon, FranceEDF, GDF SUEZ, Renault, Saint-Gobain, Total and Véolia, with the support of RECORD and of ADEME, are the co-founders of a new non-profit Corporation, SCORE LCA, created to financially support collaborative research on LCA and related topics . SCORE-LCA will drive collaborative research thanks to the annual funding of each member, and the potential complement of public funding . The research topics will be defined and selected by SCORE LCA members, on an annual basis . An “Experts committee”(from University, Research Centers and Consultancy) and a “Scientific advisory panel”will assist the members in this selection of topics . Each research project will be done by a selected organism (or group of organisms) such as research institutes or consulting company . They will be selected by SCORE LCA members through a call for tenders . Each project will be supervised by a specific panel formed by members and selected experts from the “Experts committee” . The set up of SCORE LCA will be done before end of 2011 . The research topics that will be covered by SCORE LCA studies encompass the following points:

- LCA systems modeling, and inventories calculation, - Calculation algorithms and LCA tools, - Indicators and impacts, - Analysis and interpretation methods, - Combination of LCA and other methods to assess products and services, - Surveys . The co-founders are willing to see SCORE LCA growing thanks to new memberships from other companies . SCORE LCA is opened to any entity, and has the will to be useful to the public: most of the outputs will be disseminated, and representative of SCORE LCA will bring their contribution to other public or private organisms at European level . The interest to belong to SCORE LCA is to be able to participate in the definition of the research topics and to be able to monitor the studies . SCORE LCA is currently preparing call for tenders on two first topics . Contractors will be selected among the candidates at the beginning of 2012, and the work will start . SCORE LCA has a European vision and expects that researchers from all Europe will collaborate to fulfill these research projects . The goal of the poster is to present the activities of SCORE LCA, including the two research topics that will be covered first . Some representative will be there to answer the questions from the attendees to the conference, including from the Universities or Research Laboratories which would like to be candidates to the first call for tenders .

MOPC5 - Established and emerging footprints – striving towards a valid and comprehensive support for decision-making processes

MOPC5-1Development of a framework for assessing the chemical footprintS . Sala, D .W . PenningtonEuropean Commission- Joint Research Centre, Ispra, ItalyIn the last few years, environmental footprint concept has obtained an increasing interest by both the scientific and political communities . The chemical footprint evaluation aims at assessing at which extent actual emission of chemicals harm the ecosystems above their capability to recover (the so-called carrying capacity of the system) . The evaluation of the chemical footprint, bringing together approaches and results from various disciplines (from ecotoxicology to ecology, from ecological/human risk assessment to life cycle thinking and related tools), may support the future development of policy both for environmental protection and green economy . A review of existing approaches and methodologies able to be integrated in a chemical footprint framework will be presented and assessed . The following issues are the key elements: - Assessing the intensity of chemical pressure, in terms of release in the environment by class of chemicals, economic sector of use, typology of release (point source or diffuse) and potential harm to the environment in a life cycle perspective - Linking the release in the environment with the carrying capacity of the system (vulnerability analysis, exposed ecosystem assessment, Eco epidemiology)

MOPC5-2The footprint family: differentiation, classification, and integrationF . Kai, R . HeijungsLeiden University, Leiden, NederlandIn the past decades, a variety of footprint concepts has entered the sustainability community under intensive debate . In this paper, we first review relevant literature . The idea of footprint originates from the ecological footprint described as a biophysical assessment tool that measures the human impacts of energy and materials consumption . After that the water and carbon footprint concepts were introduced analogous to the ecological footprint, representing the freshwater required or GWP-weighted GHG emissions . Obviously there is no single indicator able to represent the full spectrum of environmental impacts, but footprint indicators rather need to be applied jointly . Attempt has been made to develop an integrated footprint assessment . The Global Footprint Network recently defined the “Footprint Family”as an aggregation of the ecological, water and carbon footprint to track human pressure . However, the definition was not yet clear and comprehensive, exclusion indicators like the chemical footprint being discussed by the SETAC, and failure to capture the rationale of Footprint . From a methodological point of view, the Footprint is a method rather than an indicator . Thereby, we provide for the first time a definition of the “Footprint”as an approach to convert the environmental impacts of human activities into a physical quantity (area, volume, weight, etc .) . In this sense, the “Footprint Family”would rephrase as a suite of indicators able to measure the environmental impacts from different angles, by representing how many resources for each kind human have appropriated . According to the ecosystem compartments where resources flow and stock, we classify the footprint indicators into four patterns, the biologic, the atmospheric, the aquatic and the geologic . For example, the ecological footprint belongs to the first pattern, the carbon footprint to the second, the water footprint to the third, and the energy footprint to the last . As for the chemical footprint, it depends on the exact pollutant pathways . A description of the boundary, function and characteristics for each pattern is given . The remainder of the paper is to identify the similarities and differences among different patterns to show how these indicators overlap, interact and complement each other . We hope this paper will serve as a reference for the footprint research, as well as a support for policy makers, as it brings clarity to the concern about what the footprint actually is .

MOPC5-3Comparison of life cycle-based methods to assess the consumption of LuxembourgB . Rugani, C .O .L .I .N Jury, M .O .R .G .A Mey, P .A .U .L .A Hild, E . BenettoPublic Research Centre Henri Tudor (CRPHT), Esch-sur-alzette, LuxembourgA plethora of decision-making tools and sustainability indicators is available today for supporting national stakeholders and public authorities in the process of implementing resource management regulations or environmental impact mitigation strategies . The reliability of these methods, as well as the requirement for data collection and impact assessment methods is widely debated, especially for policy making support purposes . In this poster presentation, we intend to discuss several methodological approaches to identify a reliable framework to support reduction strategies (of emissions and resource exploitation) at national level . More specifically, in this poster we present the assessment of a country’s net consumption (where Consumption=Production+Imports-Exports) using different life-cycle assessment (LCA)-based methods (i .e . Ecological Footprint-EF, ReCiPe and Solar Energy Demand-SED) along with different environmentally extended input-output tables (EEIOTs) of EU-27, USA and The Netherlands . The case study of Luxembourg is used to evaluate this methodological framework . By using EF, SED, and ReCiPe we cover the main environmental impacts generated by a country’s consumption . However, results highlight the need for higher consistency of EEIOTs . Indeed, there is a high uncertainty related to the type of environmental extensions (EE) coupled to each economic sector . This aspect plays a major role when applying different assessment methods, since results show important changes according to the set of resources and emissions included in the EE . However, the increase of scenarios with different EEs does not substantially influence the results at the level of economic sector’s contribution . Whatever the scenario considered and the method applied, the financial and banking sector relies to more than 45% to the consumption of Luxembourg . Direct and indirect contribution analyses by sector allow outlining strategies to reduce impacts . The strengths and weaknesses of the approaches adopted are discussed with special emphasis on the benefits for potential applications in policy support .

MOPC5-4Improving the relevance of carbon footprint calculations: temporal issues and global warming indicators

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A . Levasseur, M . Margni, R . SamsonCIRAIG - École Polytechnique de Montréal, Montréal, CanadaCarbon footprint calculations are widely used to compare the impact of different products or projects on global warming . An increasing number of standards and guidelines based on life cycle assessment (LCA) are developed to provide a framework for those calculations . There is a broad consensus to multiply each greenhouse gas (GHG) emission by its GWP (Global Warming Potential) value, calculated for a chosen time horizon, and to add them up to get a carbon footprint expressed in kg CO2-eq . However, this approach implies some assumptions and simplifications that can bias the results and the conclusions drawn from them .

Recent papers have shown that the use of GWPs calculated for a given time horizon can lead to inconsistent analysis, and that the global warming impact can be very sensitive to different metrics and time-related issues . The use of a time horizon is a value-laden choice that can have significant impacts on the decisions taken from carbon footprint results . There is also an increasing tendency to consider some time-related phenomena such as storing carbon in biomass or delaying GHG emissions . Some researchers have shown that temporary carbon storage can have a beneficial impact on climate by decreasing cumulative radiative forcing over a given time horizon, but can cause an increase in temperature right after the end of the storage period . In this paper, the dynamic LCA approach is used on a fictitious case study comparing the impact on global warming of two objects with a 50-year lifetime: one made of wood, and a second made of a non biomass-based material . The dynamic LCA approach is applied to determine the time-dependent cumulative radiative forcing for each product . A new dynamic characterization factor is also developed to calculate the instantaneous increase in temperature caused by the life cycle emissions . This characterization factor is based on the concept of GTP (Global Temperature Potential), an analytical expression to calculate the temperature increase at any time following a GHG pulse-emission . The results show that the choice of a time horizon for the analysis or the use of an alternative metrics can completely change the conclusions of a comparative carbon footprint study . Carbon footprint calculations are often used to guide policies, consumer choices and corporate decisions . To improve decision-making, these aspects should be considered for cases where GHG emissions and sequestrations take place over a long period of time .

MOPC5-5Ecological footprint of the Port of Vigo (NW Spain)T . Antelo1, C .A .R .L .A Lopes2, A . Franco-Uría1, A . Alonso1

1IIM - CSIC, Vigo, Spain2Instituto de investigaciones Marinas - CSIC, Vigo, SpainSustainability of the fisheries sector is nowadays a key issue, since current practices of this activity are depleting marine resources and endangering biodiversity . Besides, indirect impacts, like those originated from related activities and services must also be addressed . For assessing the global impacts that this sector may have on the environment, the indicator Ecological Footprint (EF) can be used . This tool considers the energy and raw materials fluxes to and from any defined system or economy, transforming them into areas of land or water required by nature for generating and/or absorbing these fluxes . One of the main advantages of EF is its ability to inform general public about the impact an activity has on the world’s biocapacity . EF analysis has been applied to nations, regions and to a wide variety of sectors and activities . Pressure of nations on marine ecosystems has also been assessed by this methodology . Galicia is the main fishing region in Spain and one of the largest in the European Union . In fact, the Port of Vigo (SW Galicia) is the biggest fishing port of the world . Therefore, a representative part of the fishing extractive sector relies on the port activities . However, there are other important activities within the port which also requires resources consumption and thus, need to be evaluated . In this study, the environmental impact of the global activity (fishing and transportation) of the Port of Vigo was assessed by EF . The results obtained will provide information to the Port Authority on the main impact categories, in order to take the necessary measures to improve its environmental management strategy .

MOPC5-6Ore grade decrease as an indicator to address mineral resource scarcity in life cycle assessmentM .D .M . Vieira1, M . Goedkoop1, P . Storm2, M . Huijbregts3

1PRé Consultants, Amersfoort, Nederland2Raw Materials Group, Solna, Sweden3Radboud University Nijmegen, Nijmegen, NederlandResource use is an increasingly important topic on the political agenda . Mineral resources have dissipative use meaning that they are available on Earth but often degraded or dispersed . This way, mineral resources extraction will invariably continue and so will the decline of ore grades for all minerals worldwide, given no new discoveries (Mudd, 2009) . Consequently, in order to extract the same amount of mineral, higher volumes will be mined . Ore grade is an inherent property of mineral resources and yet it also expresses the scarcity of a resource . In Life Cycle Assessment, however, ore grade decrease has not been used as an indicator to address resource scarcity up to now . The goal of this paper is to develop a meaningful life cycle impact assessment method to assess mineral resource scarcity and to calculate characterization factors for ten minerals . The method here proposed expresses the decrease in ore grade as response to an increase in production . Characterization factors for mineral resource use were defined as the marginal change in average ore grade of mineral x (fraction) as response to the additional amount extracted of this mineral (kg) . We calculated for the following ten element(-group)s characterization factors: cobalt (Co), copper (Cu), diamond (C), gold (Au), iron ore (Fe), lead (Pb), molybdenum, (Mo), nickel (Ni), silver (Ag), zinc (Zn) and platinum-group metals . Production volumes and ore grade data per mine between 1998 and 2010 were retrieved from the Raw Materials Database developed by the Raw Materials Group . Results are presented in fraction of ore grade decrease per mass of mineral extracted . A life cycle study will be included to illustrate the applicability of the method proposed .

MOPC5-7Applicability of land use impact assessment methods in LCIA - findings from practitioners’ perspectiveT . Helin1, T . Mattila2, K . Antikainen3

1VTT Technical Research Centre of Finland, Vtt, espoo, Finland2Finnish Environment Institute SYKE, Helsinki, Finland3Finnish Environment Institute, Helsinki, FinlandInclusion of land-use related environmental aspects into LCA has been under active development in recent years . Many indicators have been developed and proposed for different aspects of land use but have, as yet, not been tested and compared in LCA applications . Aim of this study was to test the applicability of the general framework and several land use LCIA indicators in an illustrative case study from practitioners’ perspective . Nine different indicators were selected to represent three different impact endpoints of land use: resource depletion, soil quality and biodiversity . The case study covered a beer production system with all the life-cycle stages from barley cultivation to the serving of beer at restaurant . Three optional system expansions were studied to estimate the possible impacts of substituting feed protein (soybean, rapeseed and silage) with mash co-product from brewing . A comparison with wine production was also made for illustrative purposes . All tested land-use indicators were applicable in LCIA . Some indicators were found to be highly sensitive to assumptions on land transformation, which sets high requirements for LCI data quality . Challenges were faced in obtaining reliable data in the inventory and impact assessment phase . The majority of the land-use impacts occurred in the cultivation phase, but significant impacts were also found far down the supply chain . Scarcity of land-use LCI data sources limits validation and cross-comparison . Interpretation of indicator results is complicated due to the limited understanding of the environmental impact pathways of land use . Most of the land-use indicators led to results that were consistent with each other . None of the tested indicators describes the full range of environmental impacts caused by land use, nor fulfils all the criteria for an effective ecological indicator . Many well-known general LCIA method sets include an impact category ‘land use’, but fail to cover all three impact endpoints . We recommend presenting land occupation and transformation LCI results, the ecological footprint and at least one of the biodiversity indicators . Regarding soil quality, the lack of reliable regional data currently limits application of the proposed methods . The criteria of effective ecological indicators should be reflected in further work in indicator development . Development of regionalized characterization factors is of key importance to include land use in LCA .

MOPC5-8Development of biodiversity and resources indicators assessing potential impacts throughout the life cycle of products, processes and servicesL . Hamon1, A . de Schryver1, B . Chappert1, E . Aoustin2, J .B . Bayart3, M . Bugel3, A . Flesch3, J . Serre4, C . Guignard1, A . Bouter1, S . Humbert1

1Quantis, Paris, France2Veolia Environnement, Paris, France3Veolia Environnement Recherche et Innovation, Rueil-malmaison, France4Volia Environnement - Research and Development, Rueil-malmaison, FranceWhile carbon and water footprint indicators begin to be fully integrated in the decision making process of industrial stakeholders, the question is still effective for other potential environmental impacts since environmental burdens could be shifted to other environmental stressors such as biodiversity and resources . Within Life Cycle Assessment (LCA), existing impact assessment methods do not fully address this issue when communicating to non-LCA practitioners, because of complex units and only partly integrated issues (e .g . arable land as a resource, not integrated in impact assessment methods so far) . In this context, an operational methodological framework has been established to assess the environmental performances of products, processes or services using two indicators: resources and biodiversity . The biodiversity indicator contains aquatic ecotoxicity (USEtox), terrestrial ecotoxicity, aquatic eutrophication, aquatic acidification, terrestrial acidification/nitrification (IMPACT 2002+), impacts of turbined water, cooling water, and a new framework for land occupation and transformation . The resources indicator includes the impact on the stock of the extraction of minerals, fossil fuels, peat, and overexploitation of wood and arable land . Developments of new characterization factors for land occupation and transformation are based on the work of Koellner et al . (2008) . The CML approach and country-specific data have been combined to obtain characterization factors for the resources indicator, developed in line with the substance flows of the ecoinvent database . The potential impacts of the 4000 ecoinvent processes have been calculated and statistically analysed to point out any inadequacy in the methodology regarding the operationalization . Case studies have been conducted within Veolia Environment’s services and processes, showing the strengths and potential for future development of the indicators created . Finally, the potential benefits in terms of helping decision making process when combined with other environmental indicators have been identified . The main advantage of this new approach is to propose a transparent method, easy adaptable in the future, and which covers impacts usually not included in LCA, such as regionalized arable land depletion, wood use and water . To provide a comprehensive picture of the system assessed, it is recommended to combine those indicators with both the water and carbon footprint indicators already existing .

2�6 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

MOPC6 - Guidance documents and guidelines for environmental risk assessment (ERA): needs, developments and progress

MOPC6-1A Setac pellston workshop on the risks from Pesticides to pollinators: feedback and basis for the futureA .O . Alix1, P .D . Delorme2, T . Steeger3, T . Moriarty3, D . Fischer4, M . Coulson5, J . Frazier6, C . Lee Steere7, F . Streissl8, J . Pettis9, J . Pflugfelder10, M . Vaughan11, J . Wisk12

1Dow AgroSciences, Abingdon, United Kingdom2Pest Management Regulatory Agency, Ottawa, Canada3US Environmental Protection Agency, Washington, United States of America4BayerCropscience, Washington, United States of America5Syngenta Ltd, Bracknell, United Kingdom6Pennsylvania University, Pennsylvannia, United States of America7Australian Environment Agency, Barton, Australia8EFSA, Parma, Italy9USDA, Baltimore, United States of America10Swiss Bee Research Center, Switzerland, Switzerland11Xerces Society for Invertebrate Conservation, Portland, United States of America12BASF, Us, United States of AmericaThere have been reports, in recent years, of declines in native and managed pollinators in several regions of the world . Modern crop management practices, progressively implemented in order to allow the extension of cropped areas and ensure a proper control of pest populations and diseases have often been pointed as a factor responsible of this decline . Plant Protection Products (pesticides) are part of these management practices . Thus regulatory texts have been adopted all around the world, requiring an assessment of the impact of these products on the agricultural environment, and among others on arthropod and pollinating species . Concern regarding the extent to which the use of pesticides in crop protection affects pollinators varies among countries . The perception we have on pollinating species appears to results in first instance of our knowledge on their implication in crop pollination and related consequences of a temporary decline on the crops of concern . With regard to evaluating the potential impact of pesticides on pollinating species, these efforts have progressively focused on key indicator species, like the honey bee (Apis mellifera), primarily since the test guidelines for assessing the effects of pesticides on non-target terrestrial invertebrates have been developed mainly on this species, for practical reasons related to rearing facilities [8] . In Europe as in North America, the risk assessment for pollinators has focused on the honey bee; however, the extent to which these studies are used in risk assessment varies, which thus appears as the only species for which a dedicated risk assessment is performed, the other non target organisms being assessed at the level of trophic groups (i .e . fish, aquatic invertebrates, algae, aquatic plants, birds etc) . A global Pellston SETAC workshop on estimating the potential risks of plant protection products to insect pollinators was organized in January 2011, to bring together the best available science regarding exposure and effects assessment methodologies for Apis and non-Apis species, and harmonize further the risk assessment approaches among North and South America, Europe, Asia and Africa for a global improvement of the protection of insect pollinators in cropped area . The proceedings of this workshop will be finalized in the coming weeks and this presentation aims at offering the audience an outline of these proceedings .

MOPC6-2Activities of the OECD expert group on pesticide effects on insect pollinators in the context of the risk assessment for pesticidesA .O . Alix1, A .P . Pease2, P .D . Delorme3, C .H . Hart3, S .P . Poret4

1Dow AgroSciences, Abingdon, United Kingdom2US Environmental Protection Agency, Washington, United States of America3Pest Management Regulatory Agency, Ottawa, Canada4Organisation for Economic Cooperation and Development, Paris, FranceThere have been reports, in recent years, of declines in native and managed pollinators in several regions of the world . Modern crop management practices, progressively implemented in order to allow the extension of cropped areas and ensure a proper control of pest populations and diseases have often been pointed as a factor responsible of this decline . Agricultural pesticides (plant protection products) are part of these management practices . Regulatory texts have been adopted all around the world, requiring an assessment of the impact of these products on the agricultural environment, and among others on arthropod and pollinating species . As a complement to this assessment, appropriate feedback from the field as an indicator of possible side-effects of pesticides on pollinating species and of conditions of pesticide use that ensure limited exposure is essential to evaluate the effectiveness of risk management measures . Finally, early alerts and clear communication of key research findings related to pollinators are also needed to allow fast and effective decisions . In this context, the creation of a dedicated working group was approved by the OECD Working Group on Pesticides (WGP) in May 2010 . The objective given to this “Pesticides Effects on Insect Pollinators”(PEIP) Expert Group is to develop a toolbox for risk assessment, risk management, incident reporting, and research about pollinators, focusing on honey bees and non-Apis bees and on the specific case of pesticides as potential factor of stress .

MOPC6-3EFSA Opinion on the science behind the development of a risk assessment guidance of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees)F . Streissl1, R .L . Luttik2, C .S . Szentes1, D . Auteri1

1EFSA, Parma, Italy2RIVM, Bilthoven, NederlandA decline in pollinators was reported from several different regions of the world . Pollination is a very important ecosystem service for food production and maintainance of biodiversity . Pesticides are one of the factors that may contribute to the decline of pollinators observed worldwide . This has led to concerns that the current risk assesment for pesticides need revision . As a response to this regulatory challenge the European Commission tasked EFSA to develop an updated Guidance for pesticide risk assessment and bees . The opinion on the science behind the risk assessment for bees analysis the existing test protocols and risk assessment schemes . It gives recommendations on how to assess cumulative and synergistic effects and how to assess the risk of substances which are highly toxic to bees such as neonicotinoids . The approach of definition of specific protection goals is used the first time in the context of developing pesticide risk asssessment . The present opinion of the PPR panel provides the scientific basis for the final Guidance Document of EFSA .

MOPC6-4Activities of ICPBR in the context of the risk assessment for pesticides to beesA .O . Alix1, H .M . Thompson2, G . Lewis3, J . Pistorius4, G . Smagghe5, K . Wallner6, J . Wassenberg7

1Dow AgroSciences, Abingdon, United Kingdom2The Food and Environment Research Agency, York, United Kingdom3JSC, Harrogate, United Kingdom4Julius Kuhn Institute, Braunschweig, Germany5Ghent University, Ghent, Belgium6Universität Hohenheim, Stuttgart, Germany7CTBG, Wageningen, NederlandThe International Commission for Plant-Bee Relationships (ICP-BR) organizes working sessions in Europe on the harmonization of methods for testing the toxicity of pesticides to bees for over 30 years . ICP-BR develops the scientific process underlying decisions from European administrative authorities, EPPO (European and Mediterranean Organization for Plant Protection) and OECD (Organization for Economic Cooperation and Development) . ICP-BR Bee Protection Group symposia have acquired considerable authority in the area of legislation and regulation concerning bee protection related to the use of plant protection products, bringing together the European expertise of national authorities, industry, research and beekeepers . The Bee Protection Group held its first meeting in Wageningen in 1980 and over the subsequent 32 years has become the established expert forum for addressing the risk of pesticides to bees . It has operated by reaching consensus amongst a wide range of experts active in this field drawn from industry, regulatory authorities and research institutes across the European Union (EU) . Operating through the EPPO honey bee sub-group, it has produced the testing methodology and risk assessment guidance currently used under Directive 91/414/EEC and Regulation 1107/2009/EC . Returning to Wageningen in November 2011, this meeting was the most recent event where research in the area of pesticide effects on bees was presented and where implications for risk assessment were discussed and taken on board in working group activities . This presentation aims at giving an overview of ICP-BR activities with an emphasis on each of the seven working groups (testing effects on bee brood; semi-field and field testing; acceptability of effects in testing; statistics; risks related to sowing dusts; risks of guttation and monitoring studies) activities .

MOPC6-5Review of higher tier methods for assessment of the risk of pesticides to honey beesJ . Miles, A . Alix, V . KramerDow AgroSciences, Abingdon, United KingdomRisk assessment procedures have been developed for domesticated honeybees (Apis mellifera) potentially exposed to residues of insecticides . After initial tier I screening risk assessments further studies may be conducting for those substances for which a potential risk cannot be excluded . Higher tier studies are often performed in cages or tunnels containing colonies of honey bees which can forage on treated crops . Cage test are typically smaller in size than tunnels but allow for the testing of more treatments often with more replication than can be offered by the larger scale tunnel tests . Field tests can be conducted to establish the effects of the product under more realistic conditions but due to use of free flying colonies there is less control than in the tunnel and cage studies . Finally, post-registration monitoring can offer additional information on specified uses of the product under commercial conditions and can be used to give feedback on the outcome of the risk assessment and the effectiveness of any risk management practices put in place to protect bees . This paper will review the various higher tier methods with respect to their ability to accurately predict the potential effects of pesticides to honey bees and how they can be employed in a robust risk assessment . The range of parameters investigated typically include; mortality, foraging, behavior, brood and colony

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development . The ability of higher tier studies to predict potential adverse effects will be evaluated and how these parameters can be employed in a risk assessment with clear decision making linked to honey bee and pollinator protection goals .

MOPC6-6Assessing the comparative risk of plant protection products to honey bees, non-target arthropods and non-apis beesJ . Miles, A . AlixDow AgroSciences, Abingdon, United KingdomRisk assessments are conducted for plant protection products (PPP) with respect to potential impacts on non-target species . These include pollinators such as the honey bee but also to other non-target arthropods (NTA) . In common with other areas of ecotoxicological risk assessment sentinel species are employed aiming at ensuring a high level of protection/conservatism . Tier I screening risk assessments are intended to rapidly exclude those substances which pose a low risk to non-target organisms and to focus resources on those for which a potential risk cannot be excluded and further studies may be undertaken to characterize the conditions and occurrence of risks . In the European Union a Hazard Quotient (HQ) approach is used to assess the risk to both honey bees non-target arthropods at Tier I . This is calculated by dividing application rate by the LR50 (Lethal Rate 50) . In other regions such as North America, a contact toxicity trigger of 11 µg active substance/bee is currently employed . At the Pellston workshop it was suggested that for a risk assessment for sprayed products the honey bee could be a suitable surrogate species . However, to account for potential differences in the sensitivity between the honey bee as a test organism and other non-Apis bees a safety factor of 10 (for interspecies differences) was suggested . On the basis of ecotoxicological data of the European data sets for honey bees and NTA, the relative risk of PPP to NTA, honey bees and non Apis bees, as depicted by HQ values is compared and where possible data on NTA pollinators (e .g . Syrphids) and non-Apis bees (e .g . Bombus sp .) are also included . At tier I using HQ trigger of 50 for honey bees, 2 for NTA, in accordance with the current EU legislation, and 5 (i .e . 50 divided by the extra factor of 10 abovementioned) for non-Apis bees, the NTA scheme identified more compounds and uses to move forward for further evaluation . The suggest non-Apis HQ trigger of 5 gave a similar pass / fail rate to the NTA scheme but was slightly confounded by a lack of determined end points . However, even taking this into consideration the most restrictive tier I assessment was that for NTAs . This poster examines the potential for a tier I risk assessment to cover NTA, honey bees, non-Apis bees and other arthropod pollinators as part of a tiered risk assessment scheme and compares the empirical HQ approach with methods using more specific species exposure estimates .

ET03P - Animal Alternatives: methods, endpoints, and testing strategies

TH 001Cartilage and bone malformations in the head of 6 day old zebrafish embryos after exposure to hydrazides and hydrazinesR . Strecker, N . Demir, T . BraunbeckUniversity of Heidelberg, Heidelberg, GermanyIn terms of alternatives to animal testing, the zebrafish embryo test (ZFET) has received much attention as a refinement or even a replacement for the acute fish toxicity test (OECD TG 203) . Using the ZFET, not only acute mortality can be investigated, but also specific sublethal alterations can easily be observed . Hydrazides and hydrazines are well known for their diverse biological effects . The toxicological and medical potential of both chemical classes on animals and humans was already identified at the beginning of the 20th century . Fertilized zebrafish embryos were exposed semi-statically to isoniazid, benzhydrazide, ben-zylhydrazine and phenylhydrazine for 6 days, fixed in paraformaldehyde and stained as whole mounts . The range of toxicity is very broad; after 144 h, the LC50 of isoniazid is about 4 g/L, whereas those of benzhydrazide, benzylhydrazine and phenylhydrazine are about 50, 10 and 1 mg/L, respectively . All four chemicals caused wave-like deformations of the notochord and cartilage malformations at sublethal concentrations . Even at the lowest test concentrations, exposure to all test chemicals resulted in notochord breaks, whereas the amount of breaks diminished with increasing concentrations, and the rate of undulations of the notochord became more pronounced . Despite the different variable extent of acute toxicity, the dose-dependent malformation of cartilage and decrease of ossification were comparable between all test substances . The cartilages of the neurocranium, e .g . the ethmoid and basal plate, proved to be more stable than cartilages of the pharyngeal skeleton such as, e .g ., Meckel’s cartilage and the ceratohyal . Ossifications are much more susceptible to the test compounds than cartilages, with reduction of bone mass as the most prominent alteration .

TH 002Use of ‘moribund’ stage in the OECD fish acute toxicity test and its effects on suffering and LC50-valuesH . RufliEcotoxsolutions, Basel, SwitzerlandIn the acute fish toxicity test according to OECD (Organisation for Economic Cooperation and Development) Guideline 203, LC50 is assessed in terms of the concentration of a test substance at which 50% of the fish die within an exposure period of 96h . The criterion for death is defined as (§ 19): ‘no visible movement, no response when touching the tail .’ By using the criterion of ‘moribund’ to replace the ‘death’ criterion, the duration of suffering could be reduced . Fish with severe sublethal effects might be declared ‘moribund’ (there is no official definition for “moribund’ stage in fish), and are removed from the test as soon as this occurs (premature discontinuation of experiment) . The criterion has already been introduced to ecotoxicological testing in the UK, and is being used by some laboratories in Germany and Switzerland . “Moribund”fish affect main study outcomes as the LC50 derived on fish declared as “moribund”, the LC50 moribund, may be lower than the conventional LC50 . This was evaluated by a retrospective analysis of 328 fish acute toxicity tests of an industry laboratory based on five different definitions of ‘moribund’, and of 111 tests from ten other laboratories from Europe and the US . Using the criterion of ‘moribund’ affected up to 79% of the studies . The LC50 moribund was lower than the conventional LC50 in up to 52% of the studies depending on the definitions of “moribund” . Based on the results of the present study, a successful inclusion of the ‘moribund’ criterion in an updated OECD guideline for the acute fish toxicity test would reduce the period of suffering by up to 92h and lower the value of the main toxicity endpoint generally by a factor of about 2; maximal by a factor of about 16 .

TH 003Screening of the estrogenic activity of environmental samples using transgenic cyp19a1b-GFP zebrafish embryosE . Fetter1, S . Scholz1, M . Krauss1, W . Brack1, K . Petersen2, K .E . Tollefsen2, F . Brion3, O . Kah4

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2Norwegian Institute for Water Research, Oslo, Norway3INERIS, Verneuil-en-halatte, France4University of Rennes, Rennes, FranceDue to the potential environmental hazards on reproduction and population development in the aquatic ecosystem by estrogenic substances, their hazard assessment play a paramount role for regulation of chemicals, biocides, pesticides and pharmaceuticals in the EU and worldwide . Furthermore, they are also highly relevant for biomonitoring within the EU Water Framework Directive . For the assessment of their potential adverse effects on the environment, biological test systems play an essential role . The identification of endocrine disrupters with the chemical analysis is limited due to the low but still biologically relevant concentration in the surface waters . Besides, assessing the environmental hazard of mixtures of endocrine disrupters through classical monitoring is not only expensive but also not easily predictable . For this reason, effect-directed analysis using bioassays can be an excellent supplement of the classical monitoring of aquatic ecosystems . Transgenic models appear to be a powerful tool for high- throughput screening of changes in the gene expression induced by estrogenic chemicals . Using the zebrafish cyp19a1b-GFP transgenic embryo, which expresses GFP under the control of the zebrafish cyp19a1b promoter, adds the complexity of a full organism to the simplicity and reproducibility of cellular reporter gene assays . In this study we present the first results using this transgenic line for effect-directed analysis of a contaminated sediment sample . In this sample from the Czech river Bilina a previous analysis using the yeast screening assay has identified fractions containing estrogenic compounds . We were able to confirm this study by means of the transgenic cyp19a1b-GFP zebrafish strain . The results were also validated by artificial mixtures of compounds, which were identified by the effect-directed analysis, resembling the compositions of the fractions . Our study also includes data on the validation of the transgenic zebrafish strain with known estrogenic substances and its confirmation using molecular and an in-vitro method . The transgenic cyp19a1b-GFP in-vivo assay proved to be an excellent screening method with high sensitivity and reproducibility for environmental samples .

TH 004Micronucleus and alkaline comet assays in Japanese Medaka larvae. Integrative methods for sensitive detection of genotoxicants in aquatic ecosystemsF . Le Bihanic1, A . Pichon1, B . Morin1, S . Berrada2, J . Cachot1

1EPOC, Talence, France2Atoxigen, Pessac, FrancePollutants accumulated in aquatic compartment can induce several adverse effects on living organisms . Among these, some are known to be genotoxic and able to induce mutations and tumour development . To assess and monitor aquatic pollutants and their effects, fish embryos and larvae are suitable models due to their chemical sensitivity and easiness to breed . Micronucleus and alkaline comet assays are two genotoxicity assays allowing DNA damage analysis at different steps of mutagenesis . Comet assay detects repairable DNA damage as single/double DNA strand breaks and alkali labile sites . Micronucleus assays (MN) detects non-repairable DNA damage such as aneugenic and clastogenic lesions . This work develops an integrative and sensitive micronucleus assay in Japanese medaka at larval stage . MN responses were compared to alkaline comet assay responses . Japanese medaka, Oryzias latipes embryos were exposed by immersion during all embryonic development to different concentrations of three model genotoxic compounds: cyclophosphamide, benzo(a)pyrene and cadmium chloride . Twelve 2dph-larvae per replicate were used to prepare cell suspension using enzymatic and mechanic dissociation . Both assays were performed on the same cell suspension . Comet assay was processed according to a previously validated protocol on this species . MN assay protocol was optimized to reduce number of larvae required and improve cell isolation, viability and concentration . MN slides were stained with acridine orange . Compared to most of the MN assays performed on erythrocytes, this method is integrative and takes into account the whole organism response to genotoxic compounds . It enables an overview of genetic damages accumulated throughout the all embryonic development and in the all body . Moreover, as ethical future of animal health and welfare, number of individual required for experiment is small and assay is performed at sac-fry stage (not consider by EU animal experimentation legislation) . This assay can be easily included in the Japanese medaka embryo-larval assay MELA .

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TH 005Effects of propylparaben during medaka (Oryzias latipes) embryo development using in vivo morphometrical/quantitative approachesM . Gonzalez-Doncel1, J .E . Garcia-Mauriño2, L . San-Segundo1, S . Sastre1, C . Fernandez1

1INIA, Madrid, Spain2School of Medicine, Complutense University, Madrid, Spain Parabens are antimicrobial preservatives widely used in personal care and food products . Among them, the most used are generally methyl and propylpareben (PB) which in turn seem to be the most detected in aquatic ecosystems . Besides the numerous reports about the in vitro and in vivo estrogenic activity of PB and other parabens, relatively few studies have been conducted that determine sublethal effects to aquatic organisms . In this sense, the use of fish embryos and eleutheroembryos are promising alternatives to existing ecotoxicity tests without reducing the validity of the risk assessment . These stages also provide the application of in vivo methods for the analysis and quantitation of biomarkers indicative of exposure/toxicity . Hence, we propose a battery of non-invasive morphometrical tools to examine physiological and anatomical features in embryos and eleutheroembryos of medaka (Oryzias latipes) so the effects of waterborne exposure to PB (40-4000 µg/L) can be investigated . Preliminary findings show PB ≥ 400 µg/L resulting in significant quantitative differences, although these exposure effects may not necessarily compromise fish normal development . Further studies will be needed to ascertain whether these effects may result in long term toxicity . This work is being made possible thanks to Spanish Government Grants RTA2010-00004-C02 and CTM2010 19779-C02 .

TH 006Evaluation of genotoxicity of sediment samples using zebrafisch embryo and comet assayS . Gartiser1, M . Garcia-Kaeufer1, C . Hafner1, S .H . Keiter2, H . Hollert2

1Hydrotox GmbH, Freiburg i .br ., Germany2RWTH Aachen University, Aachen, GermanyGenotoxic hazard estimation of sediment-bound anthropogenic pollutants is still a challenge since many genotoxicants tend to adsorb onto particulate matter . Testing of extracts usually overestimate risk because bioavailability of the compounds is not considered and enrichment processes like Soxhlet extraction might lead to false positive results due to artifact formation . For genotoxic risk assessment often biomonitoring experiments with in the field caught fish are performed . The in vitro zebrafish embryo model represents an attractive alternative since it offers the possibility to simulate in situ exposure scenarios and is considered as a replacement to animal experiments according to current legislation . In this work the novel method for determination of genotoxicity induced by particle-bound pollutants is presented . Therefore zebrafish embryos were exposed to whole sediments and DNA damage was assessed by means of the comet assay in whole body cell suspensions (Kosmehl 2006) . Within the joint research project DanTox, funded by the German Federal Ministry of Education and Research (http://www .bio5 .rwth-aachen .de/DanTox, 2009 - 2012), freeze dried sediment samples from the Rhine River and outer Vering Canal in Hamburg were investigated . Various exposure scenarios were applied and varied from 0 to 72 hours post fertilization . Preliminary results showed a high intra-assay variability of DNA damage since a heterogeneous cell mixture from homogenized tissues was obtained . Nevertheless, the in-vitro fishembryo model turned out to be a sensitive tool for detecting total genotoxic hazardous potential of sediments . Even though it should be considered that the egg chorion represents a selective barrier for toxic compounds . As well, the metabolic competence of the embryos, which is closely related to the stage of development, demands further investigations by varying different exposure windows .

TH 007Modeling concentration-response relationships of quantitative gene expression in zebrafish embryo and its application to predict mixture effectsP . Renner1, N . Kluever1, D . Zitzkat1, R . Altenburger1, D . Jungmann2, T .U . Berendonk2, S . Scholz1

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2Technische Universität Dresden, Dresden, GermanyChemicals in the environment are rarely found as single compounds but rather as part of (complex) mixtures . Mixtures may provoke effects which are difficult to describe based on the knowledge of effects for single compounds . Over the last decades several models (concentration addition, independent action) have been tested and applied to predict mixtures using apical endpoints like lethality or growth inhibition . However, the effects of mixtures at a molecular level are still poorly understood . Using a modeling approach we aim to investigate mixture effects on gene expression patterns in zebrafish embryos . Initially we analyzed effects of single substances on the gene expression of specific genes to model concentration-response relationships . Based on these results we will conduct mixture experiments investigating whether or not existing models for apical endpoints can be applied for molecular responses as well . Finally, the modeling approach will be extended to the entire transcriptome level by analyzing the effect of mixtures using microarrays . The overall goal is to apply the findings for a better interpretation of gene expression patterns provoked by exposure to environmental samples . First results clearly show that gene expression follows monotonous concentration-response relationships . However, at higher concentrations (above LC10) we noticed an inhibitory effect and/or highly variable responses . We compared classical toxicological models such as the sigmoidal Hill-slope to peak and other alternative models . To date we have completed the detailed analyses of 4 different chemicals that have been identified by a previous microarray analysis as sensitive and specific markers for exposure to chemicals with different modes of action .

TH 008An evaluation of the zebrafish embryo model for estrogenicity testing of wastewater treatment plant effluentsA . Jonáš, A . Vítecková Wünschová, I . Sovadinová, B . Jarošová, L . BláhaResearch Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech RepublicWastewater treatment plants are known sources of water pollution which may cause endocrine disruption in fish and other aquatic biota . Wastewater treatment plant effluents contain complex mixtures of compounds with various estrogens, xenoestrogens, antiandrogens, and other compounds which may potentiate each other . Therefore to access the overall potential estrogenic effects in fish, it is necessary to test the whole mixture (rather than assessing individual chemicals alone) . To measure estrogenic effects in the effluents, we have combined zebrafish embryo model with quantitative RT-PCR . Modulations of three estrogen responsive genes have been evaluated including specifically aromatase B, vitellogenin 1, and estrogen receptor α . The effects have been tested with series of waste water treatment plant effluent samples collected during the pan-European monitoring campaign (coordinated by the JRC Ispra, Italy), and the results obtained with the zebrafish bioassay have been compared to comprehensive analytical data of various contaminants . The present work contributes to the characterization of the zebrafish model as a tool for investigation of in vivo estrogenicity in complex contaminated samples . [The work has been supported by Brno PhD Talent Financial Aid to Adam Jonáš and the projects INCHEMBIOL (MSM0021622412) and CETOCOEN (CZ .1 .05/2 .1 .00/01 .0001)] .

TH 009The normal development of the fathead minnowS . Böhler, R . Strecker, B . Kais, T . BraunbeckUniversity of Heidelberg, Heidelberg, GermanyThe fathead minnow (Pimephales promelas), the zebrafish (Danio rerio) and the Japanese medaka (Oryzias latipes) range among the most commonly used fish species in aquatic toxicity testing . However, acute fish tests (OECD TG 203) require the use of large numbers of test animals . In order to reduce the number of experimental animals, attempts are underway to establish the zebrafish embryo test (ZFET) as an alternative to conventional acute fish toxicity testing . Whereas the ZFET is currently undergoing OECD validation, fathead minnow embryos have only scarcely been used for embryo toxicity testing . Prior to the use of the fathead minnow in a standardized embryo test, it is important to clearly identify those stages during development from fertilization to hatching and beyond which are of toxicological relevance . Based on fathead minnow-specific developmental data, the embryo toxicity test was adapted and refined for the fathead minnow embryo . Fathead minnow staging was performed in petri dishes containing dilution water (prepared according to OECD TG 203 Annex 2, 1992) at 25 ± 1 °C . Pictures were taken every 5 minutes between 0 - 3 hpf and every 30 minutes from 3 to 12 hpf . Pictures of embryos older than 12 hpf were taken every 60 minutes . After initiation of spontaneous movements (approx . 26 hours post-fertilization (hpf)), the embryos were dechorionated using 2 mg/ml pronase solution and anaesthetized with 0 .016 % tricaine solution . In fact, it turned out that most of experimental parameters could be kept identical to those used in the ZFET . Using the fathead minnow embryo test, LC50 data were generated for cadmium chloride, a substituted aniline (3,4-dichloroaniline, which is currently used as the positive control in the ZFET) and a substituted phenol (2,4-dichlorophenol) . Fathead minnow embryos proved to be slightly less sensitive for cadmium chloride and definitely less sensitive for 3,4-DCA than adult fathead minnow (TG 203), whereas for 2,4-DCP no difference in sensitivity could be detected . However, the comparison with the ZFET showed that zebrafish embryos are significantly more sensitive . For all three substances, toxicity increased after hatching (≥ 96 and 120hpf) . Differences in toxicity the fathead minnow and the zebrafish embryo tests might be due to differences in the chorion (thickness, structure, quantity and size of pores) .

TH 010A modified approach for the determination of bioconcentration factors (BCF) in fishW . Mailahn1, D . Schnee1, C .U . Polleichtner2, C . Kussatz2, R . Schmidt3, E . Küster4

1Federal Environment Agency, Berlin, Germany2German Federal Environment Agency, Berlin, Germany3Umweltbundesamt, Berlin, Germany4UFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyThe method for the determination of bioconcentration factors (BCF) given in OECD guideline 305 builds on the exposure of adult fish to a dilute aqueous solution of chemicals at a constant concentration . Thus, numerous samples of water and fish tissue have to be analyzed to calculate the BCF . The number of fish killed during the test is high, the test can take several weeks and is expensive . Due to the high number of fish used for BCF determination, many alternative methods have been developed with the aim to facilitate the screening for BCF, e .g . with fish larvae or with fish eggs and 14C-labeled compounds . In the ProDarT project funded by the German Federal Ministry of Education and Research (BMBF, project no . 315399), the Helmholtz Centre for Environmental Research (UFZ) and the laboratories of the Federal Environment Agency (UBA) made a further attempt to determine BCF and to further reduce both effort and the total amount of test animals . Moreover, a method of sample preparation prior to gas chromatography-mass spectrometry analysis (GC-MS) was developed . So far the approach has been tested

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with phenanthrene, chlorpyrifos and methyl triclosan . Aqueous stock solutions of the chemicals were prepared in pure water . An aliquot of each stock solution was diluted in well aerated artificial freshwater according to DIN EN ISO 7346-3 . Fertilized eggs of the zebrafish (Danio rerio) were exposed in the dilute solutions at 26°C . At the start of exposure and also 1, 6, 24, 30, and 48 hours later the fish embryos were investigated for lethal and sublethal toxicological endpoints . Furthermore, samples of the aqueous medium were taken . The compounds were extracted by solid phase extraction (SPE), and analyzed with GC-MS . Samples of fish eggs were taken at 24 and 48 hours after the start of exposure . The eggs were smashed in ethanol and treated in an ultrasonic bath . These extracts were concentrated and analyzed with GC-MS . Concentrations in the aqueous medium were compared with the residues detected in fish eggs and degradation curves were calculated .

TH 011Exploring animal alternatives: seeking a replacement for whole effluent toxicity testing in fishN .H . Conoan, M .K . Jeffries, J .T . OrisMiami University, Oxford, United States of America In the United States (US), any facility discharging effluent directly to surface waters must conduct periodic evaluations of whole effluent toxicity (WET) . One method commonly used to obtain WET data is the 7 day larval growth and survival test (LGS) in which newly hatched fathead minnows (FHMs, Pimephales promelas) are exposed to effluents for 7 days with mortality as an endpoint . Given the importance of animal welfare in toxicity testing, the current study aimed to identify a test method that is able to fulfill the regulatory requirements for WET data, while minimizing the use of protected organisms or life stages . One possible alternative is the fish embryo toxicity test (FET) . The FET has already been sanctioned for WET testing in the European Union (EU) using embryonic zebrafish (ZF, Danio rerio); however, for the FET to be adopted in the United States, it must be applicable to species commonly used in contract laboratories (i .e ., FHMs) and demonstrate comparable sensitivity to the LGS . Thus, the main objectives of this study were 1) to develop test methods that harmonize the FHM-FET with the ZF-FET and the ZF-LGS with the FHM-LGS and 2) to compare the sensitivity of the FET and the LGS using both FHMs and ZF . To accomplish the first objective, tests using existing protocols for the FET and LGS were carried out with both species under different temperature and feeding regimes in order to determine optimal test conditions . The existing protocols were then appropriately modified and applied to the remaining experiments . To address the second objective, lethal (e .g ., NOEC, LOEC, EC50) and sublethal (e .g ., length, weight, deformity frequency, molecular) endpoints were evaluated following FET and LGS testing of each species using the reference toxicant 3,4-dichloroaniline . These results provide evidence for the ability of the FHM-FET to serve as an alternative to the FHM-LGS for WET testing carried out in the US . The adoption of FET testing for this purpose would result in the use of an earlier life stage to obtain sub-chronic toxicity data, representing a refinement of current test methods in the spirit of the three R’s .

TH 012Final results of the OECD validation study on the transferability, intra- and inter-laboratory reproducibility of the Zebrafish EmbryoToxicity TestM . Halder1, S . Belanger2, T . Braunbeck3, F . Busquet4, G . Carr2, A . Lillicrap5, J . Rawlings2, R . Strecker3, S . Walter-Rohde6, A . Gourmelon7

1European Commission, Ispra, Italy2Procter & Gamble, Cincinnati, United States of America3University of Heidelberg, Heidelberg, Germany4European Commission, Joint Research Centre, Ispra, Italy5NIVA, Oslo, Norway6UBA, Dessau-rosslau, Germany7OECD, Paris, FranceThe OECD Acute Fish Toxicity Test Guideline (TG 203) is an integral component in the environmental safety assessment of industrial chemicals, agrochemicals, pharmaceuticals, feed stuffs, and biocides . One of the most promising alternative approaches to the acute fish toxicity test is based on the use of zebrafish embryos . In 2005, the German Federal Environment Agency submitted the draft TG on ‘Fish Embryo Toxicity test (FET)’ to the OECD Test Guideline Program and a supportive Draft Detailed Review Paper . Subsequently, the OECD established the ad hoc Expert Group on the Fish Embryo Toxicity Test . Based on the outcome of expert meetings, OECD decided to perform a validation study (coordinated by ECVAM and steered by a validation management group) . The validation study aims at evaluating the transferability, and the intra-/interlaboratory reproducibility of the Zebrafish FET (ZFET) . Newly fertilised zebrafish eggs (20/concentration and control) are exposed for up to 96h to chemicals . Four apical endpoints are recorded daily as indicators of acute lethality in fish: coagulation of the egg, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heart-beat . LC50 values are calculated for 48h and 96h exposure . During the validation, 20 chemicals were tested at 5 different concentrations in 3 independent runs in at least 3 laboratories with appropriate controls . Stock solutions and test concentrations were analytically confirmed for 11 chemicals . The poster will give an overview on the validation study design, the results and the correlation of the ZFET with acute fish LC50 data . Acknowledgements to the participating scientists (laboratories): Edward Salinas (BASF, Germany), Fernando Martinez-Jeronimo (Instituto Politecnico, Mexico), Przemysław Fochtman & Helena Rzodeczko (Ipo-Pszczyna, Poland), Juliette Legler & Peter Cenijn (IVM, The Netherlands), Nicole Huebler & Stefan Weigt (Merck Serono, Germany), Leo van der Ven & Evert Jan van den Brandhof (RIVM, The Netherlands), Carola Kussatz & Christian Polleichtner (UBA, Germany), Stefan Scholz (UFZ, Germany), Hilda Witters (VITO, Belgium) “Disclaimer: The opinions expressed and the arguments employed herein are those of the authors and do not necessarily reflect the official views of the OECD or of the governments of its member countries, or the European Commission’

TH 013Genotoxic effects of sediment extracts and native sediments from Hamburg harbor and the Rhine River in Danio rerio-embryosS . Peddinghaus1, S . Steffens2, L . Loibel2, J . Bräunig2, H . Hollert2, S .H . Keiter2

1Institute for environmental research, Aachen, Germany2Institute for environmental research-RWTH Aachen, Aachen, GermanyThe European Water Framework Directive (EWFD) aims to achieve a good ecological and chemical status in the surface water of European rivers . However, since particulate matters and sediments are highly relevant as secondary sources for environmental pollutants, applied sediment toxicology plays an important role to ensure the successful implementation of the goals of the EWFD . The present study is part of the joint research project DanTox, which - among other specific endpoints - investigates genotoxic effects of sediment extracts in zebrafish (Danio rerio) embryos . The comet assay is a widely used bioassay gaining increasing popularity as a screening test for genotoxicity . However, since the ecological significance of the comet assay results in not clear yet further correlations with biotests with definite endpoints such as the micronucleus assay are recommended . Within this study both biotests will be compared, in order to gain a reliable genotoxic assessment of environmental samples . Sediment samples were taken from the Rhine River (Ehrenbreitstein and Altrip) and Hamburg Harbor (outer Vering Canal) . After Soxhlet extraction of freeze dried sediments embryos were exposed to the samples for 24, 48 and 72 hours post fertilization (hpf) as well as for a time-window between 72 and 96 hpf according to the standard fish embryo test DIN 38415-6 . Single cells were isolated from zebrafish embryos using a glass/glass tissue grinder and used in the comet assay as well as in the micronucleus assay . Maximum test concentrations causing no lethality were derived before using the fish embryo toxicity test . The results of the comet assay showed a significant increase of DNA-damage for both sediment extracts compared to the negative control after 72 hpf exposure . At present it was neither possible to determine a time-depending difference nor to measure a concentration-depending reaction . The results of the micronucleus assay will be available at the SETAC world-meeting in Berlin . To achieve the goals of the European Framework Directive it is of particular interest to establish a combined vertebrate-based sediment contact assay with different biological endpoints (e .g . embryotoxicity, genotoxicity and AhR-mediated toxicity) to determine the ecotoxicological effects of sediment-bound pollutants . This part of the DanTox project will help to identify and characterize genotoxicity of sediment extracts from the Rhine River and Hamburg Harbor .

TH 014The threshold approach for acute fish toxicity testing of formulated plant protection products: a proposed frameworkS . Creton1, J .R . Wheeler2

1NC3Rs, London, United Kingdom2Syngenta, Bracknell, United KingdomThe OECD has recently published guidance on the use of the threshold approach for fish acute toxicity testing . Essentially the approach uses a limit test at a single threshold concentration determined by the results of Daphnia and algae tests . If no mortality is observed in the limit test the fish acute value can be expressed as greater than the threshold value . However, if mortality is observed a full concentration-response test is triggered . To assess applicability to plant protection formulated products, Syngenta constructed a database of 185 products (fish, Daphnia and algae endpoints) and retrospectively applied the threshold approach . This representative dataset demonstrates that a reduction of approximately 38% of the fish might be achieved . Furthermore, the approach also provides a refinement as in 76% of threshold tests no mortality would be observed and in 64% of cases there would be no toxic effects at all, including sub-lethal symptoms . However, there is a need for regulatory acceptance of this method particularly in relation to the use of the data in risk assessment, where a fish specific assessment is required and approaches may require a better defined estimate of fish acute toxicity . Consequently, the UK NC3Rs held a workshop in December 2010, bringing together industry and a number of European regulators to discuss the applicability of the approach . The workshop discussed a potential framework for applying a threshold type approach, incorporating all the available information including relative fish sensitivity, data on the component active ingredients, formulated product information (Daphnia and algae) and potentially the Predicted Environment Concentrations . Such an approach may be used to better define a fish threshold approach that is workable within the EU regulatory framework for Plant Protection Products .

TH 015Different test conditions for hydrophobic and fugitive substances in the Fish Embryo Toxicity Test for a reliable risk assessmentA .C . Pott, S . Peddinhaus, H . Hollert, S .H . KeiterInstitute for Environmental Research, RWTH Aachen, Aachen, GermanyBackground: This study is part of the DanTox project, which aims to identify specific toxicity and molecular modes of action of sediment-bound pollutants and selected primary pollutants using zebrafish embryos . Ecotoxicological investigations of volatile and hydrophobic chemicals in tests with aquatic organisms are difficult to conduct, since they evaporate from the water and/or adsorb to plastic surfaces . This could lead to substance loss during exposure, which can cause an underestimation of their ecotoxicological hazard potential . In order to optimize test conditions for such chemicals, the aim of this study was to compare embryotoxic effects caused by selected chemicals under static and semi-static test conditions in glass and plastic vessels .

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Material and methods: The embryotoxicity of three selected substances (benzofurane, quinoline and chlorpyrifos) were tested in plastic and glass vessels under static and semi-static conditions using the fish embryo toxicity test with zebrafisch (Danio rerio) . For semi-static tests, the solutions were exchanged every 24 h . Embryotoxicity were observed after 24, 48, 72 and 96 h post fertilization (hpf) . Results and discussion: Benzofurane showed in glass vessels a significant higher mortality under static than under semi-static conditions after 72 hpf . In contrast, tests in plastic vessels had a slightly higher toxicity under semi-static conditions, which was not expected . Quinoline showed in glass vessels a slightly higher toxicity under static than under semi-static conditions and a slightly higher toxicity under semi-static conditions in tests conducted in plastic vessels . In contrast to benzofurane, quinoline showed minor differences in toxicity with increasing exposure time . Differences in toxicity between quinoline and benzofurane are probably based to different vapour pressure as well as Henry`s law constant . Chlorpyrifos showed only a low mortality (< 30 %) under all test conditions . Conclusion: Only some significant differences, but mostly just tendencies between the different test conditions were recorded . Thus, no precise recommendation can be given for a reliable risk assessment for selected volatile and adsorptive heterocycles . However, for further investigations chemical analyses are required to determine the difference between nominal and measured concentrations under all test conditions . Acknowledgment - the authors thank the Federal Ministry of Education and Research for financing this project .

TH 016A quantitative and mechanism specific toxicity assessment for the fish embryo test, using fluorescent zebrafishV . Delov1, E . Muth-Köhne2, A . Wichmann2, V . Schiller2, C . Schäfers2, M . Fenske1

1Fraunhofer IME, Aachen, Germany2Fraunhofer Institute IME, Aachen, Germany The zebrafish embryo toxicity test zFET is currently the most advocated animal alternative test in ecotoxicology . To date, the application of the zebrafish embryo toxicity test zFET has focused on acute toxicity assessment, where only lethal morphological effects are accounted for . An application of the zFET beyond acute toxicity, however, necessitates the establishment of more refined and quantifiable toxicological endpoints . A valuable tool in this context is the use of gene expression-dependent fluorescent markers that can even be measured in-vivo . The fluorescence visualises specific cells or morphological structures, which can help elucidating the underlying toxicological mechanisms and enhance the detection of adverse effects and the sensitivity and specificity of the conventional zFET . This project investigates the benefits of using fluorescent marker applications on zebrafish to study chemical toxicity on vascular, myotomal and neuronal development, thereby improving the zFET . The transgenic line Tg(fli1:EGFP)y1 expresses enhanced GFP in the entire vasculature under the control of the fli1 promoter, and thus enables the visualisation of vascular defects in live zebrafish embryos . We assess the fli1 driven EGFP-expression qualitatively and quantitatively, and found an exposure concentration related increase in vascular damage for chemicals like triclosan, cartap and genistein . The effects are now being confirmed by QPCR . We additionally explored whole-mount immunofluorescence to visualise myotomal defects, using a monoclonal antibody staining specifically myosin in the embryonic muscle fibres . These investigations showed a chemical concentration depended change in muscle morphology for cartap and genistein, what also could be quantified and declared as a robust parameter . Presently, another transgenic zebrafish line is being evaluated, expressing GFP coupled to the glial fibrillary acidic protein (GFAP) gene . The Tg(gfap:GFP) embryos promise the identification of neurotoxic chemicals in the zFET in-vivo, and first data will be presented . The use of fluorescent transgenic lines and antibody staining enables the detection of chemically induced vascular, myotomal and neuronal malformations in the embryos, which were otherwise not visible in bright field assessment . The fluorescent signal based methods allow a sensitive and quantitative effect assessment, which will broaden the scope and minimize the data variability of the zFET .

TH 017Toxicity of chlorine-produced oxidants to Arabian killifish embryos for acute developmental stage exposures S .S . Saeed, A .N . al-Naema, E .R . FebboExxonMobil Research Qatar, Doha, QatarFish embryos are excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible acute and chronic effects . Therefore, we present the use of Arabian killifish (Aphanius dispar) embryos, an indigenous species to the Arabian Gulf, to study the effect of chlorine-produced oxidants to marine organisms in the Qatari Coastal Area . The objective of this study is to develop chlorine toxicity data for the marine Fish Embryo Toxicity (mFET) test . The test is designed as a means to replace or refine the use of marine juvenile and/or adult fish in standard approaches evaluating toxicity of chemicals and effluents . Embryos were collected from a breeding stock of sexually mature Arabian killifish . Testing was initiated as soon as possible after fertilization of the eggs with exposure to aqueous concentrations of calcium hypochlorite for up to 240 hours . The investigated endpoints included; coagulated eggs, somite development, heartbeat, tail detachment, hatchability and post hatch mortality . The results showed a developmental stage-dependent response to chlorine . During earlier developmental stages, chlorine had reduced effects on the embryos and the survival rate and hatchability were high, even at relatively high concentrations . In later developmental stages (pre to post hatch, eleutheroembryo), the embryos were significantly more sensitive to chlorine than in the early stages of development . The chorion, (membrane surrounding the egg), is believed to provide a barrier against chlorine in early stages of embryo development . Taking into account the findings presented here, Killifish embryos exhibit the ability to be an indicator organism for environmental risk assessments of the Qatari Coastal Area . Benefits include, animal alternative, ease of fish breeding, clarity of the embryos, reduced sample size, reduced waste generation and shorter study duration .

TH 018Sub lethal effects of methyltestosterone in adult zebrafish (Danio rerio)C . Rivero-Wendt1, R . Oliveira2, T .P .S . Galindo2, I . Domingues2, M . Monteiro2, A .M .V .M . Soares3, C .K . Grisolia1

1Universidade de Brasília, Brasília, Brazil2Universidade de Aveiro, Aveiro, Portugal3University of Aveiro & CESAM, Aveiro, Portugal Methyltestosterone (MT) is a synthetic hormone used in fish-farming to induce male monosex in Oreochromis niloticus . In Brazil, the commercial hormone is illegally used as it is not regulated by national authorities for these purposes . In this study, we aim to evaluate the sub lethal toxicity of commercial MT (cMT) and its active ingredient 17α-methyltestosterone (aMT) to adult zebrafish . Comparison between the two compounds (cMT and aMT) is relevant for the regulation of MT in Brazilian aquaculture . For this purpose, micronucleus and other nuclear abnormalities were chosen as genotoxicity markers and measured on peripheral erythrocytes cells of zebrafish and cholinesterase- ChE, lactate dehydrogenase- LDH and glutathione-S-transferase- GST were chosen as biochemical markers and measured in different fish organs . Fishes were exposed during 4 days at concentrations between 4 and 5000 ug/L of cMT or aMT .

Results showed that the most responsive biomarker tested was GST with a dose-dependent inhibition verified in several organs . GST, as a family of enzymes involved in the general process of detoxification of xenobiotics with electrophilic centers, is expected to be induced under situations of stress; however inhibition, as verified in the present study, is also described in literature for several types of compounds . Differences between males and females observed concern not only the type of tissue that was responsive but also the sensitivity (in general males were more sensitive than females) . Regarding MT types, cMT seems to elicit a response at lower doses . This trend was also observed for head ChE for which an inhibition was observed (indicating a possible disruption of nervous system by MT) although a dose dependent pattern was not observed . LDH response presented high variability and thus, no conclusions can be drawn on the effects of MT in the anaerobic pathway of energy production . Overall, results indicate that important effects of MT exposure are observed at sub lethal level that should be assessed in more detail in a perspective of risk assessment of MT use to non-target organisms . Moreover, differences between males and females responses should be taken into account, since different physiological mechanisms of response may be occurring in aquatic organisms affected by MT effluents . Finally, the apparent higher effectiveness of cMT should be carefully assessed so that proper legislation can be established for MT use in aquacultures .

TH 019Comparative sensitivity of different early life stages of zebrafish to copper and NaCLT .S . Andrade1, B . Santos1, I . Lopes1, A .M .V .M . Soares2, I . Domingues1

1Universidade de Aveiro, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, Portugal Aquatic contamination is a major constrain to the health of fish in freshwater ecosystems . Especially metals, such as copper, can be extremely toxic even at low concentrations . Additionally, due to climate change, future environmental alterations are expected as it is the case of increasing salinity derived from sea level rise, which can also affect aquatic ecosystems equilibrium . Furthermore, during their early development fish suffer several morphological and physiological modifications and thus, it is crucial to make a proper evaluation to investigate how these stress factors can affect the different life stages . Accordingly, this work aimed to assess the sensitivities of different early life-stages of Danio rerio to copper and to NaCl (to simulate increased salinity) . For this, embryos (2h hpf) and larvae (96h hpf) of zebrafish (D . rerio) were exposed to copper sulphate and NaCl and several morphological and functional endpoints were monitored during a 96h period . In the first experiment, embryos were exposed to copper (4 .78 - 39 .88µM) and NaCl (1 .85 - 26 .27g/L); in the second experiment, larvae were exposed to the following range concentrations of copper (0 .45 - 4 .7µM) and NaCl (5 - 8 .75g/L) . Dose dependent mortality was observed for both stages under exposure to copper and NaCl, however, the larval stage was more sensitive to copper and to NaCl than embryos . Moreover, in the embryo assay with NaCl, it was only observed significant mortality after the 96h . The protective enclosure of embryos could be one of the major factors contributing for their highest resistance . Even so, both copper and NaCl were responsible for other significant sublethal effects on embryos . Copper had an inhibitory effect on hatching and caused a reduction on body length and on the other hand, NaCl caused several tail deformities and edemas . These adverse effects on embryos can, as mortality, compromise the long term survival of these organisms . It is important to note that copper was the stress factor where the differences between sensitivity of embryos and larvae were more pronounced . Obtained results suggest that toxicity of copper and NaCl can be very different depending on the life stage . Also, several sublethal effects must be taken into account to better evaluated the real toxicity of these compounds to zebrafish .

TH 020Arabian killifish: An indicator model for laboratory environmental risk assessments of the Qatari coastal areaN .M . al-Naema, S .S . Saeed, E .F . FebboExxonMobil Research Qatar, Doha, Qatar

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Fish embryo toxicity (FET) studies can provide an alternative to traditional fish acute toxicity testing . FET methods offer many benefits; they are considered animal alternatives, they require relatively small sample size and reduced time to determine toxicity endpoints . The use of Arabian killifish (Aphanius dispar) embryos is proposed as an alternative to using juvenile or adult fish for environmental risk studies in the state of Qatar . The Arabian killifish are small fish about (5 -6 cm) total length, from the family cyprinodontidae . Given the lack of data regarding Arabian killifish embryology and breeding behavior, research was initiated in early 2010 to establish a laboratory fish culture capable of producing high quality embryos in sufficient quantity for toxicity studies . Another important objective was to identify the developmental stages for the Arabian killifish and to understand their relative sensitivity to more established FET species . The standard FET approach that uses Zebrafish (Daniorerio) or Medaka was adapted to the Arabian killifish . Behavioral patterns, feeding optimization, diurnal cycle, male to female ratio as well as other factors were studied in the laboratory . Embryo development was observed and documented from fertilization to hatch . Reference toxicant studies were performed and the results compared to other species . A reproducing laboratory culture was established . Embryo development was studied and the key development stages were identified . The results of the reference toxicant testing confirmed similar sensitivity with other species . We have been successful in the production of quality embryos for testing, both in terms of viability (fertilization) and quantity released . The developmental stages of the Arabian killifish have been identified to provide an array of potential endpoints for ecotoxicity studies . Organism sensitivity appears to be within the desired range for vertebrate species . We conclude that the Arabian killifish exhibits the traits needed for successful FET testing and that they have utility as a vertebrate indicator species .

TH 021Exploring the relationship between initial test medium concentrations and accumulation of organic chemicals in cells/tissues in in vitro toxicity experiments using an equilibrium partitioning modelJ .M . Armitage, J .A . Arnot, F . WaniaUniversity of Toronto at Scarborough, Toronto, CanadaReliable toxicity data are a crucial requirement for conducting hazard/risk assessment of contaminants in the environment . However, data are limited or non-existent for a large number of contaminants of potential concern (1) . Due to the high cost, time requirements and ethical considerations surrounding in vivo animal experiments, it is quite likely that in vitro toxicity data will play an increasingly prominent role in risk assessment and setting of exposure guidelines in the future (2,3) . While in vitro data can almost always provide important mechanistic insight (e .g . mode of action), direct use of such data for assessing the toxicological implications of measured contaminant levels in humans and wildlife requires that cellular concentrations be characterized (as opposed to the initial test medium concentrations, which are typically reported) . Several in vitro studies have noted that measured cellular concentrations of hydrophobic chemicals are substantially higher than the initial test medium concentrations and stress the importance of relating external to internal levels (4-7) . These findings can be rationalized in the context of the phenomenon of bioconcentration, whereby organic chemicals partition preferentially into biological phases (such as lipids) at the expense of the aqueous phase . Other partitioning behaviours of potential relevance in the context of an in vitro toxicity test include sorption to the vessel walls, volatilization into headspace and interactions with serum constituents (e .g . proteins) or other dissolved organic matter . To explore the behaviour of organic chemicals in such test systems, these sorption relationships were integrated into a mass balance equilibrium partitioning model which was then applied to a generic test system for chemicals covering a range of physical-chemical property values . Key model outputs include mass distribution, cellular concentrations, aqueous phase concentrations (in comparison to water solubility limit) and external:internal concentration enhancement factors as a function of hydrophobicity . The influence of assumptions regarding the affinity of cells for chemicals (i .e . sorption capacity), and mass of cells:volume of aqueous phase were also explored . While the results are illustrative in nature (i .e . cannot be directly extrapolated to any particular study), they provide useful insights into experimental design (e .g . how to avoid super-saturating the aqueous phase) and interpretation of results .

TH 022Quantification of chemical concentration in multiwell plate-based assays: requirements, possibilities and challengesK . Tanneberger1, M . Knöbel1, F .J .M . Busser2, J .L .M . Hermens2, S . Scholz3, K . Schirmer1

1Eawag - Swiss Federal Insitute of Aquatic Science and Technology, Dübendorf, Switzerland2Institute for Risk Assessment Sciences, University of Utrecht, Utrecht, Nederland3UFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyThe use of animals for experimental studies, e .g . toxicity testing of chemicals, gives rise to serious ethical concerns . The development of new alternative procedures and models is therefore timely . Among the most promising alternative methods are cell line- and fish embryo-based assays . Both approaches are commonly conducted using multi-well plates, making them amenable to high throughput screening . Many replicates and very little testing material is required . However, these formats also come with caveats . Because of the small volume, even minute manipulations within culture wells, such as the addition of a test chemical, can be expected to have a significant impact on the dynamics within the well . The high surface to volume ratio, combined with the open nature of the systems, favor losses of test chemicals due to sorption and/or evaporation, leading to a reduced bioavailability of test chemicals . Until today, confirmation of chemical concentrations in culture wells has rarely been done . Although, e .g . the OECD draft guideline for the fish embryo toxicity test requires to check that stable chemical concentrations are maintained during the whole exposure duration . Therefore, we set out to design protocols for sampling, sample preparation and chemical analysis applicable to the small volumes present in multi-well plates for cell line- and embryo-based toxicity assays . Chemical concentrations were determined for stock solutions, dosing mixtures and the exposure medium at the beginning and at the end of exposure taken directly out of the wells . In general, a volume of 500 µL was required for chemical analysis and 24-well plates have been found very suitable to comply to the requirement of sufficient exposure medium . Analytical protocols were developed for 30 industrial organic chemicals . For 20% of the tested compounds the samples could be subjected directly to quantification based on high-pressure liquid chromatography (HPLC) without any further treatment required . For the remaining compounds, simple liquid-liquid extraction of the aqueous sample with cyclohexane was performed prior to analysis by either gas chromatography, HPLC or mass spectrometry . For acute exposures of cells or embryos, accounting for measured chemical concentrations helped to correct for the reduced chemical bioavailability . This resulted in an improved absolute relationship between cell- and embryo-based assays versus acute fish toxicity .

TH 023Characterising the bioavailability, toxicity and metabolites of benzo[a]pyrene using animal alternatives bioassayM .T . Hultman1, K . Schirmer2, I . Allan1, B .O . Rosseland3, K .E . Tollefsen1

1NIVA, Oslo, Norway2EAWAG, Dübendorf, Switzerland3UMB, Ås, NorwayIn vitro methods is anticipated to provide alternatives to animal testing by implementing the 3R`s (reduction, refinement and replacement) . Several in vitro methods have proved to be versatile tools for toxicity assessment and have shown a potential for high throughput screening of chemicals . Primary hepatocytes isolated from livers are commonly used in toxicity testing and are being used for assessment of the bioaccumulation properties of chemicals due to retaining inate cellular properties such a toxicity targets and biotransformation capability . Although the cells may represent alternatives to use of whole animals in some respects, culturing and exposure of cells in high-throughput formats devoid of the natural toxicokinetics and partition behaviour may introduce experimental artefacts giving rise to deviations from in vivo conditions . This applies in particular to compounds that are volatile, hydrophobic and require metabolic activation (bioactivation) which today represent challenges to in vitro toxicity assessment . The aim of the present work was to assess the role of bioassay conditions on the bioavailability and toxicity of benzo[a]pyrene (BaP) in a primary culture of rainbow trout (Onchorhyncus mykiss) hepatocytes . Assessment of cytotoxicity and classical biomarkers such as EROD induction and CYP1A gene and protein expression was used to characterise the responses to this hydrophobic pro-mutagen and carcinogen under different bioassay conditions . 14C-labelled BaP was used to determine the partition behaviour and truly bioavailable fraction of BAP under the different bioassay conditions used .

TH 024Cytotoxic, mutagenic and genotoxic effects of sediments from Czech river basinM . Benisek1, K . Hilscherova1, L . Blaha1, H . Hollert2

1Research Centre for Toxic Compounds in the Environment (RECETOX), Brno, Czech Republic2Institute for Environmental Research (Biology V), RWTH Aachen, Aachen, GermanyThis study summarizes various effects of 14 sediments collected from rivers in Southern Bohemia (Czech Republic) . After Soxhlet extraction, chemical analyzes of sediment extracts was performed and extracts were further used in toxicity experiments . Fish hepatoma cell line RTL-W1 was used for neutral red cytotoxicity assay and for micronucleus test of genotoxicity . Mutagenicity of tested samples was studied by Ames fluctuation assay . Most of studied sediment extracts was cytotoxic for RTL-W1 cells, however, only one of them was significantly genotoxic in micronucleus test . Some of tested sediment extracts were mutagenic in Ames fluctuation assay with added S9 fraction . No or only minor effects were seen in this test without S9 addition . Results from toxicological experiments will be further compared with chemical data of sediments and then full results of this work will be presented . Supported by the projects CETOCOEN (CZ .1 .05/2 .1 .00/01 .0001) and by DAAD

TH 025Development and characterization of a piscine intestinal barrier model using the fish cell line RTgutGCC . Drieschner, N . Bramaz, K . SchirmerEawag, Dübendorf, SwitzerlandBackground . At present, fish in large number serve as experimental animals for the testing of chemicals and water samples . According to the 3R principles (replace, reduce, refine), our aim is to develop in vitro systems which are based on continuous cell lines from rainbow trout (Onchorynchus mykiss) . For the present study, the intestinal cell line, RTgutGC, was used to develop an intestinal barrier model for fish . Therefore, cells were grown on filter membrane inserts such that they face two compartments . The compartmentalization allows to create asymmetric exposure conditions as found in vivo to provide an environment in favor of cell polarization and differentiation into mature enterocytes . Enterocytes is the main cell type of the intestinal epithelium and responsible for the absorption of nutrients . The cells are characterized by the expression of a brush border membrane and tight junctions in vivo . Tight junctions are proteins which join adjacent cells and thus create tight epithelia which present a limiting factor for intercellular toxicant transport . Methods . The tightness of the developed epithelium was judged by means of transepithelial electrical resistance measurement (TEER) . The presence of tight junction proteins was analyzed by immunostaining . Electron microscopy served to prove the state of differentiation of the cells cultured according to different protocols . Results . Cells were found to build up a multilayer when seeded at high densities (446429 cells/cm2) while seeding of low densities (62500 cells/cm2) led to the desired

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monolayer formation . TEER values were shown to depend on cell density . For seeding of high densities, values were in a range between 30 to 70 Ω*cm2 . For seeding of low densities, TEER measurement resulted in values between 10 to 30 Ω*cm2 . Immunofluorescence labeling of ZO-1, a protein of the tight junction protein complex, was successful . Cell differentiation, as judged by morphological features, was however not achieved by culturing the cells under asymmetric conditions and by providing them with a selection of substances described to be involved in intestinal cell differentiation . Outlook . Future studies will focus on strategies to induce cellular differentiation in RTgutGC . The starting-point will be the development of a fish cell specific culture medium, because the currently used culture medium is based on mammalian serum which might inhibit piscine cell differentiation .

TH 026DNA repair capacities in fish cell lines RTL-W1 and RTGill-W1 and application of a base excision repair comet assayA .E . Kienzler1, X . Tronchère1, A . Devaux2, S . Bony2

1Université de Lyon, UMR LEHNA 5023, Vaulx-en-velin, France2USC INRA IGH, ENTPE, Vaulx-en-velin, FranceIn vitro models such as fish cell lines are possible alternative to in vivo fish testing, and new toxicity endpoints such as genotoxicity deserve to be considered in these models . However fish cell lines are suspected to possess low DNA repair activities, which could influence their sensitivity in genotoxicity testing . The aim of this study was to evaluate in RTL-W1 and RTGill-W1 cell lines, the repair kinetics of alkylation damage following exposure to methyl methane sulfonate and recovery with or without 3-aminobenzamide (3AB), a BER inhibitor . Both standard and Fpg-modified comet assay have been used . In a second step, a Base Excision Repair comet assay (BERc) has been applied and validated in those cell lines . Its principle is to evaluate the BER activity of cell extract by applying them after the lysis step of the comet assay on a substrate DNA containing a high amount of BER specific lesions . The ability of the extract to generate additional breaks in the DNA at damaged sites is a measurement of the BER activity in the cell extracts . A significant difference in the level of residual damage in cells recovering with or without 3AB demonstrated BER capacities in both cell lines . The time-course of SSB and DSB repair was similar in the two cell lines and showed a return to the background DNA damage level within 24 hours . The repair of the Fpg-specific lesion follows a similar time-course . Preliminary experiments allowed us to establish optimum conditions for the BERc assay (density of cell extract, digestion step duration, adequate freezing and storing conditions) . The application of the established protocol to both cell lines revealed the efficiency of the initial BER step in those cell lines without significant difference . Exposure to 3AB generated a significant decrease in the incision capacities of cell extracts confirming the specificity of the assay regarding BER activity measurement . Results show that RTL-W1 and RTG-W1 exhibit measurable BER capacities in the same range, and that repair kinetics followed by standard and Fpg-modified comet assay as well as BERc assay are appropriate methods to measure DNA repair in fish cell lines . The BERc assay is however much easier to perform, although it allows to measure only the initial step of DNA repair . Complementary work is in progress in order to validate the use of the BERc as a new genotoxicity biomarker allowing the assessment of the impact of pollutant exposure on DNA repair capacities .

TH 027Application of PLHC-1 as in vitro bioassay for Ah Receptor active compounds in environmental matricesC . Della Torre1, I . Corsi1, M . Mariottini1, A . Malysheva1, S . Focardi2

1Siena University, Siena, Italy2University of Siena, Siena, ItalyThe present study investigates CYP1A response in Poeciliopsis lucida hepatoma cell line (PLHC-1) exposed to soils extracts by looking at gene transcription and EROD activity . The aim is to evaluate the potential application of this fish cell lines as in vitro model for the toxicological screening of environmental samples . Soil samples were collected in two sites (A, B) close to an industrial incinerator . Samplese were extracted two fractions (Non Purified and Purified) were collected . GC/MS analysis was performed to detect levels of priority pollutants and TEQs were calculated . Modulation of cyp1a gene transcription was measured in cells exposed for 24 h to a model CYP1A inducer as TCDD (1,10,50 pM) and to two dilution of tested exctracts (1,10 mg/ml for P and 0 .5, 5 mg/ml for NP) . EROD activities were measured in cells exposed for 24 h to a range of dilution of TCDD (0-10 nM) and to tested extracts (0-50 mg/ml for P and 0-10 mg/ml for NP) dissolved in DMSO (max 1%) . EROD TEQ values were also calculated .TCDD determined a dose-dependent up-regulation of cyp1a gene tanscription 15-folds respect to controls at 10pM and 109-folds respect to control at 50pM, thus confirming the sensitivity of the adopted model . A significant up-regulation of cyp1a was observed only at the highest concentration for the purified fraction of both samples A and B (2 .1 and 1 .8 folds respectively) . The non purified fraction of both samples determined a significant dose dependent up-regulation of cyp1a from 2 .4 to 15-folds up-regulation for sample A and from 2 .1 to 13-folds for sample B . Concerning EROD activity the EC50 measured for TCDD was 17 .96 pM . A similar tend of EROD induction has been observed for A NP and B NP, while no modulation of EROD by A P was observed and only a slight induction was observed for B P (maximum of 7 .4 pmol/min/mg prot at 50 mg/ml) . A higher EROD TEQ values was calculated for A NP compared to B NP . Overall results confirm PLHC-1 cells as highly sensitive model to AHR active compounds as significant effects were observed even at low levels of contaminants . Results from bioassays does not agree with TEQ values calculated based on chemical analysis suggesting that compounds other than PCDDs/PCDFs heavily contribute to CYP1A induction at both gene and activity levels . At low contaminants concentrations EROD activity resulted more sensitive than cyp1a gene .

TH 028Use of AhR superinducers for potential improvement in the micro-EROD assayK .W . Schramm1, W . Levy1, B .M . Brena2, G . González-Sapienza2, S . Bernhoeft1

1Helmholtz Zentrum München, Neuherberg, Germany2University de la República, Montevideo, UruguayThe micro-EROD bioassay allows the determination of dioxin-like compounds in samples of diverse origin by induction of the cytochrome CYP1A1 activated through the Aryl hydrocarbon receptor (AhR) . Superinducers are compounds that are not able to bind to the AhR, but increase their response magnifying the expression of cytochrome P4501A1 by inducing the AhR to higher activity . In this work, three compounds known as superinducers; cycloheximide (CHX), MG132, and Dexamethasone (DEX) are studied in the EROD bioassay . The enzymatic activity of the cytochrome P4501A1 is used to detect the activation of the AhR in response to these compounds, potential improvers of the sensitivity in the micro-EROD bioassay . The bioassay is carried out with hepatic cells able to express the CYP1A1 and different incubation and pre-incubation concentrations of superinducers before following standard procedures . Additionally, RT-PCR is performed for cells incubated with CHX in order to quantify the potential CHX superactivation . The results indicate that under the studied conditions, the CHX and MG132 “superinducers”did not improve the performance of the bioassay . Nonetheless, DEX showed to be a promising compound promoting the CYP1A1 expression in the EROD bioassay . Further studies in the signalling mechanism of DEX in the AhR response and its possible improvement in the micro-EROD bioassay sensitivity are now remaining to be performed .

TH 029Evaluation of epigenetic DNA methylation in a zebrafish (Danio rerio) cell line modelE .F . Farmen, M .T . Hultman, M . Anglès d’Auriac, K .E . TollefsenNorwegian Institute for Water Research, Oslo, NorwayGenetic imprinting has been shown to be an early molecular event with strong correlation to adverse disease, thereby emphasizing the potential of DNA methylation as a potential environmental biomarker . However, the study of multigenerational epigenetic DNA modifications is a novel approach in ecotoxicology, and experiments can be both time and labour consuming . Therefore the potential of a high throughput in vitro screening method was assessed by exposing a zebrafish liver cell line ZF-L to various non-mutagenic model substances . Methylation of CpG islands of selected genes were monitored by bisulphite conversion and high resolution melt analysis after short term exposure to sub-lethal concentrations . The basal methylation level in unexposed ZF-L cells was close to the 100 % standard for some genes and close to 0 % for others . Exposure of ZF-L cells to 0 .5 µM 5’-Azacitidine lead to hypomethylation of genes with both low and high basal methylation, indicating similarity to mechanism in mammals . The genes selected for analyses were genes with putative importance in different cancers, and the significantly hypomethylated genes were in insulin like growth factor binding protein 1b, angiopoietin-like 3, glutathione S-transferase P1 and cyclin-dependent kinase inhibitor 2A . The sensitivity of the HRM methylation analysis was shown to detect methylation differences down to one CpG, based on clear separation of 0 % and 10 % methylation in an amplicon containing 10 CpG sites . Validation of the HRM assay will be shown by comparing to sequencing of the amplicons from bisulfite treated templates . It is concluded that cell line exposure in combination with HRM may provide an initial screening assay for CpG methylation with high throughput capacity .

TH 030Development of 3D fish hepatocyte spheroid cultures as an in vitro tool for environmental chemical toxicityG .J . Baron1, N . Jha1, M . Purcell1, K . Jackson1, F . Owen2

1Plymouth University, Plymouth, United Kingdom2Brixham Environmental Laboratory, Brixham, United KingdomThree-dimensional organotypic cell culture models (spheroids) provide significant advantages in pre-clinical and toxicological research . Specifically in the fields of pharmaco-toxicology and carcinogenesis, the model offers key aspects of the in vivo environment that are superior to standard monolayer models . While spheroid technology is now established for mammalian toxicity studies, to date it has not been considered for environmental applications in model aquatic species . Currently, whole organisms (fish) are a mainstay of environmental toxicity monitoring . While fish primary cells and cell lines are relevant to the assessment of chemical toxicity and the evaluation of environmental contaminants, monolayer cultures of these cells have significant limitations such as short culture periods and loss of functionality . The present study describes the development and application of 3D fish hepatocyte spheroid cultures for the assessment of toxic environmental contaminants . The liver-specific functions and toxicity endpoints of this model suggest that fish spheroids could be suitable as a new organotypic in vitro screening tool for use in environmental toxicological monitoring .

TH 031In vitro kinetics of chlorpromazine in cytotoxicity assays with Caco-2, Balb/c 3T3 and HepaRG cellsJ .J .W . Broeders, N . Timmer, B .J . Blaauboer, J .L .M . HermensInstitute for Risk Assessment Sciences - Utrecht University, Utrecht, NederlandDifferent in vitro alternatives are available to test the toxicity of compounds . For these assays, different cell types can be used . Cell viability is usually determined after the cells have been exposed for 24-72 hours . However, the different cell types need different culture and assay conditions, including the addition of serum to the medium . The

24�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

aim of this project, part of the European project Predict-IV, was to study the biokinetics of the pharmaceutical chlorpromazine (CPZ) in three different cell assays .The cells used for the assays were i) the human intestinal cell line Caco-2 cells, ii) mouse fibroblast Balb/c 3T3 cells and iii) the human liver cell line HepaRG cells . The Balb/c 3T3 cells were cultured for 24 hours and the Caco-2 cells for 1 week . The HepaRG cells were cultured for 4 weeks, with 2%DMSO in the medium during the last 2 weeks . The cells were exposed to different concentrations of CPZ (98 .8% ionized at a physiological pH) for 48 hours . After different time points, samples were taken from the medium, cells and well plastic . In addition, the viability of the cells was measured by the Alamar Blue method . The freely available CPZ concentration was measured by nd-SPME and all samples were measured by HPLC-UV .In the medium samples, a decrease in the amount of CPZ was seen over time; the most pronounced decrease was seen in the metabolically competent HepaRG cells . Plastic binding of CPZ was negligible in all three cell systems . A complete recovery of CPZ was found in the Caco-2 and Balb/c 3T3 cells . In the HepaRG cell assay, less parent compound was recovered .The Alamar Blue assay showed that the Balb/c 3T3 cells were the most sensitive to CPZ based on the nominal concentration . However, protein binding influences these results; therefore, the free concentration is an important factor .In conclusion, chlorpromazine showed differences in biokinetics in the three cell assays . Thus, this study shows that it is important to take the freely available concentration of the compound and its biokinetics into account for a reliable interpretation of cytotoxicity results . TH 032The luminescent bacteria toxicity assay: 20 years of interlaboratory round-robinsM . Ribo1, M .C .A .R .M Riva2

1TEchnical University of Catalonia (UPC), Terrassa, Spain2Technical University of Catalonia (UPC), Terrasa, SpainThe luminescent bacteria toxicity assay was developed in the early 1980s, to provide a reliable system able to confirm compliance of wastewater discharges . Since the early days, numerous studies were done and have been published to demonstrate correlation of traditional aquatic toxicity results with results from this assay . The assay has been used to establish a coherent database of comparable toxicity data that has been published, proved of practical use, and also used in Quantitative Structure-Toxicity Relationships worldwide . The assay is based on the inhibition of bacterial luminescence caused by the presence of contaminants in a water sample . Turbidity and color may interfere in the luminescence detection, and water samples must be clear, colorless and transparent . In spite of this disadvantages, the assay is being used worldwide for water quality monitoring . As in other toxicity bioassays, in this case different types of contaminants affect differently the enzymatic inhibition resulting in the reduction of light emitted by the bacteria . It has been observed that some considered chemical contaminants may potentiate the bacterial thus resulting in a “negative toxicity” . The observed light reductions may be the result of several different biological mechanisms depending on the chemical nature of the contaminant . In view of all this questions we started in 1993, the organization of a yearly interlaboratory comparison exercise linked to an open forum, to improve the existing knowledge on the advantages and restrictions of this assay, and its response when used to determine the aquatic toxicity of different types of contaminants . An output from these interlaboratory round-robins has been the study of the inter- and intra- laboratory variability of the results obtained, proving the reliability of the assay, and contributing to the quality assurance practices of the luminescent bacteria assay users . In the course of these round-robins the toxicity of several reference substances was determined using the luminescence bacteria assay and in this communication we present a summary of the results collected . Several hundreds of values from toxicity determinations confirm that this assay provides reliable results in a simple, quick and inexpensive way .

TH 033The on-line water toxicity monitoring system based on the luminescent bacteria toxicity assayM . Ribo1, J . Appels2, S . Gonzalez3, R . Lopez-Roldan3, M .C .A .R .M Riva4

1TEchnical University of Catalonia (UPC), Terrassa, Spain2Microlan, BV, Waalwijk, Nederland3Cetaqua, Cornella, Spain4Technical University of Catalonia (UPC), Terrasa, SpainWater quality monitoring has been a topic of many studies and discussions regarding the virtues and disadvantages of analytical chemistry and biological tests . Every day smaller amounts of more and more substances are being detected in water samples, providing increasing numbers of analytical data, whereas aquatic toxicity bioassays simply determine the combined biological effect that water contaminants may cause to laboratory test organisms . Although continuous physicochemical analysis is nowadays possible using commercial equipment, water monitoring results are usually based on discrete or continuous samples taken periodically to the testing laboratory . In this work we present the results of the study on the performance and reliability of an on-line, continuous biomonitoring water system based on the bacterial luminescence toxicity assay . The Microtox Toxicity Analyzer® uses, since the 1980s, luminescent bacteria as test organism and has proven a simple, reliable, and quick method to evaluate the toxicity of aqueous samples . The TOXcontrol biomonitoring system® (developed by microloan BV in 2006) uses also luminescent bacteria as the test organism to evaluate, on-line, the toxicity of a flowing water supply . The system examines every 30 minutes a sample of the flowing water and collects data on its toxic effect to the bacteria . The software can also measure toxicity of a reference sample collecting EC50 values of chemical contaminants, in a similar way as the Microtox Toxicity Analyzer . Experimental studies were performed to verify and validate the results produced by the TOXcontrol system, in comparison with results from the Microtox Toxicity Analyzer, using samples of several reference substances and also water from the intake of a drinking water treatment plant (DWTP) and also the effluent from a wastewater treatment plant . As expected, water samples showed low toxicity to luminescent bacteria in both systems, but the TOXcontrol ® online system can detect peaks of unwanted levels of toxicity as a warning system in both cases . It can successfully prevent low quality water from entering the DWTP, and the discharge of treated wastewater to receiving water when not in compliance with current regulations . The work done with several reference substances, and comparison with the results from the Microtox Toxicity Analyzer, confirms the reliability and acceptable variability of the toxicity data collected by the TOXcontrol system .

TH 034Estimation of pectins to be used as sorbents towards bacterial biosensor A . Kaniskin1, A . Terekhova2, G . Zakharova3, M . Aytkuliev1, A . Kydralieva4

1Moscow State University, Moscow, Russian Federation2Institute of Ecology and Evolution RAS, Moscow, Russian Federation3Moscow Aviation Institute, Moscow, Russian Federation4Institute of Chemistry & Chemical Technology, Bishkek, KyrgyzstanNatural polymeric material like chitosan, humics and pectin have been gaining interest as potential adsorbents of heavy metals because of their nature-abundant and biodegradable (1-3) . Moreover, due to the polyfunctionality they can be used in the chelation remediation in the treatment of metal intoxication . Owing to the presence of free carboxyl groups in the monomeric fragments of the polymer backbone, the pectin polysaccharides react with metals to form stable low-dissociating compounds, chelates . But, pectin to be used as a chelating/binding agent in the treatment of metals should be beforehand estimated on own toxic side effects . This study is aimed to estimate the toxicity for two samples of pectins - beet- and apple-derived - in the bioassay test towards luminescence bacteria . Luminescence bacteria bioassay is a rapid, inexpensive (in contrary of the test on living animals), reliable method to identify toxic . Toxicity analysis of the pectin samples were conducted following the standardized Ecolum protocol (analog of Microtox®) in which genetically modified E. coli cells were exposed to serial dilutions of pectin solutions . The luminescence inhibition after 30 min . was measured by the Biotox-10M analyzer (NERA, Moscow, RF) . Toxicity indices (T) were calculated with the formula T=(I0-I/ I0)x100, where I0-bioluminescence in control probe, I - in the testing probe . The data obtained was used to calculate the EC50- the median pectin concentration that caused a 50% reduction in bioluminescence, and NOEL (no observed effect level)- the pectin concentration that caused a reduction bioluminescence up to T<20 . Both the studied pectin preparations in concentrations >0,05% decreased bioluminescence intensity . At concentrations 0 .05% (ADS mass wt .) for apple-derived pectin and 0 .005% (ADS mass . wt) beet-derived pectin no toxicity were registered . Among samples examined, beet-derived pectin showed the higher toxifying ability . NOEL value of beet-derived pectin as 0,0086% was lower than NOEL value of the apple-derived pectin as 0,17% . This fact was seemingly connected with content of functional groups, esterification degree and molecular mass of the used preparations . Acknowledgements . The authors thank the ISTC KR-1880 and FTP MES RF-GK 02 .740 .11 .0693 for financial support . (1) S .K . Bajpai, M .K . Armo . 2009 . Journal of Applied Polymer Science .114:475-483 (2) F . Nuria, et al . 2005 . Sep Sci Technol . 40:1013 (3) G . Karthikeyan, et al . 2005 . J Chem Sci . 117:663

TH 035A resin buffered nutrient solution for controlling metal speciation in the algal bottle assayE . Smolders1, L . Verheyen2, R . Merckx2

1K .U .Leuven, Heverlee, Belgium2Division of Soil and Water Management, K .U .Leuven, Heverlee, BelgiumThe metal speciation in solution is uncontrolled during algal growth in the traditional algal bottle assay . The metal speciation will vary during the test as a result of the uptake of the metal by the green algae . A resin buffered nutrient solution was used to address the issue . This method was applied to test the effect of complexes on cadmium (Cd) uptake by the green algae Pseudokirchneriella subcapitata . Standard nutrient solution was enriched with the anorganic ligand chloride (40 mM) or with synthetic ligands . A reference nutrient solution with the same ionic strength and the same basic composition was used . The chloride treatments were prepared with the cation exchange resins Amberlite-CG120 (Rohm & Haas comp ., 4 .4 mmolc g-1) to contain equal Cd2+ (pCd=10; pCd=-log(Cd2+)) but varying dissolved Cd . The synthetic ligand treatments were prepared in the presence of Chelex®-100 (Bio-rad, sodium form, 0 .62 mmolc (g wet weight)-1) to obtain equal free Cd2+ activities (pCd=8 .2) in solution . The nutrient solutions and its references were subsequently used in an algal assay in 100 mL beakers containing solution with a cation exchange resin (+R) as a deposit at the bottom of the beaker . Beakers containing solution only (-R) were taken for the chloride treatments . The resin had been equilibrated with the solution prior to the assay . Pseudokirchneriella subcapitata was grown for 72h in stagnant solution and shaken three times a day . Growth was unaffected by the presence of the resin (p>0 .05) . The Cd

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concentration in solution of the -R devices decreased 50-58 % of initial values . The Cd uptake by the green algae was two times higher in the chloride -R treatment than in its reference solution . The metal uptake in the cells in the +R treatments was unaffected, despite a larger total Cd concentration in solution in the ligand containing solutions . This confirms that Cd2+ is the preferred ion and that Cd complexes do not contribute . In contrast, Cd uptake in the -R devices was two-fold larger in the NaCl treatment than that in the NaNO3 treatment (p<0 .001), suggesting that CdCln2-n complexes are bioavailable . The bioavailability of the complexes in the traditional algal assay is apparent only because the complexes act as a buffer of the free metal ion in solution . Resin buffered solutions may be advocated to better identify the role of metal complexes on bioavailability to primary producers because free metal ion activity is buffered by the resin .

TH 036Effects of heavy metals on free-living nematodes: a multifaceted approach using growth, reproduction and behavioral assaysL .M . Monteiro1, M .B . Brinke2, G .A .P . Dos Santos3, W . Traunspurger4, T .M . Moens5

1Bielefeld & Gent University, Gent, Belgium2University of Bielefeld, Department of Animal Ecology, Bielefeld, Germany3Federal University of Pernambuco, Centre of Biological Sciences, Recife, Brazil4University of Bielefeld, Dept of Animal Ecology, Bielefeld, Germany5Gent University, Department of Biology, Marine Biology Section, Gent, Belgium Toxicity tests, using single species bioassays, are a promising way to understand the biological effects of toxicants . The aims of this study are: (a) to assess sublethal (0 .01 to 1mg/L) effect concentrations of lead, zinc and nickel for the free-living nematode Caenorhabditis elegans using a liquid assay focusing on growth and reproduction as endpoints . And (b) to test a behavioral assay with taxis to food as an endpoint, focusing on the same metals (lethal and sublethal concentrations) . The liquid assays with C. elegans showed that only the highest tested concentrations of Pb, Zn and Ni caused a significant negative effect on growth and reproduction . For the behavioral assays, we compared an approach in which the pollutant was only present in the food spots with one where the pollutant was homogenously distributed in the agar medium . The results at lethal metal doses differed according to the metal as well as to the type of assay . For instance, C.elegans almost completely avoided Zn-contaminated food patches, and less nematodes reached the attractant spots on the polluted agar experiment . Sublethal concentrations of Ni resulted in a preference for contaminated food-spots than to “clean”E . coli . Our results show that behavioral assays may provide important insights complementary to those of survival, growth and reproduction tests .

TH 037Screening of soils using an avoidance assay with springtailsM . Ribo1, J .A . Domenech2, S . Latres3, M .C .A .R .M Riva4

1TEchnical University of Catalonia (UPC), Terrassa, Spain2Waste Agency of Catalonia ., Barcelona, Spain3Waste Agency of Catalonia, Barcelona, Spain4Technical University of Catalonia (UPC), Terrasa, SpainSoil contamination is of increasing concern due to the continued use of chemicals . Moreover, current regulations require that the potential for soil contamination must be reported for registration of chemical substances . On the other hand, classical toxicity tests require exposition of the suspected soil to soil-dwelling organisms for a period of time to assess the biological impact of contaminants . Reproduction and mortality have been the effects studied on earthworms, springtails and other organisms as indicators of the soil health . Most of the standard assays call for an exposure time of several days (from 48 hours to 21 days), but regulators require quick teats that quickly assess the quality of soils allegedly contaminated enabling them to take correct decisions . The behavior of soil organisms exposed to chemical contaminants in soil is used as the end point in the avoidance test . Soil organisms move away from contaminated soil and the degree of avoidance is related to the amount of pollution in the soil . Since several years we have studied the avoidance effect caused by chemical contaminants in soil to earthworms and springtails, with the aim of developing a quick and practical assay to screen large numbers of potentially contaminated soil samples capable to support decision taking on soil quality and remediation practices . The avoidance test can provide toxicity results based on the behavior of test organisms after exposure times from 2 to 48 hours . Recently, the avoidance test with collembolans has been standardized (ISO 17512-2:2011) . This endpoint does not imply mortality or reproduction inhibition, and collembolans are organisms widely present in all territories therefore providing a good indicator of soil health . In this communication we present the results of the collembolans avoidance test in the evaluation of the environmental quality of soil samples from known contaminated sites in Catalonia and of soil samples artificially contaminated with recognized soil pollutants . The work done confirms that results from this assay are in good correlation with results obtained with other soil toxicity tests and show also a good relationship with analytical chemistry results . Since the test provides reliable and quick results, it can be widely applied to the evaluation of the soil environmental quality in areas such as contaminated sites, remediation processes, application of wastes to agricultural soils, sediments, monitoring studies .

TH 038Behaviour response of the nematode, Caenorhabditis elegans after the treatments of BenzeneJ . Choi1, S .J . Lee2, H . Kim2, T .S . Chon2, H .J . Eom1

1University of Seoul, Seoul, South-Korea (Rep)2Pusan National University, Busan, South-Korea (Rep)Behaviour change has been considered important indicator of chemical toxicity, as it reflects integrated physiological alteration due to chemical exposure and thus is directly related to important ecotoxicological parameters such as reproduction . However due to difficulty of monitoring, it has not been widely used . In this study, response behaviour of Caenorhabditis elegans was observed after benzene was treated at 0 .5 ppm under air exposure conditions in wildtype and three stress response mutants (cyp35a2, pmk-1, cep-1) . The movement patterns were analyzed with different parameters before (3 hours) and after (3 hours) the treatments . The wild type (N2) showed decrease in speed and acceleration, while stop duration and turning rate increased after the treatments . Stop number and turning rate appeared to increase in mutants, but different mutant strains were also characteristic in showing different parameters in response to the treatments . The cyp35a2 mutant was contrasting to N2, showing low level of stop duration and high level of stop number . The pmk-1 mutant was differentiated from cyp35a2 with increase in stop duration and turning rate after the treatments . The cep-1 mutant showed a similar trend to N2, but stop number was higher and stop duration was shorter than the parameters obtained with N2 after the treatments . This study suggest C. elegans behavior changes to chemical exposure have a potential of being used as toxicity indicator and the response of different C. elegans mutant can provide an insight of the relationship between toxic mechanism and higher level effect . Acknowledgments : This work was supported by the “ The Eco-technopia 21 project”of Korean Ministry of Environment through and by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2011-0003214)

TH 039Differences in the number of micronucleated red blood cells and other nuclear abnormalities in Ciconiiformes: potential bioindicators of genotoxicity in birdsC .S .A . Santos1, M .S . Monteiro1, A .M .V .M . Soares2, S . Loureiro2

1Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, PortugalOver the past decades, the presence of micronucleated blood cells has been used as a widespread tool to detect genotoxic effects of xenobiotics in mammals, fish, amphibians and birds . In order to: (i) investigate the usefulness of the micronucleus test in Ciconiiformes and (ii) monitor the occurrence of genotoxic effects in Portuguese populations of Ciconiiformes, the frequency of micronucleus (MN) and other nuclear abnormalities (lobed nuclei, segmented nuclei, kidney shaped nuclei and notched nuclei) was assessed in wild individuals of Ciconia ciconia, Ardea cinerea and Ardea purpurea . About 80 blood smears, collected between 2007 and 2011 in birds from different geographic origins, were analysed and statistical tests were performed to determine eventual temporal and geographical differences in the frequency of MN and other nuclear abnormalities . Results showed elevated levels of MN and other nuclear abnormalities, and the sum of frequencies of all other abnormalities was generally higher than MN on the samples studied . Significant differences in the frequency of total nuclear abnormalities were observed also between different years . These results suggest that the assessment of total nuclear abnormalities, rather than only MN frequencies, might be a useful tool to complement the study of induced genotoxicity in birds .

TH 040Impact of thiol reactivity on the excess toxicity of a,β-Unsaturated Ketones, Esters, and AldehydesA . Böhme, D . Thaens, A . Paschke, G . SchüürmannUFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyIn aquatic toxicology, the minimum (narcosis-level) toxicity of an organic compound can be well estimated applying hydrophobicity-based quantitative-structure activity relationships (QSARs), addressing its´ membrane affinity . By contrast, electrophilic compounds may undergo chemical reactions with nucleophilic target sites (e .g . ‘SH and ‘NHR) of glutathione (GSH), proteins and DNA, leading to enhanced toxicity (quantified by the toxicity enhancement Te) as compared to iso-hydrophobic narcotics . For α,β-unsaturated carbonyls as one class of electrophiles, Michael-type addition forms a prominent route to cause excess toxicity toward aquatic organisms . Here, the kinetic glutathione chemoassay, employing GSH as a surrogate for thiol groups in vivo, allows one to quantify chemical reactivity in terms of the second-order rate constants kGSH, and thus may form a non-animal component of integrated testing strategies (ITS) for REACH and the new European cosmetic guideline . In this communication, the kinetic GSH chemoassay is employed for assessing the electrophilic reactivity in terms of kGSH of 52 α,β-unsaturated ketones, esters, and aldehydes . Comparison with toxicity data in terms of 48-h 50 % growth inhibition of Tetrahymena pyriformis resulted in structural alerts for the predictive identification of excess-toxic compounds . Moreover, regression models for predicting both log EC50 and log Te were derived, using log kGSH and log Kow as parameters . Financial support was provided by the European Commission through the project OSIRIS (Optimized Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Test Information, EU Contract No . GOCE-CT-2007-037017), which is gratefully acknowledged .

TH 041Excess toxicity of organic electrophiles in the Tetrahymena pyriformis bioassay informs about reactive modes of actionA . Laqua, A . Paschke, G . SchüürmannUFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany

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In recent years, the concept of integrated testing strategies (ITS) has been developed as a practical approach toward the 3R goals of reducing, refining and replacing animal testing for the toxicological and ecotoxicological assessment of industrial chemicals under the European Directive REACH . The goal is to combine complementary non-animal (in chemico, in vitro, omics) and non-test (in silico) methods with a strong emphasis on mechanisms of action, thus compensating for the reduction in adverse outcome pathways associated with every individual alternative method . In this context, organic electrophiles represent a prominent group of particularly toxic contaminants that cover various classes of chemical substances, and may also be generated in vivo through metabolic processes . Their high intrinsic toxicological potential results from their ability to deplete the anti-oxidant glutathione, and to undergo chemical reactions with nucleophilic sites of proteins and the DNA . In this study, a simple in vitro approach is presented that enables a targeted analysis of electrophilic modes of action of organic compounds . To this end, 50% growth inhibition (EC50) of the unicellular ciliates Tetrahymena pyriformis of a given compound is determined and compared with the corresponding narcosis-level toxicity of an isohydrophobic counterpart . The resultant toxicity enhancement Te, derived as ratio of narcosis-level over actual EC50, informs about the lack (Te < 10) or presence (Te ≥ 10) of reactive mechanisms as major determinant of the observed in vitro toxicity, and can be used to derive respective structural alerts for a predictive toxicity assessment . Moreover, different mechanisms of reactive toxicity may translate into different level ranges of the toxicity enhancement, thus providing pertinent information in the context of ITS schemes . The approach is illustrated with results for quinones and hydroquinones as two compound classes that may generate reactive oxygen species (ROS) through redox cycling, and - without (quinone) or after metabolic oxidation (from hydroquinone to quinone) - chemically modify endogenous nucleophiles through Michael-acceptor addition reactions . Financial support from the EU project OSIRIS (Optimized Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Testing Information), contract no .: 037017, is gratefully acknowledged .

TH 042Predictive assessment of the aquatic toxicity of Michael acceptors using a kinetic chemoassay with 4-nitrobenzenethiol (NBT)R . Hiltrop, K . Eismann, A . Paschke, G . SchüürmannUFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyAccording to the European Directive REACH, the hazard and risk assessment of chemical substances should be performed with as little animal use as possible, following the 3R principle and its goal to reduce, refine and replace animal testing in the interest of animal welfare . In this context, the concept of integrated testing strategies (ITS) has been developed, exploiting different types of non-animal data such as in vitro, in chemico and in silico information . In aquatic toxicity, the well-accepted narcosis paradigm states that every chemical substance exerts a minimum toxicity proportional to its hydrophobicity . Accordingly, excess toxicity - identified through a toxicity enhancement over baseline narcosis - is expected for those compounds where reactive or specific modes of action govern the adverse effect . Here, organic electrophiles form an important group of potentially excess-toxic contaminants because of their chemical reactivity toward nucleophilic sites of glutathione as well as endogenous macromolecules (protein, DNA) . An efficient non-animal approach for screening the excess toxicity of organic electrophiles is to measure their reactivity toward model nucleophiles, employing so-called chemoassays (in chemico) . In this work, 4-nitrobenzenethiol (NBT) is taken as a model for endogenous thiol groups . It has the advantage to be directly detectable by UV-VIS absorption, enabling a fast and low-cost quantification of the toxicologically relevant reactivity . Using this approach, second-order reaction rate constants of reaction with NBT, kNBT, have been determined for a number of α,β-unsaturated carbonyls (Michael acceptors) . These electrophiles are able to react irreversibly with nucleophilic groups through a Michael-type addition mechanism, and therefore have the potential to exert substantial excess toxicities . Our results demonstrate a good correlation between log kNBT and the extent of the excess toxicity (the latter of which was derived from in vitro results of the growth inhibition bioassay with the ciliates Tetrahymena pyriformis) . Thus, the NBT chemoassay is found to be a simple and promising tool for the predictive assessment of the aquatic toxicity of organic electrophiles . This work was financially supported by the European Union project OSIRIS (Optimized Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Testing Information), contract no . GOCE-CT-2007-037017 .

TH 043Linking high-throughput In Vitro Screens with EDSP assays: the causal relationship challengeE .M . Mihaich1, C .J . Borgert2, L .S . Ortego3, B .H . Neal4, M .S . Marty5

1Environmental & Regulatory Resources, Durham, nc, United States of America2Applied Pharmacology & Toxicology Inc ., Gainesville, fl, United States of America3Bayer CropScience, Research triangle park, nc, United States of America4Exponent, Alexandria, va, United States of America5The Dow Chemical Company, Midland, mi, United States of AmericaOne step in complying with Test Orders under the US EPA’s Endocrine Disruptor Screening Program was compilation of Other Scientifically Relevant Information (OSRI) . The extent and quality of OSRI was evaluated to determine what requirements were met by existing data, and hence, which of the 11 Tier 1 Endocrine Screening Battery (ESB) assays were needed . Many of the List 1 chemicals under ESB Test Orders were analyzed in the EPA’s ToxCast Program, hence ToxCast data were submitted as OSRI in lieu of conducting many similar ESB in vitro assays, and to help support waivers for some in vivo assays . ToxCast seeks to predict human toxicity by measuring the activity of chemicals in a suite of some 500 cellular, biochemical and molecular assays conducted in high-throughput mode . The concept presumes that these assays can identify a chemical’s ability to activate so-called ‘toxicity pathways .’ In evaluating acceptability of OSRI, EPA rejected ToxCast results claiming that the reliability, responsiveness and relevance of ToxCast were undetermined and that ToxCast does not evaluate all known endocrine targets . In rejecting ToxCast data, EPA claimed its validation status was inferior to that of the individual ESB assays . However, even the predictive value of the in vitro ESB assays for in vivo endocrine activity is still unclear at this time . Although ToxCast data was rejected as OSRI for the ESB, ToxCast data was used to assess and deem acceptable the risks of oil dispersants used for the Deep Water Horizon spill . These decisions have significant implications for the use of screening assays in regulatory programs, yet inconsistencies in application confound what those implications might be . As both the ESB and ToxCast programs appear to rely on correlative analyses rather than dispositive evidence that assays individually or in combination measure precursor events or frank toxicity, the use of high-throughput assays, like ToxCast, to screen chemicals for further testing may be no less probative than some of the ESB battery . However, despite potential advantages offered by high-throughput methods to quickly screen for possible modes of action, it will be necessary to elucidate aspects of biological and physiological function as well as causal relationships between biochemical events and adverse effects before these data can offer improvements in the assessment of endocrine-mediated toxicity .

TH 044Study of key characteristics using multicriteria analysis for development of chronic inherited toxicity testC . Vaz1, R .F .S . Böhm1, R .A . Gonçalves1, E .C . Spitzner1, T . Kleine1, T .S . Tortelli1, T .M .N . Oliveira1, W .G . Matias2

1UNIVILLE, Joinville, Brazil2UFSC, Florianópolis, BrazilThe multicriteria analysis is a useful tool for comparing different conditions and the results are normally oriented to the operational decisions . The development of methods that identify environmental risks and detection of compounds that cause deleterious effects to living organisms in an area at low cost, reproducibility, ease of implementation and speed of analysis are relevance to the promotion of activities that assist the sustainable development and minimize impacts to the ecosystems of developing countries . This study is aim at developing a multicriteria analysis to define key characteristics to design a method for chronic toxicological evaluation, considering aspects of the complete life cycle of organisms, as well as the inherited effects in tropical countries . A multicriteria matrix was prepared based on Quality Function Deployment model where each key characteristic was evaluated taking into consideration the interrelationships between them . In order to establish the criteria to be used in the matrix factors for assessing the response time, technical difficulty, cost, reproductivity and number of citations in the literature were defined . The total score was calculated by the sum of scores, multiplied by the corresponding weights . The criteria technical difficulty is of great importance in this study as being proposed for use in developing countries, where technological development is generally behind . In line with results, the key features with the highest score and also considered for the development of a chronic toxicity test were reproduction, mortality, time to first reproduction and total size of the organisms .

ET06P - Ecotoxicology of amphibian and reptiles . Novel approaches for linking contaminant effects with population declines

TH 049Relevant lizard species for the risk assessment of plant protection products in Northern and Central EuropeO . Körner, N . Lutzmann, J .D . Ludwigs, G . Vogel, C . DietzenRIFCon GmbH, Hirschberg, GermanyAccording to the new data requirements under the EU regulation 1107/2009, reptiles come into question regarding risk assessments of plant protection products (PPP) . Although no specific data requirements on reptiles are stipulated in the respective EU documents (e .g . SANCO 11802/2010/rev July 2010) some toxicity data are available in the open literature . These data are intended to be used in the risk assessment . One potential approach for reptile risk assessment may be the way how it is successfully established for birds and mammals . The major routes of exposure in that case are diet and drinking water . In order to provide a scientific basis for reptile risk assessment, a detailed literature survey on habitat use and the biology of lizards occurring in North and Central Europe (geographical zones according to SANCO/6896/2009 rev 1) was conducted . The study focused on the habitat use of the different species, but also evaluates the different diets as a source of exposure . The data obtained may allow the determination of relevant species at risk and how they can be linked to potential exposure in agricultural practice .

TH 050Relevant snakes and turtle species for the risk assessment of plant protection products in Northern and Central EuropeO . Körner, N . Lutzmann, G . Vogel, C . Dietzen, J .D . LudwigsRIFCon GmbH, Hirschberg, GermanyAccording to the new data requirements under the EU regulation 1107/2009, reptiles come into question regarding risk assessments of plant protection products (PPP) . Although no specific data requirements on reptiles are stipulated in the respective EU documents (e .g . SANCO 11802/2010/rev July 2010) some toxicity data are available in the open literature . These data are intended to be used in the risk assessment . One potential approach for reptile risk assessment may be the way how it is successfully established for birds and mammals . The major routes of exposure in that case are diet and drinking water . In order to provide a scientific basis for reptile risk assessment, a detailed literature survey on habitat use and the biology of snakes and turtles occurring in North and Central Europe (geographical zones according to SANCO/6896/2009

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rev 1) was conducted . The study focused on the habitat use of the different species, but also evaluates the different diets as a source of exposure . The data obtained may allow the determination of relevant species at risk and how they can be linked to potential exposure in agricultural practice .

TH 051Relevant lizard species for the risk assessment of plant protection products in Southern EuropeN . Lutzmann, C . Dietzen, J . Gerlach, O . Körner, G . VogelRIFCON GmbH, Hirschberg, GermanyAccording to the new data requirements under the EU regulation 1107/2009, reptiles come into question regarding risk assessments of plant protection products (PPP) . Although no specific data requirements on reptiles are stipulated in the respective EU documents (e .g . SANCO 11802/2010/rev July 2010) some toxicity data are available in the open literature . These data are intended to be used in the risk assessment . One potential approach for reptile risk assessment may be the way how it is successfully established for birds and mammals . The major routes of exposure in that case are diet and drinking water . In order to provide a scientific basis for reptile risk assessment, a detailed literature survey on habitat use and the biology of lizards occurring in South Europe (geographical zones according to SANCO/6896/2009 rev 1) was conducted . The study focused on the habitat use of the different species, but also evaluates the different diets as a source of exposure . The data obtained may allow the determination of relevant species at risk and how they can be linked to potential exposure in agricultural practice .

TH 052Are the Mediterranean chameleon species possible focal species for risk assessments of PPPs in Europe?N . Lutzmann1, G . Vogel1, W . Böhme2

1RIFCON GmbH, Hirschberg, Germany2Zool . Research Museum A . Koenig, Bonn, GermanyAccording to the new data requirements under the EU regulation 1107/2009 reptiles and amphibians have to be addressed in the risk assessment of plant protection products (PPP) . One necessary background for such an addressing are reliable data on the exposure of PPP . For reptiles generic data recorded in agricultural landscapes are nearly completely missing . In a five months field study about the distribution and ecology of the chameleons of Egypt it was prooved that both European chameleons are species which thrive in areas developed by man and are candidates as focal species for risk assessments for PPP in fruit orchards and olive grooves in their distribution areas in Europe . In 83 night-searches with torches of at least 2 hours duration each chameleons could be detected in 18 of 28 vegetated locations, but only two of them were natural vegetated . No chameleons were found in mixed vegetation (urban, natural and agricultural habitats in close environment) . In the agricultural habitats half of the Chamaeleo africanus were observed inside the crop and 40% of the Ch. chamaeleon, respectively . All other specimens used the direct surroundings, like hedges or or isolated trees between fields . These findings in Egypt are supported by first field data on habitat selection of chameleons in Europe (Greece, Spain and Portugal) . A higher percentage of the adults were found inside the crop than in the direct surrounding vegetation, but the majority of juveniles were found in the surrounding vegetation .

TH 053Relevant snake species for the risk assessment of plant protection products in Southern EuropeN . Lutzmann, C . Dietzen, J . Gerlach, O . Körner, G . VogelRIFCON GmbH, Hirschberg, GermanyAccording to the new data requirements under the EU regulation 1107/2009, reptiles come into question regarding risk assessments of plant protection products (PPP) . Although no specific data requirements on reptiles are stipulated in the respective EU documents (e .g . SANCO 11802/2010/rev July 2010) some toxicity data are available in the open literature . These data are intended to be used in the risk assessment . One potential approach for reptile risk assessment may be the way how it is successfully established for birds and mammals . The major routes of exposure in that case are diet and drinking water . In order to provide a scientific basis for reptile risk assessment, a detailed literature survey on habitat use and the biology of snakes occurring in South Europe (geographical zones according to SANCO/6896/2009 rev 1) was conducted . The study focused on the habitat use of the different species, but also evaluates the different diets as a source of exposure . The data obtained may allow the determination of relevant species at risk and how they can be linked to potential exposure in agricultural practice .

TH 054Plant protection products and juvenile amphibians: no matter of concern or unexpected risk?T .W . Schmidt1, A . Alscher1, S . Hoeger1, S . Pieper2, C . Bruehl3

1Harlan Laboratories Ltd ., Itingen, Switzerland2Federal Environment Agency, Dessau, Germany3University Koblenz-Landau, Landau, GermanyThe current risk assessment of plant protection products requires a diversity of tests with vertebrate species, especially fish and the African Clawed Frog (Xenopus laevis) from aquatic environments, and birds and mammals from terrestrial environments . Concerning amphibians, many studies are available concerning the influence of plant protection products on survival and growth of aquatic larval stages, but the knowledge on toxicology and ecotoxicology of adult frogs is scarce . This is surprising since juvenile and adult frogs are supposed to be at risk of pesticide uptake due to their highly permeable skin which is only a weak barrier against plant protection products . Additionally, many frog species are exposed to a variety of plant protection products, since they migrate through agricultural fields either from their larval aquatic habitats to over-wintering sites in autumn or back in spring for egg-laying .

In order to fill the data gap of the potential risk of plant protection products on the terrestrial life stage of amphibians, approx . 1-2 months old juveniles of the Common Frog (Rana temporaria) were caught in the field and exposed to six different plant protection products in the laboratory on bare soil for one week . For each plant protection product three application rates, including the practically used field application rate, were selected to determine the LR50 . Our results show that the majority of the plant protection products caused a mortality between 20-100% at field application rates . Therefore, our data demonstrate that the current risk assessment is not protective for amphibians .

TH 055Assessing atrazine toxicity to the Australian striped marsh frog, Limnodynastes peroniiK .A . Siddiqua1, S .P . Wilson1, R . Alquezar2

1CQUniversity, Gladstone, Australia2Vision Australia Queensland, Gladstone, AustraliaAbstract

Atrazine is one of the most extensively applied herbicides in Australia and frequently detected in surface and ground water samples . In overseas studies, it has been reported that atrazine can cause reproductive and developmental abnormalities in exposed amphibians . Limited studies have been carried out in Australia, resulting in subsequent gaps in knowledge particularly among Australian species . To explore the effects of atrazine to native frogs, the striped marsh frog, Limnodynastes peronii, was exposed to a range of low environmentally relevant concentrations of atrazine, including, control, solvent control, 0 .1, 1, and 10 µg/L for 21 days . Results showed that there were no significant differences in total length, snout vent length, body weight and time to metamorphosis across the treatments (p> 0 .05) . No mortality was observed in any treatments . Only 4 .4% of tadpoles exhibited abnormal kinky tails in 1 µg/L and 2 .2% in 10 µg/L atrazine . No such abnormalities were found either in controls or solvent controls . Histological analysis of gonads is underway and the implications for management of atrazine impacts to native amphibians will be further discussed .

TH 056Toxicity of eucalypt and pine leaf leachates in zebrafish (Danio rerio) and iberian frog (Pelophylax perezi) early-life stagesJ . Medeiros1, K . Monaghan2, A .M .V .M . Soares3, M .S . Monteiro4

1CESAM, Universidade Aveiro, Aveiro, Portugal2CESAM, Dep Biologa, Universidade de Aveiro, Aveiro, Portugal3University of Aveiro & CESAM, Aveiro, Portugal4CESAM, Dep Biologia, Universidade de Aveiro, Aveiro, PortugalWhilst deforestation of natural forests represents one of the major causes of biodiversity loss, the extent of plantation forests has increased, especially in Mediterranean countries . Represented by vast areas of monoculture this trend in afforestation has raised concern for biodiversity conservation . Afforestation of Pine and Eucalypt is associated with increased stream acidity and a reduction in base cations whilst the short rotation harvesting of trees deprives stream systems of inputs of large woody debris . In the present study, the toxicity of eucalypt and pine leaf leachates that may be particularly damaging in intermittent streams, was evaluated using embryo toxicity testing with Danio rerio, a tropical cyprinid used in toxicological research, based on the OECD draft guideline on Fish Embryo Toxicity (FET) and with the iberian frog Pelophylax Perezi based on the ASTM guideline on Frog Embryo Teratogenesis Assay-Xenopus (FETAX) . The endpoints monitored included mortality and sub-lethal parameters in embryo development such as pericardial edema, tail deformation and hatching rate . Five concentrations of each leachate were tested, ranging from 0 .64 to 20,48 and 1,6 to 32 g L-1 of Eucalypt in zebrafish and P. perezi, respectively; and from 1,6 to 32 g L-1 of Pine in both species . Simultaneously, the water physico-chemical parameters and phenolic content were evaluated in eucalypt and pine leachates . In general, leachates were found to reduce water dissolved oxygen and pH, and increase dissolved phenolics and conductivity . The results obtained suggest higher toxicity of eucalypt leachates for zebrafish (LC50-96 h = 3,32 and 12,25 g L-1 of eucalypt and pine leaves, respectively), while for embryo frogs the different leachates presented similar toxicity (LC50-96 h = 14,97 and 13,26 g L-1 of eucalypt and pine leaves, respectively) . The embryo developmental alterations registered in both species will be presented and discussed considering the type of leaf leachate tested and their respective water physico-chemical parameter characteristics and phenolic content . These data demonstrate that leachate toxicity may contribute to the impact of pine and eucalypt plantations on aquatic biodiversity .

TH 057Alterations of embryonic development and mRNA expression in Xenopus laevis caused by perfluorooctane sulfonate (PFOS)L . San Segundo1, L . Guimarães2, G . Carbonell1, C . Fernández1, L . Guilhermino3, M .V . Pablos1

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1INIA, Madrid, Spain2University of Porto: CIIMAR, Porto, Portugal3University of Porto: ICBAS & CIIMAR, Porto, PortugalPerfluorooctane sulfonate (PFOS) is a syntetic chemical that has been used in industrial activities for decades . This compound has been detected throughout the world in numerous environmental samples and is considered a pollutant of concern because of its potencial toxic effects, persistence and bioacumulation properties . Nevertheless, few data exist about the effects of PFOS on amphibians . In this study, Xenopus laevis embryos were exposed for 96 hours to various concentrations of PFOS (0 .5, 6, 12, 24, 48 y 96 mg/L), from early gastrula stage to larvae . Survival and deformities rates were monitored daily during the test and head-tail length was measured at the end of the assay as an indicator of growth . In addition, at 24 and 96 hours of exposure, mRNA expression levels of genetic markers involved in general stress responses (hsp70, hsp47, crf and urocortin), apoptosis (p53 and bax), oxidative stress (cat and sod), and differentiation and lipid accumulation (pparβ) were analysed using RT-qPCR . Deformities rate was significantly higher in embryos exposed to the highest concentration of PFOS than in controls, at 48 (+43%), 72 (+49%) and 96 (+60%) hours of exposure . The most common deformity found was axial shortening . Growth inhibition was observed in embryos exposed to PFOS concentrations ≥ 48 mg/L . After 24 hours of exposure significantly higher expression levels were found for hsp70 at concentrations ≥ 24 mg/L, for hsp47 at concentrations ≥ 12 mg/L, and for p53 at concentrations ≥ 48 mg/L, relative to controls . After 96 hours of exposure, significant increases in expression levels were found for hsp70 at concentrations ≥ 12 mg/L, for hsp47 and crf at concentrations ≥ 0 .5 mg/L, and for p53 at concentrations of 12 and 48 mg/L . Alterations in mRNA expression indicated a stress response to PFOS at 24 and 96 hours of exposure, as well as alterations in apoptosis regulation . This work was supported by the projects CTM2010 19779-C02 and RTA2010-00004-C02-01 . LSS acknowledges her FPI-INIA fellowship .

TH 058Assessment of potential toxicity of wastewater treatment plant effluents in a Xenopus laevis embryo test using gene expressions as early response biomarkersL . San Segundo, F . Martini, G . Carbonell, C . Fernández, M .V . PablosINIA, Madrid, SpainThis study assessed the potential toxicity of wastewater treatment plants (WWTPs) effluents with classical endpoints, such as mortality, malformations rate and growth, as well as evaluating the expression levels of several genes as response biomarkers of early effects of complex samples in Xenopus laevis embryos . Sixteen samples from WWTPs effluents around the city of Madrid (Spain) were evaluated using a Xenopus laevis embryo test . The embryos were exposed to three dilutions of each effluent sample (1, 10 and 25%) for 96 hours . At the end of the assay, mortality and malformations rate were recorded and larval length was measured . Embryos for genetic analysis were collected after 24 and 96 hours of exposure and the mRNA expression levels of genes involved in embryonic development and chemical stress were investigated using semiquantitative RT-PCR . To value the sensitivity of this gene expression approach, the transcriptomic results were analysed and compared with morphological abnormalities rate and length embryo data . This work has been funded by CSD2006-00044 -CONSOLIDER- and RTA2010-00004-C02-01 projects . LSS was supported by an FPI-INIA fellowship .

TH 059Effects of larval estrogen exposure on adult reproductive potential in the amphibian model, Silurana tropicalisB . Pickford, S . LarrozeBrunel University, Uxbridge, United Kingdom Amphibians are receiving increasing attention in test method development for chemical safety assessment, in particular with respect to detection of endocrine disrupting activity . While there is evidence that exposure to exogenous estrogens in the larval phase can cause aberrant sexual differentiation in anuran amphibians, consequences for reproductive fitness in the adult phase remain unclear . Assessing impacts of gonadal abnormalities, such as testicular oocytes, requires models in which incidence of these phenomena at individual level can be correlated with apical endpoints indicative of reproductive fitness that might have population level consequences . We used an emerging amphibian model, the West African clawed frog (Silurana tropicalis) in which the adult phase is relatively short, to assess the impacts of larval exposure to ethinyl estradiol (EE2; 10, 50, 100ng, 500, 3000 ng l-1), on metamorphic and adult reproductive endpoints . Exposure of larvae to EE2 from stage 51/52 until stage 66 (completion of metamorphosis) at two temperatures (25°C and 28°C) skewed the sex ratio toward females in the exposed groups, though at completion of metamorphosis there were a significant number of undifferentiated individuals in all treatment groups . At sexual maturity, sex ratio, based on gonad histopathology, was female-biased in EE2 exposed treatment groups, and absence of one or both oviducts was also observed in female frogs that had been exposed to EE2 at concentrations > 50 ng l-1 . Regressed testicular oocytes were observed in males from all treatment groups (including control) and incidence appeared to be influenced by parentage not EE2 exposure . Mature testicular oocytes and presence of oviduct-like ducts in frogs with testes were only seen in treatment groups exposed to EE2 > 10 ng l-1 . Cluster analysis of computerised sperm motility measurements indicated presence of two sub-populations of sperm in frogs from all groups: one group typified by lower VSL and STR/higher VAP (slow and circular); the other by higher VSL and STR/lower VAP (fast and straight) . Frogs exposed to 10 ng l-1 EE2 had significantly more sperm in the former group (slow and circular) than other treatment groups . These findings suggest that further development of partial life cycle methods employing Silurana tropicalis, including sperm analysis, may be warranted in order to support identification and characterization of endocrine disrupters in chemical safety testing .

TH 060Exposure to cadmium: evaluation of the effects on the larval epidermis of Lithobates catesbeianusC .S . Carvalho, I .M . Soriano, M .J . Costa, M .N . MarisaUniversidade Federal de São Carlos, Sorocaba, BrazilSince amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the acute toxicity of the cadmium (Cd, 1ppb-48h) on the skin of bullfrog tadpoles (25 Gosner stage, Gosner, 1960), Lithobates catesbeianus . Enzyme activities (CAT, catalase and GST, gluthatione S-transferase), and also LPO (lipid peroxidation), MT (metallothionein) and GSH (gluthatione reduced) levels were evaluated . Tadpoles were randomly divided into 2 groups: controls, exposed to water free of cadmium (n=8), and cadmium-exposed animals: exposed to CdCL2, 1ppb-48h (n=8) . After exposure, tadpoles were sacrificed and the skin was removed to assays . LPO (7 .33±1 .2 nmol/mg), MT (100,13 µmolSH/mg protein) and GSH (0,08 mM) concentrations and CAT (26 .49±2 .37 UB/mg protein) and GST (244 .59±15 .22 mU/mg of protein) activities were significantly higher were than those from the control group . The results obtained were of ecological relevance, suggesting an adaptation to stress resulting from acute exposure to metal that directly or indirectly, generates reactive oxygen species (ROS) . In contrast, an antioxidant system deficiency will induce a precarious state, making biological species more susceptible to toxic agents . GSH and MT, which represents the bulk of non-protein thiols, are synthesized in the liver and exported to other tissues functioning in the detoxification of H2O2, other peroxides and free radicals, being considered the predominant defense against ROS . MT has an important role in the detoxification of toxic metals such as Cd, and in the maintenance of the homeostasis of essential metals . The increased antioxidant enzyme’s activity suggests that bullfrog tadpoles can develop adaptations to the pro-oxidant challenges provoked by environmental contaminants signaling increased H2O2 and LPO and/or other organic xenobiotics levels . These results indicate frog’s epidermis can function as an adaptive system equipped with chemical and structural supports that can counteract, within certain limits, the effects of exposure to some environmental pollutants such as, cadmium . Considering that the skin represents the organ which is more directly exposed to environmental toxicants, the effects of cadmium on tadpoles are more likely to be due to injuries induced by the toxicant on the liver and the kidney, considering that these organs, usually involved in metabolizing and excreting harmful substances, are not completely developed at that stage .

TH 061Integrated assessment of the effects posed by historical exposure to chemical contamination in natural populations of the Perez’s frog, Pelophylax perezi (Seoane, 1885)I . Lopes1, B . Santos2, P . Coelho3, I . Domingues2, A . Faustino4, G . Lopes4, M .S . Monteiro2, E . Pereira3, M . Ortiz-Santaliestra5, R . Pereira6, R .M . Silva7, A .M .V .M . Soares8

1University of Aveiro, Aveiro, Portugal2Departamento de Biologia & CESAM, University of Aveiro, Aveiro, Portugal3Departamento de Química & CESAM, University of Aveiro, Aveiro, Portugal4ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal5Instituto de Investigación en Recursos Cinegéticos (IREC) CSIC-UCLM-JCCM, Ciudad real, Spain6Departamento de Biologia, Universidade do Porto, Porto, Portugal7IPATIMUP, Universidade do Porto, Porto, Portugal8University of Aveiro & CESAM, Aveiro, PortugalMost works assessing the effects that chemical contamination may pose to amphibians have been centered on laboratory assays with organisms originated from non-impacted populations . However, amphibians may respond differently when exposed to contaminants in field or in laboratory . In addition, more holistic and integrated studies are essential to promote ecological relevance of generated knowledge . This work aimed at an interdisciplinary assessment of the effects caused by a long-tem exposure to chemical contamination on adults of Pelophylax perezi . For this, four populations were sampled: two at chemically contaminated sites and two at reference sites . Sixteen adult organisms (8 females; 8 males) were captured at each site and transported chilled to the laboratory, where were euthanized with MS222 and its morphometric parameters measured . Afterwards organisms were dissected to collect gonads, kidney, liver, muscles, spleen, lungs and bones, which were deep frozen for posterior analysis . For each organism, biomarkers of oxidative stress (catalase, glutathione S transferase, lactate dehydrogenate) and endocrine disruption (vitellogenin-VTG), metallothioneins (MT), molecular markers, histological effects and metal levels in tissues were analyzed . Chemical analysis of surface waters revealed higher values of some elements (B, Be, Ca, K, Li, Mg, Mn, Na, Sr) at the contaminated sites comparatively with the reference ones . Metal analyses in sediment, soil and tissue samples are still being processed . Significant differences in enzymatic activities and in VTG levels were detected among the four populations, but could not be associated with different levels of the metal contamination in water . Histological analysis on male gonads revealed testicular oocytes (TO) in the impacted populations (25% and 12 .5%) while no TO were detected in reference populations . Differences in the mean seminiferous lobule (MSL) diameter were observed between reference and contaminated populations, and as well between the two contaminated populations . Analysis of MT and histology of spleen, liver, lungs and kidney are underway . Also, the genetic diversity within and between populations is being addressed using a combination of neutral, non-neutral, genomic and mitochondrial DNA markers . All these data will be integrated and presented in the poster .

TH 062Development of new non-destructive biomarkers in the loggerhead sea turtle: first evidence of induction of CYP1A in skin biopsy slices treated with PAHs, OCs and PBDEsM . Giannetti, S . Casini, L . Marsili, S . Maltese, T . Campani, L . Carletti, M .C . FossiUniversity of Siena, Siena, ItalyInduction of CYP1A has been proven by a large number of studies to be a sensitive and specific biomarker of exposure to some classes of lipophilic contaminants . However,

24� SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

the use of this biomarker has been mostly destructive, since the liver is the main target organ for analysis . This study is part of a larger project aimed at developing non-invasive biomarkers in the endangered species of C . caretta in the Mediterranean . In particular it focuses on developing and validating the induction of CYP1A as a non-invasive biomarker of exposure in skin biopsy slices .During the first part of the study, the Western blot method to evaluate protein expression of CYP1A in this species was set up and applied in liver and skin sampled from two specimens that died in a turtle rescue center . We detected a strong band at about 59 KDa of molecular weight in both tissues, in accordance with the findings in liver of the freshwater C . picta picta by Yawetz et al in 1998 . In order to semi-quantify CYP1A, a series of CYP450 calibration curves from the liver (ranging from 0 .5 to 5 .0 pmol of CYP450) of Caretta caretta were analysed . In addition a skin standard was analyzed within the curves and maintained as an internal standard in subsequent WBs . Semi quantitative analysis was performed for each WB with Quantity-One software (BioRad) using Adjusted Volume (Intensity*mm2) as a quantitative parameter .In the second part of the study, skin biopsy samples were taken from 7 free-ranging specimens, and subaliquots (slices) were incubated in vitro for 24 hours with different doses of PAHs (0 .5 μM BaP + 10 μM BnF and 12 .5 μM BaP + 250 μM BnF, plus a acetone (0 .1%) control), OCs (0 .01 µg/ml, 1 µg/ml and 25 µg/ml, plus a DMSO (0 .05%) control) and PBDEs (0 .1 µg/ml and 0 .05 µg/ml, plus a nonane (0 .01 µg/ml) control) in order to investigate the potential for CYP1A induction . Western blotting of CYP1A showed a dose-related induction in skin biopsy slices treated with the different compounds . A higher induction was found after treatment with PBDEs, indicating a strong potential of these emerging chemicals to interfere with bioactivation enzymatic systems in C . caretta .In conclusion this study, which we believe is the first of its kind in the literature, demonstrated the presence and inducibility of CYP1A in skin biopsies of C . caretta, and validated this non-destructive biomarker for application in field studies .

TH 063Effects of lead shot pellets contamination on oxidative stress biomarkers and plasma biochemistry in turtlesM . Martinez-Haro1, A .J . Green2, R . Mateo3

1IMAR, Coimbra,, Portugal2Department of Wetland Ecology, Estación Biológica de Doñana (CSIC), Seville, Spain3Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC-UCLM-JCCM), Ciudad real, SpainLead (Pb) shot pellets spent in hunting activities have been accumulated in wetlands worldwide, with densities in the upper 20 cm of wetland sediments above 100 shot/m2 at many locations . Currently, a large number of countries are making significant efforts to reduce or eliminate the input of Pb shot in most wetlands . However, the problem often persists because spent Pb shot remains in soil for 100-300 years . During this time the pellets are slowly buried by sedimentary accumulation, or dissolve over time to disappear, affecting local sediment Pb concentrations . Although studies carried out on the effect of Pb exposure in wildlife through ingestion of pellets accumulated in sediments are numerous, those that address the effect of Pb exposure due to contamination associated with its dissolution in the environment are scarce . In order to fill this gap, we used non-destructive biomarkers to study the exposure and effect of Pb on turtles (Mauremys leprosa and Emys orbicularis) from two Spanish wetlands . The Medina lagoon (ML), where the highest densities of Pb shot pellets of Spain has been described, and Dulce lagoon (DL), a non-contaminated lagoon located in Doñana National Park . The mean whole blood Pb concentration (PbB) from ML turtles was higher than in DL (F1,97=42 .541, p<0 .01; 29 and 5 ng/ml, respectively), both in males and females (F1,45=20 .130, p<0 .01; F1,50=26 .704, p<0 .01, respectively) . The С-aminolevulinic acid dehydratase (ALAD) ratio was negatively related with PbB (F1,93=4 .661, p<0 .05), regardless of the lagoon or sex . Plasma Total Antioxidant Status (TAS) was positively related with PbB (F1,81=6 .723, p<0 .05), and also differed with lagoon (F1,81=4 .333, p<0 .05; higher in DL), and sex (F1,81=5 .683, p<0 .05, higher in females) . Finally, three biomarkers of oxidative stress in red blood cells statistically differed with PbB and lagoon: lipid peroxidation, estimated as thiobarbituric acid-reactive substances (F1,67=7 .450, p<0 .01; F1,67=14 .523, p<0 .01, respectively); total sulfhydryl (F1,77=6 .387, p<0 .05; F1,77=19 .982, p<0 .01, respectively); and glutathione in oxidized form (F1,63=12 .004, p<0 .01; F1,63=6 .469, p<0 .05, respectively) . Additionally, the ratio reduced/oxidized glutathione was negatively associated with PbB (F1,63=5 .138, p<0 .05) . Our results evidenced that turtle living in Pb shot contaminated wetlands showed sublethal effects . Nonetheless, further reseach is needed to elucidate Pb effects on individual and population health and dynamics .

TH 064Transition metals contamination on marine turtle eggshell from ThailandM . Areekijseree1, T . Chuen-Im2, K . Sribuddhachart3, C . Thepsithar2

1Silpakorn University, Nakorn pathom, Thailand2Department of BiologyFaculty of Science, Silpakorn University, Nakorn Pathom 730, Nakorn pathom, Thailand3Department of BiologyFaculty of Science, Silpakorn University, Nakorn pathom, ThailandElemental analysis of eggshells in 3 marine turtle eggshell from Thailand; green turtle (Chelonia mydas), hawksbill turtle (Eretmochelys imbricata), and leatherback turtle (Dermochelys coriacea), were studied by scanning electron microscope (SEM) with secondary electron imaging (SEI), backscattered electron imaging (BEI), and energy dispersive X-ray analysis (EDX) detectors . Results of elemental analysis in all 3 marine turtle types showed mainly contained carbon (C), oxygen (O), calcium (Ca), sulfur (S), sodium (Na), chlorine (Cl), magnesium (Mg), silicon (Si), potassium (K), aluminum (Al) fluorine (F) and phosphorus (P) . Among elements detected, carbon, oxygen, and calcium were found at high percentages . It was found that calcium was accumulated in the outer cuticle layers (P<0 .05) . Interestingly, it was found contamination of hazardous metals and non metals in turtle eggshell samples . Ttransition metals and other metals; including lead (Pb), palladium (Pd), and molybdenum (Mo), were detected in all 3 eggshell types at the percentages of 0 .1-0 .5% . Also, copper (Cu) contaminated in leatherback eggshells and cadmium (Cd) was found in green turtle samples . Contaminations of these heavy metals were always seen in the outer layers except for Mo . Finally, there were other elements found in the eggshells which were bromine (Br) and Iodine (I) . This work showed the merit of SEM . It is a powerful and comfortable to analyze percentage of trace elements accumulation and transition metals contamination . Moreover, this finding would be the fundamental data for further works to management of marine turtle conservation .

Acknowledgement: This research was funded by grants from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand . We are also grateful to the Sea Turtle Conservation Centre, the Air and Coastal Defense Command, the Sattahip Naval Base for their kind support in providing marine eggshell turtle samples .

TH 065Analytical microstructure on eggshells of green turtle (Chelonia mydas), hawksbill turtle (Eretmochelys imbricata), and leatherback turtleM . Areekijseree1, T . Chuen-Im2, K . Sribuddhachart3, C . Thepsithar2

1Silpakorn University, Nakorn pathom, Thailand2Department of BiologyFaculty of Science, Silpakorn University, Nakorn Pathom 730, Nakorn pathom, Thailand3Department of BiologyFaculty of Science, Silpakorn University, Nakorn pathom, ThailandAnalytical microstructure on eggshells of green turtle (Chelonia mydas), hawksbill turtle (Eretmochelys imbricata), and leatherback turtle, were studied by scanning electron microscope (SEM) with secondary electron imaging (SEI) detectors . All marine turtle eggshells composed of 3 layers, which were outer cuticle layer or calcareous layer, middle layer or middle multistrata layer, and inner fibrous layer . Generally, morphology of the outer layer (cuticle layers) that composed of the aragonite form of calcium carbonate (CaCO3) . It is thick and porosity . The middle layer is compact thick that composed of CaCO3 . While, the inner layer is compact thin . However, there also were different characteristics among 3 eggshell types . The cuticle layers of green turtle eggshell contained loose nodular units forming as clusters of branching needle-like crystals without interlocking attachments . The leatherback turtle eggshells had a cuticle layer which was similar to the green turtle but their crystals were more tapering and smaller . For hawksbill turtles, the cuticle layers contained ball-shape and needle-like crystals . The middle layers and the inner layers consisted of fibers that were firmly forming as network thorough the layers . For the middle layers, sodium chloride was also observed as complexes in the compositions . The outer layer thickness of green turtles, hawksbill turtles, and leatherback turtles were 129 .88±14 .22, 117 .51±16 .19, and 83 .67±17 .15 m, respectively . While the middle layer thickness were 108 .06±13 .00, 118 .32±20 .06, and 144 .20±2 .20 m, respectively, the inner layer thickness of green turtles, hawksbill turtles, and leatherback turtles were 11 .38±3 .07, 3 .72±0 .86, and 9 .69±2 .53 m, respectively . This work reviewed the utilization of SEM that classified marine turtle eggshell for management of marine turtle conservation .

Acknowledgement: This research was funded by grants from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand . We are also grateful to the Sea Turtle Conservation Centre, the Air and Coastal Defense Command, the Sattahip Naval Base for their kind support in providing marine eggshell turtle samples .

TH 066Will global warming have an influence on the adverse effects caused by pesticides? A first approach using amphibians as a modelE .G .M . García-Muñoz1, I .L . Lopes1, M .A .C . Carretero2, A .M .V .M . Soares3

1CESAM, Aveiro, Portugal2CIBIO, Vairao, Portugal3University of Aveiro & CESAM, Aveiro, PortugalOver the past thirty years, climate change has produced an enormous biodiversity loss as well as a shift in species distribution ranges . Amphibians epitomize the modern biodiversity crisis, and global climate change may be directly and indirectly responsible for amphibian’s decline . Although amphibians are considered less sensitive to direct temperature shifts than other groups, small changes can trigger cascade effects when thermal disturbance is combined with pollutants . Among these indirect consequences, temperature changes have been demonstrated to modify the lethal and sublethal effects of contaminants in biota . Most studies addressing the adverse effects posed by contaminants to several amphibians species have been performed at moderate, constant ambient temperature of approximately 20 ºC, which is an over simplification of what happens in the environment (for example, temperatures as high as 40ºC may occur, with diary variations of 5ºC) . In fact, there is fragmental but strong evidence suggesting that temperature induces changes in acute toxicity of chemicals, hence, the effect under laboratory conditions may substantially differ from that observed in real ecosystems .

The aim of this project, addresses the question: Could be that changes in environmental temperature regimes, due to climate changes, influence the adverse effects caused by pesticides on amphibians?

Future results from small scale experiments (laboratory experiments) may be validated by conducting studies at large spatial and temporal scales with a higher ecological realism (mesocosms) . The underlying mechanisms responsible for the changes observed in experimental systems at a large scale, not replicable, may be examined at lower levels, such as subcellular levels (biochemical biomarkers), individual level (survival, growth/development, behaviour) or population level (recruitment) .

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As a further step, studies as the one proposed here, exploring the interactions between climate change and pesticides contamination at different hierarchical levels, will provide a better understanding of the responses of species to novel situations, such as global warming and, hence, the basis for prevention, management and mitigation measures .

EC02P -Contaminant pathways, trends and biological effects in a warmer Arctic

TH 073Occurrence and seasonality of cyclic volatile methyl siloxanes in Arctic airI .S . Krogseth1, K . Breivik1, M . Schlabach1, A . Kierkegaard2, M .S . Mclachlan2

1Norwegian Institute for Air Research, Kjeller, Norway2Stockholm University, Stockholm, SwedenCyclic volatile methyl siloxanes (cVMS) are high-volume production chemicals occurring in a wide range of technical applications and personal care products . Due to the high vapor pressures of cVMS, volatilization to the atmosphere is their main mode of emission to the environment . In the atmosphere, cVMS have half-lives ranging from 6 .9 to 10 .6 days due to reaction with hydroxyl radicals, indicating that they are persistent in the atmosphere according to the definition by US EPA . In addition, model simulations have predicted cVMS to have potential for long-range atmospheric transport . However, measurements of cVMS in the atmosphere of remote areas, such as the Arctic, remain scarce . Thus, there is a critical need for more empirical data to assess their long-range atmospheric transport potential and atmospheric behavior . At high latitudes large seasonal variations in the amount of sunlight are followed by corresponding large variations in atmospheric hydroxyl radical concentrations . In addition, cold temperatures in the winter season slows down chemical reaction rates . As a consequence, air concentrations of cVMS are expected to vary substantially in the Arctic atmosphere, with relatively high concentrations in winter (polar night) and low concentrations in summer (midnight sun period) . This has been predicted by model simulations, but still remains to be validated by observations . In this study an active air sampling method using solid phase extraction cartridges, previously validated and applied for decamethylcyclopentasiloxane (D5) in Sweden, was adapted for Arctic conditions and further evaluated for hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4) and dodecamethylcyclohexasiloxane (D6) in addition to D5 . The method was applied to collect air samples at the Zeppelin observatory in Ny Ålesund (79°N), Spitsbergen, Norway, to assess the atmospheric burdens of cVMS and to investigate if the predicted seasonality is occurring . Results from various sampling campaigns will be presented and discussed, including results from method validation .

TH 074Emerging persistent organic pollutants in arctic air, surface water and snow in Ny-Alesund, SvalbardZ . Xie, A . Moeller, G . Zhong, Z . Zhao, R . Sturm, R . EbinghausHelmholtz-Zentrum Geesthacht, Geesthacht, GermanyEmerging persistent organic pollutants (ePOP) may reach ecologically sensitive Arctic environment via atmospheric and/or oceanic long range transport . They are subject to a variety of processes in the Arctic environment such as degradation, bioaccumulation and interaction between the atmosphere, snow, water and soil . Additionally, climate change may significantly influence the transport and environment fate of ePOPs in the Arctic . As a part of collaborative German-French program at joint French-German Arctic Research Base (AWIPEV) in Ny-Alesund, Svalbard, this project is proposed to investigate the occurrence and long term trends of ePOPs in arctic air, water and snow .

Integrated high-volume air samples were taken on the roof of IPEV station using a high-volume pump operated for 7 days to obtain a volume of ~2000 m3 . A glass fiber filter is used to trap the airborne particles and the gaseous contaminants are collected with a PUF/XAD-2 resin column . Surface snow samples were collected on the glaciers in Ny-Alesund and seawater samples were obtained in Kongs Fjord in May of 2011 . All samples were analyzed for neutral and ionic perfluorianted compounds, brominated flame retardants, dechlorane plus and current-use pesticides . Data achieved from this study may improve models to predict the environmental progression and assess the effect of climate change on the long-range transport and the fate of the ePOPs in the Arctic ecosystem .

TH 075Brominated flame retardants in ice cores from Svalbard: an east-west comparisonM .H . Hermanson1, E . Isaksson2, C . Teixeira3, O . Brandt2, S . Forström2, T . Aasen2, D .C .G . Muir3

1University Center on Svalbard, Longyearbyen, Norway2Norwegian Polar Institute, Tromsø, Norway3Environment Canada, Burlington, CanadaIce cores from Svalbard have proven to be useful indicators of contaminant conditions in the European Arctic . Our previous work has shown that ice cores from eastern Svalbard (the Austfonna site, 79 .48oN, 24oE, 740 m .a .s .l .) shows a larger number of current-use pesticides in its profile than observed in western Svalbard (the Holtedahlfonna site, 79 .13oN, 13 .27oE, 1150 m .a .s .l .) . Since our earlier work, comparisons between these sites for other contaminants have been of interest . In 2007 we drilled a short core (12 m) at Austfonna in order to identify the burden and inputs of brominated flame retardants at that site, providing comparable data for Holtedahlfonna . Two of the most abundant BFRs to appear in marine and glacial samples from Svalbard include decabromodiphenyl ether (BDE-209) and hexachlorocyclododecane (HBCD) . The results for these BFRs at Holtedahlfonna show growing inputs over time since the 1970s, reaching 322 pg cm-2 yr-1 and 910 pg cm-2 yr-1 between 1995 and 2005 . Results from Austfonna cover 1995 - 2007 . Because of larger sample size, we were able to obtain 3 samples over this period from 1995 - 2000, 2000 - 2004, and 2004 - 2007 . Our results for BDE-209 show fluxes of 1 .95, 47 .9 and 28 .3 pg cm-2 yr-1 from 1995 and ending in 2007, the surface about 10% the input found at Holtedahlfonna . For HBCD, the Austfonna inputs over the same time intervals are 272, 315 and 804 pg cm-2 yr-1 over the intervals from 1995 - 2007 . The HBCD results at Austfonna are lower, but between 2004 and 2007 are about 88% of the Holtedahlfonna flux from 1995 - 2005 (910 pg cm-2 yr-1) . The burden of BDE-209 at Austfonna is 466 pg cm-2 while at Holtedahlfonna over similar time period it is 3232 pg cm-2 yr-1, about 7 times more . The burden of HBCD is 8348 pg/cm2 at Austfonna (1995 - 2007), while at Holtedahlfonna it is 9078 pg cm-2 yr-1, about 9% more . These results clearly show that our earlier observations of a greater pesticide input at Austfonna than Holtedahlfonna are not true for BFRs, at least after 1995 . Different classes of compounds likely have different sources in the region; pesticides may be delivered in the gas phase while BFRs are certain to be on particles . These and other characteristics of long-range atmospheric transport in this region of the Arctic likely play a role in the contrasting observations we have made between pesticides and BFRs . TH 076Atmospheric deposition of legacy and emerging persistent organic pollutants in Northern SwedenS .R . Newton1, T .F . Bidleman2, M . Tysklind2, K . Wiberg3

1Stockholm University, Stockholm, Sweden2Umeå University, Umeå, Sweden3Swedish University of Agricultural Sciences, Uppsala, SwedenThe Arctic is an ideal indicator region for Persistent Organic Pollutants (POPs) because it is far from sources of these contaminants . To reach remote areas such as the arctic, POPs undergo long range transport via multiple cycles of atmospheric deposition and re-volatilization, known as the grasshopper effect . Thus, atmospheric deposition is believed to be an important pathway for arctic contamination . The aim of this study was to quantify the flux of legacy POPs from atmosphere to land in the Arctic as well as screen for emerging POPs new to the arctic . Bulk atmospheric deposition samples using Amberlite IRA-743 as an adsorbent for hydrophobic pollutants were taken every two months for one year at two different locations in northern Sweden . The first sampling site was a subarctic site approximately 60 km from the city of Umeå . The second site was within the Arctic Circle, in a remote area near Abisko National Park . A method was developed for the analysis of various legacy and emerging POPs in these samples based on GC-HRMS . Two current use pesticides and four flame retardants, which have scarcely been detected in the arctic, were identified in the samples: trifluralin, chlorothalonil, TBECH, hexabromobenzene, BTBPE, and Dechlorane Plus . Hexabromobenzene, BTBPE, and Dechlorane Plus were detected in all samples and TBECH and Trifluralin were detected in more than half of the samples . The ratio of the two isomers of Dechlorane Plus was similar to the technical product at the first site; however, the more remote site exhibited a reversal in the dominant isomer indicating isomer selective degradation during long range transport . Legacy compounds including isomers of HCH, PCBs, DDT and metabolites, and chlordane related compounds were quantified in levels comparable to similar studies . PCB congener profiles differed between the two sites as well, with a lower proportion of PCB 180 and a higher proportion of PCB 52 depositing at the remote site . Nearly all compounds at both sites showed an increase in deposition during the months of June and July correlating with increased precipitation .

TH 077Assessing the potential influence of global climate change on the long-range transport and accumulation of organic contaminants in the Arctic environment in relation to physical-chemical propertiesJ .M . Armitage, F . WaniaUniversity of Toronto at Scarborough, Toronto, CanadaThe potential influence of global climate change (GCC) on the fate, transport, bioavailability and bioaccumulation of organic contaminants is rapidly becoming an area of interest for scientists engaged in environmental chemistry and toxicology research . The Arctic region is of particular concern due to changes to the cryosphere that are already occurring (e .g . reduction in sea-ice cover and volume, melting of permafrost and glaciers), the fact that climate change models project the greatest warming to occur in this region in the future and finally that human inhabitants of this region harvesting local food resources (e .g . seals) have an elevated exposure to many organic contaminants of concern (i .e . POPs) (1-3) . Global climate change could potentially influence exposure to organic contaminants via direct (e .g . altered distribution of chemicals in the environment) and indirect (e .g . increased primary productivity = altered organic carbon cycling = altered fate in aqueous environments) pathways (1) . For human beings, lifestyle changes (e .g . dietary transition) facilitated by changes in the environment (reduced sea-ice = increased delivery of imported food items) could also be highly influential in the long-term (4) . The purpose of this investigation is to explore the potential influence of changes to the physical environment (e .g . temperature, precipitation rate, sea-ice cover, primary productivity) on the long-range transport and accumulation of neutral organic compounds in the Arctic using a global-scale fate and transport model . Simulations were conducted for hypothetical chemicals covering a wide range of partitioning properties for a 20-year constant emission period followed by a 20-year depuration (i .e . zero primary emission) period . While sensitivities to the GCC scenario parameterizations considered here are relatively low (typically within a factor of two), responses can differ substantially depending on i) partitioning properties, ii) compartment (e .g . air, water, soil) and iii) stage of emission (uptake vs . depuration) . 1 . Macdonald RW, Harner T, Fyfe, J . 2005 . Sci . Tot . Environ . 342: 5 - 86 .

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2 . Arctic Climate Impact Assessment (ACIA) . 2005 . Arctic Climate Impact Assessment . Cambridge University Press, Cambridge, UK . 3 . Donaldson SG et al . 2010 . Sci . Tot . Environ . 408: 5165 - 5234 . 4 . Armitage, J .M .; Quinn, C .L .; Wania, F . J . Environ . Monit ., 2011, 13, 1532-1546 .

TH 078Increasing environmental concentrations of POPs in the Arctic in a future warmer climateK .M . Hansen, J .H . Christensen, J . Brandt, L .M . Frohn, C . Geels, A . Gross, A .B . Hansen, G .B . Hedegaard, C .A . SkjøthAarhus University, Roskilde, DenmarkThe Danish Eulerian Hemispheric Model (DEHM) was applied to study the impact of climate change on contaminant levels in the Arctic . DEHM is a 3-D atmospheric chemistry-transport model developed over the last 20 years for studying the long-range transport of SO2, SO4, and Pb to the Arctic . All important source regions for the Arctic are included in the model domain, which covers the Northern Hemisphere . This model has been developed further to include mercury species/chemistry, and Persistent Organic Pollutants (POPs) . The model has a spatially detailed 3-D atmosphere up to 15 km over the surface . In addition, it has four surface compartments: a 75 m thick ocean layer, a 15 cm thick soil layer, and dynamically evolving vegetation and seasonal snowpack compartments . The model system has been run with meteorology obtained from ECHAM5/MPI-OM (SRES A1B scenario) for two different decades: 1990-1999 and 2090-2099 . In this climate scenario the global temperature is predicted to increase continually with 3 ºC by the end of 2100 relative to the period 1971-2000 . The model system was run initially for a period in order to spin-up the concentrations of POPs in the ocean and soil compartments . The concentrations in air, ocean water and soil of all the species from this simulation where used as initial concentrations for three different model runs with constant emissions (from year 2000) for the decades: 1990-1999, 2090-2099 and 2190-2199 using input from the ECHAM5/MPI-OM climate model . Simulations were also performed without spin-up concentrations . The differences between the results for the three decades are hence only due to changes in the meteorological as predicted by the ECHAM5/MPI-OM model system . The results produced with the DEHM model system shows that the atmospheric concentrations of the studied POPs will increase . As the surface concentrations (soil and ocean water) also increases, this indicates that there will be an increased transport to the Arctic in a future warmer climate . This study has shown that it is useful to use meteorological output from climate models to investigate the exposure levels of contaminants under future changed climate conditions .

TH 079Concentrations and potential sources of some semi-volatile organic contaminants in the air at Little Fox Lake, Yukon Territory, CanadaJ .N . Westgate1, U .M . Sofowote2, P . Roach3, P . Fellin4, I . d’Sa5, E . Sverko5, Y . Su5, F . Wania1, H . Hung5

1University of Toronto Scarborough, Toronto, Canada2Ontario Ministry of the Environment, Toronto, Canada3Aboriginal Affairs and Northern Development Canada, Whitehorse, Canada4AirZOne, Mississauga, Canada5Environment Canada, Burlington, CanadaIn an investigation of the levels and potential sources of Semi-Volatile Organic Contaminants (SVOCs) at Little Fox Lake (LFL) in Canada’s Yukon Territory, air samples were collected from 2007 to 2009 . Air was drawn through a PM10 sampler inlet to exclude snow and large particles, then a Glass Fibre Filter (GFF) and dual Poly-Urethane Foam (PUF) plugs to trap the analytes . The sampling media were changed every 24 hours over two two-week periods in the late summer of 2007 and 2008, while week-long samples were collected the remainder of the deployment . After solvent extraction, column clean-up and fractionation, samples were analyzed by GC-ECD and GC-NCI-MS for a suite of SVOCs including some chlorinated pesticides, benzenes and industrial by-products . The median detection frequency for all compounds was 11%, ranging from 1% to 99% . Concentrations of most substances appeared to be independent of one another, but a weak but significant correlation appeared between levels of α-Endosulfan and trans-Nonachlor (concentrations were log-transformed, r2 = 0 .50, p < 0 .001) . After correcting for sample volume, concentrations of analytes ranged up to 22 pg m-3 for α-Hexachlorocyclohexane (α-HCH), considerably lower than the levels of α-HCH reported for the same station in 2000-2003, which was detected in 97 percent of samples (%OS) . This was followed by pentachloroanisole (19 pg m-3, 19% OS), pentachlorobenzene (17 pg m-3, 99% OS), α-Endosulfan (10 pg m-3, 58% OS), trichloroveratrole (9 pg m-3, 97% OS) and a handful of others with low concentrations or low detection frequency . Hexachlorobenzene was present in nearly all samples but could not be quantified due to PUF breakthrough . The measured concentrations were combined with back-trajectories to elucidate potential source areas of these contaminants . The method is conceptually a Concentration Weighted Trajectory analysis, but trajectory endpoints above 700m elevation were filtered out and a ‘geodesic’ grid was employed to avoid skewing near Earth’s poles . Most analytes were not revealed to have specific source regions . The method does suggest that the primary source of γ-HCH to the air at LFL is the Bering Strait, but the Chukotka region in Easternmost Siberia was highlighted as well . Tri- and tetra-chlorinated benzenes correlated with air-masses that originate in Inuvik, Northwest Territories .

TH 080Heavy metals and nutrients in soils of the Yamal Peninsula (West Siberia) as indicator of ecosystem stabilityD .V . MoskovchenkoInstitute of Northern Development, Tyumen, Russian Federation The development of gas fields on the Yamal peninsula (West Siberian Arctic) leads to disturbances of landscapes . Vegetation of the Arctic tundra is the most changeable component prone to surficial and subsurface impacts . Vegetation and related peat stabilize the thermal regime of Arctic soils and thus prevent permafrost from degradation . The patterns of vegetation and recovery depend directly on soil chemistry . Vegetation on soils depleted in major and trace elements is poor, and such landscapes are weakly sustainable . Thus, the sustainability of plant-soil systems largely depends on their chemical composition . The biogeochemistry of permafrost plant-soil systems has been studied in soil and plant samples collected through environmental profiling in the Arctic tundra of the Yamal Peninsula . Sampling was along profiles that traverse different geomorphic levels and different types of disturbances (drilling sites, staffsettlements, quarries of mineral ground) . Soils in northern West Siberia are deficient in many plant nutrients necessary for rapid and efficient revegetation in disturbed areas . Disturbance to peat topsoil reduces the store of organic matter, potassium, and nitrogen . Mechanic damage and pollution cause degradation or extermination of plant communities, while the remediation efficiency depends on the availability of mineral nutrients . Technogenic loads at development sites change dramatically the geochemical framework of landscapes . Specifically, soils at drilling sites are contaminated with Ba, Sr, Pb and petroleum hydrocarbons . The chemical composition of different types of soils varies greatly . Zonal tundra peat-gley soils are depleted in P and have low cation exchange capacity (CEC) and total absorbed bases (TAB), alluvial soils have highest P and K enrichment, while peat in bog-tundra and fen soils is the richest in nitrogen . The sustainability as an ability of rapid recovery after disturbances (“elasticity”) is the maximum in alluvial ecosystems and depends on the contents of plant nutrients in soils .

TH 081The 2012 assessment report on persistent organic pollutants in the Canadian ArcticP .B . Kurt-Karakus1, D .C .G . Muir2

1Bahcesehir University, Besiktas-istanbul, Turkey2Environment Canada, Burlington, on, Canada The Northern Contaminants Program (NCP) has been documenting levels and trends of persistent organic pollutants (POPs) in the Canadian Arctic since 1991 . The program includes measurements of legacy and new POPs in high volume air samples at Alert on Northern Ellesmere Island as well as more limited measurements using passive air sampling . The same chemicals are determined in samples of arctic biota (seabirds, beluga, seals, lake trout, burbot, arctic char) from multiple locations in Nunavut, the Northwest Territories and the Yukon . As a result of annual sampling of selected species initiated in 2004, temporal trend data sets for legacy and new POPs in biota are improving in statistical power . Scientific information about legacy and new persistent organic contaminants in the Arctic that is generated under the NCP has been contributed to circumpolar assessments of the Arctic Monitoring and Assessment Program (AMAP) . These arctic data have played an important role on addition of new substances to the Stockholm Convention and the Convention on Long-Range Transboundary Air Pollution, and the implementation of these international agreements, particularly for the purpose of assessing their effectiveness . Because contaminants are accumulated in the Arctic primarily from long-range transport, data on new chemicals from arctic monitoring is considered as critical evidence when assessing the need to add new substances to these conventions . The issues have been addressed in the NCP have also provided the scientific base for Canada to evaluate the significance of the issue and it equipped the Government of Canada to advocate on the international stage for legal agreements on ban or phase out of POPs that end up in the Arctic . In this study, we will present highlights from the recent assessment of spatial and temporal trends of POPs in Canadian Arctic that assessed results over the period 2003-2010 . This assessment noted declining concentrations of legacy POPs such as chlordane, DDT, HCH and toxaphene and more limited declines of PCBs and HCB in the atmosphere and in biota . Trends of new and emerging POPs during 2003-2010 varied with increases (HBCD, BTBPE), plateauing levels ( PBDEs) and peaks followed by declines (perfluorinated chemicals; PFCs) in biota and slow increases of PBDEs and PFCs in air .

TH 082POPs in the Arctic & climate change, Part C: hemispheric transport of air pollution, UNECEP .W . Bartlett1, T . Harner2, H . Hung2, G . Lammel3, R . Guardans4, A . Gusev5, Y .F . Li2, J . Ma2, R . Macdonald6, V . Shatalov5

1Saint Peter’s College, Jersey city, United States of America2Environment Canada, Toronto, Canada3Max Planck Institute for Chemistry & Masaryk University, Mainz, Germany4Ministry of the Environment and Rural and Marine Affairs, Madrid, Spain5Meteorological Synthesizing Centre-East, Moscow, Russian Federation6Fisheries and Oceans Canada, Sydney, CanadaThe Arctic region is ideal to observe intercontinental transport of Persistent Organic Pollutants (POPs), since it has few regional POPs sources . Conversely, it is crucial to understand the emissions of POPs from Northern Hemisphere continents and transport to the Arctic before projecting exposures to toxics within the Arctic . We present here key findings of an assessment of the transport of air pollutants across the Northern Hemisphere conducted by the Task Force on Hemispheric Transport of Air Pollution (TF HTAP) for the consideration of the Convention on the Long Range Transport of Air Pollutants . This presentation will emphasize the TF HTAP integrated approach to measurements, modeling, emissions and impacts, but focus on measurements and modeling, with special attention to climate change, which may alter POPs cycling between environmental compartments . Most scientific projects are not coordinated in a way to facilitate

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synergy and integration . The TF HTAP collaboration enabled communication between specialists and identification of the most important cross-dependencies and research gaps . Furthermore, the review permitted the collating and sharing of an immense amount of data, among which recent IPY observations played a crucial role . Modeling enabled comparison between diverse and, sometimes, competing approaches with the benefit that model parameterizations could be exchanged . This intercomparison led to better appreciations of the robustness of models and the importance of uncertainties . Mindful of overlap with other international organizations, the TF HTAP is initiating a new four-year work plan with a multi-pollutant co-benefit approach that harmonizes experiments, model parameterization and exploitation of results . Collaborations are voluntary, and key areas, like global POPs emission estimates, remain bottlenecks for further progress . Global estimates have often been provided in forms that cannot be used by models . For instance some emission estimates under UNEP have been reported by country, not location, substances are sometimes grouped together, and sometimes emissions are given without uncertainties . Continued monitoring will be essential to discriminate between the effects of climate change and progress resulting from emission regulations . The five TF HTAP volumes provide an extensive synthesis of the state-of-science and a guide to future research and policy needs . http://www .htap .org/activities/2010_Final_Report .htm

TH 083Brominated flame retardants and polychlorinated biphenyls in the Antarctic food webH . Wolschke1, A . Möller2

1Helmholtz-Zentrum Geesthacht, Institute of Coastal Research, Geesthacht, Germany2Helmholtz-Zentrum Geesthacht, Geesthacht, GermanyAntarctica is a pristine desert continent on which industrial and agriculture activities never existed . Therefore, contaminants primarily reach the continent by long-range atmospheric transport (LRAT) . Since semi-volatile persistent organic pollutants have a documented LRAT potential these substances are of special importance for Antarctic research . Their lipophilic and persistent nature allows accumulation in organisms and magnification throughout the food web . In this study various organisms have been analysed for the dioxin-like PCB congeners, polybrominated diphenyl ethers, alternative brominated flame retardants as well as Dechlorane Plus in order to investigate the contamination in the food web in Antarctica and estimate the risk for the species analysed imposed by PCBs (calculation of the toxic equivalents -WHO) . The samples were collected during in November 2010 to January 2011 around the Chinese “Greatwall-Station”on King George Island . The freeze-dried biota samples were extracted in a Soxhlet apparatus using dichloromethane . Extracts were purified by gel permeation chromatography to remove large molecules such as fatty acids, and further by silica-gel clean-up to remove polar compounds . Gas chromatography coupled with mass spectrometry was used for instrumental analysis . The results indicate biomagnification throughout the food web from the herbivores to the carnivores . The detected concentrations were in the high pg/g to the low ng/g wet weight range .

TH 084POPs affect the plasma steroid levels in polar bears, Ursus maritimusB . Styrishave1, M . Hansen2, J . Bytingsvik3, T . Ciesielski3, I .T . Hansen3, L . Gustavson3, J . Aars4, E . Lie5, B .M . Jenssen3

1Section of Toxicology, Copenhagen, Denmark2Section of Toxicology, University of Copenhagen, Copenhagen, Denmark3Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway4Norwegian Polar Institute, Polarmiljøsenteret, Tromsø, Norway5Norwegian School of Veterinary Science, Oslo, NorwayThe Polar bears, Ursus maritimus, is the ultimate top predator of the arctic food chain . Due to biomagnification, polar bears are therefore exposed to high levels of persistent organic pollutants (POPs) . Such POPs may potentially exert endocrine effects on sensitive pathways such as the steroidogenesis . Genital deformities in polar bears from the Svalbard region indicate that vital processes such as growth and reproduction may be impaired . Such impairments may potentially be caused by POPs . We have implemented a recently developed GC-MS/MS method for the simultaneous detection of 9 steroids (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol, and 17β-estradiol) in plasma from 35 adult and subadult polar bears (3-23 years) collected in April 2008, Svalbard, Norway . POPs and their metabolites levels (HCB, α-HCH, β-HCH, oxychlordane, trans-nonachlor, mirex, p,p’-DDE, p,p’-DDT, PCBs, OH-PCBs, BDE-47) were also analysed in plasma . This allows for the first time an assessment of the impact of POPs on the overall polar bear steroidogenesis . Relationships were investigated using Principal Component Analysis . Significant impact of POPs on steroid plasma levels was observed . The coupling between POPs and steroid hormone levels may have serious implication for reproduction in polar bears . Thus, combined with climate change, POP exposure may exert detrimental impact on polar bear growth and reproduction .

TH 085Temporal trends of legacy and new persistent organic pollutants in landlocked char in High Arctic Lakes in CanadaC .G . Muir1, G . Köck2, X . Wang1, D . Iqaluk3, M . Williamson1, G . Lescord4, K . Kidd4, E . Barresi1, E . Sverko1

1Environment Canada, Burlington, Canada2Austrian Academy of Sciences, Vienna, Austria3Resolute Bay, Resolute bay, Canada4University of New Brunswick, St . john, CanadaLandlocked char are important sentinel species in Arctic lakes . As the only top predators in most high latitude Arctic lakes, char are good indicators of changes in inputs of bioaccumulative contaminants such as persistent organic pollutants (POPs) . In this long term study landlocked Arctic char are collected annually from three lakes near the community of Resolute Bay ( 74° 42’ N; 94° 51’W) on Cornwallis Island (Amituk, Char and Resolute) and in Lake Hazen (81o 49’N; 70o 42’ W in Quttinirpaaq National Park on Ellesmere Island . Results from 10 (Char) to 15 (Resolute) years of sampling are available with collection numbers ranging from 7 to 25 adult fish (>200 g) per lake except in Char Lake where the range has been 3 to 10 fish annually . Homogenized muscle (+skin) samples have been analysed for PCBs, chlorobenzenes, DDTs, chlordanes, dieldrin, and toxaphene, as well as new POPs (endosulfan, HBCD, HCHs, hexachlorobutadiene, PBDEs, PFOS) to assess trends over time . ΣPCB concentrations declined significantly in Char Lake (1993-2010), Amituk (1992-2010) and Hazen (1990-2010), but not in Resolute (1997-2010) . ΣHCH also declined significantly and at a more rapid rate than other POPs . Toxaphene concentrations have also declined although the trend is significant only in Lake Hazen . Endosulfan (including endosulfan sulfate) concentrations increased during the 1990s until about 2004, particularly in Amituk and Resolute Lakes and have since declined . Concentrations of tetra- and penta-BDE congeners increased significantly and rapidly in most of the lakes during the 1990s-early 00s . The year for maximum concentrations varied from 2003 in Amituk, 2005 in Resolute to 2008 in Lake Hazen . While local contamination, i .e . from waste waters or dumpsites, does not appear to have influenced levels of most POPs, this is not the case for PFOS and related chemicals used in fire fighting foams in Resolute Lake . This lake is downstream of the Resolute airport where these chemicals appear to have been used in the past . On the other hand the perfluorocarboxylates (PFCAs), were present at similar low concentrations in all lakes . Preliminary temporal trend results suggest that PFC concentrations have plateaued in Amituk, Char and Hazen lakes and are now declining .

TH 086Monitoring and modelling reveals time trends of POPs in Arctic biotaF . de Laender1, J . Hammer2, A .J . Hendriks3, K . Soetaert4, C .R . Janssen1

1Ghent University, Ghent, Belgium2University of Amsterdam, Amsterdam, Nederland3Radboud University, Nijmegen, Nederland4NIOO CEME, Yerseke, NederlandProtecting Arctic ecosystems against potential adverse effects from anthropogenic activities is recognized as a top priority . In particular, understanding the accumulation and effects of persistent organic pollutants (POPs) in these otherwise pristine ecosystems remains a scientific challenge . A typical feature of data bases that contain monitoring data on tissue concentrations of POPs in Arctic biota is that these are fragmentary, i .e . not all POPs have been measured in all species . Here, we present a new approach to predict tissue concentrations in species for which no data exist by using the available tissue concentrations, a bioaccumulation model and time trend modelling . We demonstrate this approach for the Barents/Norwegian Sea ecosystem and show that the legacy-POPs decreased by a factor of 10 between 1985 and 2010, which reflects regulatory efforts to restrict these substances . In contrast, concentrations of fossil fuel derived PAHs in lower trophic levels (invertebrates and fish) increased 10 to 30 fold over the past 25 years and now dominate the summed POP burden in these biota . Our findings indicate that the debate on the environmental impacts of fossil fuel burning should move beyond the expected sea water temperature increase and examine the possible environmental impact of fossil fuel derived PAHs .

TH 087Persistent organic pollutants in Antactic Krill (Euphasia superba)C .J . Galbán-Malagón, G . Hernán, N . Berrojálbiz, M .J . Ojeda, M .J . Abalos, E . Abad, J . DachsIDAEA-CSIC, Barcelona, Spain Krill is a key node in the Antarctic ecosystem and the most important source of food to higher trophic levels in the Antarctic Region . Few Studies have reported legacy and emerging POPs concentrations in krill samples and when done it has for a reduced number of samples and covering a limited spatial extension . Hexachlorocyclohexanes (HCHs), Hexachlorobenzene (HCB), Polychlorinated biphenyls (PCBs), dibenzo dioxins and furans (PCDDFs) and Polybrominated diphenyl ethers (PBDEs) concentrations were reported previously in the bibliography . The objective of this work is to report concentrations of PCBs, HCHs, HCB, PBDEs and PCDDFs in Antarctic Krill (Euphasia superba) . Samples were taken in February-March 2009 in the Weddel Sea, Bransfield Strait and Bellinghausen Sea around the Antarctic Peninsula . The common pattern between the different families of POPs and the influence of biogeochemical factors and proximate ice regions will be discussed .

TH 088Effects of a warmer climate on temporal trends of persistent organic pollutants in Greenland biotaK . Vorkamp, R . Dietz, F .F . RigétAarhus University, Roskilde, DenmarkPersistent organic pollutants (POPs) have been used extensively in industry, agriculture and consumer products and have reached the Arctic via atmospheric and ocean transport . They include organochlorine compounds such as polychlorinated biphenyls (PCBs) and DDT, which are regulated by the Stockholm Convention and were banned in most industrialised countries about 30 years ago, as well as fluorinated and brominated compounds, such as perfluorooctane sulfonate (PFOS) or hexabromocyclododecane

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(HBCD), recently phased out or still unregulated . These compounds have in common to bioaccumulate and biomagnify through the Arctic food chain, with biomagnification factors depending on the properties of the compound and species-specific metabolic capacities . In Greenland, systematic monitoring of POPs has covered the last 16 and 28 years for organochlorines and polybrominated diphenyl ethers (PBDEs), respectively . In general, organochlorines have decreased in biota of the marine and the freshwater environment, however, with indications of stabilisation since approximately 2000 . Brominated compounds have been shown to increase, following their increasing use in industry and a wide range of consumer products, but decreasing PBDE concentrations have been observed in adult seals from East Greenland over the last 5-10 years, a trend which might also be expected for other species, due to recent regulations . Climate change and its consequences for the Arctic might also have an impact on the fate of POPs, in terms of e .g . changes in transport pathways and feeding behaviour of Arctic wildlife . POP time series in ringed seals from Disko Island (West Greenland) are currently being studied, in combination with climate information and data on trophic level . Local climate indices include winter ice coverage in Disko Bay and summer water temperatures and salinity at Fylla Banke near Nuuk . In addition, the Atlantic Oscillation index is used as a more global climate index . Furthermore, data are included on stable nitrogen isotopes in ringed seal muscle, to give information on trophic variability .

Acknowledgements The authors wish to thank Arctic Station and the University of Copenhagen for providing the data on daily ice coverage in Disko Bay . We also thank Mads Hvid Ribergaard, Danish Meteorological Institute (DMI) for providing the data on sea water temperature and salinity at the top of Fyllas Banke .

TH 089Impact of dense shelf water cascading in the transfer of organohalogenated contaminants offshore Svalbard, Arctic OceanJ .A . Salvadó1, J . Grimalt1, C . Canals2

1Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, Spain2GRC-Marine Geosciences, University of Barcelona, Barcelona, SpainThe formation of dense shelf water cascading depends on the evaporation or cooling of the sea surface, which depends on air temperatures . Only if the air-sea surface gradients become large enough, sea-ice freezing may lead to the formation of dense waters at Storfjorden (Svalbard Archipelago) . The Arctic Ocean warming may have an influence on the production of cascading water masses flowing from the Storfjorden shelf area to the continental slope, which may contribute to the rapid transfer of persistent organic pollutants into the open marine sediments . To date no studies have considered the influence of these cascading processes in the transport of contaminants from the continental shelves to the deep marine environments, and there are no records of organohalogenated contaminats in open sea sediments from the Arctic Ocean . For this purpose, sediment cores were collected at 500, 1000, 1250, 1500 and 2000 m water depths along the Storfjorden area and the continental slope during two oceanic cruises (July 2010 and July 2011) . These organohalogenated compounds encompass organochlorine pesticides and industrial products, as polychlorobiphenyls and polybrominated diphenyl ethers (PBDEs) . The results show detectable values of all analysed compounds, with higher concentrations of HCB and 4,4’-DDE considering organochlrine compounds, and higher levels of BDE-47 and BDE-100 congeners, relating PBDEs .

TH 090Biogeochemical and physical controls on legacy persistent organic pollutants in the Antarctic atmosphere, seawater and planktonC .J . Galbán-Malagón, N . Berrojálbiz, M .J . Ojeda, J . DachsIDAEA-CSIC, Barcelona, SpainLegacy Organochlorine compounds including Polychlorinated Biphenyls (PCBs), Hexachlorobenzene (HCB) and Hexachlorocyclohexane (HCs) are ubiquitous pollutants that reach remote ecosystems through atmospheric transport and deposition . Persistent organic pollutants were detected in Antarctica for the first time in the 60s by Risebrough and coworkers, but despite these pioneering work few studies have been centered in the marine environment . Antarctic studies have reported occurrence of some of these chemicals in seawater, the atmosphere, and in zooplankton but no data are published for phytoplankton . In addition, previous studies did not assessed their simultaneous occurrence in air, water and plankton and the fluxes between these compartments . Current paradigms of the controls on the cycling and sinks of POPs in remote areas take into account the effect of temperature and organic matter cycling . Lately, the biological pump and physical pump (subduction of water masses) have also been postulated as important processes affecting the inputs of POPs to polar environments even though large uncertainties remain on the relative importance of all these processes affecting the occurrence of POPs in Antarctic waters and biota . The objectives of this work are: i) to measure the simultaneous occurrence of Legacy POPs in gas and aerosol phase, dissolved seawater and phytoplankton concentrations around the Antarctic Peninsula (Weddel Sea, Bransfield Strait and Bellinghausen Sea) and ii) to assess the physical and biogeochemical controls driving their occurrence in Antarctic seawater and plankton . This was carried out through two sampling campaigns in the austral summers of 2007/8 and 2008/9 . The results show low levels in all phases, but the lower levels in plankton are observed in the high productivity regions of the Weddel and Bellinghausen sea . The potential role of ice melting plays on levels in air, seawater and plankton was assessed at the ice margin role of the ¨Weddel Sea and Wilkins platform .

TH 091Relationships between mercury, organic matter, and climate changeH .S . Sanei, P . Outridge, G . Stern, J . Carrie, F . WangUniversity of Manitoba, Calgary, CanadaRecent studies in the remote pristine lakes in the Canadian High Arctic has shown that temporal trends of mercury (Hg) may significantly correlate with the labile, algal-derived organic matter (OM) over past decades and centuries . This correlation is attributed to increase in phytoplankton productivity during the latter half of the 20th Century; suggesting that the dynamics and fate of Hg in High Arctic lakes may have been seriously confounded by increased algal productivity as the result of recent climate change . The important questions facing this hypothesis are (1) the degree that climate change and resulting algal productivity influence the Hg flux into the aquatic systems (2) whether or not this phenomena is limited to Arctic region and what is the role of depositional environment in the observed pattern (3) how atmospheric flux of Hg as related to the local/regional anthropogenic source(s) may influence the Hg-OM correlation observed in the sedimentary records .

This study examines the historical records of Hg and OM from 40 high-resolution lacustine/marine sediment cores representing a wide range of depositional environment, climatic, and geographical conditions across Canada . The results show that the nature of the relationship between sedimentary Hg and OM may significantly change depending on the degree of Hg influx, and quantity and composition of the OM content of the sediments . It appears that OM, in particular, labile, mainly algal-derived OM primarily controls the Hg distribution in the sediments when the Hg influx (atmospheric deposition, runoff, etc) is within or close to background levels (e .g ., as in Arctic lakes) . When the Hg influx exhausts the capacity of OM scavenging (in the case of high anthropogenic input), excess Hg can be found in sediments, showing a clearly different temporal pattern to its organic counterpart . Only in these cases will sedimentary Hg profiles follow the history of anthropogenic Hg deposition . Given the relative homogeneity and paucity of OM in many Arctic and Sub-arctic lakes, small changes in climate may induce dramatic shifts in the rate of suspended OM scavenging of Hg, which is reflected in the sedimentary record of Hg .

TH 092Temporal trends of mercury in landlocked char in High Arctic Lakes in Canada; Possible influence of climate warming?C .G . Muir1, X . Wang1, G . Köck2, D . Iqaluk3, A . Sett1, P . Drevnick4, G . Lescord5, K . Kidd5, K . Gantner6, B . Francoeur1, G . Sardella1

1Environment Canada, Burlington, Canada2Austrian Academy of Sciences, Vienna, Austria3Resolute Bay, Resolute bay, Canada4Université du Québec, Québec, Canada5University of New Brunswick, St . john, Canada6University of Victoria, Victoria, CanadaAs the only top predators in most high latitude Arctic lakes, char are good sentinels for changes in inputs of bioaccumulative contaminants such as methyl mercury . In this long term study, landlocked Arctic char have been collected annually from three lakes near the community of Resolute Bay ( 74° 42’ N; 94° 51’W) on Cornwallis Island (Amituk, Char and Resolute) and in Lake Hazen (81° 49’N; 70° 42’ W in Quttinirpaaq National Park on Ellesmere Island . Results from 10 (Char) to 15 (Resolute) years of sampling are available with collection numbers ranging from 7 to 25 adult fish (>200 g) per lake except in Char Lake where the range has been 3 to 10 fish annually . Char muscle samples have been analysed for total mercury (THg) by cold vapour atomic absorption spectrometry after acid digestion in a high pressure microwave oven or by Direct Mercury Analyser . In addition the tissue digest was analysed for selenium and 30 other elements using ICP-MS . Geometric mean (length adjusted) concentrations of THg ranged from 0 .12 to 0 .30 ug/g (wet wt) in Resolute and Hazen lakes and from 0 .5 to 1 .6 ug/g ww in Char and Amituk Lakes Although THg levels were generally higher in samples from the mid-00s compared to the 1990s, no statistically significant trends were found in all 4 lakes using length adjusted geomeans for each sampling year . Results for Resolute Lake, with 15 sampling years, were closest to achieving the desired goal of detecting a 5% change (with a power of 80% at P=0 .05) at 16 yrs and power of 65% . These trends are in contrast to those observed in lake trout and burbot in Canadian sub-arctic lakes and rivers where increasing THg has been observed, and also to some mid-latitude lakes in the Great Lakes region where THg has been shown to be declining in fish . Preliminary comparisons with climate related data for the period 1990-2011 showed that THg in char in Resolute Lake were weakly correlated with average maximum air temperature in June and July with higher temperatures and average THg in 1997-98 and in 2005-06 . However additional annual sampling is needed to further evaluate this tentative relationship .

TH 093Temporal trends of cadmium in landlocked Arctic Char (Salvelinus alpinus) in the Canadian High Arctic: the possible influence of climate changeC .G . Muir1, G . Köck2, X . Wang1, D . Iqaluk3, P . Drevnick4, G . Lescord5, J . Reist6, J . Babaluk6, M . Flannigan7, H . Niederstätter8

1Environment Canada, Burlington, Canada2Austrian Academy of Sciences, Vienna, Austria3Resolute Bay, Resolute bay, Canada4Université du Québec, Québec, Canada5University of New Brunswick, St . john, Canada6Dept of Fisheries and Oceans, Winnipeg, Canada

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7Canadian Forest Service, Sault ste marie, Canada8Medical University Innsbruck, Innsbruck, AustriaThe Austrian-Canadian research cooperation “HighArctic 1997-2010”investigated the effects of short-term and long-term climate change on freshwater ecosystems in the Canadian High Arctic and in the Austrian Alps . The ongoing multi-year study has shown that global warming may endanger fish populations from Arctic and alpine lakes by leading to an increase of both metal accumulation and stress . Comparison of metal levels (e .g ., cadmium, zinc) and biochemical stress indicators in Arctic char collected from Canadian Arctic lakes revealed marked seasonal and inter-annual trends in the turnover of metals, as well as stress responses in the liver . Results indicate metal accumulation and level of stress to be higher the warmer the summers are in the Canadian Arctic . Predictive relationships between lake temperature and metal uptake were similar for high Arctic lakes and previously studied Austrian high mountain lakes, thus confirming water temperature to be a driving force of metal accumulation in Arctic char from these sensitive ecosystems . The observed effects provide clues as to what would happen to the extremely vulnerable land-locked char populations in the event of a longer-term, synoptic warming trend associated with global climate change . We speculate that the projected warming conditions could be a serious threat to the stability of Arctic char populations in high latitude and high altitude lakes . The results illustrate that fish from high latitude and high altitude lakes appear to be sensitive bioindicators of the interactive effects of pollution and global climate change

TH 094Modeling pathways, processes and trends of mercury contamination in the ArcticA .P . Dastoor1, D .D . Durnford2, A .R . Ryzhkov3

1Environment Canada, Dorval, Canada2Reed Harris Inc, Montreal, Canada3Nexio Inc, Montreal, CanadaA three dimensional process based global mercury model - GRAHM (Global/Regional Atmospheric Heavy Metals Model) is applied to study the pathways, processes and trends of mercury in the Arctic . Environment Canada’s mercury model GRAHM simulates meteorological processes and Hg related physico-chemical processes in the atmosphere . The model simulates transport and transformations of mercury in the atmosphere and its exchange with Earth’s surfaces on global scale . Hg emissions in the model include global anthropogenic, natural and reemission of previously deposited Hg from land and oceans . The model includes mercury transport, chemical reactions and bi-directional surface exchanges of mercury . Proper treatment of the transformations of mercury deposited onto snowpacks is important to model the impact of changing climate in the Arctic . In the springtime, Atmospheric Mercury Depletion Events in the Arctic are accompanied by high levels of deposition of mercury to the cryosphere . A significant portion of the deposited mercury to the snowpacks is observed to be revolatilized rapidly depending on the surface characteristics and meteorological conditions . A combination of physical and chemical environmental factors may result in trapping or losing a substantial portion of the deposited mercury within the cryosphere . This mercury may enter the meltwater and make it to the oceans or other aquatic bodies . A comprehensive dynamic scheme of air-ice-snowpack-melt water exchange of mercury based on extensive review of measured data was developed and implemented in the model to specifically address the impact of warming Arctic on mercury accumulation in the Arctic . The new version of the model is applied to determine the net accumulation of atmospheric mercury to the Arctic soils and Ocean . Relevance of mercury deposition to the snowpacks in the Arctic was analyzed . Atmospheric pathways, source attribution and trends of mercury concentrations and deposition were analyzed . The model description and results will be presented at the conference .

TH 095Identification of degradation products of quinalphos in rats: an in vivo studyM .R . Rani, B .G . Gupta, R .K . KumarIndian Institute of Technology Roorkee, Roorkee, IndiaPesticides are among the few toxic materials deliberately disbursed into the environment to preserve the agricultural produce . Besides their use as agrochemicals the pesticides are known homicidal and suicidal agents because of their easy availability . According to a WHO estimate as many as three million people per year are poisoned by pesticides resulting in around two hundred thousand deaths . Therefore, the various monitoring programmes need to fulfil the obligation to know as to what is the behaviour of pesticide in the human system . In order to evaluate the deleterious effects of exposure to quinalphos a comprehensive study was planned to follow the pharmacokinetic in vivo degradation in rat . A rapid HPLC procedure has been developed employing an octadecyl endcapped RP-C18 column and an ultraviolet detector . Several procedures for the extraction of quinalphos from biological samples were scanned and judged on the basis of recovery . Methanol was found to be the most suitable extractant with a recovery of 95±5% . All the samples viz . blood serum and urine collected at various time points were lyophilized, extracted and analyzed . Also the metabolic intermediates of quinalphos in serum and in urine of albino rats at different time intervals were identified using GC-MS after dosing the animals with 5 mg kg-1 body weight . The decay was measured for a maximum of twenty four hours in rats . Control studies show that the degradation of the pesticide followed a first order kinetics . The profiles of concentration observed in biological fluids suggest that quinalphos is rapidly absorbed and reaches a peak concentration in 2 hours after dosing and thereafter it is metabolized in blood and urine with a half life of 3 .8 and 4 hours, respectively . Among the important metabolites identified in vivo, a majority of them appear to be formed mainly through hydrolysis and oxidation . In each case quinalphos oxon, O-ethyl-O-quinoxalin-2-yl phosphoric acid, 2-hydroxy quinoxaline and ethyl phosphoric acid are formed . The results on metabolites may be helpful in assigning possible contributors towards the toxicity of quinalphos in addition to parent pesticide . The results further indicate that the metabolites, 2-hydroxy quinoxaline and oxon which are more toxic than the parent compound seem to persist for a longer time . The presence of these metabolites in the concerned biological fluids can help in the identification of quinalphos as the source of poisoning .

EC03P - In situ remediation of metal(loid) contaminated soils: exploration of emerging technologies and long-term effects of previously remediated sites

TH 097Helianthus tuberosus L. grown with multiple pollution in greenhouse conditionsB . Montalbán, M .C . Lobo, A .E . Pradas del Real, A . Plaza, A . Pérez-SanzIMIDRA, Madrid, alcalá de henares, SpainHelianthus tuberosus L . (Compositae) offers wide technological interest as a bio-ethanol due to its high potential for biomass production . Recently studies showed the tolerance of this crop to heavy metals . These characteristics make it a potential candidate plant for phytoremediation of soils and to obtain renewable energy in polluted areas with moderate levels of heavy metals that can no longer be used for agriculture applications . The present work reports data concerning to the tolerance of two cultivar-clones of H . tuberosus (VR and D19) grown in hydroponic culture with a multipolluted solution of heavy metals, in order to evaluate their use as energetic crop in polluted sites . The experiment was carried out in a greenhouse under controlled conditions . Tubers were recollected in field and transferred to plastic trays filled with turf during two weeks . Then, the plants were carefully rinsed in distilled water and transferred to hydroponic culture with coarse perlite . Plants were fertilized with a modified Hoagland’s solution and treated with heavy metals: (i) Treatment 1: 30 mg-L-1 of As (V), Cd, Cr (VI) and Ni; (ii) Treatment 2: 30 mg-L-1 of Cu, Zn, Pb and Cd; (iii) Treatment 3: 30 mg-L-1 of As (V), Cd and Ni . Control plants were fertilized only with nutrient solution . The plants were harvested after two weeks, separated into leaves, stems, roots and tubers, dried in an oven at 70 ºC for 72 h, and dry weight was recorded . Results showed the leaf, stem and root dry weight were significantly reduced in both cultivar-clones as comparison of the control when plants grown in presence of a mixture of As (VI), Cd, Cr (VI) and Ni . Biomass of both cultivar-clones showed no significant differences when treatment with Cu, Zn, Pb and Cd was applied . VR was not affected by As (V), Cd and Ni, while D19 biomass was significantly reduced with this treatment, in the case of leaves and roots by 50% in comparison to control . From this preliminary experiment we concluded that both cultivar-clones of H . tuberosus seem to tolerate mixtures of Cu, Zn, Pb and Cd . Related to cultivar-clones, VR tolerated concentrations of As (V), Cd and Ni in solution better than D19, and both cultivar-clones were sensible to the presence of Cr (VI) . Acknowledgements: EIADES PROGRAM S2009/AMB-1478, RTA000150-00-00-INIA; Contratación de Doctores INIA-CCAA and FPI-INIA to support the predoctoral grant Montalbán B .

TH 098Numerical modeling of a Phyto-extraction processF . LugliCOPPE / Federal University of Rio de Janeiro, Rio de janeiro, BrazilThe phyto-extraction is an in situ remediation technique involving plants that extract contaminants and accumulate them in plant tissues . Despite its low cost and low impact presents difficulties in estimating the efficiency of the process and the time required for remediation . The results depend on a complex network of interactions between plants, soil and atmosphere in the presence of contaminants . This research aims to estimate the time required for remediation of soil . Pedologic and climatic data from Rio de Janeiro were used . The initial contamination levels were determined based on real cases of industrial activities and waste disposal . The root contaminant uptake model was previously calibrated using data from greenhouse experiments . The simulation was organized in three phases: pre-contamination (determination of hydrological conditions and water table), contamination (evolution of the plume of contamination before planting), remediation (remediation process simulation) . Although the results were specific to the chosen scenario, it was observed that the efficiency of the phyto-extraction decreased with depth reached by the plume of contamination . The proposed methodology, despite the need for calibration enhancements and models for vegetation, provided indications that may be useful in the design of phyto-extraction project .

TH 099Immobilization of metals in contaminated sites with use of abundant waste materials exemplified in Cu and Cd binding onto sewage sludgeI . Twardowska, E . Miszczak, S . StefaniakPolish Academy of Sciences, Zabrze, PolandViable remediation of sites contaminated with metal(loids), should fulfil several basic requirements, of these cost-efficiency, effectiveness, abundance of a material used for remediation, easiness of management and temporal sustainability are the major ones . In these terms, stabilized sewage sludge (SS) seems to be particularly promising . In this study, binding properties of SS from a tertiary sewage treatment plant for metals, exemplified in Cu and Cd sorption, in view of SS application for remediation of sulfidic extraction waste dumps generating metal-rich Acid Rock Drainage (ARD) has been presented . Freshly generated SS was moderately acidic (pH 5 .55), of hydraulic conductivity 109-1010 m/s, specific surface with respect to polar adsorbate (H2O) 216 m2/g, CECt=258cmol/kg, with predominance of Ca and Mg as exchangeable ions, OM 51% and initial content of trace metals from 4 .82 mg/kg d .m . (Cd) to 3790 mg/kg d .m . (Zn), with Cu content 222 mg /kg d .m .Sorption capacity for studied metals

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assessed in batch mono-metallic Me-SO4 system at pH 4 .0 accounted for 40600 mg Cu /kg and 39000 mg Cd/kg and was comparable with natural sorbents (e,g . peat) . In the environmentally relevant systems, such as ARD generating sites, a number of factors may influence sorption capacity of materials, of these pH, competitive effect of other ions, adsorbent-adsorbate contact mode, dominating anions or cations in solution and the temporal transformation of adsorbent are the most important ones . Sorption experiments taking into consideration all these factors were thus conducted . The following systems were studied: (a) sorption of metals from Me-Cl, Me-SO4 and Me-NO3 solution, in monometallic and binary batch systems, at pH 4 .0; (b) comparative Me binding onto freshly generated and weathered SS; (c) sorption in flow-through systems, in mono-, binary and polymetallic systems, also at strong domination of Fe ion, at pH 3 .0 and 1 .5; (d) application of a simulated “worst case”polymetallic ARD onto freshly generated and weathered SS in a flow-through system . The aforementioned factors appeared to exert a strong reducing effect on the SS sorption capacity, of these pH, Fe predominance in adsorbate and weathering transformations of adsorbent (SS) were the most important ones, able to reduce its sorption capacity up to 2 orders of magnitude . Despite this, even in the worst case, sorption capacity of SS was relatively high and allowed to substantially attenuate metal mobility . TH 100Bioremediation of contaminated soils by biosurfactants produced in co-culture fermentation processesJ .M . Domínguez, R .O .D .R .Í Noelia, S .A .L .G .A José Manuel, R .O .D .R .Í Raquel, C .O .R .T .É SandraVigo University, Ourense, South AfricaCoculture fermentations, instead of single cultivation can be utilized advantageously, in industrial biotechnology in the production of chemicals, particularly during the use of lignocellulosic material as feedstock, since both cellulosic and hemicellulosic fractions can be employed . The use of Lactobacillus pentosus and Lactobacillus rhamnosus allow producing biosurfactants from both fractions . Biosurfactants are amphiphilic compounds of microbial origin with a pronounced surface activity and with several advantages over chemical surfactants including lower toxicity, higher biodegradability, and effectiveness at extreme temperatures or pH . Biosuractants are potential candidates for many commercial applications in the biomedical, petroleum, and food processing industries .

This work deals with the application of biosurfactants obtained from different agroindustrial wastes, during cocultures processes, in the bioremediation of soils contaminated with heavy metals .

TH 101Biosorption of heavy metals from gold mine wastewaters by Penicillium simplicissimum immobilized on zeolite: kinetic, equilibrium and thermodynamic studiesH . Tutu, E .N . Bakatula, E .M . Cukrowska, C .J . Straker, I .M . WeiersbyeUniversity of the Witwatersrand, Johannesburg, South AfricaA biosorbent based on zeolite and Penicillium simplicissimum (both living and heat-killed fungal biomass) was developed for the clean up of heavy metals from gold mine wastewaters . With an initial concentration of 500 mg l-1 at pH 3 - 4 for a single component system, 99% adsorption was observed for: Cu2+, Co2+, Cr3+, Fe2+, Ni2+, Zn2+, Hg2+ . Immobilisation of fungi on zeolite yielded higher biomass, showing the potential of this study towards remediation of polluted mine sites . Desorption results showed that the adsorbent could be reusable .

TH 102Mercury immobilization using modified mackinawite as reactive layer in in-situ cappingM .R .M . Chaves1, K .T . Valsaraj2, R .P . Gambrell2, R .D . Delaune2, P .M . Buchler1

1University of Sao Paulo, Sao paulo, Brazil2Lousiana State University, Baton rouge, United States of AmericaMercury and methyl mercury adversely affect human, birds, and mammals health . The effects of mercury contamination are cause of global concern; thus remediation of contaminated sites is a high priority issue . This study investigated the mercury immobilization using mackinawite modified with L-cysteine as an active layer in in-situ capping . Mackinawite (FeS) is a promise material to be used in in-situ capping due its excellent capacity for mercury uptake from anoxic contaminated sediments . The surface modification with L-cysteine increases the mackinawite oxidation resistance, becoming suitable its use in field conditions . Modified mackinawite as a reactive capping layer was studied in lab scale simulator systems using lake sediment contaminated with mercury . The mercury profile towards the cells showed that modified mackinawite is an excellent material to be used as reactive layer in in-situ capping . It was observed that the mackinawite modification have little or no influence on the mercury uptake from the contaminated sediment, in comparison to the unmodified mackinawite . The double layer system (sand layer over the modified mackinawite layer) was the best design observed for in-situ capping . Incubation experiments showed that modified mackinawite has higher mercury methylation inhibition capacity, than the observed from unmodified mackinawite .

TH 103In situ arsenic and lead stabilization and remediation in acidic soils using green chemistry and nano iron welding fumesB .M . Miller1, K .G .S . Kirk G .1, T .L . Todd1, A .Z . Zimmer2

1U . S . Environmental Protection Agency, Cincinnati, United States of America2U .S . Public Health Service Officer, Cincinnati, United States of AmericaArsenic (As) and lead (Pb) are highly toxic naturally occurring elements found in soils worldwide and is recognized as a carcinogen responsible for neurological, gastrointestinal, cardiovascular, and reproductive illnesses . Toxic concentrations of As can be released into drinking water supplies as a result of mineral weathering and human activities . Soils with toxic accumulations of As and Pb pose a public health threat particularly to children because of their tendency to consume soil via hand-to-mouth contact (USEPA, 2000) and, to a lesser extent, adults with home gardens and occupational exposures . When pseudo total As contamination levels are ≤ 2000 mg kg-1 in situ remediation with Fe amendments has been successful in preventing As leaching (Akhter et al ., 2000) and may under appropriate conditions be an effective long-term strategy . Likewise, the transformation of Pb to pyromorphite at low levels of contamination by the addition of phosphorus (P) compounds has become an approved strategy to protect human health in the US . However, many soils, such as orchard soils, are contaminated with As and Pb due to historical applications of the insecticide lead arsenate . In these soils, the addition of P solutions is not feasible due to the risk of As leaching into ground water or streams . The goal of this project is to demonstrate physiochemical technology responsible for the immediate in situ long-term stabilization and remediation of contaminated soils by iron (Fe) occlusion . Arsenic and Pb remediation is achieved by occlusion of As within newly precipitated Fe (hydr)oxides minerals . The risk for As leaching will be reduced or eliminated by the application of Fe nanoparticles applied to the soil with a “green”nonionic surfactant and wetting agent derived from citrus fruits . We will present results of ligand-exchangeable and ligand-dissolvable As and Pb pools, citrate bicarbonate dithionite extractable Al, As, Fe, and Pb, as well an As and Pb X-ray Absorption Near Edge Spectroscopy . Remediation efforts that sequester As and Pb within recalcitrant occluded Fe minerals while controlling soil physiochemical factors could give risk assessors and stakeholders confidence that the danger to human health has been significantly reduced or eliminated . Concurrently, this remediation technology may achieve substantial cost savings in comparison to the standard practice of soil excavation and off site landfill deposition (Scheckel et al ., 2009) . TH 104Influence of humic-based sorbents on bioavailability of lead in soilM .A . Pukalchik1, V .A . Terekhova1, A .A . Izosimov1, O .S . Yakimenko1, K .A . Kydralieva2

1Lomonosov Moscow State University, Moscow, Russian Federation2Institute of Problem of Chemical Tecnology,, Byskek, KyrgyzstanHeavy metals are major pollutants in industrial soils . This study focuses on in-situ remediation of lead’s soil polution in model experiment . Commercially available potassium humate BC-HumNa produced by Russian company from brown coal as well as magnetoactive humic-based nanocomposite Fe3O4/HA formulated by coprecipitation method in situ were tested . Fe3O4 nanoparticles are spherical and their diameters vary from 7 to 16 nm . Modeling of lead pollution was conducted in pots on samples of the model soil (100 g soil; Pb 320mg/kg) . Humic sorbents was introduced to achieve a concentration of 0 .0025 and 0 .01% (by weight) . Lead water-soluble fraction was measured using Atomic Absorption Spectrophotometry, 10 days after . Toxicity was valued in relation to standardized test-cultures of living organisms (representatives of higher plants, invertebrates crustacean, and bacteria) . Results are presented and discussed, indicating that both humic-based sorbents modified lead bioavailability in soil . Apparently changing in the content of water-soluble lead, and the responses of test organisms on the impact of aqueous extracts of contaminated soil humate nanostructured substances showed a higher detoxifying potential and were more effective than sodium humate .

TH 105Preliminary results of the zinc, copper and iron content in the feral oyster Striostrea margaritacea and the effects of them on the lysosome membranes Y . Slabber, R .G . Snyman, J . OdendaalCape Peninsula University of Technology (CPUT), Cape town, South AfricaThe causes, the monitoring and the control of marine pollution, particularly heavy-metal pollution, have become a very important subject to a great deal of research in recent years . The aims of the present study is to determine the degree of metal contamination in the wild oysters, Striostrea margaritacea, as well as in the water and sediment from selected polluted (Wilderness, Witsand) and unpolluted (Goukamma MPA, Knysna) sites on the South African Coast and also to determine the toxicity of selected metals to the oysters, by using a biomarker (lysosome destabilization) as tool . The biomarker will be tested using the neutral red retention time assay . A primary screening has revealed high amounts of Zinc, Copper, Iron and Aluminum in oysters from the polluted areas . Results of this study will possibly be used to introduce a more advanced monitoring program using bivalves as bioindicators .

TH 106Uranium fate in crayfish after different conditions of exposure. Focus on U accumulation and speciation in the cytosolic fraction of 2 target organs: gills and digestive gland. S . Frelon1, S . Mounicou2, R . Gilbin3, O . Simon3

1IRSN, St paul lez durance, France2IPREM / LCABIE, Pau, France3IRSN/DEI/SECRE/LRE, St paul lez durance, FranceWhereas uranium speciation in water column is quite well studied by modelling in correlation to its bioavailability, its speciation within living aquatic organism is poorly described despite a wide coordination chemistry and affinity for proteins . Uranium analysis at the organ and sub-cellular levels is necessary to understand biodistribution,

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precipitation and specific metal interaction with biomolecules during the fixation, transport and incorporation into a biological cell or tissue . So, such information is fundamental to better explain uranium bioavailability, accumulation after direct and trophic exposures and toxicity . In the case of uranium the elucidation of its toxicity does not only go through the characterization of target-biomolecules involved in metabolic pathways but also goes through the determination of the sequestrated fractions likely to emit alpha rays in the cell . This study deals with the uranium fate in two target organs of crayfish, i .e . gills and digestive gland, after different levels of direct exposure . As mainly considered as chemiotoxic, focus was done of the soluble fraction of U in these organs . Thus, subcellular distribution of uranium was performed using ultracentrifugation followed by uranium ICP-MS measurement . Following this step, uranium distribution within cytosolic proteins was assessed using hyphenated HPLC SEC-ICPMS . Results show that in crayfish, U accumulation in gills is higher than in digestive gland after direct exposure . Only 25 to 5% of U are in the soluble fraction of gill cells and can pretend to be studied for U speciation and the assessment of the chemiotoxic fraction of this element . In digestive gland, the soluble fraction represents 60 to 30% of the uranium content . The ICP MS chromatogram of DG cytosols shows similar patterns for control samples and contaminated samples while intensity of 3 fractions at 4, 7 and 40 kDa seems to be enhanced with the contamination . Same patterns and trends were observed with the UV detection at 280 nm . To conclude, Hyphenated techniques were used to assess U recovery and U distribution within cytosolic proteins of cells from gills and digestive gland of crayfish after exposure . Three main protein peaks of interest have been identified and will further be analyzed to identify molecules complexed with U . These speciation studies correlated to biological effects are a key step to elucidate the chemiotoxicity of this element .

TH 107Determination of Se species in fish tissues by HPLC-ICPMSR .U . Jagtap1, W .A . Maher1, F . Krikowa1, S . Foster1, M . Ellwood2

1University of Canberra, Canberra, Australia2Australian National University, Canberra, AustraliaSe plays a major nutritional role in humans and animals as it is an essential component of a number of enzymes including glutathione peroxidase . The narrow margin between the beneficial and toxic levels of Se has important implications on human health . Due to its dual role, Se has been widely studied in the environment . The release of Se from geological sources has been accelerated due to industrial and agricultural activities making it bioavailable to fish and other organisms in aquatic ecosystems, hence, Se has surfaced as an element of primary concern . Se is present in many chemical forms in the environment: from inorganic Se (IV) and Se (VI) to the organic amino acids such as selenocysteine and selenomethionine . The toxicity of Se is related to its chemical form with the inorganic forms being more toxic than the organic from in general . Many enzymes and other proteins require Se for their activity and the forms required by animals are the Selenoaminoacids or Se containing proteins . Since, selenoaminoacids are mainly associated with proteins in the living organism, to release the Se species incorporated into proteins, the peptide bond needs to be broken in a way that releases the intact Se containing amino acids or their derivatives . Although, the measurement of total Se is essential to provide elemental concentrations, it is insufficient information as the biological actions of Se are dependent on the physical properties of its various chemical forms . Speciation information is therefore required to gain an understanding of its biogeochemical cycling and metabolism . This project investigates the extraction and determination of Se species by HPLC-ICPMS after proteolytic digestion and the derivatisation of selenoaminoacids . Low efficiency of proteolytic extractions reported in the literature could be a consequence of incomplete digestion of tissue due to insufficient unfolding of proteins, hence the inaccessibility of the enzymes to the target peptide bonds . Fish tissues are extracted using urea to denature proteins followed by reaction with dithiothretol to break the Se-Se and S-Se bonds and alkylation with iodoacetamide to derivatize selenomethionine and selenocysteine to more stable carboxymethylated forms . This allows for the enzymatic digestion of the stabilised carbamidomethyled selenocysteine and selenomethionine allowing proteolytic digestion to extract Se species .

TH 108Measurement of methyl mercury and inorganic mercury in sediments and fish tissues by HPLC-ICPMSR .U . Jagtap1, F . Krikowa1, W .A . Maher1, S . Foster1, M . Ellwood2

1University of Canberra, Canberra, Australia2Australian National University, Canberra, AustraliaMercury is considered to be a major environmental contaminant on a global scale and therefore has inspired the development of methods for the determination of mercury species in the wide range of environmental matrices . While natural inputs of mercury occur, anthropogenic inputs from combustion of coal and petroleum products account for more than 50% of the global mass of mercury . The elemental mercury produced being highly volatile is transported in the atmosphere over long distances and in the earth’s crust and oceans, wet deposition occur in the form of inorganic mercury . Dissolved inorganic mercury shows a high tendency to absorb onto suspended particulate matter and consequently, gets accumulated in the top of sediments . Environmental and health risks for mercury derive from methyl mercury that is formed by biomethylation of inorganic mercury by microorganisms in sediment and soil . Methyl mercury is a neurotoxin that readily crosses biological membranes which accumulates to harmful concentrations in exposed organisms and biomagnifies in aquatic food webs to concentrations of toxicological concern . Developing a sensitive, reliable and cost effective method to measure mercury species is important for monitoring mercury concentrations in biota and sediments in order to avoid ecotoxicological risk and to understand the biogeochemical cycling of mercury species in the environment . Since methyl mercury is the most toxic species, routine analysis of this species is increasingly in demand . This project describes a procedure for the extraction and determination of methyl mercury and inorganic mercury in sediments and fish muscle tissues using HPLC-ICPMS after extraction using 2-mercaptoethanol . A Perkin-Elmer 3 µm C8 (30 mm x 3 mm) HPLC column and mobile phase containing 0 .5% v/v 2-mercaptoethanol and 5% v/v methanol ( pH 5 .5) at a flow rate of 1 .5 ml min-1, 250C was used for the separation of mercury species . The developed extraction procedure of mercury species from the fish tissues was compared against an enzymatic extraction using Protease type XIV and similar results were obtained for both extraction procedures . The methyl mercury concentration of sediment reference material ERM CC 580 and biological certified reference materials, NRCC DORM-2 Dogfish muscle, NRCC Dolt-3 Dogfish liver, NIST RM Albacore tuna and IRMM IMEP-20 tuna fish were in agreement with the certified values .

TH 109EDTA: a marker of anthropogenic pollution in the Suquía River basinM .E . Valdés1, M .V . Monferrán2, M .A . Bistoni3, D .A . Wunderlin1

1Facultad de Ciencias Químicas,Universidad Nacional de Córdoba, Córdoba, Argentina2ISIDSA-ICYTAC, UNC-CONICET ., Córdoba, Argentina3Diversidad Animal II .Fac . Cs . Exactas,Fís . y Naturales, Univ . Nac . de Córdoba, Córdoba, ArgentinaAminopolycarboxylates, such as ethylenediaminetetraacetic acid (EDTA) have been extensively used as chelating agents in many industrial and domestic applications for years . EDTA is released to the aquatic environment mainly via runoffs of sewage treatment plants . Due to high consumption and slow biodegradability, EDTA has been found in surface waters of different industrialized countries . Our main goal was determining the presence of EDTA as a marker of anthropogenic influence along the Suquia river basin (Córdoba-Argentina) . To verify the pollution we also measured concentration of metals (Li, Be, B, Mg, Al, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ag, Cd, Ba, Tl, Pb and Bi), pH, conductivity and dissolved oxygen (DO) at eight monitoring stations with different pollution degree from the upper to the lower river basin . Sampling was carried out during 2011, considering both dry and wet seasons . EDTA was analyzed by HPLC coupled to ESI-HRMS (QTOF), while metals were assayed by ICP-MS . EDTA was found in at least 3 locations . Amounts of EDTA varied along the basin, showing absence of EDTA in the high basin, trace amounts in the middle basin, while the highest values were observed at the lower basin . So far, it is possible to differentiate 3 areas in accordance with anthropogenic inputs: a quasi pristine area in the upper basin, a medium polluted zone surrounded by small cities in the medium basin, and a high impacted area, downstream the sewage discharge of the main city (Cordoba) . Some metals (Be, Cr, Co, Ni, Se, Mo, Ag, Cd, Tl and Bi) were below LOD throughout the basin . Conductivity, DO and other metals (Li, K, Ca, Mg, Ga, Sr, Ba) showed a pattern coincident with EDTA concentration, with increased values as anthropogenic influence rose . Additionally, concentrations of Al, Mn, Fe, Cu and Zn increased progressively after Córdoba sewage discharge . Concentrations were generally higher during the dry season . Our current results demonstrate that EDTA can be used as an efficient marker of anthropogenic pollution, in addition to metals and other water qualities parameters . TH 110Speciation and bioavailability of arsenic and heavy metals in contaminated stream sedimentsP .J .C . FavasUniversity of Trás-os-Montes e Alto Douro, Vila real, PortugalThe application of the Selective Chemical Extraction technique on the sediments of the stream that drains the “Vale das Gatas”Mines area, allowed us to notice that: 1) the most part of the contents of Cr, W, Sn, Ni and Mn in the residual fraction, is mainly constituted by silicate minerals, non bioavailable; 2) part of the metals (Fe, Mn, Cu, Zn, Cd and Pb) are easily bioavailable in all sampling sites in large percentages; 3) all the researched metals are also potentially bioavailable, that is, they occur in organic, reducible and carbonate phases, susceptible of being set free depending on some changes of water stream physical-chemical conditions . Cu, Zn, As, Cd, and Pb are the most remarkable examples . The characteristics of the main contamination focus, also appear as an important aspect, conditioning the support phases of the researched chemical elements . Thus, in station where the water stream contamination is essentially owed to the Acid Mine Drainage (AMD), a great part of the elements appear preferentially associated to exchangeable and moderately reducible fractions . That is a result of the affinity of these elements in being adsorbate and its easiness of coprecipitating with Fe oxides; while in stations where the contamination is owed not only to the AMD of tailing but also to solid load transportation, exchangeable and moderately reducible fractions lose some importance, increasing the tendency of association with fraction 5 (sulphides) and with fraction 6 (residual) .

TH 111The occurrence of Arsenic-Glutathione complexes in Chlorella vulgaris: off-line elemental (ICP-MS) and molecular (ES-MS) mass spectrometric detection for liquid chromatographic separationL .P . Pantoja Munoz, D .P . Purchase, H .J . Jones, H .G . Garelick

2�6 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

Middlesex University, London, United KingdomOne response of plants to exposure to arsenic is the production of Glutathione (GSH), although its part in the detoxification mechanism is still unclear . Due to the strong affinity of arsenic for sulphur, glutathione (GSH) and phytochelatins (PC) tend to form complexes with As(III) and to a lesser extent, As(V) . However, the identification and quantification of these complexes has proven to be elusive due to the instability of the metalloid complexes and it represents a challenge for modern speciation techniques . Arsenic speciation instrumentation has received much attention recently and reliable speciation has been achieved . In contrast, relatively little progress has been made to improve sample storage and extraction protocols . In particular, extraction of As in the microalgae Chlorella vulgaris remains poor impeding As-GSH complexes identification in these cells . The aim of this investigation was to determine the presence of As-GSH complexes in Chlorella vulgaris after exposure to arsenic and contribute to elucidate the mechanisms of arsenic detoxification used by microalgae . For this reason synthetic As-GSH complexes were prepared . Chlorella vulgaris cells were exposed to As(III) and As(V), concentration ranging from 0-200mg/L . The quantification and determination were performed by extraction with HPLC-ICPMS and HPLC-ESI-MS . The same chromatographic conditions were used for both instruments . Quantification by ICPMS does not ensure unequivocal identification of molecules but allows the determination at low detection limits and is not significantly affected by matrix interference . The use of ESI-MS alongside ICPMS allows both low concentration quantification and unequivocal identifications of molecules of interest . In order to understand the mechanism of detoxification employed by living cells exposed to inorganic arsenic, reliable methods for extraction, quantification and detection must be developed . In this study, sample preparation and extraction procedure appropriate for the nature of the analyte has been applied to minimise oxidation and preparation time . Trace analyte quantification with low limits of detection and low matrix effects by ICP-MS and correct identification of the As-GSH with ESI-MS was achieved in Chlorella vulgaris cells exposed to inorganic arsenic .

TH 112LC-ICP-MS speciation of selenium compounds in Se-enriched green algaeJ . Kuta1, J . Machát1, M . Vítová2

1Masaryk University, Brno, Czech Republic2Institute of Microbiology, Academy of Science, Trebon, Czech RepublicSelenium is an essential element and has been known to be a necessary component of the human diet . It is important e .g . for protection of cells against effects of free radicals and for the normal function of immune system . Selenium is also toxic and its toxicity and bioavailability is related to concrete selenium compound . Usually organic selenium species (Se-amino acids) exhibit higher bioavailability and lower toxicity than inorganic selenium compounds . Se-enriched biomass of green algae Scenedesmus and Chlorella might become a source of organic bound selenium . The aim of this work is to develop a method for identification and quantification of selenium species in algal biomass . For this purpose various types of extracts were studied by coupling of anion exchange liquid chromatography with inductively coupled plasma mass spectrometry . Acid and enzymatic hydrolysis of proteins in algal biomass was applied for determination of total content of selenomethionine and selenocysteine . Extraction with buffers and alkaline media was used for identification of non-protein bound selenium amino acids and inorganic ions . Supported by CETOCOEN (CZ .1 .05/2 .1 .00/01 .0001) project granted by the European Union and administered by the Ministry of Education, Youth and Sports of the Czech Republic . The authors also are grateful for financial support of MŠMT (MSM0021622412) .

TH 113Speciation of arsenic and selenium in surface waterM .C . Kotte, R . Breidenbach, I . Bakker, H . van den Heuvel, O .J . EpemaRWS Centre for Watermanagement, Lelystad, NederlandWithin the framework of various international River basin programs arsenic pollution is monitored in Dutch surface waters, among other matrices . Selenium is monitored on a national basis . To determine the impact on the aqueous environment of the amounts present (national) environmental quality standards are used . Current research into the speciation of both elements should lead to a better understanding of the extent of pollution, because toxicity of the various species differs extensively . This approach is expected to be incorporated into future legislation frameworks . TH 114Ion chromatography with XSERIES 2 for metal speciation applicationsD .K . Kutscher, J . WillisThermo Fisher Scientific, Bremen, Germany1. Introduction Here, the coupling of the Dionex ICS-5000 ion chromatography system to XSERIES 2 is described . This combination provides a single powerful, flexible solution for both ion chromatograph and ion exchange chromatography applications as well traditional HPLC methods . The combined system is ideally suited for trace metal speciation as it provides a completely metal free pathway reducing contamination risks and improving robustness when used with potentially aggressive mobile phases . Together with the wide selection of chromatographic columns applicable, appropriate solutions for all types of trace metal speciation studies can be found . 2. Materials and methods In this study, speciation analysis of different trace metals was carried out using hyphenation of the Dionex ICS-5000 chromatography system coupled to XSERIES 2 . Different analytical columns were used including anion exchange chromatography and reversed phase chromatography . 3. Results and discussion Different application examples of the system will be shown, including from ion chromatography (e .g . Cr speciation), ion exchange chromatography (speciation of different As compounds), and also reversed phase chromatography for the separation and quantification of organotin species . Particular emphasis will made on the use of smaller internal diameter columns that significantly lower the consumption of mobile phase (and therefore sample) without compromising separation efficiency . 4. Conclusions The results show the possibilities offered by the hyphenation of the Dionex ICS-5000 LC system to XSERIES 2 for trace metal speciation analysis . Due to the high flexibility offered by the system, different applications could be succesfully run on the same instrument and showed high separation efficiency together with reduced sample and mobile phase consumption .

TH 115Speciation of Bromine in water samples using HPLC-ICP-MSR .C .H . ChemnitzerBruker Daltonics GmbH, Berlin, GermanyFoods and agricultural products contain various concentrations of nutrients, which can range from trace amounts to percentage levels . However, whilst most of their nutritional content is intended for maintaining good health, the benefits of the desired mineral content of such products can be compromised by concentrations of elements deemed to be toxic to humans and/or animals . ICP-MS is a flexible tool for matrix, minor and trace elemental analysis . In combination with separation techniques, ICP-MS allows the determination of the chemical form of elements present in the sample and thereby a differentiation between essential nutrient and toxic to human or animals . For elements such as As, Cr, Se and Br it is well known that it depends on the oxidation state or the present compound if an element is harmless or toxic to humans . Bromate e .g . is formed by the ozonation of water in purification processes . Bromate is potential carcinogen whereas bromide is essentially non-toxic . Therefore monitoring of the total bromine concentration in water will not indicate the risk for human health . The coupling of a chromatography technique to an ICP-MS provides a powerful and very sensitive tool to separate bromine and bromate . The presentation will imply the required sample preparation steps, flexibility and sensitivity for the determination of different bromine species in water samples using the Bruker auroraM90 ICP-MS . The capability of ICP-MS for this kind of analysis will be shown with the help of different sample types .

TH 116New approaches for the traceable determination of PBDEs (in coastal water samples) according to the Water Framework Directive (WFD)A . Gonzalez-Gago1, D . Pröfrock1, A . Prange1, J .I . Garcia Alonso2, C . Swart3, D . Schiel3, R . Philipp4

1Helmholtz-Zentrum Geesthacht/Institute for Coastal Research, Geesthacht, Germany2University of Oviedo/Department of Physical and Analytical Chemistry, Oviedo, Spain3Physikalisch-Technische Bundesanstalt (PTB)/Department 3 .1, Metrology in Chemis, Braunschweig, Germany4Bundesanstalt für Materialforschung und -prüfung (BAM)/Department 1, Analytical, Berlin, GermanyPolybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in polymeric materials . Owing to the adverse effects linked to the exposure to these compounds, the EU has included PBDEs (congeners 28, 47, 99, 100 153 and 154) in the list of priority substances that need to be measured in all surface waters covered by the WFD . Environmental Quality Standards (EQS), equivalent to the maximum allowable concentrations, have been defined at very low levels for PBDEs (ΣPBDEs ≤ 0 .5 ng/L) . Analytical methods intended for their monitoring must meet certain minimum performance criteria in terms of uncertainty (≤ 50% at EQS, 95% confidence) and limits of quantification (LOQ ≤ 30% of EQS) for reliable measurements of PBDEs in complex environmental water matrix at EQS level . The overall low concentrations of the target analytes in water together with the challenging requirements defined by the WFD necessitates the development of suitable methodologies combining sensitive detection approaches with accurate and precise analytical techniques . In this regard, inductively coupled plasma mass spectrometry (ICP-MS) or electron capture negative ionization (ECNI) mass spectrometry provide interesting possibilities as it offers high sensitivity for the detection of heteroatoms such as bromine, especially when using gas chromatography (GC) as sample introduction technique . Furthermore, ICP-MS has been widely used in combination with Isotope Dilution Mass Spectrometry (IDMS) for the accurate and precise determination of other priority pollutants . In this work, some preliminary results on the determination of PBDEs in water by elemental IDMS are shown . Ultrapure water samples fortified at the EQS level were spiked with 81Br-labelled PBDEs, liquid-liquid extracted with isooctane and injected in a GC-(ECNI)MS . Obtained results met the uncertainty requirements of the WFD in river water samples but LOQs still need to be further improved . Instrumental settings of the GC-ICP-MS coupling were optimised for the highest bromine sensitivity for its future use in IDMS in an attempt to meet the WFD requirements . This work has been performed within the scope of an EMRP Researcher Grant for the development of a traceable measurement approach for monitoring PBDEs in coastal water, awarded in accordance with the EURAMET process to complement the JRP “Traceable measurements for monitoring critical pollutants under the European Water

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Framework Directive” .

TH 117Improved extraction, preservation and analysis of hexavalent Chromium from soil extracts and waterR .F . Jack1, C .G . Giuriati2, L .B . Basumalik1, F .A . Abballe1, R .J . Rohrer1

1Thermo Fisher Scientific, Sunnyvale, United States of America2Caterina Giuriati, Ferrara, ItalyChromates are oxyanions (e .g . CrO42’, Cr2O72’) of chromium in oxidation state +6 . They are strong oxidizing agents that are considered toxic and potentially carcinogenic . Hence, they are regulated in the environment and as a primary drinking water contaminant in the US . Drinking water standards are regularly re-evaluated by the US EPA . In 2008, they conducted a comprehensive review of the health effects of chromate based on toxicity studies done by the National Toxicology Program . In September 2010, the EPA released the Toxicological Review of Hexavalent Chromium . Based on that report the state of California’s Office of Environmental Health Hazard Assessment in the recently issued a new public health goal (PHG) for chromate in drinking water of 0 .02 μg/L . Currently, dissolved hexavalent chromium is measured according to EPA Method 218 .6 . This method is based on anion exchange chromatography on an IonPac AS7 column (4 mm format) and detection after postcolumn reaction with diphenylcarbazide to yield a compound with visible absorbance at 530 nm . This permits a method detection limit (MDL) for chromate in drinking water at 0 .3 μg/L . Modifications including lower eluent and postcolumn reagent flow rates, larger reaction coil, and larger injection volume significantly increased the sensitivity over Method 218 .6, resulting in an MDL of 0 .02 μg/L . However, the modified method does not allow sufficient sensitivity for routine analysis at the proposed PHG (California) level of 0 .02 μg/L . This presentation describes modifications including the use of the column in the 2 mm format and a smaller reaction coil to increase method sensitivity . The modified method permits a MDL of 0 .001 μg/L, and a quantitation limit of 0 .003 μg/L, which is more than sufficient for analysis at the proposed California PHG level . It also discusses alternatives for delivering of the post column reagent and other eluents that have been proposed . We are working with the US EPA Office of Ground Water and Drinking Water to create a new EPA method based on these final modifications, EPA 218 .7

TH 121Class 1 and 2 integrons, sul resistance genes and antibiotic resistance in Escherichia coli isolated from the East River, South ChinaG .G . YingGuangzhou Institute of Geochemistry, Guangzhou, ChinaExtensive use of antibiotics by humans and animals have resulted in the development of antibiotic resistance in various bacteria and dissemination of antibiotic resistance genes (ARGs) into the environment . The objective of this study was to investigate antibiotic resistance profiles in Escherichia coli isolated from surface water of the East River catchment in both dry and wet seasons, and to characterize selected ARGs including sul genes and integrons to better understand the dissemination of resistance genes and integrons in surface water . 89 .1% of the isolates were resistant and 87 .5% showed resistance to at least three antibiotics . Tetracycline and streptomycin had the highest resistance frequencies of 60 .1% and 56 .3%, respectively . Multiple antibiotic resistances (MAR) index ranged from 0 .16 to 0 .54 in different parts of the East River catchment . sul2 was detected most frequently in 89 .2% of 1403 SXT (sulfamethoxazole/trimethoprim)-resistant isolates . The presence of integrons (class 1 and 2) was frequently observed (82 .3%) while no class 3 integron was found . In these integrons, 21 resistance genes of 14 gene cassette arrays and 10 different families of resistance genes were identified . Three gene cassette arrays, aac(6’)-Ib-cr-aar-3-dfrA27-aadA16, aacA4-catB3-dfrA1 and aadA2-lnuF, were detected for the first time in surface water . Integrons could play a key role in developing multiple resistance amongst microorganisms by transferring various resistance genes simultaneously, which is clearly demonstrated by a strong correlation between the MAR index and number of integrons (p<0 .0001) .Principle component analysis and cluster analysis demonstrated that human activities, especially discharge of wastewater are the main factor leading to the wide dissemination of resistance bacteria and genes in the aquatic environment . The findings reflect the importance of surface water as a reservoir and as a medium for the dissemination of antibiotic resistance genes and resistant bacteria in the environment .

EP01P - Antimicrobial resistance in the environment

TH 122Antibiotic contamination and promotion of antibiotic resistance in microbial communities of three reservoirsB . Huerta1, E .M . Marti1, M .G . Gros1, S .R .M . Rodríguez-Mozaz1, J .L .B . Balcázar1, D . Barceló2, R .M . Marcé1

1Catalan Institute for Water Research, Girona, Spain2IDAEA-CSIC, Girona, SpainPharmaceuticals are continuously released in the environment from discharges of wastewater treatment plants or trough run-off from fields after application of sewage sludge and animal manure . They attract increasing attention as contaminants because of their potential threat to the ecosystems and public health, even at their very low concentrations and persistence rates . Among these compounds, antibiotics may pose a risk to the environment since they are designed to have a biochemical effect in the microorganisms, and thus can have a significant impact in the processes controlled by them in aquatic ecosystems . One of the greatest concerns about the presence of antibiotics in the environment is the escalation of antibiotic resistance as a consequence of chronic exposure of naturally occurring bacteria to sub-lethal doses of these compounds and the potential impact in public health . The objectives of this study were first to determine the levels of antibiotics in different environmental compartments (water, sediments and fish) of three reservoirs in Catalonia: La Llosa reservoir was chosen for its pristine condition, whereas Sau and Foix are respectively a moderate and a highly polluted reservoir . Antibiotic concentrations were correlated with the composition and functioning of the natural bacterial communities in the reservoirs as well as with the presence of antibiotic resistance genes, not only in water and sediment but also in fish intestine . A general chemical characterization showed a clear difference between the reservoirs in terms of eutrophication and chemical pollution, with exceptional high values in Foix reservoir . Foix samples showed the highest antibiotic content in water and sediment, as it was expected according to the pollution gradient of the target reservoirs . Load was particularly high for macrolides family, which is consistent with data related to antibiotic consumption last years . Antibiotic concentration found was consistent with the quantity of qnrS and sull gene copies (index of antibiotic resistance for the quinolone and sulfamide family, respectively) found in the samples . Resistance genes were detected in Foix Reservoir at much higher levels than in the other two, less affected by anthropogenic contamination . The combination of chemical and biological data provided a broader perspective to assess the impact of antibiotics released into the environment and their potential effect as promoters of antibiotic resistance .

TH 123Persistence of heavy metal and antibiotic resistance downstream a point source pollution of a river ecosystemC . Seiler1, W . Von Tümpling2, A . Lupo1, T .U . Berendonk1

1Dresden University of Technology, Dresden, Germany2UFZ - Helmholtz Centre for Environmental Research, Magdeburg, GermanyThe spread and evolution of antibiotic resistance in the natural environment is still not well understood . There is evidence that heavy metal contaminations contribute to the spread and persistence of antibiotic resistance genes in the natural environment . Lab experiments identified heavy metals as selective agents for the co-selection of antibiotic and heavy metal resistance due to co- and cross-resistance . However, field studies are rare and the results of some field studies did not support the hypothesis that heavy metals co-select for antibiotic resistance . Thus, the impact of heavy metal pollution on the persistence of antibiotic resistance in the environment needs further investigation . The Western Bug River (Ukraine), a river ecosystem that was supposed to be highly contaminated by a heavy metal point source due to industrial and urban waste water was chosen to investigate the persistence of antibiotic and heavy metal resistance along a heavy metal gradient . Therefore, the heavy metal concentrations of water and sediment samples from ten sampling sites along the Western Bug River were analysed . Furthermore, sediment and freshwater bacteria were isolated and identified . Microbes belonging to the genera Aeromonas and Pseudomonas were tested to their susceptibility towards a wide range of antibiotics and heavy metals . First results showed that elevated concentrations of the heavy metals Cu, Cd, Cr, Ni and Pb were detected over a long distance downstream the source . Furthermore, the bacterial resistance to Cu, Cd and Ni correlate to the analysed concentrations of the corresponding metals in the river . The concentration of Cu and Cd also correlate to the bacterial tolerance to the antibiotics cefotaxime and cefepime . Those first results lead to the conclusion that the wide ranging contaminations of copper and cadmium might support the persistence of the tolerance to the respective metals as well as to the antibiotics cefotaxime and cefepime . Investigations on the genes responsible for the resistance as well as the location of those genes within the genome will contribute to a deeper understanding of the co-occurrence of antibiotic and heavy metal resistance genes in the Western Bug River .

TH 124Organic pollution and spread of multi-drug resistant Escherichia coli in the Bug River, UkraineA . Lupo1, M . Coci2, N .I .C .O .L Scheifhacken1, A .M . Ertel1, S . Stefani2, T .U . Berendonk1

1Technical University of Dresden, Dresden, Germany2University of Catania, Catania, ItalyThe emergence and the dissemination of antibiotic resistances constitute a global threat for public health and an emergent environmental problem . Rivers and streams constitute a reactor for evolution of new resistances because bacteria from different sources such as urban, industrial waste water, or farms, are mixed with indigenous species . Genetic exchange between allochthonous and indigenous bacteria appear to be promoted by inorganic and organic pollution; however current knowledge is largely incomplete . As a case study, we investigated the antibiotic resistance features of the Gram-negative populations of the Western Bug River (Ukraine) and its main tributary Poltva, a waste water treatment plant effluent . Hydro-chemical criteria, benthos abundance and Escherichia coli load, determined in five sampling sites, highlighted a high organic load from the urban area . A total of 247 isolates were studied for their susceptibility to the clinically relevant ß-lactams, aminoglycosides and ciprofloxacin . Generally higher resistances were found in the most polluted sites, thus in the Poltva and, downstream, in the urban area of the Bug . Nineteen drug resistant and three multi-drug resistant E . coli were isolated . The genetic analysis of the acquired resistances showed the presence of blaTEM-1 in 18 out of the 19 resistant E . coli . In the Poltva and downstream in the Bug, four β-lactams resistant E . coli harbouring blaCTX-M-14 and one blaCTX-M-1 were found . Additionally, the acquired fluoroquinolones resistance gene qnrS was detected in three isolates . Class 1 integrons were present in 16 of the resistant E . coli . For two isolates, the insertion sequence ISEcp1, involved in

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the mobilisation of blaCTX-M genes, was found ahead of blaCTX-M-14 gene . RAPD analysis indicated a relatedness between four E . coli harbouring blaCTX-M-14 of which two were isolated from different sampling sites . The MLST profile indicated that the E . coli harbouring blaCTX-M-14 belonged to the ST131 and the one harbouring blaCTX-M-1 belonged to ST156 . This study, integrating environmental and clinical approaches, is the first report of antibiotic resistance species isolated from water bodies in the Ukraine . Furthermore, this is an important case study demonstrating how organic pollution of rivers may represent a clinical risk: the water acts like an infectious sources and reservoir of antibiotic resistances .

TH 125Distribution of novel beta-lactam resistance genes of ampC family among environmental aquatic bacteriaV .E .L .J .O Kisand, V . VoolaidUniversity of Tartu, Tartu, EstoniaIn addition to medical setting, antibiotic resistance is wide spread in environmental bacteria . One factor contributing to rapid spread of antibiotic resistance is horizontal gene transfer . For detecting the horizontal transfer, phylogeny of the resistance genes has to be compared to the phylogeny of the hosts . In the current work we focused on the ampC genes in environmental aquatic bacteria and detected several events of potential horizontal gene transfer . Presence of beta-lactamases was tested among bacteria isolated from a lake and its run off river . The bacteria were isolated on antibiotic (including ampicillin) containing selective media . The isolate collection was obtained from samples collected in years 2005 to 2008 . The lake is potentially exposed to several antibiotics from use in farming and diffuse countryside settlements while there is a larger town (100 000 inhabitants) at the runoff river . The isolates were phylogenetically characterized by 16S rRNA gene sequencing and presence of various ampC subclasses (LAT, DHA, ACC, ACT, FOX) as tested using multiplex PCR . Only the FOX subclass gave PCR signal . This subclass was found among Gammaproteobacteria and one Betaproteobacterium . The total number of FOX containing isolates was 44 . The most prominent group of FOX gene carriers was genus Aeromonas (n = 26), most of them the fish pathogen Aeromonas salmonida . On the other hand, not all isolated Aeromonas bacteria (n ~ 60) had FOX genes . In addition, the FOX gene was abundant among Pseudomonas . According to the sequences the FOX genes found clustered to form a novel subclass, related to the plasmid borne FOX (FOX-3, -4, -8 and -9) sequences found in the Enterobacteria Escherichia coli and Klebsiella spp . We also found that the phylogeny of FOX genes does not coincide with the phylogeny of the host bacteria . In conclusion, we have identified a new subclass of ampC genes where transfer between different bacterial groups has been relatively frequent .

TH 126Emergence of antibiotic resistant bacteria in Baltic Sea aquacultureI . Muziasari1, A .N .T .T .I Karkman1, O . Mitsuko2, S . Suzuki2, M .A .R .K .O Virta1

1University of Helsinki, Helsinki, Finland2Center for Marine Environmental Studies, Matsuyama, Japan Aquaculture environment has been suggested to be a reservoir of antibiotic resistance genes due to prophylactic and therapeutic use of antibiotics . It has been observed that antibiotic resistance genes are persistent in the sediment below aquaculture farms even in the absence of selection pressure . Here, HPLC analyses were conducted to monitor the selection pressure in the aquaculture farm sediments . Sediment samples were collected from two medium sizes of marine aquaculture and pristine areas in the northern Baltic Sea during summer time for six successive years . HPLC measurements revealed a high concentration of oxytetracycline in the sediment samples of 2011 . There is a therefore a high selection pressure in the aquaculture farm sediment . These results suggest a mechanism for the emergence of antibiotic resistant bacteria in the Baltic Sea aquaculture environment . In future studies, resistant bacteria from sediment, fish feeds, and fish guts will be isolated by using dependent-bacterial culture methods to further clarify the relation between resistant bacteria and fish pathogens . In addition, molecular methods such as PCR and qPCR will be performed to qualitatively and quantitatively analyze antibiotic resistance genes, respectively .

TH 127Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tapC .A . Narciso Da Rocha1, I . Vaz-Moreira1, L . Svensson-Stadler2, E .R .B . Moore2, C .M . Manaia1

1Escola Superior de Biotecnologia - Universidade Católica Portuguesa, Porto, Portugal2Culture Collection University of Gothenburg and Department of Infectious Disease, Gothenburg, SwedenAcinetobacter spp . are ubiquitous bacteria in the environment and hypothetic reservoirs of antibiotic resistance in drinking water . Acinetobacter spp . isolated from a drinking water treatment plant and from tap water, served by that plant, were identified and typed based on the analysis of the rpoB, gyrB and recA partial gene sequences . Antibiotic resistance was characterized, using the disc diffusion method, and each isolate was classified as wild or non-wild type, according to the observed phenotype . The strains of Acinetobacter spp . could be affiliated to 11 different species, four of which represent presumably new species . Most of the isolates were related to the species A. johnsonii and A. lwoffii . These two groups, as well as others related to the species A. parvus and A. tjernbergiae, were detected both in the water treatment plant and the tap water . Other strains, related to the species A. pittii and A. beijerinckii, were isolated only from tap water . Most of the isolates (80 %) were susceptible to all of the 12 antibiotics tested . In contrast, non-wild phenotypes for tetracycline, meropenem, ceftazidime, streptomycin, among others, were observed in water-treatment plant samples and/or in the tap water samples . Although, in general, this study reports a low antibiotic resistance risk of the Acinetobacter spp . in water, the potential of these bacteria to acquire and disseminate resistance via drinking water is demonstrated .

TH 128Resistance to broad-spectrum antibiotics in aquatic systems: do anthropogenic activities affect the dissemination?M . Tacão, I . Henriques, A . CorreiaUniversity of Aveiro, Aveiro, PortugalSignificant amounts of antibiotics and antibiotic-resistant bacteria are released daily into the environment . The prevalence and molecular diversity of resistance genes may be an indicator of water quality, useful to assess the risk of environmental resistance to public health . Production of extended-spectrum beta-lactamases (ESBLs) is one of the major resistance mechanisms that Gram-negative bacteria have developed against broad-spectrum antibiotics such as cefotaxime (CTX) . The association of ESBL genes with genetic elements that potentiate their capture and expression have contributed to their highly efficient mobilization and expansion, being reported frequently worldwide and thus becoming a serious clinical threat . The aim of this study was to compare the prevalence and diversity of CTX-resistant (CTXR) bacteria within polluted (P) and unpolluted (UP) ecosystems . Twelve rivers from the Vouga River basin (Portugal) were sampled and physicochemical and microbiological parameters of water quality were determined . Results classified 9 rivers as UP and 3 as P . Bacterial counts were 1 .9 X 106 CFU/L in P and 0 .68 X 106 CFU/L in UP streams, of which 8 .8% and 0 .6%, respectively, grew on CTX-supplemented agar . Antimicrobial susceptibility testing against 16 antibiotics of 6 different classes revealed that 60% (P) and 40% (UP) strains were multiresistant . ESBL production was detected on 39 strains (27P and 12UP) . ESBL genes were screened by PCR and blaCTX-M was the most frequently detected . Analysis of the blaCTX-M genetic context revealed the presence of different mobile genetic elements previously described in clinical strains . Clone libraries of blaCTX-M amplified from environmental DNA were constructed to compare the diversity of these genes in P and UP sites . Clear differences were detected with the P library presenting a much higher diversity of CTX-M-like variants (14 in P and 4 UP) . Also, UP library was mainly composed by sequences that affiliated with chromosomal ESBLs . In conclusion, results showed clear differences between P and UP lotic ecosystems, concerning prevalence, phylogenetic diversity and antimicrobial susceptibility profiles of CTXR bacteria and production of clinically relevant ESBL genes . This validates the hypothesis that anthropogenic activities modulate the environmental resistance gene pool and are promoting antibiotic resistance dissemination . Also, it suggests that blaCTX-M-like genes may constitute pollution indicators .

TH 129Abundance of class 1 integrons and diversity of their associated gene cassettes in hospital and urban effluentsT .S . Stalder1, O . Barraud2, M .C . Casellas3, C .D . Dagot1, M .C .P . Ploy1

1University of Limoges, Limoges cedex, France2Universite de Limoges, Limoges cedex, France3GRESE, Limoges, FranceThe spread of antibiotic-resistant bacteria is a growing problem and a public health issue . Wastewaters are considered to be sources for antibiotic resistance dissemination by horizontal gene transfer . Mobile integrons (MI) are genetic elements that acquire, exchange and express antibiotic-resistance genes embedded within gene cassettes (GC) . Class 1 MI are the most frequent with more than 130 GC conferring resistance to almost all known antibiotics . The aim of this study was to assess the class 1 MI relative abundance and their GC composition in an hospital and an urban effluents, as well as in the influent of the WWTP receiving sewer from the urban area, comprising the hospital & the urban effluent . Effluents from a 2000-bed French hospital, effluent from an urban area and the wastewater treatment plant (WWTP) influents receiving wastewater from 280,000 inhabitants equivalent were sampled . After a 24h flow proportionate sampling, effluents were concentrated and total genomic DNA was extracted using the PowerWaterTM DNA isolation kit (MoBio) . Class 1 MI were quantified using a qPCR, and normalized by the 16S DNA gene . The GC variable region of class 1 MI was PCR amplified from total DNA and cloned in pGEM-T easy vector (Promega) to construct a library from which 60 clones, chosen at random, were sequenced for characterization . The relative abundance of class 1 MI in hospital effluents was 9 and 7 fold higher than in urban effluents, suggesting that hospital effluents are an important source of bacteria harbouring class 1 MI . GC harbored by the class 1 MI belonged to GC involved in resistances to aminoglycoside, beta-lactams, trimethoprim, chloramphenicol, erythromycin, and quaternary ammonium compounds . The Chao abundance based Jaccard similarity index was used to compare the shared GC pool between samples . Indeed, the gene cassette composition of the urban effluent and the WWTP influent was the most closer . These results suggest that the influent from the WWTP is more influenced by the urban effluent than the hospital effluent . To our knowledge, this is the first study which has assessed the impact of hospitals effluents on the WWTP . Even if the prevalence of MI was higher in hospital effluents, it appeared that this impact on the WWTP influent was attenuated by the dilution effect in the sewage network

TH 130Occurrence of antimicrobial resistance among aquatic environmental bacteria of fish farms and pond sedimentsQ .A . Shah1, T .M .L . l ‘Abée-Lund1, F .C . Cabello2, D .J . Colquhoun3, H .S . Sørum1

1Norwegian School of veterinary Science, Oslo, Norway2Departments of microbiology and Immunology New York Medical College, Valhalla, ny 10595, United States of America

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3Norwegian veterinary Institute, Oslo, NorwayAntibiotics are the most important therapeutic agents used in control of bacterial infectious diseases . The use of a wide variety of antibiotics in human medicine and animal husbandry, including aquaculture, has led to the emergence of antibiotic resistant pathogens . In the present study bacteria from water and sediments related to aquaculture were collected from Chile (200 isolates), Norway (100 isolates), Pakistan (127 isolates) and Tanzania (135 isolates) . The bacterial isolates were screened for the presence of prevalent antibiotic resistance genes by a disc diffusion assay, followed by Southern hybridization and further confirmation by PCR and gene sequencing . The marine aquaculture industry in Chile uses antibiotics extensively while the marine aquaculture industry in Norway uses low levels of antibiotic treatment . Freshwater aquaculture in Pakistan and Tanzania has no history of antibiotic use . The frequency of resistance genes was, however, higher in fresh water isolates than in marine isolates . The prominent resistance genes found in the bacterial isolates studied were Tet A, Tet G and Tet E operons (7 .6 % of isolates), sul1, and sul2 (23 .5 % of isolates), dfrA1, dfrA5, dfrA7, dfrA12 and dfrA15 (56 .4 %), blaTEM (10 .2 %), strA-strB, and aadA1 (10 .7 %), CAT-1 (2 .1 %) and mefA (1 .5 %), causing resistance against tetracycline, sulphonamides, trimethoprim, amoxicillin, streptomycin, chloramphenicol and erythromycin, respectively . The Int1 gene was also found at all four sites in association with gene cassettes . Carbapeneme resistant fresh water environmental isolates were screened for the presence of the blaNDM-1 (New Dehli metallo-β-lactamase) gene, but blaNDM-1 was not found . All isolates were also negative for plasmid mediated qnrA, qnrB and qnrS genes . Extensive use of some antibiotics in Chilean marine aquaculture and in Norwegian fish farming during the 1980s and early 1990s appear to have altered the aquatic environment by selecting antibiotic resistance genes . Use of manure and poultry waste is common in freshwater aquaculture in South East Asia . We assume that in Pakistan integrated freshwater fish farming practices utilizing domestic farm and poultry waste along with antibiotic residues may have contributed to creation of a pool of resistance genes in the aquaculture facilities . These antimicrobial resistance genes persist in aquatic environmental bacteria and help in further dissemination of resistance genes .

TH 131Increased antibiotic resistance levels in Swiss lakes are related to human activitiesN . Czekalski, R . Sigdel, H . BürgmannEawag, Kastanienbaum, SwitzerlandAntibiotic resistance in clinical environments is a major concern in human and veterinary settings as it interferes with infectious disease treatment and raises health costs . Moreover, antibiotics and antibiotic resistant bacteria are currently discussed as a new class of environmental pollutants as they are released in high amounts from treated animals and waste water facilities into natural environments, including natural waters, where their fate and impact are yet unknown . Their elevated discharge has likely lead to an increase in the natural resistance background level of the exposed environments, such as surface waters and soils . The aim of the presented study is to evaluate the prevalence of antibiotic resistance genes in different lakes in Switzerland, a country with comparatively low antibiotic use . Furthermore, we want to investigate the correlation between increased level of resistance and measures of human impact on the lakes, e .g . degree of eutrophication, presence of intensive agriculture or waste water treatment plants in the watershed, etc . . We have sampled 20 Swiss lakes varying in size, trophic status (P content) and land use pattern which are currently evaluated for the prevalence of several antibiotic resistance genes, including sulfonanmides (sul1, sul2), tetracyclines (tetB, tetM, tetW) and fluoroquinolones (qnrA) using solely non-culture-based approaches . The respective target genes are amplified directly by PCR and qPCR in environmental DNA extracts . As a reference gene we amplified 16s ribosomal gene fragments . It is hypothesized that an increased occurrence of antibiotic resistance genes in lakes is due to increasing anthropogenic activities surrounding the lake and that the more eutrophic the lake (highest phosphorus concentration) the higher the prevalence density of resistance genes . The dataset will provide a basis to determine background levels of the natural antibiotic resistance versus antibiotics resistance pollution and to propose suitable indicators .

TH 132Differential gene expression and biofilm formation of multiple antibiotic-resistant Pseudomonas aeruginosa in municipal wastewaterT . Schwartz, S . Kirchen, J . Bruchmann, A . Babu VangalaKarlsruhe Institute of Technology / Institute of Functional Interfaces, Eggenstein-leopoldshafen, GermanyAs antibiotic resistance is commonly found in wastewater systems, its persistence seems to be obvious . The factors determining the persistence of antibiotic resistance are largely unknown . Antibiotics or substances with a likewise mode of action may provide selection advantages for resistant bacteria . Pseudomonas aeruginosa is a clinically important, opportunistic pathogen, which exhibits high intrinsic resistance to numerous antimicrobial agents . The persistence of multi-resistant Pseudomonas aeruginosa carrying up to seven acquired therapeutically relevant antibiotic resistance mechanisms was studied . Such multi-resistant P . aeruginosa were enriched by clinical as well as municipal wastewaters . Agar diffusion testing and molecular biology confirmed their multi-resistances . Pulsed Field Gel Electrophoresis (PFGE) genotyping resulted in different groups of P . aeruginosa isolates depending on sampling points and numbers of resistances . Differential gene expression analyses via quantitative SyBrGreen PCR were performed to demonstrate wastewater matrix dependent impacts on molecular responses in P . aeruginosa . A number of different genes were selected being involved in biofilm formation, signal molecule syntheses, antibiotic resistance, and virulence . The expression of these gene targets were analysed in presence of wastewater or tap water as a control . Additionally, epifluorescence microscopy analyses in combination with different staining methods were applied to quantify live/dead percentages in P . aeruginosa biofilms and visualized differences in biofilm formation (biomass, structure) under wastewater influence . The results are important to understand selective pressures of wastewater xenobiotics on P . aeruginosa regarding resistance as well as persistence in aquatic environments .

TH 133Occurrence of antibiotic resistance genes in wastewater used for irrigation in the Mézquital ValleyM .M . Broszat, J . Hübner, E . GrohmannUniversity Medical Centre Freiburg, Freiburg, GermanyWastewater (WW) reuse for irrigation is widely practised in agriculture to alleviate water shortages . WW contains a huge amount of potentially harmful compounds, such as pharmaceuticals and microbial pathogens . We investigated the risks of WW irrigation in the Mézquital Valley (60 km north of Mexico City) for environment and human health . The Mézquital Valley is the world´s largest WW irrigation field . WW from Mexico City is reused for crop irrigation, which poses risks for farmers and the consumers of the agricultural products . We followed the pathway of the WW from Mexico City to the Mézquital, starting with the WW effluent of two hospitals in Mexico City . In addition we took samples from the two major WW channels which transport the WW out of the city to the Mézquital . The influent and effluent of a dam in the Mézquital, and the reclaimed water after passing through the soil were sampled subsequently . Sampling was done in March and September 2011 to study differences between dry and rainy seasons . We selected two frequently used antibiotics, sulfamethoxazole and ciprofloxacin, and five antibiotic resistance genes (sul1, sul2, qnrA, qnrB, qnrS) that mediate resistance against these antibiotics . Furthermore, the blaSHV5 gene that is frequently found in bacterial isolates from patients in Mexican hospitals was selected . This gene encodes for Extended-Spectrum Beta-Lactamases (ESBLs) . Furthermore, we determined the concentration of the faecal indicator Enterococcus spp . and the total bacterial concentration . Total DNA was extracted from all samples and was used for quantitative PCR specific for each resistance gene, total bacteria (16S rRNA gene) and Enterococcus spp . All of the resistance genes were present in high copy numbers (103-107 gene copies/100 ml water) in the WW samples . Two antibiotic resistance genes (qnrA and qnrB) were completely removed after soil passage, but the other four genes were still found in the reclaimed water: qnrS decreased by 4 logs from 105 to 101 copies/100 ml, sul1 decreased by 5 logs from 109 to 104 copies/100 ml, sul2 and blaSHV5 both decreased by 5 logs from 107 to 102 copies/100 ml . The concentration of total bacteria decreased by 3 logs from 109 to 106 copies/100 ml and the concentration of Enterococcus spp . by 4 logs from 107 to 103 copies/100 ml . The recovered water still contains a high concentration of total bacteria and faecal indicators . Therefore, without further treatment it is not suited for crop irrigation .

TH 134Development of treatment methods for the degradation of antimicrobial compounds present in wastewatersA .C . Reis1, C .M . Manaia2, O .C . Nunes1

1Faculdade de Engenharia, Universidade do Porto, Porto, Portugal2Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalThe intensive use of antibiotics and disinfectants has been leading to the environmental contamination with these micropollutants or with their toxic degradation products . The widespread distribution of these contaminants may have harmful effects, such as the reduction of microbial diversity and the spreading of antimicrobial resistance (AR) . The increase of AR leads to a generalized claim that micropollutants with antimicrobial activity must be removed from industrial, hospital and domestic wastewaters . Among the different mechanisms that bacteria can use to resist the antibiotic action, degradation is one of the most often used to tolerate beta-lactams . The diversification of beta-lactamases in the environment has been observed over the last years and, curiously, such a property can also be of interest to attenuate antibiotics contamination . Indeed, the implementation of degradation methods is urgently needed . The present study aims at developing low cost efficient treatment systems to remove beta-lactams from wastewaters, by using microorganisms able to degrade these antibiotics . .Biodegraders thriving in wastewater treatment plants have been enriched usingin amoxicillin supplemented culture media . Mixed cultures able to transform approximately 100 % of 30 mg L-1 amoxicillin to non-active degradation products were obtained . The members of these cultures were identified and their antibiotic resistance phenotype was determined . The genetic determinants responsible for their degrading activity were also characterized . The diversity of bacteria and of genes involved in these processes will be also discussed .

TH 135From the hospital effluent to the municipal wastewater treatment plant: bacterial communities and antibiotic resistanceA .R . Varela1, C .M . Manaia1, O .C . Nunes2

1Escola Superior de Biotecnologia - Universidade Católica Portuguesa, Porto, Portugal2LEPAE-Dep . Engenharia Química, Faculdade de Engenharia, Universidade do Porto, Porto, Portugal Given the intensive use of antibiotics, hospital effluents, which often do not have any dedicated treatment and are discharged into the municipal collectors, are suspected to contribute to the dissemination of AR bacteria into the environment . In spite of this suspect, studies on the fate of AR bacteria from the hospital effluents to the receiving sewage treatment plants and the environment are scant . The main objective of this work was to assess the impact of the bacterial load from a raw Hospital effluent in the municipal wastewater treatment plant (WWTP) into which it is discharged . Antibiotic resistance was assessed through successive sampling campaigns of raw hospital effluent and raw and treated wastewater of the municipal wastewater treatment plant to which the hospital effluent is directed . The presence of antibiotics and heavy metals in the samples was determined in the different samples . The microbiological analyses comprised the enumeration of total heterotrophs, enterobacteria and aeromonads on different culture supplemented or not with amoxicillin or ciprofloxacin (1) .

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Furthermore, the influence of the hospital effluents on the bacterial communities of the WWTP was investigated using 16S rRNA PCR DGGE . Cell counts and amoxicillin resistance rates were similar between the hospital discharge and the point of inflow to the WWTP . In contrast, ciprofloxacin resistance was detected to be about three times higher in the hospital effluent than in the raw municipal wastewater . The hospital discharge seems to have a significant impact on the levels of ciprofloxacin resistance that enter the WWTP . The treated effluent displayed a reduced cell density of about 100-1000 times for both total and AR bacteria . However, the treatment did not cause significant variations on the rates of amoxicillin and ciprofloxacin resistance . This study confirms hospital discharge as a significant source of AR into the environment . The variation in bacterial communities in the hospital effluent and in the WWTP raw and treated effluent is analyzed . 1 . Novo, A and Manaia, CM, (2010), Factors influencing antibiotic resistance burden in municipal wastewater treatment plants, Applied Microbiology and Biotechnology, 87:1157 - 1166 . <br type=’_moz’ /> TH 136Phylogenetic diversity of quinolone resistant Escherichia coli isolated from wastewatersA .R . Varela, M . Rebelo, J . Azevedo, C .M . ManaiaEscola Superior de Biotecnologia - Universidade Católica Portuguesa, Porto, PortugalAntibiotic resistance dissemination has become more important in the last decades . The species Escherichia coli comprises different subgroups which may have different roles in antimicrobial resistance dissemination . In previous studies, wastewater treatment was observed to promote an increase on quinolone resistance, presumably due to the differential removal rates of distinct genotypic groups (Figueira et al ., 2011) . The current work aimed at investigating the relationship between quinolone-resistance genes and the phylogenetic group of the bacteria . Seventy Escherichia coli strains isolated from raw and treated municipal wastewaters were characterized based on a multi locus sequence typing (MLST) approach . The presence of quinolone resistance determinants was assessed based on the detection of mutations in the gyrA and parC genes and on the presence of the genes qnrA, qnrB, qnrS, aac6, qepA, qnrC . The distribution of the MLST groups and the type of quinolone resistance genetic determinants is discussed .

TH 137Antibiotic resistance and bacterial communities in a wastewater treatment plantA . Novo1, L . Barreiros2, P . Viana3, S . André3, O .C . Nunes2, C .M . Manaia1

1Escola Superior de Biotecnologia, Porto, Portugal2LEPAE/DEQ, Faculdade de Engenharia da Universidade do Porto, Porto, Portugal3Agência Portuguesa do Ambiente, Amadora, PortugalAlthough it is still difficult to establish clear cause-effect relationships, it is widely accepted that chemical pollution in the environment can influence the spread of antibiotic resistance among bacterial communities . Domestic wastewater is a complex mixture of environmental and human commensal bacteria, which come in contact with a considerable amount of antimicrobial agents and other substances with unknown effects on microbial communities and on bacterial metabolism . In this work we studied the bacterial communities, based on DGGE patterning and used multivariate analysis to infer about the influence of some environmental parameters (antimicrobials concentration, percentages of antibiotic resistance among culturable bacteria, and climate or plant operation conditions) . This study was designed to identify the factors observed to influence or be interdependent of either the bacterial communities and/or the antibiotic resistance rates . A total of 14 composite samples of raw and treated wastewater were collected from an activated sludge wastewater treatment plant over a period of about six months . Total DNA was extracted from raw and treated wastewater and the bacterial communities were compared using DGGE analysis . Antibiotics and heavy metals were determined in the inflow and in the treated effluent . The culturable antibiotic resistant rates were documented on Novo & Manaia, 2010 study . Some of the most relevant DGGE bands were selected for further identification . Based on the obtained DGGE profiles, bacterial communities formed coherent groups according to the time of the year . Among the bacterial taxa varying over these periods were, for example, Arcobacter, Streptobacillus, Firmicutes and Clostridiales in raw wastewater and Sulfurimonas, Bacteroidetes, Leadbeterella, Gammaproteobacteria, Arcobacter and Streptobacillus in treated wastewater . After Canonical Correspondence Analysis, it was possible to assess the influence of some factors and to find significant relationships between DGGE bands, antibiotic resistance rates and antimicrobials concentrations . However, the direct influence of a specific antibiotic resistant bacterial group or antimicrobial could not be identified in the community structure . The results suggest that the wastewater communities are affected by the presence of antimicrobial agents and that these substances affect the bacterial metabolism which may be related to antibiotic resistance dissemination .

TH 138Quantification of resistance genes and isolation of resistant bacteria for last resort antibiotics from wastewater treatment plantsA .V . Karkman, S . Pussila, M . VirtaUniversity of Helsinki, University of helsinki, Finland Antibiotic resistance is increasing rapidly among bacteria as antibiotics are used in increasing quantities in treating humans and animals . Due to bacterial multiresistance to different antibiotics, we are heading back to pre-antibiotic era where there is no treatment for common bacterial infections . One major concern is the microbial resistance for the last resort antibiotics, such as vancomysin, carbapenems and quinolones . In wastewater treatment plants antibiotics, other pharmaceuticals, heavy metals and xenobiotics mix up with bacteria from human, animal and environmental origin . Wastewater treatment plants are known reservoirs for various resistance determinants and serve as hotspots for horizontal gene transfer . Resistance genes for different antibiotics are usually associated with mobile genetic elements that are able to horizontally transfer between different bacteria . Dense bacterial populations and biofilms in the sludge promote horizontal gene transfer and different chemicals can pose a selection pressure promoting the survival of the resistant bacteria . Horizontal transfer of certain mobile genetic elements, such as Tn916, has also been shown to be inducible by antibiotics . The resistant bacteria from the wastewater treatment plants may eventually end up in the environment either in the effluents or in the sludge . In this study we quantified different resistance genes for the last resort antibiotics, such as carbapenems, 3rd & 4th generation cephalosporines, quinolones and vancomysin, from different compartments of the wastewater treatment plant using quantitative PCR . Moreover resistant bacteria were cultured on ciprofloxacin and meropenem containig media and the resistance pheno- and genotypes were characterised . Our results show that many resistance determinants and resistant bacteria can be found from the wastewater treatment plants . Resistance genes and resistant bacteria are even found from the effluents via which they are released to the environment . TH 139Vancomycin and ciprofloxacin resistance in enterococci from a Hospital effluent and in the receiving Municipal Wastewater Treatment PlantA .R . Varela1, G . Ferro1, J . Vredenburg1, M . Yanik1, L . Rizzo2, C .M . Manaia1

1Escola Superior de Biotecnologia - Universidade Católica Portuguesa, Porto, Portugal2University of Salerno, Fisciano, Italy The increasing incidence of resistance to a wide range of antibiotic agents in human commensal and pathogenic bacteria has become a major concern over the last decades . Enterococci, important human commensal bacteria are also frequently associated with nosocomial infections . Given the intensive use of antibiotics, untreated hospital effluents released to the municipal sewage collectors are suspected to constitute a major source of antibiotic resistances . In spite of this suspect, the influence of hospital effluents on the levels of antibiotic resistance in the municipal wastewater treatment plants is not very well understood . This study was designed to verify if hospital effluents may be a possible source of ciprofloxacin and vancomycin resistant enterococci and how these resistances are affected by wastewater treatment in municipal wastewater treatment plants . Raw hospital effluent and raw and treated wastewater of the municipal wastewater treatment plant to which the hospital effluent is directed were collected and analyzed during successive campaigns . The analyses included the enumeration and isolation of enterococci on appropriate culture media supplemented or not with vancomycin or ciprofloxacin . The resistance phenotypes of the isolates were determined by diffusion agar method, using the antibiotics amoxicillin, gentamicin, ciprofloxacin, tetracycline, erythromycin and vancomycin . Isolates were also examined for the presence genes possibly responsible for the observed phenotypes . The percentage of resistance to ciprofloxacin was generally higher than that to vancomycin and both decreased from the hospital effluent to the wastewater treatment plant raw inflow, presumably due to dilution . The wastewater treatment in the municipal facility also decreased the prevalence of ciprofloxacin and vancomycin resistances . Frequently, vancomycin resistance was not detected in the final effluent .The impact of hospital effluents in the prevalence of antibiotic resistant Enterococci and antibiotic resistant determinants in the environment is discussed .

TH 140Occurrence of beta-lactamases, namely GES-5 carbapenemase, among Gram-negative isolates from wastewater samples in Northern PortugalM .C . Caniça1, V .M . Manageiro1, E .F . Ferreira1, V .F . Figueira2, C .M . Manaia2

1National Institute of Health, Lisbon, Portugal2CBQ - Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Oporto, PortugalBackground: Antimicrobial resistant pathogens are profoundly relevant to human health and many were the studies that focused on their spread . However, natural and human associated environmental reservoirs of antibiotic resistance (AR) are yet poorly understood . The main goal of this study was to evaluate some AR mechanisms in Gram-negative bacteria isolates from surface and raw and treated waste water environments . Methods: Water samples were collected from different aquatic environments within an urban water cycle in the region of Northern Portugal (1, 2) . Screening of antimicrobial susceptibility of 48 Gram-negative isolates (20 Escherichia coli, 8 Citrobacter spp ., 7 Klebsiella spp ., 6 Kluyvera spp ., 2 Enterobacter spp ., 1 Hafnia alvei, 1 Pantoea agglomerans, 1 Pseudomonas luteola, 1 Roultella ornithinolytica, 1 Serratia spp .) was performed by disk diffusion method . The search for antibiotic resistant genes was based on the interpretative reading of susceptibility profiles . PCR and sequencing were used to screen and identify bla and plasmid-mediated quinolone resistance (PMQRs) genes . All isolates were also screened for the presence of class 1 integrons . Results: Overall, 13 isolates were multidrug resistant, suggesting a great diversity of AR mechanisms . Two isolates showed non-susceptibility to carbapenems, one of the last resorts on the antimicrobial therapy . Their phenotypic and molecular characterization revealed the expression of a chromosomal metallo-beta-lactamase in P. luteola and the presence of a GES-5 encoding gene in a Klebsiella pneumoniae isolate . Furthermore, a variety of beta-lactamase encoding genes were observed, specifically 12 blaTEM-1 with

261SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

distinct promoters, 4 blaSHV (2 blaSHV-1 and 2 blaSHV-11) and also different chromosomal AmpC beta-lactamases, namely CMY-65 . Class 1 integrons were detected among 6 of TEM-1-producing isolates . Together, these beta-lactamases explain the level of beta-lactam resistance . None PMQR genes were detected . Conclusion: In conclusion, this study provides the first description of a class A carbapenemase in an environmental setting in Portugal, in addition to several other beta-lactam resistance mechanisms . The study highlights the need of surveillance of these AR mechanisms in environmental backgrounds, since it represents a liable reservoir of potential pathogenic resistant bacteria . 1-Figueira V et al . 2011 . Sci Total Environ, 409:1017-23 . 2-Figueira V et al . 2011 . J Water Health doi:10 .2166/wh .2011 .002 .

TH 141Pseudomonas spp. in drinking waters - diversity and antibiotic resistanceI . Vaz-Moreira1, O .C . Nunes2, C .M . Manaia1

1Escola Superior de Biotecnologia, Porto, Portugal2LEPAE - Dept .Engenharia Química, Faculdade de Engenharia, Univ . Porto, Porto, Portugal Pseudomonas are common inhabitants of aquatic environments, including drinking water . The problems associated with multi-resistance in clinical isolates of P . aeruginosa are well known, but a lack exists in the study of the presence of antibiotic resistance phenotypes in other species and in environmental isolates of this genus . This study aimed to evaluate the diversity and antibiotic resistance phenotypes of Pseudomonas spp . present in drinking water . Isolates from a drinking water treatment plant, household taps, cup fillers of dental chairs, a drinking water biofilm and bottled mineral waters were examined . Based on the analysis of the 16S rRNA gene sequence, it was observed that the isolates belonged to 20 species . Taxonomic diversity was further inferred based on the analysis of six housekeeping genes (16S rRNA, rpoD, rpoB, gyrB, recA, ITS) and PFGE profiles . Susceptibility to eight classes of antibiotics was tested using the ATB PSE(08) and disk diffusion method . Pseudomonas spp . were not detected immediately after the chlorination steps, in some taps and in a brand of mineral water . Twenty eight different sequence types (ST) were observed by the housekeeping genes analysis, with the genes rpoD and gyrB showing the best performance in the species differentiation . Although the same species have been isolated from different samples, the same ST was never observed in different types of water . Nevertheless, the same clone was detected in different taps . The prevalence of antibiotic resistance was, in general, low and related with the species or even with the ST . Resistance to ticarcillin, fosfomycin and cotrimoxazole were the most prevalent . Ceftazidime resistance was very rare, but was present in all the isolates identified as P . simiae . Resistance to nalidixic acid was observed in P . chlororaphis, P . nitroreducens, and P . aeruginosa recovered from tap water but not from biofilm . Intrinsic resistance was observed to cephalothin, and no resistance phenotypes were observed to ciprofloxacin, tetracycline, piperacillin, imipenem, meropenem and colistin . This study shows that the Pseudomonas spp . observed in the taps may not have origin on the drinking water treatment plant . Antibiotic resistance was observed to be mainly species, rather than site-related . In general, Pseudomonas spp . are confirmed as potential vectors of antibiotic resistance in drinking waters, with the selection of some strains representing an important role of resistance spreading .

TH 142Bottled mineral water as a potential source of antibiotic resistant bacteriaM .F . Falcone-Dias1, I . Vaz-Moreira2, C .M . Manaia2

1Ministry of Education of Brazil, Brasília-df, Brazil2CBQF - Escola Superior de Biotecnologia, Univ . Católica Portuguesa, Porto, Portugal The hypothesis that bottled mineral water may represent a supplier of antibiotic resistant bacteria to humans was tested . The antibiotic resistance phenotypes of the cultivable bacteria present in nine batches of two Portuguese and one French brands of commercially available mineral waters were examined . Among the 238 isolates recovered on R2A and PIA and on these culture media supplemented with amoxicillin or ciprofloxacin, most were identified (based on 16S rRNA gene sequence analysis) as Proteobacteria of the divisions Beta, Gamma and Alpha . Also present, were members of the phyla Firmicutes, Actinobacteria and Bacteroides . Bacteria resistant to more than three distinct classes of antibiotics were detected in the all batches of the three water brands at densities up to 102 CFU/mL . In the whole set of isolates, it was observed resistance against all the 22 antimicrobials tested (ATB, BioMerieux and disc diffusion), with most of the species showing resistance to three or more classes of antibiotics . Bacteria with the highest multi-resistance indices were members of the genera Variovorax, Bosea, Ralstonia, Curvibacter, Afipia and Pedobacter, yielding resistance up to seven classes of antibiotics . Presumable episodes of resistance acquisition in the aquifer or during bottling are supported by the fact that members of the same species isolated from different brands exhibited distinct antibiotic resistance profiles . The Actinobacteria presented high resistance levels to the antibiotics ceftazidime, meropenem and ciprofloxacin according to minimum inhibitory concentrations (Etest and M .I .C . Evaluator) . Bottled mineral water was confirmed as a source of antibiotic resistance mechanisms, with the potential to be transmitted to humans .

TH 143Important vectors of antibiotic resistance genes in agricultural systemsK . Smalla, C .T . Binh, S . Jechalke, C . Kopmann, U . Zimmerling, E . Krögerrecklenfort, H .H . HeuerJKI, Braunschweig, GermanySpreading manure on agricultural soils was recently shown to promote spreading of transferrable antibiotic resistances and residual veterinary medicines in agricultural soils . Treatment of soil with manure spiked with the antibiotic sulfadiazine (SDZ) or manure from pigs treated with SDZ resulted in significantly higher transfer frequencies compared to manure not containing SDZ . In the present study we aimed to characterize plasmids which were captured into E . coli recipients from soil samples of microcosm, mesocosm and field experiments by probing and antibiotic resistances conferred . The plasmids captured were mainly assigned to LowGC and IncP-1ε plasmids . The diversity of antibiotic resistance genes carried on these plasmid vectors was remarkable . Our data showed that the selection of bacterial populations carrying sul genes often will co-select a wide range of other antibiotic resistance genes . The diversity of antibiotic resistance gene cassettes reported in IncP-1ε, the ability to efficiently transfer under soil conditions and the wide host range of IncP-1ε plasmids strongly suggest that these plasmids are important vectors for spreading antibiotic resistances in the agro-ecosystem . LowGC plasmids have in contrast to IncP-1ε plasmids a much more narrow host range but their relative abundance is one to two orders of magnitude higher, and they are important as putative hosts are emerging pathogens of the genus Acinetobacter .

TH 144Spread of antibiotic resistance determinants in wastewater irrigated soils in the Mézquital Valley, Mexico M .M . Broszat1, B . Prado2, C . Siebe2, J . Siemens3, J . Huebner1, E . Grohmann1

1University Medical Centre Freiburg, Freiburg, Germany2Universidad Nacional Autónoma de México, Institute of Geology, Ciudad Universita, Mexico city, Mexico3University Bonn, Institute of Crop Science and Resource Conservation, Bonn, GermanyWe investigated the spread of antibiotic resistance determinants by horizontal gene transfer (HGT) in wastewater (WW)-irrigated soils from the Mézquital Valley (60 km north of Mexico City), the world´s largest WW irrigation area . There, untreated WW from Mexico City is reused for crop irrigation . WW contains a huge amount of potentially harmful components such as resistance determinants, pathogens and antibiotics . WW-irrigated soils are a potential reservoir for multiresistant organisms, which might pose risks for field workers and consumers of the agricultural products . In the Mézquital, fields have been irrigated with WW for different time periods, from 1 year up to more than 100 years . We studied the possible correlation between the irrigation time and the concentration and dissemination of resistance genes . Furthermore, we performed soil column experiments with two different types of soil (soil irrigated with WW for 100 years and rain-fed soil), to test our hypothesis that WW irrigation leads to spread of resistance genes in soils . Gene transfer scenarios were performed as follows: an Enterococcus faecalis donor harbouring a mobilizable broad host range resistance plasmid labelled with the Green Fluorescent Protein (GFP) [1] and a second non-mobilizable plasmid labelled with the Red Fluorescent Protein (RFP) were added to rain-fed and 100 years-irrigated soil, each in soil columns of 20 cm height and 15 cm diameter . At each irrigation 109 donors were applied to the columns . The rain-fed soil was irrigated with artificial rainwater and the WW-irrigated soil was irrigated with WW once a week, in total three times . The duration of the experiment was 4 weeks . Samples were taken daily from top soil and at the end of the experiment from different heights (every 2 .5 cm) . During irrigation leachate water was collected and pore water was sampled at 4 heights with suction cups . Bacteria in soil and water which have acquired the mobilizable resistance plasmid via plasmid transfer are detectable by green fluorescence . Donors are identified by their green and red fluorescence . Transfer rates for both types of soil and in water will be presented . Furthermore gfp and rfp will be quantified by real-time PCR . The soil column experiment will help assess the risk posed by HGT of resistance determinants in WW-irrigated soil .

TH 145Development of micro-scale leucine incorporation assay and its application for analysis of co-selected antibiotic resistance patterns in Cu-contaminated soilJ . Modrzynski, K .K . Brandt, J . Berg, O . NybroeUniversity of Copenhagen, Faculty of Life Sciences, Frederiksberg, DenmarkIt is well known that Cu selects for Cu tolerance in soil microbial communities, but recently it has been documented that Cu may also co-select for antibiotic resistance . Due to the ongoing Cu accumulation in agricultural soils and the risk of human exposure to soil microorganisms, this co-selection phenomenon deserves further study . Soil is a spatially heterogeneous matrix and we thus asked whether co-selected antibiotic resistance is a general phenomenon in the entire soil matrix or whether this phenomenon can be localized to specific micro-habitats in the same long-term (+85 years) Cu-contaminated soil as used for a previous co-selection study (Berg et al ., 2010; Environ Sci Technol 44:8724-8728) . To analyze spatial heterogeneity of community-level tolerance to Cu and antibiotics, we first developed a novel micro-scale [3H]leucine incorporation assay for measuring growth rates of bacteria extracted from soil . The assay allowed us to analyze [3H]leucine incorporation rates in 5-µL soil bacterial suspensions with retained sensitivity and performance as compared to the original protocol based on larger sample sizes (Bååth et al ., 2001; Soil Biol Biochem 33:1571-1574) . When combined with an optimized extraction and soil handling protocol, we were able to perform 96 replicate [3H]leucine incorporation incubations based on a single 5-mg soil sample . The optimized micro-scale [3H]leucine incorporation assay was subsequently used to measure pollution-induced community tolerance to Cu and co-tolerance to selected antibiotics in 5-mg Cu-contaminated soil samples and corresponding non-contaminated control soil samples . Cu bioavailability was analyzed by a downscaled version of an existing whole-cell bacterial biosensor assay (Brandt et al ., 2008; Environ Sci Technol 42:3102-3108) . A low extent of spatial heterogeneity was found for both Cu tolerance and Cu bioavailability, whereas co-selected antibiotic resistance patterns exhibited a highly heterogeneous distribution (i .e . co-selection in some 5-mg soil spots, but not in others) . Our studies indicate that field exposure to Cu may constitute a persistent selection pressure for the expansion of the soil bacterial resistome, but also that co-selection

262 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

phenomena are complex and difficult to predict at the bacterial community level . The developed micro-scale [3H]leucine incorporation assay should have several possible applications within soil microbial ecology and ecotoxicology .

TH 146Distribution of minimum inhibitory concentrations to antibiotics in E.coli and Enterococci isolated from wastewater effluents and surface watersR .J . Tuckwell, M .I .K .E . Revitt, H .E .M .D .A GarelickMiddlesex University, London, United KingdomBacterial resistance to antibiotics increases the difficulty in treating both hospital and community acquired infections and are therefore of great concern to public health . Consequently, there has been extensive research in the clinical domain . There are however, few studies investigating environmental compartments, most of which have defined antibiotic resistance in terms of clinical break points . In order to understand the mechanism of resistance in environmental isolates, a more detailed study profiling the minimum inhibitory concentration (MIC) is required . Wastewater treatment plants are a reservoir of antibiotic residues and resistant bacteria, providing probable hotspots for the dissemination of antibiotic resistance, potentially influencing the environment when wastewater effluents are discharged into receiving waters . This study presents the surveillance of MICs to amoxicllin, ciprofloxacin and clarithromycin expressed by E .coli and Enterococci isolated from effluents of a large urban treatment plant (employing activated sludge) as well as surface waters from up and down-stream of the effluent discharge . E .coli and Enterococcoi were selected as they are important human commensal bacteria, commonly found in the environment and frequently associated with nosocomial infections . Standard culture methods were used to detect, enumerate and isolate bacteria from the collected samples . Bacteria isolates were identified with API identification systems . The MICs to amoxicillin, ciprofloxacin and clarithromycin the isolates expressed were measured using antibiotic gradient strips . E .coli isolated from receiving surface waters, up and down-stream of the effluent discharge, expressed MICs to amoxicillin ranging from 2-256 µg/mL, . However, only 30 % are considered resistant according to clinical break points (of 8 µg/mL) . Entercocci isolates did not have a resistant phenotype to amoxicillin according to clinical break points(of 8µg/mL) .However, the MICs ranged from 0 .062-0 .50 µg/mL . The possible significance of the MIC distribution and its relevance to environmental microbial communities is explored .

TH 147Aspects of antibiotics resistance in Salmonella spp. isolatesS . Purkrtová, K . Koukalová, J . Pazlarová, K . DemnerováInstitute of Chemical Technology Prague, Prague 6, Czech RepublicSalmonella spp ., mainly some serovars of Salmonella enterica as Enteritidis or Typhimurium, are etiological agents of salmonelosis, the second common gastrointestinal disease in the industrially developped countries . While most cases do not require the antibiotics treatment, it can be neccassary for immunosuppressed, elder or child patients . The increasing resistance of Salmonella spp . to antibiotics can cause complications in such cases, as well as to support the horizontal transfer of antibiotics resistance genes in the microbial community . The development and the acquistance of these genes can occur at different places under different conditions - for example by overusing/misusing antibiotics in veterinary and medical practice or in wastewater treatment plants, supported by biofilm formation . The aim of this work was to study the different aspects of antibiotics resistance in 50 strains of Salmonella spp . isolated in Czech Republic from different sources (wastewater treatment plant, food and clinical isolates) . The isolates were typed by ERIC-PCR and REP-PCR methods and studied for their plasmids profile . Then they were tested by disc diffusion method for the resistance to 12 antibiotics (amoxicillin, ampicillin, cefotaxime, gentamicin, streptomycin, ciprofloxacin, nalidixic acid, trimethoprim, tetracycline, chloramphenicol, nitrofurantoin, kanamycin, sulfonamide) . The occurrence of tet genes (tetA, tetB, tetC, tetG) was detected by PCR method together with the cluster SGI-1 (Salmonella Genomic Island-1) . The strains resistant minimally to one antibiotics (tetracycline, ampicillin, streptomycine, nalidixic acid, ciprofloxacine or chloramphenicol) were examined for the ability of biofilm formation in microtiter plates in different media (LB, BHI, BHI + 2 % glucose) and at different temperatures (25 °C, 37 °C) . This work was supported by the project LD11048 of Ministry of Education, Youth and Sports of Czech Republic .

TH 148Adaption of bacteria to ameliorate the metabolic burden of carrying antibiotic resistance genes (a systems bioecology modeling analysis)F .L . HellwegerNortheastern University, Boston, United States of AmericaThe spread of antibiotic resistance is an important problem, and it is unclear what leads to the relatively high abundance of resistant bacteria in the ambient environment . Mathematical models are useful tools for research and management in this area . However, although many of the mechanisms underlying antibiotic action, resistance and cost of resistance are understood at the molecular level, models typically describe these processes using system-level parameters (e .g ., cost of resistance) . This constitutes a disconnect that limits the utility of modeling . Here we investigate if these behaviors can be predicted from basic molecular biology . A mathematical model for E . coli and tetracycline is developed based on existing models of metabolism, plasmid and antibiotic behavior . The model explicitly accounts for the action (i .e . binding to ribosome) and resistance (Tet efflux pump), and the metabolic cost of resistance, including copying the plasmid DNA, transcribing the plasmid genes, translating the plasmid proteins and the energy consumption of the efflux pump (i .e . ATP requirement to maintain membrane charge) . To investigate the fitness of different strains under various conditions, individual cells are simulated using an agent-based approach . The concept of combining biological modeling (systems biology) and ecological modeling (systems ecology) in this manner is referred to as “systems bioecology” . The model reproduces observations from the literature, including growth rate vs . substrate concentration, competition between resistant and susceptible strains in the absence of tetracycline, intracellular tetracycline concentrations of wildtype and resistant strains, growth rate under various tetracycline concentrations and the response of tetracycline resistance in the human gut to a 5-day tetracycline treatment . The results show that these behaviors can be predicted from molecular mechanisms . The model is then used to investigate the role of potassium uptake by the Tet efflux pump . When this feature is included, a mutation in the endogenous uptake system (Trk) reduces the cost of carrying the resistance plasmid . Carrying the plasmid is then beneficial even in the absence of the antibiotic, which is consistent with observations in laboratory evolution experiments . This finding suggests the resistance may not be reduced when antibiotic use or discharge is stopped, an important implications for the ecology of antibiotic resistance in the environment .

EP03P2 - Environmental characterisation, fate/detection, exposure, effect and risk of nanomaterials

TH 157Toxicity of silver nanoparticles to rainbow trout - a toxicogenomic approachF .O . Gagné1, C .A . André1, M .G . Gélinas2, J .A . Auclair2, R .S . Skirrow1, G .V .A . van Aggelebn2, P .T . Turcotte2, C .G . Gagnon2

1Environment Canada, Montreal, Canada2Environnment Canada, Montreal, CanadaSilver (Ag) nanoparticles possess antimicrobial properties which makes them convenient adjuvant in consumer products such as clothes and medical devices . NanoAg could leach out from these products and pervade the our environment with perhaps unsuspected impacts . The purpose of this study was to examine and compare the sublethal effects of nanoAg and dissolved Ag in rainbow trout . Changes in hepatic gene expression were monitored to provide insight on the mode of action of both forms of Ag . Oncorhynchus mykiss were exposed to increasing concentrations of nanoAg (20 nm) and AgNO3 for 96 h at 15oC . The liver were also analyzed with a suite of biochemical markers of toxicity . Gene expression analysis was performed using a DNA microarray comprised of 207 stress-related genes followed by quantitative polymerase chain reaction on a selection of genes for validation . The biochemical markers consisted in the determination of labile zinc, metallothioneins, DNA strand breaks, lipid peroxidation and vitellogenin-like proteins . The analysis of total Ag in the aquarium water revealed that Ag was mostly aggregated where 1 % of the total Ag was truly dissolved . Exposure to both forms of Ag led to significant changes in gene expression for 13 % of tested gene targets . About 12 % of genes responded specifically to nanoAg while 10 % of total gene targets responded specifically to dissolved Ag . The levels of vitellogenin-like proteins and DNA strand breaks were significantly reduced by both forms of Ag but DNA breaks levels were lower with nanoAg and could not be explained by the presence of ionic Ag . Labile zinc and the oxidized fraction of metallothioneins were increased by both forms of Ag but LPO was significantly induced by nanoAg only . Discriminant function analysis revealed that the responses obtained by biochemical markers and a selection of 10 target genes were able to discriminate completely (100%) the effects of both forms of Ag . It was found that genes involved in oxidative stress, protein stability and inflammation responses were able to discriminate each forms of Ag where nanoAg influenced more genes involved in inflammation and dissolved silver involved more oxidative stress and protein stability .

TH 158Gold ingegnerized nanoparticles (Au NPs): first investigation of toxicological effects in fibroblast cell cultures of cetaceansS . Maltese1, T . Renieri2, D . Coppola1, T . Campani3, M . Giannetti3, L . Mazzi2, G . Terzuoli2, I . Caliani3, L . Carletti3, G . Baldi4, M .C . Fossi1, L . Marsili3

1University of Siena, Siena, Italy2Department of Biomedical Sciences, University of Siena, Siena, Italy3Department of Environmental Sciences, University of Siena, Siena, Italy4Cericol Colorobbia Research Center, Sovigliana, ItalyNanoparticles (NPs ) are produced intentionally with the aim of developing new materials that exhibit certain specific properties . These properties are related to at least one of their dimensions, which must be less than 100 nanometers (nm) . Their use has been proposed in many fields of science and industry . Several studies exist on the toxicological properties of NPs . Although the various toxicological aspects and the diversity of the NPs assessed are just beginning, many deleterious effects have been documented, particularly in animals . However, no data are available for marine top predators of conservational interest, such as toothed whales (Odontoceti) . Gold (Au) NPs, also called gold colloids, are the most stable metal nanoparticles . Recently the photothermal conversion property of Au NPs has attracted a keen interest from the scientific community . In fact the Au NPs that are irradiated by a laser beam can efficiently convert the photon energy to thermal energy, which leads to the destruction of biological cells such as tumor and bacteria . Despite Au NPs are considered non-toxic by several authors, there is a serious lack of information concerning their impact on the life health and the environment . The aim of this study was to evaluate the toxicological effects of the ingegnerized Au NPs in fibroblast cell cultures of two free-ranging specimens of toothed cetaceans: striped dolphin (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus), sampled in Mediterranean Sea by non destructive method represented by subcutaneous biopsy . Genotoxicity of Au NPs in cetacean fibroblasts was detected by the alkaline Comet assay . Immunofluorescence technique was used for qualitative and quantitative evaluation of cytochrome P450 1A1 (CYP1A1), cytochrome P450 2B (CYP2B) and MICA expression . The first two proteins are evaluated as target of potential indicators of man-made pollutants, and the MICA as toxicological stress marker of the immune system . Finally, the Au NPs capacity to pass through the cellular barriers, was

26�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

performed by STEM . The main results of these experiments were: 1) the increase of DNA tail % compared to the control and in function of the time of exposure (4 and 24 h); 2) no significance increase of fluorescence in relation to control in the treated cells for all proteins: P450 1A1, P450 2B and MICA; 3) the presence of Au NPs into the cells with the STEM approach .

TH 159Uptake and distribution of multiwalled carbon nanotubes in zebrafish Danio rerioF . Stibany, H .M . Maes, S . Giefers, B . Daniels, B . Deutschmann, F . Hischen, W . Baumgartner, H .T . Ratte, A . SchäfferRWTH Aachen University, Aachen, GermanyDue to its unique properties, carbon nanotubes (CNT) are considered one of the most promising materials in nanotechnology . A wide spread usage is predicted . There is a lack of data regarding the ecotoxicology of this nanomaterial, which needs to be replenished soon . We investigated the bioavailability of radiolabelled multiwalled CNT (14C-CNT) to the zebrafish Danio rerio by quantifying uptake over time at different concentrations after water exposure in absence and presence of dissolved organic carbon (DOC) as well as after dietary exposure . Subsequently, elimination of the material by the fish was measured . To investigate the possible interaction of CNT with fish tissue cells, imaging with transmission electron microscopy (TEM) was performed . 14C-CNT were synthesized by means of chemical vapour deposition of 14C-benzene in a small-scale batch reactor . Ultrasonication was used to disperse 14C-CNT in the media . Zebrafish were exposed via water (0 .1, 0 .3 and 1 .0 mg 14C-CNT/L), via water with DOC (1 .0 mg 14C-CNT/L & 8 mg DOC/L) and via a diet of 72 h preliminary exposed blackworms (Lumbriculus variegatus) . Measurements were carried out after 3, 24 and 48 h . For exposure via water in absence of DOC, an additional sampling point was set to 168 h . Elimination was measured after an exposure time of 168 h, at 48, 72 and 168 h . At sampling time, the fish were dissected . The internal radioactivity of the different organs and tissues was measured via liquid scintillation counting (LSC) after tissue solubilising . After exposure via water in absence of DOC, 14C-CNT were found in gut, gills, skin and filet of the fish . Only little radioactivity was distributed to gonads and blood . In presence of DOC, generally little lower uptake was observed, and 14C-CNT were mainly found in gut and gills, while ony low amounts were detected in the leftover fish . After dietary exposure, 14C-CNT were only present in the gut . Over the course of the elimination experiment, the 14C-CNT were largely but not completely eliminated to the water phase . By means of TEM imaging, CNT-like structures were shown to be located in the fish gut epithelium . These results show that dispersed 14C-CNT are taken up by zebrafish via water both in absence and presence of DOC and after dietary exposure . The results of TEM imaging confirm that CNT are bioavailable for D . rerio, and raise concern about possible consequences of the observed interaction of CNT with gut epithelial cells of the fish .

TH 160Co-exposure to nano-TiO2 and ultraviolet: an ecotoxicological evaluationZ . Clemente1, L .O . Feitosa2, L .F . Fraceto3, R . Lima2, C .M . Jonsson1, V .L . Castro1

1Embrapa Environment, Jaguariúna, Brazil2University of Sorocaba / Department of Biotechnology, Sorocaba, Brazil3Unesp, Sorocaba, Brazil Studies about the potential ecotoxicological risks of titanium dioxide nanoparticles (nano-TiO2) have been published but the results are still inconclusive . The properties of the diverse nano-TiO2 must be considered in order to establish experimental models to study their toxicity . TiO2 has important photocatalytic properties and its photoactivation occurs in the ultraviolet range . The aim of this study was to investigate the effects of co-exposure to nano-TiO2 and ultraviolet . A South America fish (Piaractus mesopotamicus) was exposed to nano-TiO2 Sigma Aldrich (25 nm, 100% anatase) under different illumination conditions: visible light (V) or visible light and ultraviolet A (UVA) at 2 .7 J/cm2/h . Animals (n=8) were exposed during 96h to 0, 1, 10 or 100 mg/L nano-TiO2 . The solutions were renewed daily, and the characteristics of nanoparticles in solution were: 596 .1 ± 38 nm, pdi 0 .31 ± 0 .09; zeta potential -19 .2 ± 1 mV . Protein carbonylation, lipoperoxidation and the activities of catalase, superoxide dismutase, acid phosphatase and glutathione s-transferase (GST) were analyzed in liver . Metallothionein concentration was analysed in gills . Comet and micronucleus assays were performed with blood . The LC50 was >100mg/L for both conditions tested . GST showed a significant decrease in groups exposed to 1 and 10 mg/L nano-TiO2 under UVA compared to control under V . The results of comet assay showed the greatest damage in fish exposed to 100 mg/L nano-TiO2 under V and 10 mg/L under UVA . For the other biomarkers there was no statistics difference between groups and control groups . Our results corroborate with literature, showing low acute toxicity of nano-TiO2 in fish . However, co-exposure of nano-TiO2 and UVA can cause biochemical and genetic changes . The decrease in GST activity can be related with an inhibition or reduction of expression levels, but more investigation is needed . Further studies will be carried to investigate the relevance of considering the nano-TiO2 properties in ecotoxicological studies . Supported by: FAPESP, CNPq, CAPES, Fundunesp and Embrapa (AgroNano) .

TH 161Salinity- and pH-dependent silver nano-toxicity in post-hatch embryos of Japanese medakaT . Ariyoshi, Y .N . Nakagame, T . Niwa, S . Nagasaka, H . Takei, S . KashiwadaToyo University, Itakura oura, JapanOur group has been investigated nano-sized silver toxicology using Japanese medaka embryos and elucidated that silver nanocolloids cause severe inhibition of embryogenesis and morphological changes such as blood clots, percardio vascular edema, tubular hearts, small eyes, shortened body, and spinal deformities at 0 .5 mg/L and 1 .0 mg/L . Post-hatch embryo stage and following early-life stage of fish are well known to be more susceptible to xenobiotics than chorionated-embryos . To elucidate stage-dependent toxicity of silver nanocolloids (SNC, colloids size 43 .8-66 .8 nm in ultra-pure water) between embryo and post-hatch embryo stages, post-hatch embryo medaka was exposed to SNC in ERM (embryo rearing medium, pH7) for 7 days . Unlike embryos there was no observed biological effect at 0 .5 mg/L and 1 .0 mg/L of SNC, biological effects are significantly (P<0 .05) observed at 5 .0 mg/L and 10 mg/L of SNC in day 7 . In terms of biological effects, body length and whole length were 6 .6% and 4 .5% decreased, respectively; however, tail length was 13% increased at 10 mg/L in compare with control . Eye and gallbladder sizes were also 8 .7% and 35% bigger, respectively, than control . In addition, gallbladder’s green color turned to yellowish, and liver formation got invisible in both SNC exposures . These results suggested that post-hatch and early-life stage of medaka are less-susceptible to SNC exposure than embryonic stage . Physicochemical properties of nanomaterials were considered to be changed by pH and salinity, and then, changed properties (e .g . zeta-potential and aggregation) would influence on environmental fate of nanomaterials . Tested SNC changed zeta-potential smaller in pH9 (-39 .2 mV) from pH7 (-44 .5 mV) and colloidal size larger in ERM (ca . 450 nm) from ultra-pure water (ca . 50 nm) . We will demonstrate salinity- and/or pH-dependent toxicity of SNC using post-hatch embryos as well . TH 162Development of a test chamber to enable homogenous aqueous phase dispersions of nanoparticles for testing nanoparticle toxicity in fishH . Boran1, D . Boyle2, R .D . Handy2, I . Altinok1, T .B . Henry2

1Karadeniz Technical University, Trabzon, Turkey2University of Plymouth, Plymouth, United KingdomNanoparticles have unique properties and their behaviour in water often leads to aggregation and sedimentation of NPs out of the aqueous phase . Determination of NP toxicity in fish is important, but the heterogenous nature of static aqueous NP preparations leads to difficulties in obtaining repeatable results of NP toxicity and likely explains some of the differences in NP toxicity reported in the literature . The objective of this investigation was to develop a simple standardized procedure to enable testing toxicity of homogeneous aqueous dispersions of NPs in fish (e .g ., zebrafish Danio rerio) that is repeatable and easily transferable across laboratories . The test system (2-L glass beaker) is designed to maintain homogeneous aqueous dispersions of NPs by stirring, and delivers water containing aqueous NPs to fish held in a semi-isolated chamber . With this we have conducted acute toxicity tests with larval zebrafish to establish 96-h LC50 values for TiO2-NPs, Ag-NPs, and Cu-NPs; and compared to metal ion controls Ag+ and Cu2+ . The ability of the NP dispersion chamber to maintain homogeneous aqueous NP-dispersions was tested by analytical chemistry (inductively coupled plasma mass spectrometry, for metals), which determined that homogenous dispersions were maintained during the exposure (total loss of NPs from water column <30% over 96 h) . Without use of chamber (i .e ., static exposures) the concentration of NPs (based on total metals concentration) was reduced by >90% within 24 h . The LC50 values of Ag-NPs, Cu-NPs, and TiO2-NPs were 0 .27 mg/L, 0 .51 mg/L, and 897 mg/L, respectively . Of particular importance was the reproducibility of LC50 results across triplicate independent exposures which indicated a low coefficient of variation of 14 .81%, 10 .94%, and 12 .69%, for Ag-NPs, Cu-NPs, and TiO2-NPs . This approach has potential to become a standardized test procedure for testing the toxicity of NPs in the aqueous phase and addressing the problem of the lack of comparability of NP toxicity results across laboratories .

TH 163Silver nano-toxicity and biological effects are dependent on cation contents and pH in tested waterN .Y . Nakagame1, T . Takuto1, T . Ariyoshi2, S . Nagasaka1, H . Takei1, S . Kashiwada1

1Toyo university, Itakura oura, Japan2Toyo University, Itakura oura, JapanSilver nanomaterials are one of the major components of healthcare products . Their environmental fate and ecological risk are not well understood yet . Although it is known silver ion (Ag+) has toxic effects, it has been discussed whether nano-sized silver has its own toxicity . To elucidate this problem, silver nitrate (AgNO3) has been used for Ag+ source with low pH (pH5 .63 in 0 .01 mg-AgNO3/L, pH5 .53 in 0 .1 mg-AgNO3/L) . It is also well known that low pH aqua has some toxic effects to aquatic organisms, which not allow us to use low pH ambient for aquatic toxicology . In this study, a novel method has developed and tested to elucidate which nano-sized Ag and Ag+ have more toxic effects to medaka embryos . A bottom-fixed silver nanoparticles plate (nano silver plate; NSP), which is developed in this study is utilized to test toxicity of Ag+ released from silver . This test was demonstrated in ultra-pure water or ERM (embryo rearing medium for medaka) under different pH (4, 7 and 9) . As a counterpart compound, silver nanocolloid (SNC; colloidal diameter 43 .8-66 .8 nm in ultra-pure water) was used . In NSP with ultra-pure water, embryos exposed to Ag+ at 0 .57, 0 .08 and 0 .08 mg/L at pH4, 7, and 9, respectively, showed complete lethality in 12 hr; however, embryos in ultra-pure water without Ag+ exhibited 100% and 97 .8% hatching in pH7 and pH9 although embryos in pH4 were completely killed in 4 days . Those suggested Ag+ has completed lethal toxicity at 0 .08 mg/L under less-mineral ion water condition . In contrast, in NSP with ERM, embryos exposed to Ag+ at 0 .04, 0 .04 and 0 .04 mg/L at pH4, 7 and 9, respectively, exhibited high hatching ratios at more than 95% . However, these embryos were observed hatch delay and decrease of heart rate, less-developed eyeball and body length . On the other hand, in exposure to SNC at 0 .064 mg/L, embryos in ultra-pure water showed lower hatching ratio (0 - 62 .2%) and small eyeball, shortened body length, and altered heart beat and time to hatch; and then these biological effects were moderated in ERM as well as happened in case of NSP . We estimated that embryos in ERM were relieved from biological effects of silver toxicity, which may depend on cation contents (mainly Ca++, Mg++, K+, Na+ ) . NSP appeared significant biological effects to medaka embryos rather than SNC due to released Ag+ . This study suggests that biological effects are higher by Ag+ not by nano-sized silver .

264 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

TH 164Silver nanocolloids and target genes analyses in medaka embryosN .T . Niwa, Y .N . Nakagame, T . Ariyoshi, S . KashiwadaToyo University, Itakura oura, JapanSilver nanomaterials are considered to be one of the major pollutants from nano-industries . Our group has been investigated SNC toxicology using medaka embryos and elucidated that SNC causes severe inhibition of embryogenesis and morphological changes such as blood clots, percardiovascular edema, tubular hearts, small eyes, shortened body, and spinal deformities at 0 .5 mg/L and 1 .0 mg/L . This study explores the toxic mechanisms of silver nanocolloids (SNC; average particle size 3 .6 diameter nm, purity 99 .99%, pure water solution) using medaka embryo model . From DNA microarray analyses, 118 genes are up-regulated (>2 .0 fold) and 117 genes are down-regulated (<0 .5 fold) . However, severe morphological defects including vascular defects, percardiovascular edema, tubular heart, and underdevelopment of eyes and the central nervous system were all observed in SNC-exposed medaka embryos . Therefore, we chose six genes that were relevant to vertebrate embryogenesis and/or morphogenesis and well-studied in mammalian systems . Cathepsin L (ctsl), tropomysin 1 alpha (tpm1), retinol-binding protein (rbp) and metallothionein (mt) were significantly down-regulated in SNC exposed embryos, while sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1, atp2a1) and homeobox B6b (hoxb6b) were both up-regulated . In order to understand SNC toxicology, those gene expression patterning during normal embryogenesis (from stage 12; pre-early gastrula stage, to stage 39; hatching stage) were measured with qRT-PCR . Retinol-binding protein (rbp) relative to carrier protein that bind retinol and regulate epithelial cell growth, growth of bone tissue, immune function and tumor suppression; ATPase 1(atp2a1) relative to calcium pump on endoplasmic reticulum, heart dysfunction and abnormality; and tropomysin 1 alpha (tpm1) relative to calcium dependent regulation of vertebrate striated muscle contraction, were 170, 2600, and 65000 folds expressed, respectively, according to embryonic developmental stages . Homeobox B6b (hoxb6b) relative to regulation of patterns of anatomical development (morphogenesis) was reached at peak (4 .2 folds) in stage 35 (pectoral fin blood circulation stage), and then reduced to 1 .5 folds in stage 39 . Other two genes (ctsl and mt) were constantly expressed during embryogenesis (2-3 folds) . Biological effects of SNC rely on biological development including expression of target genes . This information should help us to understand the toxic mechanism of SNC .

TH 165Carbon nanospheres, multiwalled carbon nantubes and β-cyclodextrin polymer as emerging pollutantsD .W . Nyembe1, V . Wepener1, B .B . Mamba1, N . Musee2

1University of Johannesburg, Johannesburg, South Africa2CSIR: Natural Resources and the Environment, Pretoria, South AfricaThe use of engineered nanomaterials is fast growing and the environmental concerns about them are also growing . The nano size (<100 nm) of nanomaterials which has resulted in their vast applications has since raised concerns about their toxicity . Carbon-based nanomaterials are one such group and have since secured vast applications in industry . Some of these nanomaterials have been specifically engineered for water purification purposes and hence their fate in the aquatic system is not yet fully understood . There is therefore a need for in vivo studies on aquatic organisms that are known to be good pollution indicators . The pollution indicators chosen for this study were Poecilia reticulata (fish), Daphnia pulex (water-flea), Vibrio fischeri (bacteria) and Selenastrum capricornutum (algae) and the carbon-based nanomaterials tested were multi-walled carbon nanotubes (MWCNTs), carbon nanospheres and β-cyclodextrin polymer . These nanomaterials were vastly characterized to determine their physico-chemical properties in their dry and wet state as per the techniques applied . All the exposures experiments were done in the presence and absence of humic acid (20, 10, 5, 2 .5 and 1 .25 ppm humic acid concentration) at neutral pH . Particle size and zeta potential were monitored over 96 hr (the longest time for fish exposures) . LC50 (lethal concentration) and the EC50 (effective concentration) values were calculated using the Probit method . Fish mortalities were observed at 3 g/l of pristine MWCNT and 187 .2 g/l β-cyclodextrin polyurethane; D . pulex showed an LC50 of 1 .736 mg/L with oxidized MWCNTs while bacterial and algal growth was stimulated at low concentrations (2 .50-1 .25 ppm) of MWCNTs and carbon nanospheres . In the absence of humic acid the nanomaterial either settled or agglomerated . The nanomaterials were also well dispersed in the presence of humic acid and their hydrated particle sizes were reduced . For instance, in carbon nanospheres the presence of humic acid reduced the agglomerated size from 296 nm in “no humic acid”samples to 99 .9 nm at 8 ppm humic acid and down to 31 .7 nm after 96 hr . The presence of humic acid also enhanced the toxicities, especially that of carbon nanospheres to fish . At sublethal concentrations of these nanomaterials catalase, glutathione and superoxidase dismutase activity was observed at in fish and daphnia, thus indicating oxidative stress to the tested organisms . .

TH 166Sublethal toxicity of titanium dioxide nanoparticles in the shore crab Carcinus maenasC . Vanegas, D . Boyle, T .B . Henry, R .D . HandyUniversity of Plymouth, Plymouth, United KingdomEcotoxicological studies with TiO2 nanoparticles (NP) in aquatic systems have largely focussed on freshwater biota such as crustaceans (Daphnia magna, Ceriodaphnia dubia and Thamnocephalus platyurus) and fish (Danio rerio, Oncorhynchus mykiss) . Few studies have investigated effects of TiO2-NPs in marine or estuarine fauna despite the expectation based on physicochemistry that accelerated aggregation and deposition of the NPs on substrates will occur at the freshwater-saltwater interface . Moreover, where reported, effects in marine invertebrates including mussel Mytilus galloprovincialis and polychaete Arenicola marina have been inconsistent . In particular, bioavailability of TiO2 NP is unclear and has clouded interpretation of measured molecular, biochemical and physiological effects . To our knowledge, no studies have been conducted in the benthic shore crab Carcinus maenas, a sensitive and widely used bioindicator of coastal environmental contamination . Thus, the aim of this study is to evaluate biochemical and physiological effects of TiO2 NP in C. maenas and determine NP bioavailability and accumulation in key tissues (e .g . gill and hepatopancreas) . After acclimation of 15 days, adult male crabs were exposed in a semi-static test system to 0, 0 .1, 1 .0 and 10 mg TiO2 NP/L (24 .5 ± 10 .6 nm diameter) and 10 mg/L bulk TiO2 (134 .0 ± 42 .5 nm), a particle size control . For each treatment 15 crabs (5 per replicate) were evaluated . Daily, crabs were briefly removed from exposure tanks, fed with frozen cockle (Cerastoderma edule) in clean water to avoid dietary TiO2 exposure and 100% exposure media exchanged . After exposure, biochemical responses associated with physiological function will be measured including gill Na+K+-ATPase activity, osmotic pressure and major ions in haemolymph as indicators of osmoregulatory competence; oxidized/reduced glutathione ratio and ferric antioxidant status in haemolymph as biomarkers of oxidative stress; pH and lactic acid in haemolymph and oxygen consumption as end points of respiratory metabolism and, O/N ratio and total soluble proteins in haemolymph as end points of energetic status . Gill and hepatopancreas will also be analysed for tissue Ti accumulation . Results are under analysis; however obtained information will likely add to our understanding of potential TiO2 NP toxicity in an ecologically important benthic coastal species .

TH 167Effect of silver nanoparticles on ovarian follicular cells of marine medaka (Oryzias melastigma)N . Degger, R .S .S . Wu, A . TseState Key Laboratory in Marine Pollution, Hong kong, HongkongSilver nanoparticles (AgNPs) have gained much popularity on account of their antimicrobial properties . The increase in the production of these nanoparticles and their wide occurrence has become a growing environmental concern . While toxicity effects of AgNPs on aquatic organisms have been identified, there is a lack of data concerning their effects on marine species . It was hypothesized that exposure to nanoparticles may potentially interfere with steroidogenesis and hence reproduction . An In vitro study was carried out to investigate the effects of two commercial AgNPs on ovarian follicular cells of marine medaka (Oryzias melastigma) . After exposing the follicular primary cell culture to AgNPs for 48 hours, dose-dependent cytotoxicity was evaluated by an MTT assay . Modulations of the expression of the steroidogenic genes (cyp11a . cyp19a, 3bhsd and 20bhsd) were determined by measuring the mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR) . Additionally, the level of mRNA expressions of the apoptotic and anti-apoptotic proteins (bax and bcl-2) were measured . At least three independent experiments were conducted . Our results indicate that changes in gene expression pattern are elicited by AgNPs, and differences between the two commercial AgNPs were clearly evident . The data presented here provides insight that AgNPs may affect steriodogenesis and sex hormone production in ovarian follicular cells of marine medaka, which may subsequently compromise reproduction .

TH 168Toxicity of CdSe quantum dots to marine microalgae: effects on oxidative stress, phytochelatin production and growthE . Morelli1, M . Posarelli2, E . Salvadori1, E . Gabellieri1

1National Research Council (CNR), Pisa, Italy2ENEA, Pisa, ItalyThe rapid increase of nanotechnology has raised the concern about the potential adverse effects of nanoparticles on the ecosystems and living organisms . Quantum dots (QDs) are semiconductor nanocrystals, candidate to be widely used for biomedical applications and microelectronics, due to their unique optical properties . However, their environmental impact and the mechanism of toxicity still have not been fully elucidated . Phytoplankton represents the first link of the food chain in natural waters, thus the knowledge of the mechanisms of uptake, accumulation and toxicity of nanoparticles in these unicellular organisms represents a crucial point for understanding their ecotoxicity . In this work we evaluated the effects of CdSe/ZnS QDs on the marine diatom Phaeodactylum tricornutum, which served as a model of cell toxicity and an indicator of potential impact to ecosystem health . Laboratory cultures of P. tricornutum were grown for three days in culture media at increasing concentrations of CdSe/ZnS QDs, from 0 .04 to 1 .00 nM . The results show a gradual decrease of the growth rate at concentrations of QDs higher than 0 .5 nM . At 1 nM, it was 75% of the control . At the end of exposure, we tested the cellular Cd accumulation, the synthesis of phytochelatins (PCs) as a biomarker of the bioavailability of metal ions, the production of ROS, and the activity of antioxidant enzymes as an indication of oxidative stress . The results show that P. tricornutum accumulated Cd in a dose-dependent manner, but cellular Cd was not able to stimulate a comparable PC synthesis . Since the production of PCs is a specific signal of the presence of free metal ions, our findings suggest that QDs were taken up by P . tricornutum and were not degraded inside the cell . Measurements of the activity of the antioxidant enzymes showed that superoxide dismutase (SOD) and catalase (CAT) activities were enhanced by exposure to increasing QD concentrations, whereas ascorbate peroxidase and glutathione reductase activities were not significantly affected . SOD is considered the first line of defence against ROS, followed by other H2O2 scavenging enzymes, such as CAT or peroxidases . Thereby, an increase in SOD and CAT activity can be considered a symptom of oxidative stress induced by an enhanced production of ROS . This hypothesis was confirmed by the concomitant increase in the intracellular ROS concentration . Experiments are in progress to test the response of other species of microalgae .

TH 169

26�SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

Effects of silver nanoparticles in the freshwater snail Physa acutaS . Gonçalves1, M .D . Pavlaki1, S . Loureiro2

1Universidade de Aveiro & CESAM, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, Portugal The environment is constantly being exposed to various types of contaminants due to anthropogenic activities . Recent studies showed an increase in the amount of metals, such as copper, silver or cadmium that can be found in aquatic ecosystems due to industrial effluents and domestic wastewaters disposals . Silver nanoparticles are nowadays widely being used in all kind of products, such as domestic disinfectants, pharmaceutical products, cleaning products or even contact lenses, which will lead to an increase in concentration in the environment . Therefore, this study aimed to assess the survival, reproduction and egg viability of the freshwater pulmonate snail Physa acuta after exposure to silver nanoparticles and also its counterpart silver nitrate in laboratory conditions . A 96h exposure test will be used to evaluate acute toxicity and a 14/28 days chronic test will be carried out where egg viability and the number of new born snails will be evaluated . Results showed a decrease in reproduction and egg hatching in higher concentrations . Silver nanoparticles and ions significantly affected all 3 endpoints measured .

This study highlights that silver nanoparticles are bringing new inputs of silver as contaminant (as ions and nanoparticles) into the aquatic ecosystems .

TH 170Evaluation of chronic toxicity of copper oxide nanoparticles to microcrustacean marine Mysidopsis JuniaeC . Vaz1, T .M .N . Oliveira1, R .F .S . Böhm1, R .A . Gonçalves1, E .C . Spitzner1, T . Kleine1, S .P . Melegari2, C .H . Costa2, F . Perreault3, R . Popovic3, W .G . Matias2

1UNIVILLE, Joinville, Brazil2UFSC, Florianópolis, Brazil3University of Quebec in Montreal, Montreal, CanadaThe copper oxide nanoparticles are components of antifouling paints that are used for painting the hulls of boats . Their decomposition can provide both the soluble ionic form and the NPs in the marine environment, becoming an important source of contamination of aquatic ecosystems . Chronic toxicity studies in aquatic environments is a trend in environmental toxicology, since in many cases the contaminants from this group are highly diluted and do not offer sufficient concentrations to elicit acute effects but capable of causing only long-term effects . This study was aim at assessing the chronic toxicity of copper oxide nanoparticles on the marine microcrustacean, Mysidopsis juniae . Tests were conducted with concentrations of 100, 75 and 50 µg-L-1 and 40 test organisms with sexual dimorphism were added in each of the 2 liter tanks . The organisms were exposed for 30 days to the nanoparticles . The test was carried out in semi-static environment and the water exchange occurred once a week . The test conditions were salinity 32; photoperiod 12 hours of light and 12 hours of darkness and temperature 24±1 oC . The mysids were fed daily with Artemia sp. nauplii enriched with omega 3 and cod liver oil . The parameters evaluated were mortality, reproduction and total size . The laboratory maintains control charts for monitoring the sensitivity of test organisms . The data were analyzed with the softwares Statistica 7 and Minitab 16 . In order to check the differences between the evaluated parameters Student’s t test with a confidence interval of 95% and the Dunnet test for calculating the NOEC were used . The mysids exposed to nanoparticles suffered changes in number of mortality, fertility and size in concentrations above of 50 µg-L-1 .

TH 171Effects of in vivo exposure to nTiO2 on Mytilus immune parametersC . Barmo1, R . Fabbri1, C . Ciacci2, G . Gallo1, A . Marcomini3, G . Pojana3, L . Canesi1

1University of Genoa, Genoa, Italy2University of Urbino, Urbino, Italy3University Ca’ Foscari Venice, Venice, ItalyAcute toxicity of different engineered nanoparticles (ENP) underlined the potential risk of ENPs exposure to aquatic life . Apart from traditional ecotoxicity testing, more specific assays like immunotoxicity, genotoxicity, oxidative stress, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different ENP types also in aquatic organisms . We have previously demonstrated that in the marine bivalve Mytilus galloprovincialis, short term in vivo exposure to nTiO2, in the mg/L range, induced significant effects on lysosomal and oxidative stress biomarkers in the digestive gland . Moreover, Mytilus immune cells, the hemocytes, have been shown to represent a sensitive target for the in vitro effects of different types of NPs, including nTiO2 . Biomarkers of immunotoxicity may reveal changes in the immune function induced by ENP exposure, possibly leading to changes in organisms’ health .

In this work, the in vivo effects of nTiO2 on immune function were investigated in mussels exposed to different nominal concentrations of nTiO2 (1, 10 and 100 mg/L) for 4 days . Hemolymph immune parameters were evaluated: hemocyte lysosomal membrane stability (LMS), ROS and NO production, phagocytic activity and serum lysozyme activity . Expression of selected genes was evaluated in hemocytes by RT-Q-PCR . Physico-chemical characterization of nTiO2 primary particles was performed by a combination of techniques such as SEM, TEM, XRD, BET, DLS, TGA, ICP-MS, etc . . DLS analysis revealed the formation of different nm- and mm-sized agglomerates of nTiO2 in artificial sea water (ASW) . The results showed that exposure to nTiO2 induced significant changes in different functional and molecular immune parameters in mussel hemocytes . Moreover, decreases in lysosomal biomarkers (lysosomal membrane stability and lipofuscin accumulation) were observed in the digestive gland, indicating general stress conditions . These data indicate that exposure to nTiO2, at concentrations in the low mg/L range, can affect the mussel immune function, and support the hypothesis that transfer of ENPs from the digestive system to the hemolymph and circulating hemocytes may occur . Biomarkers of immunotoxicity may represent sensitive indicators of how NPs may cause alterations in the organism’s physiology, providing an indication of the sublethal impacts of ENP exposure, as well as an “early warning”of population level impacts .

TH 172Method development to test toxicity of fullerenes (nC60) on sediment-dwelling invertebrate Chironomus riparius larvaeG .C . Waissi-Leinonen1, E .J . Petersen2, K . Pakarinen1, J . Akkanen1, M .T . Leppänen1, J .V .K . Kukkonen1

1University of Eastern Finland, Joensuu, Finland2Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, United States of AmericaThe possible ecotoxicological risks of nanoparticles (NPs) have received increasing research attention in recent years as a result of the enormous economic potential of nanotechnology-related applications and the lack of information about such risks . The extent to which current standard ecotoxicological methods can be used to accurately measure the ecological risks of NPs is largely unknown due to the unique chemical properties of nanoparticles which substantially differ from those of hydrophobic organic chemicals and dissolved metals . The aim of this study was to investigate the extent to which fullerenes impact this benthic organism Chironomus riparius using an abbreviated version of Organization for Economic Co-operation and Development (OECD) standard method 218 to test short-term effects after exposure for 10 d . First, an environmentally realistic method to test fullerene (C60) toxicity to benthic organism C. riparius was created by allowing suspended fullerenes to settle down creating a layer on top of the sediment (fullerene masses of 0 .36 to 0 .55 mg/cm2 ) . This created an environmentally realistic exposure method which simulates a sensitive exposure route for C. riparius givens its feeding habits . Second, two different food concentrations (0 .5 % and 0 .8 % Urtica sp .) in the sediment were tested to investigate the impact of this parameter on toxic responses . Thus, we hypothesized that higher food concentrations will decrease toxic effects . In the 0 .5 % food level treatments, there were significant differences in all growth-related endpoints compared to controls . Fewer effects were observed for the higher food treatment . Fullerene agglomerates were observed by electron microscopy in the gut but no absorption into the gut epithelial cells was detected . In the organisms exposed to fullerenes, microvilli were damaged and significantly shorter . The potential toxic effects of fullerene to C. riparius appear to be caused by morphological changes, thus inhibiting the larvae growth .

TH 173Multibiomarker assessment of cerium dioxide nanoparticle (nCeO2) sublethal effects on the freshwater invertebrates Dreissena polymorpha and Gammarus roeseliM . Garaud1, J . Trapp1, C . Cossu-Leguille2, V . Felten1, S . Pain-Devin1, P . Rousselle1, P . Wagner1, L . Giambérini1

1Laboratory LIEBE - CNRS UMR 7146 - Université de Lorraine (UdL), Metz, France2LIEBE - CNRS - UMR 7146 - Université de Lorraine (UdL), Metz, FranceCerium dioxide nanoparticles (nCeO2) are among the most widely used, principally as fuel additive for their catalytic properties . As a result, the release of nCeO2 and the subsequent environmental exposure are predicted to be consequent, while the risks incurred are still unknown . Most of the aquatic ecotoxicological studies published from now have focused on the acute toxicity of nCeO2, often at environmentally irrelevant concentrations, and have shown its relative innocuousness . However, with environmental concentrations in surface waters predicted to be in the low µg/L range, it is unlikely that we could extrapolate real impacts of nCeO2 on aquatic ecosystems from the results of those studies, especially given that the physico-chemical properties of nanoparticles at these concentrations are expected to be quite different . Moreover, to our knowledge, no study has ever used a multi-biomarker approach to evaluate nCeO2 impacts on aquatic ecosystems . In this work, we filled those knowledge gaps by investigating sublethal effects of nCeO2 on two aquatic invertebrates, the freshwater mussel Dreissena polymorpha and the freshwater amphipod Gammarus roeseli, at concentrations close to predicted environmental concentrations (10 and 100 µg/L) for 96 hours . A broad set of biochemical and histological biomarkers were used to evaluate nCeO2 exposure effects on 1) antioxidant and antitoxic defences (catalase, GPx, GPx-Se and GST activities, metallothionein concentration), 2) cellular damages (lipoperoxydation), 3- iono/osmoregulation (Na+/K+-ATPase activity and Na+, Cl- and Ca2+ haemolymph concentrations), 4) lysosomal system and 5) behavioural responses (locomotion and ventilation for Gammarus only) . The first results showed an absence of significant adverse effects of nCeO2 on all tested biomarkers . On the contrary, nCeO2 seems to exhibit antioxidant properties, decreasing lipid peroxydation and catalase activity, and this protective activity will need to be more thoroughly investigated .

TH 174Effects of TiO2 nanoparticles on Mytilus galloprovincialis 48 h-old larvaeG . Libralato1, D . Minetto1, C . Losso1, L . Manodori2, E . Sabbioni2, A . Volpi Ghirardini1

1University Ca’ Foscari Venice, Venice, Italy2Veneto Nanotech S .c .p .a . - ECSIN Lab, Rovigo, ItalyEngineered nanomaterials are at the forefront of ecotoxicologist agendas due to their widespread use in a broad range of industrial and domestic sectors . Actually, they are manufactured in increasing amounts year-by-year . Particularly, nanoscopic titanium dioxide (nTiO2) is used by a variety of industries mainly for catalysis and photocatalysis

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applications and as an additive in paints, papers, inks, plastics and various consumer products with a rapid increase in its production, estimated in the United States to reach 106 tons per year by 2025 . So, we expect an increased environmental exposure to nTiO2 with concentrations in different environmental compartments that may induce toxic effects to the aquatic ecosystem . Most of the literature on the ecotoxicity of nTiO2 deals with aquatic organisms, mainly from freshwaters, such as bacteria, algae, invertebrates and fish . Little information is available about saltwater species such as bivalves that, amongst environmental quality status sentinels, are considered a potential target group and bio-indicators for nanoparticles toxicity . They are widespread in all fresh, brackish and salt water environments, having a key ecological role both as filter and deposit feeders according to various species and habitats . All stages of their life cycles (gametes, embryos, larvae and adults) have been used to define an endpoint for a specific monitoring purpose (acute, sub-chronic or chronic toxicity) . Bivalve molluscs toxicity tests are generally easy to perform, highly sensitive, cost-effective and not time consuming ranging from 24 h to 48 h to obtain a first meaningful result, or even lower when some biomarker are taken into consideration . The aim of the present work is focused on the assessment of the potential toxicity of nTiO2 towards early larval stages of Mytilus galloprovincialis (LMK) - 48 h-old larvae, that are commonly known as D-shape ones . Dark and dark/light (8/16 h) scenarios were investigated to comply with standard protocol and mimic a more likely environmental exposure . Results evidenced unusual toxicity trends presenting their acmes around 6 mg/L of nTiO2 and potential defensive mechanisms in the exposed larvae .

TH 175Cellular and subcellular localization of Au nanoparticles and Au bioaccumulation in mussels exposed to Au nanoparticles of two different sizesA . Jimeno-Romero1, A . Warley2, D . Gilliland3, M .P . Cajaraville1, I . Marigómez1, M . Soto1

1UPV/EHU, Leioa, Spain2Centre for Ultrastructural Imaging, King’s College London, London, United Kingdom3European Commission Joint Research Centre, Ispra, Italy The subcellular localization of metallic nanoparticles in invertebrates has been scarcely studied and the results are inconclusive . Here, mussels, Mytilus galloprovincialis, were exposed, for 3 d, to Na- citrate stabilised Au nanoparticles (NPs) of two different sizes (5 and 40 nm), as well as to bulk and ionic (HAuCl4) Au forms . The nominal exposure concentrations were 0, 0 .75, 75 and 750 μg Au/l in the four experimental sets . No mortality was recorded in mussels exposed to the different Au forms . Au in mussel soft tissues was measured by ICP-MS . Tissue Au accumulation was dose-dependent for all treatments . Lowest Au concentrations were found in mussels exposed to bulk Au . Autometallography was applied to localize metal ions (as black silver deposits, BSD) in paraffin-embedded tissue sections of the digestive gland . Black silver deposits were mainly found in the digestive cells . The volume density of BSD (VvBSD) was quantified by image analysis and there was agreement between VvBSD values and the tissue concentration of Au . Transmission electron microscopy (TEM) was used to study the distribution of silver enhanced (Aurion R-Gen kit) Au NPs in ultrathin sections mounted on Ni grids . In mussels exposed to Au NPs of both sizes, electron dense particles were found in the digestive gland epithelium, especially within late endosomes, lysosomes and residual bodies of digestive cells . The presence of Au in these electron dense particles was confirmed after electron probe X-ray microanalysis in mussels exposed to 40 nm NPs but not in those exposed to 5 nm NPs, due to the low signal recorded (5 nm NPs were sparse) . It can be concluded that Au NPs are bioavailable for mussels, and are accumulated in the lysosomes of digestive cells . The study of the effects produced by these NPs is currently under research using a battery of selected biomarkers at molecular, cell and tissue-levels . Acknowledgements: TEM and X-ray microanalysis were carried out at the Centre for Ultrastructural Imaging, King’s College London . Work funded by the European Community’s 7th FP (FP7/2007-2013) under grant agreement n° CP-FP 214478-2 and by the Basque Government (GIC07/26-IT-393-07) . A .J-R is recipient of a pre-doctoral fellowship from the University of the Basque Country .

TH 176Metal-particle size affects metal bioaccumulation from sediment and depuration in a deposit-feeding snailC . Pang1, H . Selck1, G .T . Banta2, D . Berhanu3, S .K . Misra3, E . Valsami-Jones4, V .E . Forbes5

1Roskilde University, Roskilde, Denmark2Department of Environmental, Social and Spatial Change, Roskilde University, Roskilde, Denmark3Mineralogy, Natural History Museum, Cromwell Road, London SW7 5BD, UK, London, United Kingdom4School of Geography, Earth and Environmental Sciences, University of Brimingham, Brimingham, United Kingdom5University of Nebraska-Lincoln, Lincoln, United States of AmericaWith the increasing use of CuO nanoparticles (NPs), nano CuO may pose risks to aquatic organisms . The persistence of nano CuO in aquatic organisms has not yet been determined . In the present study, we compare the bioaccumulation and depuration of copper in the freshwater snail, Potamopyrgus antipodarum, exposed to sediment spiked with aqueous Cu, 6 nm-CuO, or 100 nm-CuO nanoparticles . Snails were exposed for 2 weeks to the control (0 µg Cu/g dry weight sediment) and three forms of Cu-spiked sediment (240 µg Cu/g dry weight sediment) and allowed to depurate for two weeks in clean natural sediment . Our results showed that the smaller size particles (6 nm-CuO) were associated with higher mortality of P . antipodarum during the exposure period, and the net uptake rates for sediment spiked with 6 nm-CuO and aqueous Cu were significantly higher (around 40~50%) than for sediment spiked with 100 nm-CuO . During two weeks of depuration, there were no significant differences in depuration rates (kd) among forms (aqueous Cu: kd = -0 .12 wk-1; 6 nm-CuO: kd = -0 .22 wk-1; 100 nm-CuO: kd = -0 .20 wk-1) . Our results suggest that particle size has an important influence on the toxicity and bioaccumulation of Cu in P . antipodarum . TH 177The ecotoxicology of engineered metal oxide (ZnO) nanoparticles with respect to the aquatic sediment dweller, Lumbriculus variegatusS .A . O’Rourke, V . Stone, T .F . FernandesHeriot Watt University, Edinburgh, United Kingdom Since the 1990’s there has been a rapid increase in the use of engineered nanomaterials and one of the side effects of this “nano-boom”is the potential for these NP’s to be released into the environment . The novel properties of nanoparticles (NPs) make them extremely useful in industry however these properties may also render them uniquely hazardous in nature . Zinc oxide NPs are used in a variety of applications including sunscreens, antibacterial lotions, lipsticks, UV stabilisers in plastics, as a catalyst for chemical reactions and as a food additive . Oligochaetes are important constituents of freshwater ecosystems as they feed on subsurface sediments which have an important role in the processing and recycling of deposited material . In areas where there is a high density of worms, reworking and recycling of the sediment materials can substantially restructure the sediment itself . Due to their feeding behaviour, Lumbriculus variegatus make excellent test organisms for studying the bioaccumulation and general effects of contaminants . To date very few published studies have used this species to investigate the environmental effects of engineered NPs . This study investigated the impact of zinc oxide nanoparticles on the mortality, reproduction and behaviour of Lumbriculus variegatus following O .E .C .D . protocol conditions . The NPs have been characterised using ESEM, XRD, BET and DLS techniques . Tests were conducted at 0, 1 .25, 2 .5, 5 and 10mg/L NP concentrations in U .S .E .P .A . media both in the presence and absence of 5mg/L Suwannee River humic acid (H .A .) . There was a concentration effect, a particle size effect and an interaction effect between concentration, humic acid addition and particle size . All concentrations were significantly different from the controls and the 1 .25mg/L concentration was significantly different in all cases from the 10mg/L concentration . The addition of humic acid did not have a significant effect on the behavioural response when compared to the tests done in the absence of humic acid . There was a significant difference between the bulk and nanoparticle effect .

TH 178Uptake, subcellular distribution and genotoxicity of nano-CuO, micron-CuO and aqueous Cu in the benthic polychaete, Nereis diversicolorA . Thit, Y . Cong, G .T . Banta, H . SelckRoskilde University, Roskilde, DenmarkEngineered metal nanoparticles (NPs), such as Copper oxide (CuO) NPs are increasingly being used in consumer products, and human and environmental exposure is likely to increase . CuO NPs may cause adverse effects as a result of their small size (< 100 nm) and high surface to volume ratio . Engineered metal NPs have been shown to cause organelle damage, DNA-damage, oxidative stress, apoptosis, up/down regulation of proteins etc . CuO NPs released into the aquatic environment will likely agglomerate and/or aggregate upon contact in the brackish water of the estuary and subsequently accumulate in the sediment compartment . Therefore the benthic environment is a likely exposure scenario for CuO NPs . Before CuO NPs can cause potential toxic effects to the organism the particles must be accessible to the organism and able to reach the site of action (e .g ., proteins and DNA) either directly or indirectly via the release of ions or production of reactive oxygen species . In this study we examined dietary uptake, intracellular distribution and genotoxicity of Cu added to sediment in different forms and particle sizes for the deposit feeding polychaete Nereis diversicolor . Cu was added to sediment as aqueous Cu (administered as CuCl2 -2H2O) or as CuO particles of different sizes; NPs (polydispersed-100 nm) and micro particles (5 µm) .

TH 179Silver nanoparticle dissolution and Ag speciation as key parameters for toxicity of AgNP to algaeL . Sigg1, F . Piccapietra1, U . Lindauer1, N . Odzak1, R . Sofield2, R . Behra1

1Eawag, Duebendorf, Switzerland2Western Washington University, Bellingham wa 98225, United States of AmericaBecause of their multiple applications in consumer products silver nanoparticles may reach the aquatic environment and affect aquatic organisms . Toxic effects of silver nanoparticles to aquatic organisms may be either due to the effects of dissolved silver ions or to specific effects of nanoparticles . Various processes need to be considered to evaluate the fate of silver nanoparticles (AgNP) in natural waters and their effects on aquatic organisms, namely agglomeration of nanoparticles, dissolution and speciation of dissolved Ag(I) . Dissolution of AgNP is of fundamental importance, as dissolved Ag+ ions appear to be the species directly causing toxicity to algae . However, upon dissolution in the presence of ligands, Ag(I) may be complexed by the ligands . In the presence of cysteine as a binding ligand, increased dissolution of AgNP over a few hours is observed to be followed by a decrease of dissolved Ag(I), probably due to slow reduction processes . Dissolved Ag(I) in the presence of an excess of cysteine is mostly present in Ag(I)-cysteine complexes, and free Ag+ is very low . In contrast, in the presence of chloride concentrations lower than the solubility limit of AgCl(s), a slow steady dissolution process of AgNP takes place . Dissolved Ag(I) is in this case partly present as Ag(I)-chloride complexes and partly as free Ag+ . Complexation of Ag(I) by fulvic and humic acids appears to be only weak . Toxic effects of AgNP to photosynthesis of algae are suppressed by complexation of Ag+ by cysteine, indicating that toxicity is mostly caused by dissolved Ag+ ions . Effects of chloride need to be carefully taken into account in growth media for algae and other organisms, due to the possible complexation and precipitation reactions . Fulvic and humic acids only slightly decrease the toxic effects of Ag+, in agreement with weaker binding by these ligands .

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TH 180Investigations on the effects of silver nanoparticles in the zebrafish embryo toxicity test, with consideration of sewage treatment processesE . Muth-Koehne1, L . Sonnack1, K . Schlich1, K . Hund-Rinke1, C . Schäfers1, R . Debus2, M . Fenske3

1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany2Hochschule RheinMain, Rüsselsheim, Germany3Fraunhofer IME, Aachen, GermanyDue to their antibacterial and desirable material properties, silver nanoparticles (AgNPs) are used in many consumables and everyday products . Critically, evidence is strengthening that AgNP possess a toxic potential, mainly due to the property to continuously release silver ions into the environment . The toxicity of the AgNPs themselves depends greatly on the environmental conditions and the susceptibility of the AgNPs to decomposition . During sewage treatment plant (STP) processes, most of the AgNPs are supposedly retained in the sewage sludge while the ionic silver may be released to the effluent . Both, sewage sludge and effluent can enter the environment, and AgNPs and their derivatives were shown to be present in surface water . The risks of AgNP release for men and environment, however, are still inadequately examined . This project aims to examine the teratogenic and organotoxic potential of AgNPs and ionic silver (in terms of AgNO3) to the model organism for aquatic vertebrates, the zebrafish embryo (Danio rerio) . Thus, fish embryos were exposed to AgNPs and AgNO3 for 48 h, examined for their lethal and sublethal effects, and subsequently whole-mount immunostained for the determination of motor neuron defects and muscle malformations . Additionally, to monitor the effects of STPs on the toxic potential of AgNPs, samples of sewage sludge treated with AgNPs and the effluents of simulated STP, were examined in the zebrafish embryo toxicity test (zFET) . AgNP- and AgNO3-treated embryos displayed teratogenic effects such as a delayed development, tail malformations and edema . Most of the embryos were smaller than the control embryos and possessed a strikingly small yolk sac . A specific organotoxic potential was not detectable . Fish embryo tests for the determination of the toxic potential of AgNPs in sewage sludge led to effects rather ascribed to oxygen deprivation . In contrast, zFETs performed with the effluent of the simulated STP resulted in the characteristic AgNP-mediated effects in higher influent concentrations . Dilutions of the samples indicated an accumulation of ionic silver in the effluents, as an increased AgNP toxicity through STP processes was observed . This study therefore provides first insights into the toxic potential and behaviour of AgNPs in sewage plant processes .

TH 181Partial bioavailability of a herbicide adsorbed to carbon nanotubes (CNT) for green algae: comparison of different CNT types with soot, kinetics, and mechanismsF . Schwab1, L . Camenzuli1, K . Knauer2, A . Magrez3, B . Nowack4, L . Sigg5, T .D . Bucheli1

1Agroscope Reckenholz Tänikon, Research Station ART, Zurich, Switzerland2Federal Office for Agriculture FOAG, 3003 bern, Switzerland3EPFL - Ecole Politechnique Fédérale de Lausanne, 1015 lausanne, Switzerland4Empa - Materials Science and Technology, 9014 st . gallen, Switzerland5Eawag - Swiss Federal Institute of Aquatic Science and Technology, 8600 dübendorf, SwitzerlandCarbon nanotubes (CNT) are increasingly manufactured and used [1] . Hence, exposure of our environment with CNT is an eventuality . The effects of CNT on green algae are currently not fully understood . CNT alone seem to be relatively non-toxic for green algae [2] . However, they are strong adsorbents for many organic compounds and may likely reduce their bioavailability . In environment, CNT will always occur together with a great number of organic micropollutants . The aim of our study was therefore to quantify the bioavailability of an herbicide in an aqueous ternary algae-nanoparticle-pollutant laboratory system . Pristine and oxidized, purified and industrial grade multi-walled CNT (MWCNT) and diesel soot reference material were used . Classical desorption and adsorption kinetics experiments were performed . Ternary batch experiments adapted from [3] were accomplished with mixtures of CNT and diuron in a concentration range covering 2-3 orders of magnitude . The mixtures were pre-equilibrated, then inoculated with an algal stock culture of Chlorella vulgaris, and incubated for 24 h . The photosynthetic activity (PS II activity) of the algae and the aqueous diuron concentration were quantified before addition of algae and during the incubation time . The findings suggest that the overall toxicity of diuron is reduced in presence of CNT due to strong sorption of the herbicide to CNT (Freundlich coefficients ranged from 5 .60 to 6 .22) . However, locally elevated diuron concentration in proximity of algal cells, in combination with fast desorption kinetics of diuron from CNT, lead to partial bioavailability of the herbicide adsorbed to CNT for green algae . [1] Hendren CO, Mesnard X, Dröge J, Wiesner MR . 2011 . Estimating production data for five engineered nanomaterials as a basis for exposure assessment . Environ Sci Technol 45:2562-2569 . [2] Schwab F, Bucheli TD, Lukhele LP, Magrez A, Nowack B, Sigg L, Knauer K . 2011 . Are carbon nanotube effects on green algae caused by shading and agglomeration? Environ Sci Technol 45:6136-6144 . [3] Knauer K, Sobek A, Bucheli TD . 2007 . Reduced toxicity of diuron to the freshwater green alga Pseudokichneriella subcaptitata in the presence of black carbon . Aquatic Tox 83:143-148 . Acknowledgement - The authors thank the Swiss National Science Foundation for the founding of the study .

TH 182Ecotoxicity of soils contaminated with zinc oxide nanoparticles to aquatic organisms M .D . Fenández1, C .O .N .C .E García-Gómez1, A .N .A . Obrador2, J .M . Álvarez3, P .I .L .A .R García1, M . Babin1

1INIA, Madrid, Spain2UPM, Madrid, Spain3ETSI Agrónomos (UPM), Madrid, SpainThe ecotoxicity and behaviour of nanoparticles in the environment is yet almost entirely unknown . Manufacturing processes, handling, use and disposal increase their appearance in air, soil, water and living matter . Nanoparticles can reach the soil through the application of sewage sludge . This agricultural practice has potential for contaminating not only soils but also waters (both surface and ground), since this compartment could be the ultimate container of contamination . The objective of this work is to evaluate whether zinc oxide nanoparticles (ZnONPs), leached from contaminated soil, exert ecotoxicological effects on aquatic organisms . To achieve this purpose, organisms pertaining to different trophic levels representative of the aquatic ecosystem, i .e . the unicellular green alga Chlorella vulgaris, the crustacean Daphnia magna and fish (in vitro test) have been exposed to extracts obtained from a soil (physical-chemical properties known) spiked with larger particles (zinc oxide), Cl2Zn and ZnONPs, and the possible noxious effects have been compared . Toxicity to Chlorella vulgaris is determined using OECD 201 algal growth inhibition test . Toxicity to Daphnia magna is determined using OECD 202 immobilization test . Toxicity to fish is evaluated for in vitro tests, using the rainbow trout gonadal cell line RTG-2 as bioindicator of cell viability, with eight indicator dyes: ethoxyresorufin O-deethylase (EROD) for cytochrome, 4-M-umbeliferona (ß-gal) for stress cellular, Alamar Blue for energy metabolism, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for membrane function, lactate dehydrogenase-catalysed conversion of pyruvate to lactate for measuring the inhibition of cell growth (total LDH) and the membrane integrity (LDH released into the medium), 2’,7’-dichloro fluorescein diacetate (H2DCF-DA) for oxidative damage, neutral red (NR) uptake for lysosomal activity and total protein assays . Generated data concerning ZnONPs will be compared with data on larger particles and Cl2Zn . This study was funded by the Spanish project RTA2010-00018-00-00 .

TH 183Are carboxyl CdSe/ZnS quantum dots taken up by bacterial cells? V .I . Slaveykova1, G . Garcia2, M . Floriani3, L . Fevrier4, R . Gilbin3

1University of Geneva, Versoix, Switzerland2Swiss Federal Institute of Technology, Lausanne, Switzerland3IRSN/DEI/SECRE/LRE CEA Cadarache, Saint-paul-lez-durance, France4I, Saint-paul-lez-durance, FranceThe ability of the engineered nanoparticles (ENPs) to interact with biological barriers is considered as one of the prerequisite for observation of the hazardous effects in the environment . In the present study we examine the interaction of the carboxyl - CdSe/ZnS quantum dots (QDs), as a model of composite water dispersible ENPs and metal resistant bacterium Cupriavidus metallidurans, largely used in metal decontaminantion . More specifically we explored the capabilities of the QDs to cross and/or damage bacterial cell membrane . QDs hydrodynamic diameter, zeta potential and dissolution in the bacterial exposure experiments were also investigated . Multiparametric flow cytometry measurements revealed increasing the fraction of bacterial cells expressing QD-florescence in QDs concentration- and exposure time - dependent pattern demonstrated the association of QDs with C . metallidurans . Transmission electron microscopy (TEM) images allowed observing electron dense agglomerates and aggregates with a size between 20 and 50 nm on the bacterial surface, periplasmic space and between individual bacterial cells . Energy dispersive spectroscopy (EDS), confirmed that the agglomerate at the surface and in the periplasmic space observed by TEM contains Zn, Cd and Se, major QD components . The presence of the QDs in the periplasmic space of this bacteria was associated with damaging of the cellular barrier as found by the 2 .5-fold increase of the cells with damaged and leaky membranes observed by FCM following the QDs-exposure to the bacteria . Overall, this work demonstrates that low levels of QDs originating from incidental release or QDs disposal can be expected to affect bacterial membrane integrity . However given the relatively low percentage of affected cells, they are not expected to significantly influence their growth and thus their use in metal decontamination .

TH 184Assessing genotoxic activity of nanosized gold, silver, zinc oxide and titanium dioxide using the SOS chromotestY .J . An, S .H . NamKonkuk University, Seoul, South-Korea (Rep)Gold nanoparticles, silver nanoparticles, zinc oxide nanoparticles, and titanium dioxide nanoparticles are included in various cosmetic products that many consumers use on a daily basis . In this study, we assessed the genotoxic activity of gold, silver, zinc oxide and titanium dioxide nanoparticles using the SOS chromotest . The SOS chromotest based on E . coli PQ37 has been used to screen for a variety of chemicals because of its practicality, however, available SOS chromotest data for nanoparticles is currently very limited . This study demonstrated that gold, silver, zinc oxide and titanium dioxide nanoparticles in a tested concentration range are classified as non-genotoxic in the SOS chromotest, evidenced by IFmax values of below 1 .5 . To the best of our knowledge, these are the first results to study the genotoxic activity of gold, silver, zinc oxide and titanium dioxide nanoparticles with the SOS chromotest . This work was supported by the National Research Foundation Grant funded by the Korean Government (NRF 2011- 0015985).

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TH 185Polyhydroxyl fullerene can mitigate toxicity effects of cadmium on yeastsA . Pradhan, S . Seena, C . Pascoal, F . CássioCBMA, University of Minho, Braga, PortugalWith inception of nanotechnology, nanomaterials are currently in the prime-line of research . Polyhydroxyl fullerene is one of the widely commercialized nanomaterials and applied in electronics and biomedical fields . Unlike fullerene, polyhydroxyl fullerene is known to be biocompatible, and recent studies reported its antioxidant properties . On the other hand, cadmium toxicity has been often attributed to the ability of this metal to induce oxidative stress . To assess antioxidant properties of polyhydroxyl fullerene, the model yeast Saccharomyces cerevisiae was exposed to cadmium (≤ 5 ppm, 3 levels) in the presence or absence of polyhydroxyl fullerene (≤ 500 ppm, 3 levels) at different pH values (5 .8, 6 .3 and 6 .8) . The size distribution and dispersion of polyhydroxyl fullerene in the stock suspension were measured with DLS (z-average 143 nm, PdI 0 .365) . Yeast growth, plasma membrane integrity and accumulation of reactive oxygen species (ROS) were investigated in the presence or absence of Cd2+ and/or polyhydroxyl fullerene . Yeast growth was inhibited up to 34% by exposure to Cd2+, but was not affected by polyhydroxyl fullerene . At pH 5 .8, the exposure to the highest concentration of Cd2+ (5 ppm) and polyhydroxyl fullerene (500 ppm) stimulated yeast growth by 59 and 61% at the exponential and late exponential growth phases, respectively . Growth increased even more when pH increased to 6 .8 . Severe plasma membrane disruption and ROS accumulation were observed after exposure to the highest concentration of Cd2+ in the absence of polyhydroxyl fullerene . Membrane disruption and ROS accumulation decreased with increasing concentration of polyhydroxyl fullerene and with the increase in pH to 6 .8 . Overall, results indicated that polyhydroxyl fullerene is not toxic to yeasts and may potentially trigger antioxidant defense mechanisms to mitigate metal-induced toxicity .

TH 186Genotoxicity of cotton cellulose nanofibersR . Lima1, L .O . Feitosa2, M . Barga1, P .A .T .R .I Yamawaki1, C .I .N .T .I Maruyama1, T . Silva1, E . Teixeira3, A . Corrêa3, L .U .I .Z .H Mattoso3, L .E .O .N .A Fraceto3

1Uniso, Sorocaba, Brazil2University of Sorocaba / Department of Biotechnology, Sorocaba, Brazil3Embrapa, São carlos, BrazilCellulose whiskers have potential for applications in the future . These include optical document security and improvement of the mechanical resistance of thin films containing polyelectrolytes in lithium batteries, as well as reinforcement in the polymeric matrices of environmentally-friendly wrapping materials and possible use in medical applications This work evaluates the genotoxicity of cotton cellulose nanofibers intended for application in the field of new nanocomposites . The emergence of proposals for the application of such materials necessitates detailed study of their toxicity, considering their safety in terms of the environment and living beings, as well as possible modifications of production techniques that could reduce any toxicity, where present . Furthermore, the use of these materials in the health sector could improve biocompatibility, which is an obligatory requirement of regulatory agencies . Nanotoxicological studies aim to ensure the safe application of these materials, hence increasing their acceptance and minimizing any future problems that might be associated with their large-scale usage . This work describes the genotoxicity evaluation of different cotton cellulosic nanofibers (white cotton, brown cotton, ruby cotton and green cotton) . In this study we investigate the genotoxicitiy by the use of Allium cepa chromosome aberration test, comet assay, animal cell cytogenetic analysis, clonogenic assay and molecular assay to measure DNA breakdown by the nanomaterials . The results showed that the effects depend on the cotton nanofiber type, being that brown cotton produces more genotoxic effects than others cotton nanofibers . In this way, the results showed that more studies are need in order to better understand their possible effects, including their environmental impacts when used in agribusiness applications . Supported by: FAPESP, CNPq, CAPES, Fundunesp, Finep and Embrapa (AgroNano) .

TH 187Modelling health effects from inhalation of nano-objectsA . Laurent1, M .Z . Hauschild1, S .I . Olsen1, O . Jolliet2

1Technical University of Denmark (DTU), Kgs . lyngby, Denmark2University of Michigan, Ann arbor, United States of AmericaThere have been many attempts to apply life cycle assessment (LCA) and risk assessment (RA) to engineered nanomaterials, i .e . products embedded with nano-objects (NO) . However, the lack of data and modelling capacity for quantifying the fate, exposure and effects of NO currently prevents LCA and RA of nanomaterials to include the assessment of the environmental and health risks as well as the toxic impacts stemming from the releases of nano-objects to environment . To help abridge some of these gaps, we draw on existing knowledge in the field of (nano)toxicology to provide an approach to model the health effects from inhalation of NO, with specific focus on TiO2 and nanosilver . We apply a physiologically-based pharmacokinetic (PBPK) model to obtain the concentrations of nanosilver and TiO2 at the different target organs in the human body after inhalation . By using data from in vitro and in vivo studies, we establish dose-response relationships, using the formation of reactive oxygen species (ROS) as a response metrics, and relate the concentrations of NO at the target organs to respective cellular responses . This enables to localize where effects occur in the human body and identify zones, where hazard may arise, based on the hierarchical oxidative stress model . Available data does not enable to translate the cellular responses to a required whole-body response, e .g . incidence of disease . We thus provide recommendations to fill in this gap and we identify important data and modelling components necessary to achieve a full damage modelling; these include the need for chronic tests and methods to extrapolate acute in vitro assay results to chronic in vivo effects for which we discuss opportunities for future incorporation into the developed modelling framework . Overall, the proposed approach thus aims to serve as a first step towards a full assessment of the effects of NO on human health . The relevancy of the work applies to both risk assessment and LCA fields although the combination of the approach with a consistent (environmental) exposure model is still required to support proper risk and impact assessment from nanomaterials .

TH 188Moving towards quantitative ecological risk assessment of engineered nanomaterialsA . Moccia, D .R . Hristozov, A . Critto, A . MarcominiUniversity Ca’ Foscari, Venice, Venice, ItalyDespite the substantial potential of engineered nanomaterials (ENMs) to contribute to sustainable innovation, there is an extensive gap of knowledge regarding both their environmental health and safety profile and the suitable tools and frameworks for assessing their risks . The scientific community has been long discussing the appropriateness of standard risk assessment approaches to apply to ENMs and it has been broadly agreed that, despite the conventional framework is suitable, some implementations are needed to adapt it to their novelties [1] . In response to this need, in the last few years several frameworks and tools aimed to support and speed up the risk assessment of ENMs have been developed, but, with few exceptions, they all lead to qualitative results . In this context, the latest frameworks and tools for Ecological Risk Assessment (ERA) of ENMs have been collated and evaluated with respect to their strengths and weaknesses according to predefined criteria . In result, the development of specific approaches for quantitative effects assessment appears necessary to complement the available toolset and allow low-tier quantitative ERA . In this context, a nanospecific Weight-of-Evidence approach for the quantitative estimation of Predicted No Effects Concentrations (PNECs) has been proposed . This new approach implements important data quality control standards and at the same time can be easily integrated in standard RA procedure . Such tool, however, would require an advanced system for screening and evaluation of the available ecotoxicity database in order to guarantee high quality the input data . Considering that still substantial epistemic and technical uncertainties influence the design and performance of ecotoxicity tests with ENMs, a key aspect in the estimation of reliable and realistic PNECs is the deep understanding of the existing studies . Here we propose an overview of all the available ecotoxicity data found in open-source databases and in peer-reviewed literature, followed by a comprehensive analysis of those aspects related to the data generation that could play a key role in determining the overall quality of the studied endpoints . References [1] OECD . 2010 . Report of the workshop on risk assessment of manufactured nanomaterials in a regulatory context . Paris, France . Acknowledgement - The authors thank the FP7-funded MARINA project

TH 189Mass balance of nanoscale titanium dioxide in laboratory wastewater treatment plants according to OECD 303AS . Gartiser1, C . Nickel2, M . Stintz3, S . Damme4, L . Erdinger5, T .A .J . Kuhlbusch2

1Hydrotox GmbH, Freiburg i .br ., Germany2Institute of Energy and Environmental Technology e .V ., Duisburg, Germany3Technische Universität Dresden, Dresden, Germany4RWTH Aachen University, Aachen, Germany5Heidelberg University Hospital, Heidelberg, GermanyTitanium dioxides are used in huge amounts as white pigment in consumer products and industrial applications . The use of nanoscale TiO2 is predicted to increase in the near future . While the clearance efficiency of microscale TiO2 (> 0 .7 µm) in sewage treatment plants (STP) has been determined as being in the order of 70-85% (Kiser et al . 2009), no data on the behaviour of nanoscale TiO2 in STP are available so far . In the study presented the fate and behaviour of TiO2 nanomaterials (P25 with an average particle size < 250 nm) in the laboratory sewage treatment plants simulation test according to OECD Guideline 303A has been investigated . The nanoscale suspensions were stabilised with sodium metaphosphate (SMP) using an ultrasonic homogeniser and added under continuously stirring in a ten-fold concentrated solution via a flexible-tube pump to reach a final concentration of 1, 5, and 10 mg L-1 TiO2 and 1 g L-1 SMP . The organic synthetic wastewater (final concentration 50 mg L-1 DOC) was added as a concentrate to the matrix of synthetic drinking water . After one week of adaptation the test was run for one week at each concentration . The TiO2 in the outflow and in the surplus sludge was measured via ICP/OES after HCl, HNO, HF digestion . The functionality of the treatment process in terms of DOC-elimination and nitrification was not affected . The overall TiO2 balance indicated that 4% (trial 1) and 3% (trial 2) of the total TiO2 added is discharged with the outflow of the STP and that more than 95% of the TiO2 is adsorbed to the activated sludge . Glass fiber filtering of samples indicated that the main part of TiO2 in the outflow has to be attributed to particles below 0 .6 µm . The TiO2 balance showed a gap of 18% (trial 1) and 37% (trial 2) probably due to the limited analytical recovery rate of TiO2 from the activated surplus sludge . The OECD Guideline 303A allows enough modifications for assessing the behaviour of nanoscale particles in STP . The calculation of an overall balance of the nanomaterial tested is recommended as a quality control . The study was sponsored by the German Federal Environment Agency (UBA) (FKZ 3709 65 417) Reference: Kiser, M . A . et al . 2009 . Nanomaterial Removal and Release from Wastewater Treatment Plants . Environ . Sci . Technol ., 43 (17), pp 6757-6763

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TH 190Bioaccumulation and effects of sediment-associated silver in different forms for the deposit-feeders, Capitella teleta and Macoma balthicaL .I .N .A . Dai1, G .T . Banta1, K .R .I .S .T Syberg1, H . Selck1, D . Gilliland2, V .E . Forbes3

1Roskilde University, Roskilde, Denmark2European Commission, Joint Research Centre, Ispra, Italy3University of Nebraska-Lincoln, Lincoln, United States of America Due to wide spread applications of nanoparticles, including Ag, in our daily life, these novel particles are receiving increasing attention by risk assessors . Many studies have been performed to test the toxicity of nanoparticles at the subcellular level . Despite these findings, there is still limited information at the whole organism level as to whether metallo-nanoparticles differ in toxicity from the same metals added to the environment in ionic or bulk form . In the present study, two organisms (i .e ., a marine polychaete, Capitella teleta and a marine bivalve, Macoma balthica) were exposed to sediment amended with Ag in various forms (ionic Ag(I), nano-sized Ag and micron-sized Ag), and toxic endpoints were assessed at the whole organism level . After exposure for several weeks, no significant toxic effects were detected (i .e ., mortality, growth rate and condition index) at the whole organism level, although all forms of metallic Ag were accumulated in our test organisms . Our results thus did not confirm findings from studies performed at the subcellular level that have found the toxicity of Ag nanoparticles was more toxic than the corresponding metallic Ag in ionic and in bulk forms . However, more studies should be performed at the whole organism level to confirm whether the toxicity of nanoparticles can be extrapolated from that of the corresponding metallic ions which have been well studied .

TH 191Silver nanoparticle toxicity in Caenorhabditis elegansJ .N . MeyerDuke University, Durham, United States of AmericaWe tested a number of nanoparticles (NPs) for toxicity in the nematode model Caenorhabditis elegans, and found that certain silver NPs were the most toxic of the NPs that we had tested . We therefore extended our studies to characterize the uptake and toxicity of AgNPs in C . elegans, as well as the role of dissolution, aggregation, and environmental variables (presence of chloride and natural organic matter in the medium, and sulfidation of the NPs) . Using six different AgNPs of different size and coating, we found a linear correlation between AgNP toxicity and oxidative dissolution, but no correlation between size and toxicity . Pharmacological rescue (using N-acetyl cysteine or trolox) and mutant analysis (using mutants sensitive to oxidative stress, sod-2 and mev-1 or deficient in metal sequestration, mtl-2 and pcs-1) experiments supported the importance of dissolution in determining the toxicity of AgNPs in C . elegans . Some AgNPs (typically less soluble due to size or coating) also cause toxicity via generation of reactive oxygen species, an effect specific to nanoparticulate silver, but this effect was masked by the toxicity of silver ions except when dissolution was very limited . Interestingly, the toxicity of the tested AgNPs was almost never greater than would result from complete dissolution of the same mass of silver . Finally, we found that higher ionic strength, the presence of natural organic matter, and sulfidation of the AgNPs (expected to occur in many environments after AgNP release) all significantly reduced Ag NP toxicity .

TH 192Impact of zero-valent iron (ZVI) nanoparticles on soil microbial structure and functionality: a molecular approachC .F . Fajardo1, M .L . Saccà2, L . Ortíz1, M .L . Rodríguez-Membibre1, M . Nande1, M .C . Lobo3, M . Martín1

1Universidad Complutense de Madrid, Madrid, Spain2Campus de Excelencia Moncloa, Madrid, Spain3Instituto Madrileño de Investigación y Desarrollo Rural Agrario y Alimentario, Madrid, SpainAnthropogenic contamination of soils by heavy metals, such as Pb, and Zn, occurs from many sources such as mining, atmospheric deposition, application of sludge, and mineral fertilizers and pesticides . Over the last decade, the use of binding agents containing nanoscale materials has led to a new generation of environmental remediation technologies that could provide cost-effective solutions to some of the most challenging environmental cleanup problems . Research has shown that nanoscale iron particles are very effective for the transformation and detoxification of a wide variety of common environmental contaminants, both organic and inorganic . The introduction of nanoparticles (NP) in natural systems (e .g . soil, groundwater, sediments) leads to their interaction with unicellular organisms (e .g . bacteria or protozoa), plants, and animals . Prior to apply a new remediation method, studies must be done to arrive at predictive information of the impact of the process on biota, at different levels of complexity . Before manifestation of acute toxic effects on cell or organism level, the initial changes appear on molecular level . Thus, the elucidation of toxicant-specific molecular responses provides information on toxicity pathways already at subtoxic exposure levels .

In this work, nanoscale zero-valent iron (NZVI) particles have been used as an immobilisation strategy to reduce Pb and Zn availability and mobility in polluted soils . The application of NZVI to two soil microcosms at a dose of 34 mg g-1 soil efficiently immobilised Pb (25%) and zinc (20%) . Exposure to NZVI had little impact on the microbial cellular viability and biological activity in the soils . Three bacterial genes (narG, nirS and gyrA) were used as treatment-related biomarkers . These biomarkers ruled out a broad bactericidal effect on the bulk soil microbial community . A transcriptome analysis of the genes did not reveal any changes in their expression ratios after the NZVI treatment: 1 .6 (narG), 0 .8 (nirS) and 0 .7 (gyrA) in the Pb microcosm and 0 .6 (narG), 1 .2 (nirS) and 0 .5 (gyrA) in the Zn microcosm . However, significant changes in the structure and composition of the soil bacteria population were detected by fluorescence in situ hybridisation . Thus, our results showed that NZVI toxicity could be highly dose and species dependent, and the effective applicability of the proposed molecular approach in assessing the impact of this immobilization strategy on soil microbial population .

TH 193Molecular ecotoxicology of nanosilver guided using in vitro prognosisY . Hayashi1, L .H . Heckmann1, P . Engelmann2, H . Autrup1, D .S . Sutherland1, J .J . Scott-Fordsmand1

1Aarhus University, Aarhus c, Denmark2University of Pécs, Pécs, HungaryTo study the molecular and cellular basis of silver nanoparticle (AgNP) toxicity, we here used a recently established in vitro model of earthworm coelomocytes in comparison to the conventional in vivo molecular ecotoxicology approach . Compared to the latter where the test organisms are exposed to NPs of interest held in an environmental matrix, in vitro models benefit from the ease of controlling exposure conditions in a defined set of biochemical milieus that NPs may encounter . The AgNPs tested in the present study originated from the same source, but to enhance the colloidal stability in the in vitro test media the NPs were pre-treated with serum proteins . In addition to physical characterisation of AgNPs, the active silver ion fraction was measured (in serum-supplemented cell culture medium and in soil pore-water) . Using flow cytometry and atomic absorption spectrophotometry, we show that a specific population of coelomocytes can accumulate AgNPs in vitro and that silver uptake from the soil environment was evident at the organismal level (measured as total accumulated silver) . For the comparison of mode-of-action of AgNPs, we have selected several biomarker genes within stress and immune pathways and assessed their transcriptional interplay at a non-lethal concentration of AgNPs over time in vitro (1-6 hours) and in vivo (1-14 days) . In vitro dataset displayed a transition from oxidative stress responses to immune signalling during the 6 hours exposure period . Similarly, a characteristic clustering pattern of oxidative stress response genes was observed in early time points of the in vivo dataset, with stronger association to immune genes at a later time point . Hence, our finding depicts a general response pattern initiating with regulation of stress genes and a subsequent shift to immune genes . This study suggests that AgNPs are likely to be recognised by the earthworm immune system gradually altering the host immune status as the exposure persists .

TH 194Zero-valent iron nanoparticles for soil remediationM . Gil-Diaz1, A . Pérez Sanz1, J . Alonso1, M . Martin2, M .C . Lobo1

1IMIDRA, Alcalá de henares (madrid), Spain2Universidad Complutense, Madrid ., SpainSoil is a non-renewable resource and needs to be managed sustainably . It is both an important reservoir of chemical elements and a living matrix, and is essential for water and ecosystem health . According the European Environment Agency, in Europe the soil contamination affects almost 250,000 sites, and is expected to continue growing . Human activities such as mining, manufacturing, or land application of urban residues, have resulted in the contamination of our ecosystem, in particular, the soils . Among other pollutants, metals and metalloids can cause significant damage to the environment and human health as a result of their mobility and solubility . So, remediation strategies must be focus on reducing the metal bioavailability in the soil . The use of zero-valent iron nanoparticles (nZVI) to remediate contaminated soil and groundwater (nanoremediation) has gained increasing amounts of attention within the last decade due to its potential for broader application, higher reactivity, and cost-effectiveness compared to conventional in situ methods . The aims of this study were, firstly, evaluate the efficiency of the remediation with nZVI to immobilize lead and zinc in two soils (acid and alkaline), and, secondly, determine the influence of this nanoremediation technology on physico-chemical and biological soil properties . Soils were artificially contaminated with lead or zinc (200 mg/kg) and treated with commercial nZVI suspension (NANOFER 25S, NANO IRON, Rajhrad, Czech Republic) . The bioavailability of these metals in soils was determined after a sequential extraction procedure, analyzing soluble, exchangeable, linked to carbonates and residual fractions . In the studied conditions, the amendment with nZVI was more effective for lead than for zinc, regardless of soil pH . The immobilization process in the alkaline soil was favoured by the carbonate content . An increase of immobilized Pb of 81 .6% and 29 .2% for acid and alkaline soil, was observed, respectively; and in the case of zinc, the immobilization increased by 51 .0% for the acid soil and 8 .3% for the alkaline one . Soil properties were not adversely affected after treatment with nZVI . These results suggest that the use of nZVI to remediate contaminated soil with lead or zinc can be proposed as a promising alternative strategy to others amendments . Acknowledgements . Projects CTM 2010-20617-C02-02 and S2009/AMB-1478 .

TH 195Phytotoxicity of nano-TiO2 and effect of humus preparationM .M . Gladkova, V .A . Terekhova

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Lomonosov Moscow State Univercity, Moscow, Russian FederationThe objective of this study was to determine the bioactivity of nano-TiO2 and to assess their toxicity in presence of humic substances (HS) which are natural components of soil necessary for higher plants growth . In this work we applied metal-containing nanomaterials - nanodioxide titanium (nano-TiO2), which are characterized <75 nm in size of particles in 10 weights % of water suspension, manufactured in the company «Sigma-Aldrich», USA . Nano-TiO2 represents oxide the titanium (IV), the mix of two known crystal modifications of dioxide titanium - anatase and rutile . Among HS we have chosen “Pow-Humus”(Le-PhK) (K-humate, originated from leonardite), manufactured by German firm ‘Humintech’ . Concentration of nano-TiO2 was varied in range 5-500 mg/l; concentration of humate Le-PhK was 5 mg/l in water medium . Bioactivity of preparations was examined by early seed root elongation assessment related to Brassica juncea L . as a test-organism . We have used a modification of the method «Phytotoxkit - Seed germination and early root growth»,developed by Belgian scientists [http://www .microbiotests .be] . The probe is considered toxic if the deviation from the control of average roots length is 20 and more % . Depressing effect of nano-TiO2 on early root growth (4 days of germination) was marked . The inhibition of root growth was showed already at the low concentration (5 mg/l) .The most effect was shown at the higher concentration: probe with nano-TiO2 500 mg/l was toxic because the deviation of average roots length was more than 25 % from control . Inhibitory effect of nano-TiO2 was reduced in presence of humate Le-PhK 5 mg/l . Samples with nano-TiO2 500 mg/l after humate application was qualified as nontoxic because the deviation of average roots length was only 14 % from control . The obtained data has shown that: 1) higher plants are sensitive to the nano-TiO2 presence in growing medium 2); the degree toxicity of nano-TiO2 depends on the concentration of nanoparticles; 3) humus substances eliminate the toxic effect of nano-TiO2 in water suspension which inhibits root growth . Further research is required for the evaluation of humus substances influence on the nanomaterials which are more and more widely distributed in the environments including the natural soils [1; 2] .

TH 196Gene expression profile of Enchytraeus albidus (Oligochaeta) in response to silver nanoparticles and silver nitrateS .I .L . Gomes1, A .M .V .M . Soares2, J .J . Scott-Fordsmand3, M .J .B . Amorim4

1Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, Portugal3Aarhus University, Silkeborg, Denmark4University of Aveiro, Dept Biology & CESAM, Aveiro, PortugalDue to its antimicrobial activity, silver nanoparticles (Ag-NPs) are among the most used NPS worldwide, yet little information is available regarding their effects, particularly in soil dwelling organisms . Enchytraeids (Oligochaeta) are important members of the soil fauna which actively contribute to the acceleration of organic matter decomposition and nutrient recycling processes . Hence, for hazard and risk assessment it is important to provide toxicity data for these organisms, and if possible to understand more in regard to the mode of action of AgNPs within organism . To study this we conducted toxicity experiments using the OECD standard test, testing AgNPs and AgNO3, and relating this to Ag+ activity of the soil water solution . Population toxicity responses were assessed showing higher toxicity for the Ag-salt . In an attempt to understand the mode of action we performed transcription profiling using the microarray . Organisms exposed to similar total concentrations (AgNPs and Ag-salt) showed different gene-expression patterns . The observed differences in toxicity between the two exposure scenarios could not be explained by dissolution of the AgNPs, as measured Ag+ activity (Ion selective electrode) in the soil water solution .

TH 197Role of P38MAPK in ROS induced DNA damage & apoptosis due to silver nanoparticle exposure in Caenorhabditis elegans and human cell line: comparative toxicity approachJ . Choi, H .J . Eom, N . Chatterjee, D .Y . Lim, J .M . AhnUniversity of Seoul, Seoul, South-Korea (Rep)Toxicity of nanomaterial studies are among the fastest growing in environmental toxicology research, because of the widespread nanotechnology . In vitro systems are widely used to derive toxic potentiality of nanomaterials . As it is complex to extrapolate the derived toxicity data of NPs from in vitro systems to humans, use of alternative in vivo model system is unavoidable to get an actual picture for human toxicity . In this study, using the nematode, Caenorhabditis elegans as in vivo model system, we conducted comparative toxicity of silver nanoparticle (AgNPs) . In our previous study, p38 MAPK mediated oxidative stress was observed as a toxicity mechanism in C.elegans and human lymphoma cell, Jurkat . In the present study, to find out the link between oxidative stress, DNA damage & apoptosis in C.elegans and human lymphoma cell, Jurkat, we hypothesised that the stress responsive protein, p38MAPK (PMK-1, a homologue in C.elegans) is conserved and unavoidable for toxic responses due to AgNPs exposure . Our approach is to investigate the role of p38MAPK pmk-1 in oxidative stress, DNA damage & apoptosis by using p38 siRNA knock down in mammalian in vitro system and/or using pmk-1 mutant C.elegans strain followed by AgNPs exposure and determine the distinguishable toxic end points . This study highlights the importance of comparative toxicity approach in extrapolation of in vitro nanotoxicity data for human and ecological health evaluation . Acknowledgments : This work was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2011-0003214)

TH 198Toxicity of ZnO nanoparticles on soil receptors compared with bulk ZnO and dissolved free ionic zincC .G . Garcia-Gomez1, M . Babín1, A .O . Obrador2, J .M . Alvarez2, C .R . del Rio1, J .L .P . Pareja1, M .D . Fernandez1

1INIA, Madrid, Spain2ETSI Agrónomos (UPM), Madrid, SpainDue to increasing proliferation of nanotechnology in industrial applications and consumer products, there has been growing concern over effects of nanoparticles (NPs) on the environment . This study aims to study the effects of ZnO nanoparticles on soil receptors . The adverse effects of NPs cannot be predicted from the bulk material as result of their small size and unique structure which give to nanoparticles very specific properties: chemical, biological and physical which affect their toxicity and environmental behavior . This work compares the ecotoxicological effects of three different forms of Zn2+ (nanoZnO, ZnO bulk, and ZnCl2 salt) using different lethal and sublethal endpoints . Biological effects of nanoZnO, ZnO bulk, and ZnCl2 salt at 1000 mg Zn2+ kg-1 soil (d .w .) concentration were assessed on three taxonomic groups: terrestrial invertebrates, plants and microorganisms . The measured effects were: lethal, body weight alterations and reproduction endpoints in Eisenia fetida, germination and growth in three terrestrial plant species and two enzymatic activities (phosphatase and deshydrogenase) and two soil functions (nitrification and respiration) in soil microorganisms . Bioaccumulation is other parameter depending on Zn forms present in the soil . In this work, accumulation of zinc in earthworms exposed to contaminated soils during 28 days was determined . In plants accumulation was studied in roots and shoots to determine effects of translocation . Differences in Zn uptake among plant species and fate of the different Zn forms studied will be reported in both earthworms and terrestrial plants . In addition chemical soil analyses were performed before and after toxicity assays, using different extractants, to determine the distribution of the Zn forms in the different soil fractions . This study was funded by the Spanish project RTA2010-00018-00-00 .

TH 199Mixture toxicity of nano-TiO2 and ingredients of personal care productsA .J . Schneider1, A . Meister-Werner2, R . Petto2, H .M . Maes1, H . Hollert1, A . Schaeffer1

1RWTH Aachen University, Aachen, Germany2Institute for Biological Analysis and Consulting (IBACON), Rossdorf, GermanyIn the last years many studies have focused on the ecotoxicology of nano-TiO2 . But only a few studies have investigated the mixture toxicity of nano-TiO2 and organic contaminants . Mixture toxicity has to be considered during risk assessment of nano-TiO2, because both substance groups often have the same entry pathway into the environment . Nano-TiO2 and biocides are both ingredients of personal care products . Consequently, they have the potential to be released into waste water treatment plants and subsequently through biosolid amendment into soils . Triclocarban (TCC, 3, 4, 4’-trichlorocarbanilide, CAS 101-20-2) is an antibacterial agent used in personal care and household products . It was identified as an emerging contaminant in biosolids and subsequently in biosolid treated soils . In this study the mixture toxicity of nano-TiO2 and TCC to terrestrial organisms is examined . The acute and chronic toxicity of nano-TiO2 (10-1000 mg/kg dw) and TCC (42-675 mg/kg dw) as single substances as well as a mixture of both (TCC 42-675 mg/kg dw together with either 400 or 1000 mg/kg dw of nano-TiO2) to Eisenia fetida is tested in accordance to OECD 207 (1984) and OECD 222 (2004) . TCC is analyzed in soil samples and worm tissues using liquid chromatography tandem mass spectrometry (LC-MS/MS) . No mortality was observed for E. fetida exposed to TCC . However in the acute toxicity test TCC decreased the biomass of E. fetida in a dose dependent manner up to 20% after 14 days . In our study TCC showed a lower acute toxicity to E. fetida as found in a study from Snyder et al . (2011, LC50 40 mg/kg) . Nano-TiO2 did not show any acute toxicity to E. fetida up to 1000 mg/kg dw . Further tests will show if the simultaneous exposure of E. fetida to both substances will lead to mixture toxicity on basis of acute or chronic toxicity . Results will be presented in the poster presentation . The investigation of mixture toxicity of nanomaterials and organic contaminants is important because only little is known about potential interactions and the related consequences for e .g . bioavailability or formation of toxic metabolites .

TH 200The influence of soil pH on the toxicity of ZnO nanoparticles to the isopod Porcellionides pruinosusS . Tourinho1, C .A .M . Van Gestel2, C . Svendsen3, A .M .V .M . Soares4, S . Loureiro4

1University of Aveiro, Aveiro, Portugal2Vrije Universiteit, Amsterdam, Nederland

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3Centre for Ecology and Hydrology, Oxfordshire, United Kingdom4University of Aveiro & CESAM, Aveiro, Portugal The stability of nanoparticles (NPs) in soils is known to be affected by soil properties, like pH, which can change NP surface charge and zeta potential . Consequently, soil pH influences the interactions between NPs and soil matrix, defining NP behaviour and bioavailability . In the present study, the effects of zinc oxide (ZnO), as NPs and non-nano sized particles, and ZnCl2 were studied in the isopod Porcellionides pruinosus upon exposure in a forest soil with different pH values . The forest soil presented a pH of 4 .5 and was amended with calcium carbonate (1 w/w %), in order to achieve an extra soil pH of 7 .3 . Soil was then spiked with ZnO NPs (30nm), non-nano ZnO (200nm) or ZnCl2, resulting in nominal concentrations of 250, 500, 1000, 2000 and 4000 mg Zn/kg dry soil . Adult isopods were exposed individually, in 10 replicates per treatment and a control for each pH set . Animals were fed with alder leaves ad libitum . After 14 days, mortality, feeding inhibition and biomass gained were calculated . The pHCaCl2 of the soil was measured at the beginning and end of experiment . The results showed that Zn concentration was affecting soil pH, but in different ways depending on the Zn used . At pH 4 .5, increasing Zn concentrations led to a pH increase in the soil spiked with ZnO 30 and 200nm, and a decrease in soil spiked with ZnCl2 . At pH 7 .3, for all treatments soil pH decreased with increasing Zn concentration . The 14 days-LC50 values found at pH 4 .5 were 1945 and 3718 mg/kg for ZnO 200nm and ZnCl2, respectively, and it was not possible to calculate the LC50 for ZnO 30nm . At pH 7 .3, the LC50 values were 1687, 1029 and 1285 mg/kg for ZnO 30nm, ZnO 200nm and ZnCl2, respectively . For the feeding activity, no effects were observed at pH 4 .5 for any of the Zn particles or salt . However, at pH 7 .3, a decrease in consumption was observed at concentrations of 2000, 1000 and 500 mg/kg for ZnO 30nm, ZnO 200nm and ZnCl2, respectively . Biomass gained was affected upon exposure to all Zn forms, with EC50 values of: 757mg/kg for ZnO 30nm, 584 mg/kg for ZnO 200nm and 315 mg/kg for ZnCl2, at pH 4 .5 . On the other hand biomass gain did not differ from the control for any treatment at pH 7 .3 . The toxicity of NPs seemed to be influenced by soil pH, and in a different way from the correspondent ionic Zn exposure based on ZnCl2 salt .

TH 201Effects of different TiO2 nanoparticles on growth of Rhizobium trifolii in liquid cultureJ . Moll, A . Gogos, M .G .A . van der Heijden, T .D . Bucheli, F . WidmerAgroscope Reckenholz-Tänikon Research Station ART, Zurich, SwitzerlandNanoparticles (NP) gain importance due to possible widespread use and great potential also in agricultural applications . Little is known on how NPs interact with plants and beneficial soil microorganisms . Several studies have shown, that some NP can enter plant roots and are spread within the plant through the vascular system . NP show also effects on bacteria - like inhibited growth, disrupted cell membrane and shift in bacterial community - and can be taken up by them . Thus, before applying NP in agriculture, e .g . as pesticides or fertilizers, it is important to test whether they may negatively influence crops and beneficial microorganisms . In a first step the influence of different NP on the nitrogen-fixing bacterium Rhizobium trifolii is tested under highly controlled conditions using a liquid medium as model system . We focus on the effects of NP on nitrogen-fixing bacteria because these bacteria play a key role in sustainable agriculture, providing up to 200 kg N per hectare per year . Here we compared the effect of four different TiO2 NP, which differ in the crystal structure or in the coating, on the growth of R. trifolii . Two of these particles consist of rutile and have an AlO3 dimethicone (silicon based organic polymer) or glycerol coating respectively, one is made of 100% anatase and the forth one contains 80% anatase and 20% rutile without coating . Initial results with two TiO2 NP - one with and one without coating, with concentrations between 200 mg/l and 1 mg/l - indicate that the different particles show similar effects on the growth of R. trifolii . Applied at concentrations between 10 mg/l and 1 mg/l, similar to those possibly used in practice, the two TiO2 NP did not show any effects on R. trifolii growth as compared to controls without NP . The objective of further experiments will be to assess possible effects of NP on two crops (clover and wheat) and on two widespread groups of beneficial soil microorganisms, rhizobia and arbuscular mycorrhizal fungi .

TH 202Impact of sewage sludge spiked with silver nanoparticles to soil microorganismsJ .M . Hänsch, C . EmmerlingUniversity of Trier, Trier, GermanyThe harmful effects of silver nanoparticles to microorganisms are commonly accepted . The application of contaminated sewage sludge to soil is the main potential pathway for the input of silver nanoparticles in soil . Several studies have shown that silver nanoparticles, which pass through a wastewater treatment plant, have been mostly modified into argentite (Ag2S) and thus, will have a very small solubility and therefore a minor toxicity compared to silver nanoparticles . But is this sufficient to claim that there will be less danger from such contaminated sludge? In our study we contaminated activated sludge with silver nanoparticles (AgNP) and silver nitrate (AgNO3), both with 1mg Ag/L for 8 days under aerobe conditions by air supply in batch reactors . Subsequently, this sludge was applied to soil and afterwards mixed with grass seed and stored in pots in a greenhouse for 2, 4 and 6 months . Additionally, we conducted two control treatments, a sludge-control and a total-control treatment, respectively . During the experimental period a series of soil parameters was analysed, such as microbial biomass-C, soil respiration, enzyme activities, phospholipid fatty acids, C-to-N ratio, and pH . Our results show that - independent from any possible detoxification due to the conversion of silver nanoparticles in the sewage treatment plant to argentite - an unknown fraction of high toxicity particles remained . As a consequence, AgNP- and AgNO3-treatments differed significantly from both control treatments over a number of months and inhibited the microbial community up to 30% .

TH 203Do nanoparticles influence cell physiology and/or function?A . Simon1, H .M . Maes2, H . Hollert2, A . Schäffer2

1RWTH Aachen University, Aachen, Germany2RWTH Aachen University,Institute for Environmental Research, Aachen, Germany Nanosciences are expanding in many fields of technology worldwide, although research on toxicological effects of these nanoparticles (NP) has just begun . To date, existing OECD guidelines are evaluated for their applicability to test the toxicity of NP . A preliminary review indicates that the tests are suitable for NP, but that sample preparation and characterization have to be described in detail . In several studies previously performed according to those guidelines, we found neither acute nor chronic reproductive or growth effects of multi-walled carbon nanotubes (CNT) on whole organisms . However, CNT were found to be incorporated in e .g . fish gut epithelial cells and algae . In the case of CNT, it is questioned if there are analogous mechanisms to those of other fibrous particles such as asbestos, which penetrate into the lung and may persist in the tissue . The environmental expert panel of the German government stated (2011) that this question especially refers to carbon nanotubes that are long and rigid . The material used in this study is however known to be short and tangle . So far uptake of CNT in cells has been observed but the relevance of this finding is still unclear . Therefore, the possible penetration in cells has to be investigated . The aim of the present study is to investigate in vitro responses of different cells after exposure to CNT . Firstly, general cytotoxicity of CNT is investigated using three different endpoints: neutral red retention, enzyme quantification (lactate dehydrogenase) and microscopic analysis on cell density and sublethal effects . Secondly, potential endocrine effects were studied using the yeast estrogen screen assisted by enzymatic digestion with lyticase (LYES) . Thirdly, the single cell gel electrophoresis assay (comet assay) is performed as a straightforward and sensitive technique for the detection of DNA damage . Finally, oxidative stress possibly caused by CNT was determined since many NP were shown to induce biological effects intracellularly . In addition to the tests with CNT material only, combination effects of CNT with triclocarban (TCC) and/or its metabolic products (3,4-dichloroaniline) are investigated in cell line tests . This work should provide more information about interactions of CNT- and combination effects of CNT- with organic contaminants at the cellular level .

TH 204Uptake routes of silver nanoparticles in earthwormsM . Diez1, C .S . Svendsen1, D .S . Spurgeon1, C .A .M . Van Gestel2

1Centre for Ecology and Hidrology, Wallingford, United Kingdom2Vrije Universiteit, Amsterdam, NederlandThis study aims at quantifying the contribution of dermal and oral uptake of silver nanoparticles (AgNP) in earthworms . With the greater production and incorporation into consumer products, it is expected that increasing amounts of AgNPs will be released to the environment . These NPs will end up in soil through wastewater and sewage sludge or may directly be deposited on land, thus being of environmental relevance for terrestrial ecosystems . To assess their risk in soil, besides their dissolution and speciation it is important to investigate the mechanisms by which the different metal species (single nanoparticles, aggregates and metal ions) interact with biological membranes and enter the body of the organisms exposed . Earthworms are representative test organisms for ecological risk assessment . They will be exposed to contaminants in soil both through the skin (dermal) and by ingestion (oral) . To assess bioavailability of metal nanoparticles in soil to earthworms, it is necessary to know the contribution of each pathway . For that reason, experiments will be performed with the earthworm Lumbricus rubellus, applying oral sealing with medical histoacryl glue to block the ingestion of soil particles . Glued and unglued earthworms will be exposed to Lufa 2 .2 soil spiked with 100 and 500 mg kg-1 dry soil AgNPs for 7 days . An exposure of glued and unglued earthworms to soil spiked with 20 and 100 mg kg-1 AgNO3 will also be included to address the interaction of silver ions with earthworm tissues . By measuring total silver body concentrations at different sampling times in both exposure scenarios it will be possible to assess the contribution of the different routes of exposure . The distribution of silver in tissues of exposed earthworms will be mapped by transmission electron microscopy (TEM) and synchrotron X-ray microanalysis .

TH 205Silver nanoparticles ecotoxicological assessment: Novel electrophysiology biomarker and the potential role of P-glycoprotein in modulating toxicity in earthworms and aquatic oligochaetesR . Gooneratne, L .O .N .G .F Mao, N .O .É .M .I Demizieux, M .A .R .T .I WellbyLincoln University, Christchurch, New ZealandSilver (Ag) nanoparticles (NPs) are the fastest growing NP class . AgNPs are a new dimension of pollutants in the environment . A novel, non-invasive electrophysiological technique was used to compare the ecotoxicity of AgNP with that of silver nitrate (AgNO3) by measuring conduction velocity (CV) in the medial giant fibres (MGF) of earthworms and aquatic oligochaete worms . In order to determine the sub-lethal dosages for the CV experiments, classical 48-h LC50 and LC10 values were determined for juvenile earthworms (A . caliginosa) and the aquatic worm (L . variegatus) . The 48-h LC10 values for adult and juvenile earthworms exposed to AgNP in filter paper studies were 7 .1 and 2 .5 µg cm-2 respectively and the corresponding values on exposure to AgNO3 were 17 .1 and 7 .9 µg cm-2 . The 48-h LC10 values for AgNP and AgNO3 in L .variegatus in water were 81 and 107 µg ml-2 respectively . Based on the LC10 and LC50 values, AgNP was 2- to 3-fold more toxic than AgNO3 and this was most marked in the juvenile earthworms .The CV in the juvenile earthworm A . caliginosa were measured at 0, 1, 3, 6, 8, 24, and 48 h following exposure to AgNP and AgNO3

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concentrations of LC0 .001, LC0 .01, LC0 .1, LC1, LC2, and L .variegatus to concentrations of LC1, LC5, LC10, and LC20, with distilled water as the negative control . MGF CV decreased progressively in L . variegatus and to a lesser extent in A . caliginosa exposed to both AgNP and AgNO3 up to 8 h and then the CV gradually increased . This could have been due either to degradation of Ag compounds or an adaptation mechanism by L . variegatus . To eliminate possible degradation of the Ag compounds, the AgNP and AgNO3 solutions were renewed every 3 h and CV measurements taken at hourly intervals for 7 h . Based on the results of these studies and our experience with P-glycoprotein (P-gp) transporters in oligochaetes (not reported here), it is proposed that the oligochaetes adapt to excessive exposure of AgNP and to a lesser extent to AgNO3via the elimination of chemicals via P-gp transporters . In conclusion, MGF CV measurements in the earthworm A . caliginosa (used to monitor soil pollution) and the aquatic worm L . variegatus (to monitor aquatic pollution) can be used as a non-invasive, sensitive, and early-warning biomarker of exposure to NP but interpretation of chronic exposure requires further evaluation in view of the role of P-gp transporters in the excretion of excess chemicals from the body .

TH 206A weight of evidence approach for occupational risk assessment of engineered nanomaterialsD .R . Hristozov1, S . Gottardo1, A . Zabeo2, A . Critto1, A . Marcomini1

1University Ca’ Foscari Venice, Venice, Italy2Venice Research Consortium, Venice-marghera, ItalyIt has been recognized that substantial limitations and uncertainties make the conventional RA infeasible to apply to ENMs today, which leaves regulators with little support in the near term . Knowledge gaps have been gradually filled by new research results and uncertainties have been reduced, but this process advances slowly and it will take decades [1], while quantitative risk assessment results are needed to support near-term risk management actions and regulatory decisions . In response to this need, a number of methodologies and tools to assess the risks from ENMs, in spite of the limitations, have been proposed . Most of them, however, are not intended to facilitate regulatory decision making, but instead to serve as preliminary hazard/risk screening and/or research prioritization tools . The present deficit of quantitative data and scientifically sound approaches will lead in the near and in the medium terms to uncertain and ambiguous, largely qualitative risk estimations based on expert judgments, which may fail to support proper risk management actions [1] . In this context a quantitative model for occupational RA and prioritization of ENMs, including probabilistic uncertainty evaluation, has been developed within the FP7-funded ENPRA project . Based on the conventional RA paradigm, it uses effects dose-response data and exposure measurements to rank and prioritize nanomaterials for further testing (in a lower tier) and qualitatively estimate occupational human health risks (in a higher tier) . In order to ensure optimal integration of all available data our approach implements some non-conventional tools like the Weight of Evidence and the Multi Criteria Decision Analysis . The model has been applied to a number of commercially available ENMs considered as case studied in the ENPRA project, i .e . titanium dioxide, zinc oxide, silver and multi-walled carbon nanotubes . In order to test its stability and ensure the robustness of the assessment results, probabilistic sensitivity analysis has been performed using the Monte Carlo approach . References [1] Hristozov D ., Gottardo S ., Critto A ., Marcomini A . Risk assessment of engineered nanomaterials: a review of available data and approaches from a regulatory perspective . Nanotoxicology (in press) . Acknowledgement - The authors thank the EU FP7-funded ENPRA project

TH 207Toxicity assessment of nanoclay Clay2, on human hepatic cell line HepG2M . Puerto1, J . Houtman2, S . Pichardo1, M . Jordá3, J .M . Bermúdez3, S . Aucejo3, A . Jos1

1University of Seville, Seville, Spain2Wageningen University and Research Centre, Wageningen, Nederland3Area of Packaging Materials and Systems, ITENE, Valencia, SpainNanoparticles (NPs) attract a great deal of attention due to their unique properties . Their high surface to volume ratios, specific chemical composition, surface structure and solubility, shape and aggregation results in materials that are qualitatively different from their bulk counterparts . These properties make them suitable for numerous applications in existing and emerging technologies . The most promising uses of NPs in the food industry are in food packaging . Dispersion of NPs in polymers increases the barrier function of plastics by creating a tortuous pathway for diffusing molecules, increasing shelf life and nutritional quality of food . Having these advantages, using NPs in food packaging seems inevitable . Since many packaging is wasted carelessly, a huge amount of these nanoparticles containing compounds might end up in the environment . As well as, possible migration from the packaging to the food product could also lead to human contact with the NPs . As earlier mentioned, the small size of NPs gives them unique properties, which includes a possible increased toxicological risk . Therefore, each nanoparticle requires its own risk assessment . In this work, the main objective was to study the in vitro effects of a newly developed nanoclay, Clay2 which is meant for use in food packaging, in order to understand the mechanism of action causing toxicity . A range of concentrations of Clay 2 (0-150 µg/mL) have been investigated in the human liver cell line (HepG2) . Different assays (total protein content, neutral red uptake, and MTS reduction), were performed, and the results showed a reduction of cell visibility with an EC50 of 88 µg/mL . Moreover, oxidative stress, genotoxicity and inflammation responses were explored . Acknowledgment: The authors wish to thank Junta de Andalucia (AGR5969) and Ministerio de Ciencia e Innovación (AGL2010-21210) for the financial support .

TH 208Characterization of aqueous metal nanoparticles and assessment of exposure on immune cell viability and effector functionA . Ortega, C . Riopelle, J . Stafford, G . GossUniversity of Alberta, Edmonton, CanadaNanotechnology is an emerging multidisciplinary field that involves the synthesis of molecules in the nanoscale (<100nm) . The small size of nanoparticles (NP) produces unique physico-chemical properties that are different from their larger bulk forms . Although impressive from a physico-chemical perspective, there is growing concern over the potential and unknown toxicity of these particles on biological systems . Since NPs can differ in their physico-chemical properties, their behavioural characteristics in solutions are typically assessed together with toxicity measurements . Thus, dynamic Light Scattering was used to characterize five aqueous metal oxide polyacrylic encapsulated NPs (TiO2, CeO, ZnO, Fe3O4 and a polyaxcrylic nanocapsule void of a metal oxide core) in our test cell media solutions . Results indicated that agglomeration was occurring between particles that increased their hydrodynamic size from their reported 5-9 nm to more than 120 nm . To measure immunotoxicity, RBL-2H3 (RBL) mast cells were exposed to NP concentrations ranging from 0, 1, 10, 50, 100 and 200 mg/L over 48 hours and were analyzed for cellular viability and proliferation using flow cytometry . Propidium iodide was used as a fluorescent marker for cell death . Results indicated a dose-dependent decrease in viability with increasing doses of some NPs . Significant changes in cell viability were only observed after two hours of exposure . We next examined the influence of NP exposure on RBL FCε receptor-mediated degranulation by dosing RBLs with both sub-lethal and lethal doses and showed that degranulation was inhibited at all doses including those that did not decrease cell viability . The IgE antibodies used to sensitize RBLs for degranulation were also exposed to several concentrations of NPs to examine the effects of NP-IgE binding on degranulation . Antibody binding affinity assays and cell fluorescent microscopy was used to supplement the results of both cell and IgE NP exposure experiments in order to demonstrate the role NPs have on influencing FCε receptor and IgE activated degranulation .

TH 209Cellular and genomic effects of nanoparticles on human cellsA .V . Viarengo, E .R . Ranzato, S .M . Martinotti, C .O . Oliveri, A .N . Negri, B .B . BurlandoUniversity of Eastern Piedmont „A . Avogadro„, Alessandria, Italy The use of nanomaterials is spreading in commercial and industrial activities . However, the interactions between these products and living systems is largely unknown . Particularly, the assessment of environment and human risks are lagging far behind the continuous development of new engineered nanomaterials, while literature data suggest that these materials represent potential environmental and health hazards . We studied the impact of nanostructured composites on human cells, by evaluations of various kinds of toxic effects and gene expression profile by DNA microarray analysis . Cells have been incubated with two different nanomaterials, which are possible candidates to develop nanocomposite products, such as Cloisite and Carbon Nanotubes (NC7000) . The experiments have been carried out on HaCaT cells, representing an in vitro model of human epidermis . The analyses have been carried out also on a mixture of Cloisite and Carbon Nanotubes . Cytotoxicity analysis of this combination on HaCaT cells was carried out by using the MixTox approach and it has revealed a biphasic effect of the binary mixtures . In fact at high concentration there is a synergistic effect but at low concentration an antagonistic effect is evident . To study this phenomenon a transcriptomic approach was used to clarify the molecular changes that led to antagonistic effects of the NOEC concentration of the mixture of the two nanomaterials . Genomic effects have been explored using a 60 k microarray . HaCaT cells were exposed for 24h alternatively to 0 .5 μg/ml Cloisite, 10 μg/ml Carbon Nanotubes, or to a mixture of the two components, each at half dose . The results demonstrate that in Cloisite exposed cells 453 genes are differentially expressed, 262 in the cells treated with nanotubes but in HaCat cells exposed to their binary mixture there are only 263 genes differentially expressed . These data confirm an antagonistic interaction of the two components . Using a blast 2go approach, the main cell functions affected by the two nanoparticles and their mixture have been also evaluated . In conclusion, by taking HaCaT as a models of human epidermis, it was possible to established that Carbon Nanotubes appear to be safe nanocompounds, whereas in contrast Cloisite is the most toxic ones . However, Carbon Nanotubes in combination with Cloisite may produce a lower impact on cellular systems with respect to the sum of single agents at least when present in the environment at sublethal concentrations .

TH 210The basic safety assessment of fullerene C60 after oral administrationK .Y . Yamashita1, Y .Y . Yasuo2, P .H . Huiyan1, O .T . Toshinobu1, T .M . Mayu1, A .M . Michihiko1, A .Y . Yasuhiro3, N .K . Kazuya3, K .H . Haruhiko3, A .H . Hisae4, T .S . Shin-Ichi3, N .H . Hiromi1, T .Y . Yoshikawa1, T .Y . Yasuo1

1Osaka University, Osaka, Japan2MEI center, Osaka University, Osaka, Japan3National Institute of Biomedical Innovation (NiBio), Osaka, Japan4Vitamin C60 BioResearch Corporation, Tokyo, Japan Summary: More recently, the development of nanomaterials with particle sizes below 100 nm is promoted extensively . These nanomaterials have been already used in various applications such as foods, medicines and cosmetics, and become essential to our daily life . Especially, fullerene C60 is one of the most promising nanomaterials as foods and medicines because of the unique chemical and physical properties . However, knowledge concerning the potential safety of fullerene C60 on human health and the environment is still fragmentary . Therefore, safety assessment of fullerene C60, following oral administration, is very important for safety assessment of food environment . Here, we

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examined the safety of fullerene C60 by oral administration in mice . BALB/c mice were orally treated with 50 mg/mouse of polyvinylpyrrolidone (PVP)-enwrapped fullerene C60 (PVP-fullerene C60), which is water-soluble fullerene, once daily for 7 consecutive days . During exposure of PVP-fullerene C60, the mice were weighted . No statistically significant differences were noted in mean body weights in the PVP-fullerene C60-treated group when compared to the control group . To further evaluate the safety of PVP-fullerene C60, we examined the hematological parameters and plasma biochemical parameters . No significant hematological change was observed in PVP-fullerene C60-treated mice . In addition, PVP-fullerene C60-treated mouse have little change in the plasma levels of liver injury parameter including AST and ALT, and renal injury parameter including BUN compared to those of control mouse . These results suggest that PVP-fullerene C60 would have no effect in oral acute high dose administration and indicate that the PVP-fullerene C60 could be safe nanomaterials for oral administration . We believe that these data provide basic information that should help to create safe and effective forms of fullerene C60 .

Acknowledgement: This study was supported in part by Health Labour Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan .

TH 211In vitro effects of amorphous nanosilica particles on the bone metabolismH . Nabeshi1, T . Yoshikawa1, T . Akase1, T . Yoshida1, T . Hirai1, M . Uji1, H . Takahashi1, K . Misato1, K . Ichihashi1, A . Udaka1, T . Mori1, Y . Yoshioka2, Y . Tsutsumi1

1Graduate school of Pharmaceutical Sciences, Osaka University, Suita, Japan2MEI center, Osaka University, Suita, JapanSummary: Recently, applications of amorphous nanosilica particles with a controlled particle size below 100 nm (nSPs) have been increasing in oral care products . nSPs exert various beneficial functions as a polishing agent and/or as a remineralization promoter for tooth, based on its unique physicochemical properties . Because high concentration of nSPs contents in oral care products (e .g . about 20%), for example, tooth paste, and we use them everyday, we are not able to avoid oral exposure of nSPs in living environment . Adversely, there are growing concerns about the possibility that unique physicochemical properties of nSPs induced health risks . However, there is little information about safety of nSPs in the context of oral exposure . Therefore, it is urgent need to identify the hazard of nSPs in oral cavity . In this study, we tried to evaluate the effects on osteoclast differentiation induced by nSPs in vitro, assuming the effects on alveolar bone metabolism, because parodontal tissue including teeth is one of the most important organs in oral cavity . Using in vitro osteoclast differentiation model, RAW267 .4 cells treated with receptor activator of nuclear factor -B ligand (RANKL), osteoclast differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity . As the results, it was revealed that at doses that did not induce cytotoxicity by nSPs, TRAP activity and the number of osteoclast cells (TRAP-positive multinucleated cells) by nSPs treatment were equivalent to non-treatment group in the presence of RANKL . Collectively, the results suggested that nSPs exerts no effect on RANKL-induced osteoclast differentiation of RAW264 .7 cell at the dose of this study . Now, we are examining the effect of nSPs on osteoblast differentiation as next step to evaluate effects on bone metabolism . We believe that applications of nSPs will extend to these new fields following further our careful safety study considering the realistic exposure amounts of nSPs . Moreover, we wish in vitro test like this is established as the alternative method of in vivo safety test in the future in consideration of animal welfare . Acknowledgement: This study was supported in part by Health Labour Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan . Reference: Nabeshi H . et al . : Effect of amorphous silica nanoparticles on RANKL-induced osteoclast differentiation in murine macrophages . Nanoscale Res . Lett ., 6(1):464-468, 2011 .

TH 212Surface interaction of coagulation factor and amorphous nanosilica particles plays critical role for acute toxicityT .Y . Yoshida1, T . Yoshikawa1, H . Nabeshi1, K . Matsuyama1, Y . Nakazato1, S . Tochigi1, T . Hirai1, M . Uji1, K . Ichihashi1, T . Akase1, Y . Yoshioka2, Y . Tsutsumi1

1Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan2MEI Center, Osaka University, Osaka, JapanSummary: Recently, the development of nanomaterials (NMs) with particle sizes below 100 nm is promoted extensively . These NMs have been already used in various applications such as cosmetics, medicines and foods . Thus, the exposure against NMs is unavoidable for us in our living environment . Under this circumstance, there is increasing concern regarding the potential health risks by the unique mechanical properties of NMs . In most cases, however, the safety evaluation of NMs has been insufficient for ensuring their safety . In this regards, using amorphous nanosilica particles (nSPs), we are studying nSPs-induced hazard identification and qualitative evaluation of exposure level, such as in vivo/in vitro distribution analysis by transmission electron microscopy . In previous study, we examined in vivo distribution of dermal administration of nSPs with particle sizes below 100 nm, because nSPs were used in cosmetic products . As a result, nSPs with particle size 70 nm (nSP70) penetrate skin barrier and move to bloodstream after dermal application . These findings mean that it is essential to identify the biological effects after systemic exposure to nSPs . In this study, we examine the biological effects after intravenous injection of nSPs . BALB/c mice were intravenously injected with nSPs of sizes 70, 300, 1000 nm and then assessed for survival, blood biochemistry and coagulation . As a result, injection of nSP70 caused fatal toxicity, liver damage, and platelet depletion, suggesting that nSP70 caused consumptive coagulopathy . Additionally, nSP70 exerts procoagulant activity in vitro, which was diminished in Factor XII-deficient plasma . Collectively, we revealed that interaction between nSP70 and intrinsic coagulation factors such as Factor XII, were related to nSP70-induced harmful effects . In other word, it is suggested that if interaction between nSP70 and coagulation factors can be suppressed, the safety of nSP70 may be ensured . Now, we evaluate quantitatively hazard effect and exposure level of nSPs in the realistic exposure pathway, such as oral or nasal route . These results would be useful for the safety/risk assessment and evaluation of NMs . Acknowledgement: This study was supported in part by Health Labour Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan .

TH 213Nanoparticle fate assessment and toxicity in the environment - Initial findings from phase 1 of the NanoFATE projectC . Svendsen1, A . Crossley2, M . Hassellöv3, S . Loureiro4, C .A .M . van Gestel5, F . Dondero6, A . Johnson1, L .A . Walker1, D . Spurgeon1

1Centre for Ecology and Hydrology, Wallingford, United Kingdom2Oxford University Begbroke Science Park, Oxford, United Kingdom3Göteborg University, Göteborg, Sweden4University of Aveiro & CESAM, Aveiro, Portugal5VU University, Amsterdam, Nederland6University of Piemonte Orientale, Alessandria, ItalyThis presentation will outline the main findings from the first 2 years of the EU FP7 project NanoFATE . The NanoFATE project was conceived to fill knowledge and methodological gaps currently impeding sound assessment of environmental risks posed by engineered nanoparticles (ENPs) . Our vision is to assess environmental ENP fate and risk in example high-volume products for which recycling is not an option, namely; fuel additive, personal care and antibacterial products . Two market ENPs from each product (CeO2, ZnO, Ag of varying size, surface and core chemistries) is being followed through their post-production life cycles i .e . from environmental entry as “spent product”, through waste treatment to their final fates and potential toxic effects . This will test the applicability of current fate and risk assessment methods and identify improvements required for assessment of ENPs at an early stage . NanoFATE focuses on developing a systematic understanding of fate and mechanisms of effects in a core set of ENPs and addressing how these may affect the application of current tools for ecological risk assessment . The ENPs we study are associated with commonly and widely used products to provide environmental and economic relevance to our work . Furthermore, the selected ENPs have different core and surface chemistry and physical properties allowing us to elaborate on current understanding of how ENP properties influence fate and behaviour in the environment, and their potential toxicity . This testing of how classical ecotox and risk assessment tools may need modifying to be “nano-applicable”will be achieved by systematically studying aspects that are related to ENP fate and toxicity and seeking to provide the knowledge needed to refine risk assessment tools and practices for use with ENPs . The presentation given will present the main results and conclusions reached and highlight the lessons learnt .

TH 214Establishing a comprehensive knowledge base on engineered nanomaterials - the project DaNaD . Kühnel1, S . Scholz1, K . Nau2, V . Richter3, H .F . Krug4, B . Mathes5, C . Steinbach5

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2Institute of Applied Computer Science, Karlsruhe Institute of Technology (KIT), Eggenstein-leopoldshafen, Germany3Hard Metals and Cerments, Fraunhofer-Institute for Ceramic Technologies and Syst, Dresden, Germany4Empa - Materials Science & Technology, St . gallen, Switzerland5Society for Chemical Engineering and Biotechnology (DECHEMA), Frankfurt am main, GermanyNumerous studies dealing with potential toxic effects of engineered nanomaterials have been published in the past years . Different types of particles in various modifications in several organisms have been investigated, making it difficult to draw general conclusions on the hazard posed by specific nanomaterials . Here, the project DaNa (funded by the German Federal Ministry of Education and Research (BMBF) and supported by the Swiss government) steps in, aiming at a comprehensive presentation of relevant material and toxicology data for nanomaterials . Further, the DaNa-project acts as an umbrella project aiming at collecting scientific results of recent and current BMBF-projects funded in the field of nanotoxicology and nanotechnology for ecological benefits . The knowledge integrated into the database could also contribute to the prioritisation of further research needs . This information is provided in the DaNa knowledge base, accessible via the internet database, www .nanoobjects .info . Data regarding application, material properties, exposure as well as human and environmental toxicology are included . From the pool of data short consumer-oriented articles are generated and published . To make the knowledge base easy to access by the user, the information is organised either according to the different types of nanomaterials or via relevant applications . Additionally, there is a glossary on nano-specific terms, as well as sections on the basics, on ongoing projects, news and FAQs . In a first step, all data and results are evaluated regarding their scientific value applying the DaNa criteria checklist, which demands specification on particle properties and behaviour as well as on toxicological tests . Besides the scientific community, we also address the interested public e .g . journalists, students, scientists from other fields or consumers by an understandable presentation of complex scientific data . An interdisciplinary group of scientists from biology, toxicology, ecotoxicology, physics and chemistry is engaged to achieve this evaluation and presentation for a broad audience .

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RA13P - Implementation of protection goals: Limits of current risk assessment and new approaches to solve old problems

TH 241Setting most robust effluent level against severe uncertainty: application of information-gap decision theory to chemical managementH . Yokomizo1, W .A .T .A .R Naito2, M .A .S .A .S Kamo2

1National Institute for Environmental Studies, Tsukuba, Japan2National Institute of Advanced Industrial Science and Technology, Tsukuba, JapanIn the ecological risk assessment, we have to make decisions based on many kinds of imperfect knowledge . For example, when we set limitations for point sources, there may be an uncertainty on relationship between a chemical concentration in effluent and that in environment, and hence it may be difficult to predict an environmental concentration using the information about effluent . Another example is that the environmental quality criteria are determined to protect the ecosystems, but the ecosystems are much more complicated and the criteria may not protect the ecosystems effectively . We apply information-gap decision theory to set effluent limitations for point sources based on scientific underpinnings rather than based on groundless predictions even under severe uncertainty . Information-gap decision theory derives the decision that is most robust to uncertainty, by guaranteeing an acceptable outcome under the largest degree of uncertainty without requiring information about the extent of parameter uncertainty at the outset . We illustrate the application of information-gap decision theory to derive a general framework to set effluent limitations of pollutants for point sources incorporating cost of reduction in chemical use and cost to wildlife species that are affected by pollutants . Our framework enables us to derive decisions to deal with severe uncertainty in ecological risk management of chemicals .

TH 242Does probabilistic approach relevant to derive Health occupational exposure limits?M . El Yamani1, R . Vincent2, R . Persoons3, D . Vernez4, B . Amzal5, A . Ces-Vlep6

1Ismael93Agence française de sécurité sanitaire (ANSES), Maisons alfort, France2INRS, Nancy, France3Université de médecine, Grenoble, France4Institut santé travail, Lausanne, Switzerland5Institut de Recherche et Developpement, Paris, France6ANSES, Maisons alfort, France Current methods for risk assessment are mostly ‘deterministic’ . This means they treat factors such as the toxicity of chemicals as if they were fixed, and precisely known . But in the real world, factors such as toxicity are not fixed but variable . For example, toxicity is measured for only a very small number of species, so scientists have to estimate toxicity to Human and in some extends to all the other species that we want to protect . Current methods for risk assessment try to allow for variability and uncertainty by using ‘fixed safety factors’, but this fails to give a complete description of the full range of the possible risks . Also, it is difficult to decide how big the safety factors should be .

Probabilistic approaches enable variation and uncertainty to be quantified, mainly by using distributions instead of fixed values in risk assessment . A distribution describes the range of possible values (e .g . for toxicity), and shows which values within the range are most likely . The result of a probabilistic risk assessment can also be shown as a distribution, showing the range of impacts that are possible, and which impacts within that range are most likely . This should provide a better basis for making decisions about chemical risks, because the full range of possible outcomes can be taken into account . For the first time to our knowledge this approach was applied to derive Occupational exposure limits professional (OEL) . OELs are the limits for concentrations of hazardous compounds in workplace air . OELs for hazardous substances represent an important tool for risk assessment and management and valuable information for occupational safety and health activities concerning hazardous substances . It is typically set by competent national authorities and enforced by legislation to protect occupational safety and health . It can be a tool in risk assessment and in the management of activities involving handling of dangerous substances .

TH 243Calibration of hazard quotient trigger values for pesticide risk assessment for non-target arthropodsK . Swarowsky1, F . Streissl2, S .K . Bopp1

1European Food Safety Authority, Parma, Italy2EFSA, Parma, ItalyThe European Food safety authority (EFSA) recently developed a methodology to define Specific protection goals (SPGs) for the ecotoxicological risk assessment of pesticides (EFSA, 2010) . The next step is the definition of specific protection goals for groups of non-target organisms in consultation with risk managers . It needs to be investigated whether the current risk assessment achieves an adequate level of protection to address the SPGs . According to EFSA (2010) “for each key driver (taxonomic group or other ecological entity) a reference tier should be identified, based on the most sophisticated experimental or modelling risk assessment method currently available that addresses the specific protection goal . This reference tier will then be used to calibrate lower tiers using simpler methods that are practical for routine use .”To apply this concept for the group of Non-Target Arthropods (NTAs), we compare the hazard quotients, which currently decide whether a higher tier risk assessment for NTAs should be performed, with the results obtained in the higher tier risk assessment taking into account the new SPGs . To do so we use published data from Draft assessment reports that have recently been summarized by EFSA in a database . Results will be presented at the conference . References: European Food Safety Authority (EFSA) (2010) . Scientific Opinion on the development of specific protection goal options for environmental risk assessment of pesticides, in particular in relation to the revision of the Guidance Documents on Aquatic and Terrestrial Ecotoxicology (SANCO/3268/2001 and SANCO/10329/2002) . EFSA Journal 2010;8(10):1821 .

TH 244How much of a difference in assessing ecological risk between effects of organism-level and population-level?B .L . LinRISS/AIST, Tsukuba, JapanMost protection goals stated in environmental regulations or policies are aimed at the population level or higher . A movement toward population-level ecological risk assessment (ERA) is gaining acceptance for support of chemical management in recent years . However, due to the reason that population-level ERA is a data-intensive analysis (it requires much more ecotoxicity data than organism-level), assessing the ecological risk of toxic chemical is mainly based on a limited number of measures of organism-level effects in laboratory toxicity tests . Thereby, there is an enormous concern in understanding how much of the difference in assessing the ecological risk between effects of organism-level and population-level . To get a rough answer to this concern, this study performs a comparative study on the difference between the derived reference values (PNECs) for risk calculation from approaches of organism-level and population-level . To derive the reference value defined as Clambda=1 for use in population-level ERA, extrapolation approach (Lin and Meng, 2009) using available acute (LC50 or EC50) and chronic (NOEC) toxicity test data is employed . From the comparative results of selected chemicals, a range of difference will be shown and discussed . This study is expected to be help in bridging the gap between the protection goals and practical ecological risk assessment .

TH 245Do we need modelling for a conservative risk assessment? An investigation on Daphnia magna populationsF . Gabsi1, M . Hammers-Wirtz2, A . Schäffer1, T .G . Preuss1

1Institute for Environmental Research- RWTH Aachen University, Aachen, Germany2Research Institute for Ecosystem Analysis and Assessment (gaiac), Aachen, GermanyIn this study, we use an individual-based model (IDamP) coupled to a TK/TD model (GUTS) to assess the risk of exposure of Daphnia magna individuals to a toxicant with several mechanisms of action (Dispersogen A), on the population level . Dispersogen A acts on reproduction by increasing the brood size at the expense of neonate length which decreases . It also affects survival, in addition to several other effects measured at higher concentrations . Effects on reproduction (neonate length and brood size) were calibrated using non linear regressions . GUTS was applied to calibrate effects on survival, using both chronic and acute datasets . Both assumptions, stochastic death (SD) and individual tolerance (IT) were tested . Validation was done using data from population tests describing population dynamics at six levels of contamination . The model was more successful in predicting population dynamics using SD approach rather than IT . The promoted reproductive effects (increase in brood size) caused a decrease in population abundance . Besides, not all effects observed on individuals were equally relevant on the population level . Whereas the promoting effects on reproduction failed alone to describe the effects on populations, the integration of both effects on reproduction and survival succeeded in describing adverse effects on populations . The additional effects observed at the higher concentrations on individuals were also important in determining population dynamics, but to a lesser extent than mortality or enhanced reproduction . Finally, population abundance was not always a direct predictor of population extinction probabilities . We conclude that using an individual-based model with an integrated TK/TD module, we could extrapolate effects of Dispersogen A from the individual to the population level for Daphnia magna . Additionally, the promoted reproduction should be considered as adverse effects on the population level in risk assessment for Daphnia . Besides, whereas effects are considered separately for risk assessment, integrating combined endpoints is essential to achieve a conservative risk assessment and this can only be done using this kind of modelling approach .

TH 246An approach to incorporate ecosystem service valuation in the assessment of protection goals: bridging the gap between risk and injuryP . Nicolette1, F . Colombo2, R . Wenning1, M . Sorensen1, S . Deacon3

1ENVIRON International Corporation, Atlanta, United States of America2ENVIRON Italy, Milan, Italy3ENVIRON, Box, United KingdomRemedial actions are typically based upon risk assessment and derived protection goals . Protection goals may or may not be overly protective . In developing a remedial action plan, it is necessary for stakeholders and decision makers to understand the potential benefits (i .e ., gains in ecosystem service value) and costs (i .e ., losses in ecosystem

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service value) associated with the implementation of various remedial alternatives based upon prescribed protection goals . In this regard, a formal quantification of the effect that remedial actions have on ecosystem service values is rarely considered . Ecosystem services concepts applied to the identification of appropriate remedial options for contamination have been evolving in the United States for many years and more recently are being included in European legislation (e .g ., Environmental Liability Directive) . Approaches and tangible metrics that can describe the costs or benefits associated with the selection of remedial alternatives, and subsequently overall site cleanup, are still evolving in Europe . Approaches which meet the goals of managing risks while concurrently creating public benefit are needed to avoid 1) creating more harm to the ecosystem than is predicted by the risk assessment that drove the remedial action in the first place or 2) providing marginal benefit for the effort expended . The SuRF UK and the NICOLE Road Map promote sustainable remediation; however, although these frameworks acknowledge ecosystem services approaches they are not covered in detail . This paper provides an overview of the use of a practical approach termed net environmental benefit analysis (NEBA) that incorporates ecosystem service valuation to better understand and quantify the potential impacts and/or benefits associated with remediating to a specified protection goal . A NEBA considers the likelihood that identified risks are associated with an injury and the potential magnitude of that injury . As such, NEBA can help to bridge the gap between risk assessment and remedial planning by answering the question, “What do the identified risks mean in relation to the protection goal?”The NEBA approach is unique and innovative in that it considers risk, cleanup and natural resource issues concurrently . The process also promotes the selection of decisions that demonstrate a balanced win for the environment and the stakeholders . Case studies will be provided .

TH 247Do threshold values for metal concentrations in soil protect the soil ecosystem properly?G . Mol1, J . Spijker2, L . Posthuma2

1Alterra, Wageningen University & Research Centre, Wageningen, Nederland2National Institute for Public Health and the Environment (RIVM), Bilthoven, NederlandRegional geochemical data of heavy metals are commonly used for environmental risk assessment and management . The data used are often (near) total concentrations of the elements (often determined with Aqua Regia), whereas the exposure of the ecosystem is determined by the available or reactive fraction . The objective of our research was to develop a wider applicable method for quantitative hazard assessment of anthropogenic soil metal contamination, based on and illustrated with data for concentrations of Cd, Cu, Pb, and Zn from 360 locations in the Netherlands covering all major Dutch soil types . Extraction of soil samples with 0 .43 M HNO3 gives a good approximation of the chemical availability of metals; the metal concentration in these extracts show a strong relation to the estimated anthropogenic enrichment, so we used them to assess the hazard of human-induced enrichment of these metals . To do this we used the toxic pressure concept, which estimates the fraction of biological species that is potentially affected due to the exposure to single metals or mixtures of metals . This is done using logistic concentration/response models parameterized with ecotoxicological effect data from toxicity tests and mixture models . Hazards varied from very low toxic pressures (lower than 0 .01) to toxic pressures just below 0 .05 (just staying within the so-called 95%-protection criterion used in some soil protection legislations) . In rare cases, the toxic pressure exceeded the value of 0 .05, to an upper limit of 0 .054 for Cd . Ranking the metals according to toxic pressure suggests that Cd enrichment induces the largest hazard increase . Ranking the soil types according to their susceptibility for toxic pressure by metals only yielded minor differences in enrichment hazards between soil types . Comparing the judgement of soils based on the current soil screening levels and based on toxic pressures that we estimated in this study showed that the soil screening values tend to be on the conservative side . Conservative soil screening values do indeed protect the soil ecosystem properly, but they do not always indicate an actual hazard or risk . When screening values are exceeded, refined hazard insights can be obtained, as illustrated in this study . These insights in the ecotoxic implications of metal concentrations in soils can provide a more refined basis for risk management decisions .

TH 248Improving exposure scenario definitions within REACH: a comparative study of sources to aggregate exposure in Korea and in DenmarkL .J .H . Jihyun, M .P . Pizzol, M . ThomsenAarhus University - National Center for Environment and Energy, Roskilde, DenmarkIn recent years, the paradigm of chemical management system has changed from being toxicity oriented and media based to being risk oriented and receptor based . This trend is evident not only regarding environmental quality standards, but also for industrial chemical regulations . Political instruments to support a precautionary chemicals management system and to protect receptor’s health have also been increasing . Since 2007, the European Union adopted REACH (the Regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals): REACH makes industry responsible for assessing and managing the risks posed by industrial chemicals and providing appropriate safety information to their users (EC, 2007) . However, to ensure a high level of protection of human health and the environment, there is a need to consider ‘aggregate exposure’ including background exposures from environment which varies in different regions . It is also needed to include information about the sustainable level of industrial emissions in REACH reporting system; this is in order to prevent chemicals from accumulating in the environmental media and biota . New approaches and frameworks are therefore needed to accomplish the goals of REACH, these at the same time can decrease historical background exposure for humans and ecosystem . A comparative review about the environmental management system such as quality goals, relevant organizations and environmental monitoring systems of hazardous chemicals in Denmark and Korea will be presented . Furthermore, the actual concentrations of selected substances (metals and POPs) in environmental media will be compared between the two countries with reference to the amount of chemicals emitted to environment by industries as reported in the national PRTR registers . Comparing the different background exposure between two countries allows in fact the definition of a common framework for improving exposure scenarios within REACH system, for monitoring environmental health, and for increasing degree of circularity of resource and substance flows . References 1 . European Commission Environment Directorate General, Oct .2007, REACH in brief

TH 249Caterpillars and protection goals: the role of field margins as habitats and the effects of pesticide applicationsM . Hahn, A . Schotthöfer, M . Geisthardt, J . Schmitz, P . Lenhardt, C .A . BrühlInstitute of Environmental Sciences, University Koblenz-Landau, Landau, GermanyBiodiversity as new protection goal is specified in a Scientific Opinion of the EFSA . In this opinion it is proposed by the EFSA PPR panel to apply the protection goals for in-crop areas also to field margins . Field margins offer a habitat for many arthropods including butterfly and moth species (Lepidoptera) but they also receive inputs of pesticides due to overspray and spray drift as a consequence of the adjoining in-field applications . Our research project focused on the assessment of field margins in an agricultural landscape and their acceptability as caterpillar habitat with a special regard of the effects of different pesticide inputs . Field margins were digitized and characterized in 4,000 ha of agricultural landscape around Landau, Germany . In a following step we recorded caterpillar communities in selected field margins of the study area . The impacts of different agrochemicals (insecticide, herbicide, fertilizer) on caterpillar occurrence were assessed in experimental field margins . Additionally, food quality for Cabbage Moth (Mamestra brassicae) caterpillars in three host plants treated with sublethal concentrations of two herbicides was analyzed in a laboratory study . Concerning their length, nearly 80% of the field margins found in the studied landscape were less than 3m wide . These small field margins were used as caterpillar habitats by a range of Lepidoptera species . Next to orchards less caterpillar individuals could be found in comparision to cereal field margins and meadows which is probably a result of higher insecticide inputs in orchards . This finding is coherent with the results of the field margin surrogates in which caterpillar occurence was reduced in plots with insecticide treatment . In the laboratory experiment, in one plant-herbicide combination the caterpillars showed statistically significantly lower weights in comparison to caterpillars feeding on untreated control plants indicating a reduced host plant quality . Lepidoptera showed a vulnerability towards different pesticides . Especially in smaller field margins pesticide inputs can lead to reduced abundances of Lepidoptera . These smaller structures constitute a high percentage of the measured semi-natural habitats occuring adjoining to agricultural sites . It therefore should be carefully considered if these field margins should be treated as in-crop area receiving a lower protection status (no habitat provision) according to the protection goals .

TH 250Bird community monitoring in chlorpyrifos-treated citrus. Results of years 1&2 of 3 year program in Valencia regionS .M . Norman1, R . Dittrich2, B . Giessing2, C . Wolf2, G . Weyman3, P . Manson4

1Dow AgroSciences, Abingdon, United Kingdom2tier3 solutions GmbH, Liechlingen, Germany3Makhteshim Agan, Thatcham, United Kingdom4Cheminova, Harrogate, United KingdomBird communities in citrus are influenced by a range of habitat features, such as the characteristics of adjacent areas . Reproductive success of birds in the field is affected by nest predation and food availability . Against this background of factors, the question is whether the insecticide chlorpyrifos has detrimental effects on the bird community including breeding success? Chlorpyrifos is applied to nearly the entire commercial citrus area in Valencia region in May-June for control of red scale . A comprehensive 3-year monitoring program in 10 citrus sites was initiated in 2010 (total area of around 100 ha) . Data are available from 2010 & 2011 . This long-term field study is a major post-registration monitoring initiative, for an insecticide which fails the standard Tier 1 risk assessment . Chlorpyrifos is the only active ingredient which controls red scale, which is a major economic pest causing downgrading of fruit to unsalable quality . This field monitoring program has Measurement Endpoints which match the Protection Goals of the regulatory assessment . Mist-netting was used throughout the season with birds being leg-ringed and their details recorded (species, age, sex, reproductive status, bodyweight) . All nests within the citrus groves were located, and their status monitored . Motion-sensitive cameras were used . Nest boxes were also installed . For Sardinian warblers radio-tracking of adults was used for nest searching . Characterisation of the ecologically relevant factors was done . Arthropod food availability was measured . All citrus orchards were treated in 2010 and 2011 with chlorpyrifos, with an application rate in 2011 of 2 .4 kg a .i ./ha . As was also found in 2010, a diverse and abundant bird community was present in 2011 . 5111 individuals of 72 species, including many small seed eaters (e .g . serin) and small insectivores (e .g . Sardinian warbler), were trapped . 60% of these (3052) were juveniles trapped during the breeding season indicating successful reproduction . In Total 306 active nests were found . The chlorpyrifos applications had no apparent effect on populations and breeding success . The diversity and abundance of birds indicated a high ‘health status’ of the bird communities . Monitoring will continue in 2012 .

TH 251Development of a ‘hair tube’ method to monitor potential effects of plant protection products on small mammal populations - application to a fungicide useF . Chiron1, R . Charge1, E . Bonneris2, R . Barfknecht3, M . Ebeling3, R . Julliard1

1CERSP -UMR 7204 - Muséum national d’Histoire naturelle - CNRS-UPMC-, Paris, France

2�6 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

2Bayer cropScience, Lyon, France3Bayer CropScience, Monheim, GermanySince 2005, an increase of requests of post-registration monitoring studies on e .g . birds, mammals, or bees is observed for plant protection products (PPP) newly (re-)evaluated in France . Actually, according to the outcome of the ecotoxicological risk assessment, a post-registration monitoring study can be requested by risk managers in France, in order to verify in practice the appropriate implementation of risk mitigation measures and get additional information on potential effects of the PPP at population level . As specified in the recently published guidance document based on the EFSA document (2009 ) concerning bird and mammals, all information coming from post-registration surveillance or monitorings can be useful to reduce the uncertainty about the level of protection provided by TER trigger values . However, at present, when post-registration monitoring studies are requested for such organisms, no standard guidelines or methods exist . Regarding small mammals, a large spectrum of methods has been applied in a variety of published studies, but these are not necessarily applicable to study the dynamic of the populations of the different species living in croplands . In this context, a simple and innovative field technique using hair tubes (Suckling, 1977 ) was further developed and applied to assess effects of a fungicide treatment on small mammal populations in cereal fields . It aimed to monitor small mammals under large scale field conditions by assessing the proportion of hair tubes visited by small mammal species (‘hair index’) living in cereal fields treated or untreated with the fungicide . Part of the work was to adapt the design of hair tubes to allow the detection of rodents and shrews living and attempted to calibrate this method against a live-trapping method . Then, the ability to detect differences of population size between fields with different treatment regimes, under a range of different sampling strategies had to be assessed using power analyses . Finally, the hair index was estimated in cereals fields of Seine-et-Marne over a-two year period and the scale of monitoring needed to detect significant changes in abundance and composition of rodents and insectivorous mammals on a short-term period was determined . The results demonstrate that this method is a very promising one able to assess small mammals’ relative abundance at large scale (i .e . in 50-100 fields) .

TH 252Screening metal impacts from tissue residues for a calibrated biomonitor using DYMBAM and monitoring data in 6 Luxembourgish riversT . Gallé, S . Massarin, R . CarafaCRP Henri Tudor, Esch-sur-alzette, LuxembourgMetals remain a pollution source for most rivers in urbanized areas although they have been pushed from the limelight by emerging compounds . The exposure of river biota to metals via food is rarely evaluated in routine risk assessments . We took the opportunity of recent development on the toxicokinetic model DYMBAM and the calibrated biomonitor for hydropsyche to evaluate the metal pressure in Luxembourgish rivers . We used extensive data on suspended matter that had been collected in the years 2002-2005 to simulate the evolution of tissue residues for hydropsyche assuming that it feeds on suspended matter (hydropsyche is a net spinning filterer) . As the uptake and elimination constants for this species are only partially known we used ranges of reported values for uptake from the water phase, ingestion rates, assimilation efficiency (as linked to suspended matter composition) and elimination rate to run an uncertainty analysis in STELLA with the DYMBAM equation for 6 differently polluted sites in Luxembourg . Results showed the dynamics of internal concentrations for the biomonitor over a life-cycle (July-June of next year) with a decrease over the winter period . Nevertheless, most sites yielded exposures to particulate metals which exceeded internal concentration thresholds for metal-sensitive species . The uncertainty of determining metal concentrations of low-flow suspended matter has a strong influence on the results . As for the DYMBAM parameters: uptake from the water phase had a negligible part in tissue residues while ingestion rate and in particular elimination rates where governing internal concentration . The approach seems interesting to evaluate the impact of metals in an isolated way . While in most of the sites classical organic pollution and eutrophication might dominate the depression of sensitive species the additive impact of metals cannot be ruled out following our calculations .

TH 253Active monitoring programs: useful tools for monitoring effects of some plant protection products on birds and mammals - Feedback on a French approachT . Quintaine1, E . Bonneris2, R . Barfknecht3, P . Radix3, A . Decors1

1ONCFS, Auffargis, France2Bayer CropScience, Lyon, France3Bayer CropScience AG, Monheim, GermanyBird and mammal risk assessments for plant protection products are carried out under EU regulation 1107/2009 using the guidance document issued by EFSA1 . This document outlines the conservative first-tier assessment procedure for a large range of crop/uses dependant scenarios and proposes different options for refinement if needed . Field studies are one of the options . In the regulatory frame, these studies are designed to detect non-intentional effects of the plant protection product in fields of a particular crop, for a particular use and on focal species of birds and mammals . For some plant protection products, post-authorization monitoring data may be additionally useful to assist in the regulatory process . This is the case of many insecticides particularly in solid formulations like granules or seed treatments where the first tier assessment usually does not match the regulatory trigger by a great extent . In this context, networks such as SAGIR2 can provide useful information on acute effects of plant protection products on birds and mammals under practical use conditions . With experienced SAGIR people, a specific bird and mammal mortality monitoring program was pro-actively launched . It has been performed for 3 years during the drilling period of methiocarb-treated rape seeds from 2009 to 2011 . According to the protocol, any dead or moribund bird or mammal in or in the vicinity (up to 500m) of oilseed rape fields was collected and analyzed for chemicals applied to determine the cause of death . From this 3-year national monitoring program, 4 positive cases were recorded with only one case attributed to methiocarb poisoning . This work using SAGIR network shows the usefulness of such existing national scheme to record effects which can be product related . Although the absence of recorded incidents does not necessarily indicate a low risk, for our case, the high theoretical predicted risk is not confirmed by any findings . Complementary tools such as field experiments or modelling would be needed to estimate quantitatively the order of magnitude of the phenomena detected . Active animation of such national network of environmental workers is an important condition to perpetuate a survey on several years . 1EFSA; Guidance Document on Risk Assessment for Birds & Mammals on request from EFSA (Sanco/10997/2009) . EFSA Journal 2009 7(12): 1-181; Available online: www .efsa .europa .eu 2French national network created by the National Hunting and Widlife Agency

TH 254Review of the causes of reported honeybee pesticide poisoning incidents in GermanyJ . PistoriusJKI, Braunschweig, GermanyIncident reporting is an important monitoring tool to receive information on the reliability of risk assessment of pesticides for bees and the effectiveness of risk mitigation measures . Learning from accidents has been proved to lead to quick reaction and adaptations of regulatory processes, e .g . following the poisoning incidents after drift of insecticidal dusts leading to contamination of flowering bee attractive plants . Even if a strong and sudden damage to bee colonies is observed, the reason for the bee incident may not be clear at the first view and the extent to which it may be due specifically to pesticides is uncertain . For several decades an analysis of bee poisoning incidents has been conducted in Germany . A system for analysis and reporting of bee poisoning incidents possibly linked to pesticide applications is well established . Biological analyses, e .g . inspection of bee diseases and, if appropriate, chemical analyses of bee, plant and other samples are conducted by the authorities to investigate the cause-effect relationship between an agricultural treatment and the incident . For the interpretation of data expert judgement is needed and all information gained on the incident needs to be considered to conclude on the real cause of the incident . Data on the numbers of honeybee poisoning incidents in Germany, pesticide residues detected and conclusions on the causes for reported incidents will be presented .

TH 255Year on year changes in Algerian mouse (Mus spretus) populations inhabiting citrus orchards treated with chlorpyrifosB . Giessing1, R . Dittrich1, S .M . Norman2, T . Städtler3, C . Wolf1

1tier3 solutions GmbH, Leichlingen, Germany2Dow AgroSciences, Abingdon, United Kingdom3RIFCON GmbH, Hirschberg, GermanyThe Algerian mouse (Mus spretus) is one of the most abundant rodents across Spain, especially preferring arid areas . Its population densities can range from 3 up to >70 individuals per ha, and fluctuate over the seasons due to phases of maximum reproductive activity in April-May and August-September . Here we present population parameters obtained during field studies in 2009 and 2011 (June to September) from local populations in citrus orchards and adjacent habitats (macchia) . Population densities over the study period differed substantially within and between the years . A potential explanation for stable and higher densities in 2011 could be that the carrying capacity of the orchards was reached early in 2011 and therefore the typical second reproductive peak in August could not be observed . Carrying capacity is dependent on the weather condition and available resources in a specific area . A population reaching the carrying capacity shows changes in population structure since i .e . birth rate slows down or the emigration rate increases . Therefore it is important to know about the population structure for interpretation of possible effects on small mammal populations through pesticide uses .

TH 256Population analysis of the insectivorous Sardinian warbler (Sylvia melanocephala) in chlorpyrifos treated citrus orchards in SpainB . Giessing1, R . Dittrich1, S .M . Norman2, T . Städtler3, C . Wolf1

1tier3 solutions GmbH, Leichlingen, Germany2Dow AgroSciences, Abingdon, United Kingdom3RIFCON GmbH, Hirschberg, GermanyThe Sardinian warbler is the smallest predominantly insectivorous bird species inhabiting conventionally treated Spanish citrus orchards, where chlorpyrifos is regularly applied . In order to investigate the importance of commercial citrus orchards during Sardinian warblers’ reproduction period, their seasonal occurrence, site fidelity and age structure was analysed . For this purpose periodically repeated standardized mist-netting was conducted in ten citrus orchards near Valencia from April to August 2011 . Sardinian warblers were present during the entire study period inside the study orchards (i .e . for reproduction) . In total 712 individuals were trapped . 154 individuals (21 .6%) were captured on at least two different days . Data gathered during a study in the same citrus orchards in 2010 was related to the 2011 results . The previous year 309 Sardinian warblers were ringed and 34 of these birds were recaptured in 2011 . The results obtained support the conclusion that chlorpyrifos treated citrus orchards offer

2��SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

habitat conditions which are obviously appropriate for breeding Sardinian warblers .

TH 257Guttation and the risk for honey bee colonies (Apis mellifera L.): is the distance of bee colonies to a treated crop a necessary and a useful risk mitigation measure?J . Pistorius1, I .P . Joachimsmeier1, U . Heimbach1, D . Schenke1, W . Kirchner2

1JKI, Braunschweig, Germany2Ruhr-Universität Bochum, Bochum, GermanyFindings of high concentrations of bee-toxic compounds in guttation fluid from young crop plants that had been seed-treated with systemic insecticides gave rise to concerns about a potential risk to honeybee colonies posed by exposure to guttation of seed-treated crops or following granular applications . As bee colonies seem to prefer water sources in the near surroundings, a field trial was set up to gain clarification about the potential risk of guttation droplets containing residues to bee colonies at different distances to seed treated crops and also if, in case effects could be observed, keeping of which distance between hives and a treated crop would be necessary for potential risk mitigation measures . The experimental field consisted of one plot planted with winter oilseed rape crop seed-treated (Elado ®, a .s . Clothianidin) and one plot with untreated winter oilseed rape . 24 bee colonies in total were set up in an untreated winter oilseed rape crop before emergence, with the hive entrances pointing towards the treated crop . 6 bee colonies each were located at different distances, 0m (field border), and also in 10m,30m and 75 m distance to treated crop . From August to November 2011 the mortality of bees was assessed with dead bee traps, bee brood and colony development assessed, until wintering of colonies . After overwintering in Spring 2012 the assessments mortality, colony size, bee brood and colony development will be continued after overwintering of colonies . During the whole observation period the occurrence of guttation was documented and, if guttation occurred, guttation droplets were sampled daily for residue analyses .

RA17P - Multiple stressors in a changing world

TH 258Regional scale risk assessment using the relative risk model of threats to the surface aquatic ecosystems of the Umvoti River catchment, South AfricaG .C . O’Brien1, N .J . Smit1, V . Wepener2

1North West University, Potchefstroom, South Africa2University of Johannesburg, Aucklandpark, South AfricaThe Umvoti River is a highly developed, highly utilised, stressed aquatic ecosystem that is often referred to as a ‘working river’ . Potential sources of impacts include numerous urban areas and associated activities, informal settlements, agricultural and forestry activities, industries, sand mining activities and recreational activities . Recent assessments show that a wide range of stressors have been identified in the lower portion of the study area in particular . Existing stressors include water quality and quantity alterations, habitat impacts and associated disturbances to wildlife impacts . Stakeholders of the catchment require a management plan that considers nature and location of multiple sources in the catchment that takes ecosystem dynamics into account and provides threat assessment of established endpoints . The aim of this study is to carry out a risk assessment of the Umvoti Catchment to identify and quantify risks to ecosystem components or habitats in accordance with existing stakeholder objectives or endpoints . In this study the relative risk model (RRM) was applied to selected sources, habitats and endpoints of the Umvoti River system to generate risk and validation hypotheses to test for the validation of the risk outcomes . Various uncertainty and sensitivity tests were applied to the model to validate the risk outcomes . Findings of the RRM showed that while the upper and middle portions of the catchment have low risks of threats impacting on the endpoints, high risks exist in the lower portion of the Umvoti Catchment . Some additional moderate risks were also identified in the extreme eastern and western parts of the catchment . Within the high risk region, sources of stressors include; sand mining activities, sugarcane activities, heavy industries, urban areas and then sugar mill activities . Endpoints are potentially threatened by water quality, water quantity and habitat impacts . Threatened endpoints of concern include the maintenance of local biodiversity, particularly in the Umvoti Estuary, the sustainable provision of ecosystem services for existing users and a safe and clean environment for local communities . Three hypotheses were developed and tested for further validation of the risk assessment . These tests confirmed the spatial projections and nature of threats established in the risk assessment and reaffirmed threats to endpoints . This risk assessment is currently being used to direct environmental management in the area .

TH 259Application of Bayesian networks for integrating multiple chemical and non-chemical stressors into a risk assessment using the South River, VA as a case studyG . Landis, K . Ayre, H .M . SummersWestern Washington University, Bellingham, United States of AmericaThe structure of the conceptual models as in the relative risk approach to regional risk assessment lends itself to the construction of Bayesian networks (BN) for the calculation of risk . BN use conditional probabilities based on available data, models or expert opinion to construct the conditional probability table . This table describes the interactions between input nodes and the resultant child node . BNs intrinsically incorporates uncertainty into each of the variables and sets specific criteria and structure for the calculation of risk . The case study we are using to demonstrate the approach is the South River in the western mountains of the state of Virginia, USA . The study area is approximately 25 km in length and includes the catchments that directly feed the river . From 1929 to the early 1950s, Hg was deposited into the river from a manufacturing process . We are now estimating risks due to Hg and other stressors up and downstream of the manufacturing site . The BNs were constructed using Netica, and the process of setting ranks and distributions will be explained in the poster . Smallmouth bass, Kingfisher and water quality parameters are the endpoints examined to date using the BN approach . An interesting result is the interaction between water temperature and contaminant loading in generating an increase in risk far downstream of the manufacturing site . Another feature of the Bayesian approach is that it allows the calculation of restoration strategies and specific management and engineering options . The desired level of risk is set and the model calculates the required values of the input nodes . BNs are a powerful tool for understanding the impacts of multiple disparate stressors over regional landscapes .

TH 260Towards a conceptual model of the impact zone ecology in riversL . Roche1, S .J . Marshall1, O .R . Price1, M .J . Whelan2, A .B . Gill2

1Unilever, Bedford, United Kingdom2Cranfield University, Bedfordshire, United KingdomDischarge of untreated domestic wastewater into rivers can result in high concentrations of unionised ammonia (NH3), nitrite (NO2), and biochemical oxygen demand (BOD) as well as chemicals used in home and personal care products (HPCPs) . In such scenarios, where aquatic communities are severely impacted by conventional waste water constituents (sanitary determinands), it may not be appropriate to conduct conventional risk assessments on HPCP chemicals . An alternative risk assessment model based on the ‘impact zone’ concept has been proposed for these direct discharge conditions . The impact zone, in this case, is the river reach downstream of a direct discharge in which concentrations of sanitary determinands are higher than their predicted no effect thresholds, resulting in potential impacts on community structure and function . Risk assessment i .e . estimation of PEC (Predicted Environmental Concentration): PNEC (Predicted No Effect Concentraiton) ratio, of HPCP chemicals is conducted at the end of the impact zone . Here, we present a conceptual modelling framework that describes how the ecosystem community composition may change through the impact zone and beyond it and postulate how this could be used to assess the potential impacts of chemicals used in HPCPs under direct discharge conditions . A key assumption is that different taxa or biological traits have differing sensitivities to various chemical stressors, which will alter the composition of the in-stream community along the stress gradient . At a basic level this can be predicted using species sensitivity distributions, but at higher tiers ecological considerations (e .g . indirect effects) are required .

TH 261Effects of toxic and non-toxic stressors on fish community in the river RhineA . Fedorenkova1, J . Vonk1, H .J .R .O .B Lenders1, A .N .T .O .N Breure2, A .J .A .N . Hendriks1, R .S .E .W . Leuven3

1Radboud University, Nijmegen, Nederland2Dutch National Institute for Public Health and the Environment, Bilthoven, Nederland3Radboud University Nijmegen, Nijmegen, NederlandPredicting river ecosystem and fish community responses to human activities is challenged by the diversity of contaminants (toxic stressors) and habitat alterations (non-toxic stressors) associated with these activities . Research on the impact of these combined stressors as well as on the ranking of impact related to individual stressors of different origin will result in improved field impact based risk management of aquatic ecosystems . The fish community is a suitable indicator for the ecological condition of a river . The river Rhine has been intensively studied and much effort has been put in the (ecological) restoration . The diversity, richness and composition of the fish community in the river Rhine has changed remarkably since the 1970s due to both the species-dependent sensitivity of fishes and the introduction of invasive species . We therefore hypothesize that the sensitivity of the fish community related to the wide range of stressors has changed over time and that invasive fish species influence the overall responses of fish community to different stressors, due to their species-specific tolerance . The present study describes the effects of toxic and non-toxic stressors on fish community in the river Rhine using monitoring data collected since the 1970s and species sensitivity distributions (SSDs) . This statistical approach enables us to quantitatively compare concentration related effects (including contaminants, metals, dissolved oxygen, and salinity) with effects related to habitat alterations (like temperature, flow rate) . Therefore, in the present study, the effects of multiple stressors on the fish community consisting of native and invasive species are analyzed . The effects of low dissolved oxygen, temperature, stream velocity, and exposure to pesticides and heavy metals are studied with the aim to: (1) identify the difference in tolerance to toxic and non-toxic stressors between native and invasive fish species, and (2) rank these stressors according to the potential risk they have on the overall fish community as well as on the native and exotic component seperately, and (3) determine possible changes over time in the sensitivity of the fish community and the relative importance of different stressors in the river Rhine . The implications of our results for risk management of the Rhine as well as management of exotic fish invaders will be discussed .

TH 262Multiple stressors in aquatic environments may profile biomarker baseline responses: a case study in Dreissena polymorpha at the population scaleN .M . Pain-Devin1, S . Devin1, C . Cossu-Leguille2, A . Geffard3, L . Giambérini1, T . Jouenne4, L . Minguez1, M . Parant1, F . Rodius1, P . Rousselle1, N . Brulé1, K . Tarnowska5, C .

2�� SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

Daguin-Thiébaud5, F . Viard5

1LIEBE - CNRS - UMR 7146, Metz, France2LIEBE - CNRS - UMR 7146 - Université de Lorraine (UdL), Metz, France3Laboratoire d’Eco-Toxicologie - Université de Reims Champagne Ardenne, Reims, France4Laboratoire Polymères Biopolymères Surfaces, CNRS FRE 3103, Université de Rouen, Rouen, France5Adaptation & Diversité en Milieu Marin, CNRS UMPC - UMR 7144, Station biologique de roscoff, FranceTo face with the multi-pollution of their environment, aquatic organisms use various systems of defense, which allow them to live and to reproduce . This statement supports the hypothesis that a natural baseline level can be identified in wild populations, which corresponds to the defense level required for living in a given environment influenced by both anthropogenic and natural stressors . It was postulated that the basal defense levels in different populations cannot be the same, because they are an adaptation and/or acclimation response under influence of both genetic parameters and environmental conditions . In this context we conducted a large and pluri-disciplinary study involving ecotoxicology, proteomics and population genetics . Eight French populations of zebra mussels were sampled and used for biometric evaluations, biomarker measurements, energetic reserve assessments, proteomic and genetic analysis . Water physico-chemistry and sediment contamination were assessed in order to describe the studied sites . Analyses of data were performed by using integrative methods in order to establish links between site, population, and biomarker typologies . At Seville in 2010, we presented the first detailed results (SETAC Abstract n°1244) of this study and today, we will present how we integrated all of these results . Altogether, the results show once again the difficulty of interpreting results of field survey and the difficulty to link biological responses to contamination data . However, we succeeded in discriminating (1) between sites based on their contamination profiles, and (2) between populations based on the shell morphology, the population genetics parameters and the proteome expression . These typologies were consistent with the biomarkers responses profile . In conclusion our results support the initial hypothesis stating that wild populations do not have the same biological baseline response as they live in different environments, which are under influence of various multiple stressors . This is to take into account when seeking to understand the impact of multiple environmental stressors on ecosystem health . This study is part of the SYDEPOP project supported by the French INSU-EC2CO-Cytrix program .

TH 263Anthropogenic contaminants exert a stronger influence on bacterial communities than environmental factors; a study in estuarine sedimentsM .Y . SunUniversity of New South Wales, Randwick, new south wales, AustraliaEstuarine environments are highly susceptible to contamination from surrounding anthropogenic activities . The most ubiquitious contaminants in the environment, metals and polycyclic aromatic hydrocarbons (PAHs), bind to fine particulates and settle to be stored in the seafloor where they interact with sediment dwelling organisms and pose a threat to organisms estuary wide during oxygenation . As contaminants continue to accumulate in estuarine systems, sensitive methods to monitor sediment contamination are required to further our understanding of contaminant effects on sediment communities and for determining levels of contamination before ecological impacts are observed . Bacterial communities are highly sensitive to the presence of contaminants, and therefore may provide early signals of sediment contamination, but until recently these changes were difficult to assess . Advances in sequencing technologies have revolutionised the way in which we can observe the microbes that represent a large portion and many functions of a system . We use 454 pyrosequencing to compare bacterial communities in estuarine sediments across a large spatial scale; from 4 contaminated (heavily modified) and 4 relatively uncontaminated (relatively unmodified) estuaries . We find that shifts in bacterial communities correlate strongly with changing contaminant concentrations and that this signature is stronger than changes associated with environmental variation . Sediment contamination was found to select for groups from the orders Rhodobacterales, Oceanospirillales and Desulfobacterales, while orders Chromatiales, Cyanobacteria and Rhizobiales experienced declines . It is likely that changes in bacterial community composition as a result of sediment contamination have implications for the functioning of estuarine environments .

TH 264Temperature-dependent toxicity: is an assessment factor of 10 appropriate for ecological risk assessment?T .C .E . Lau1, J . Li2, X .L . Yi2, N .E . Karraker1, K .M .Y . Leung2

1The University of Hong Kong, Hong kong, Hongkong2The Swire Institute of Marine Sciences, Hong kong, HongkongLaboratory derived toxicity data provide important information for ecological risk assessment (ERA) of pollutants and form the basis for deriving water quality criteria . However, toxicity tests are often conducted following standard methods and/or guidelines (e .g ., ‘U .S . EPA Ecological Effects Test Guidelines’ and ‘OECD Guidelines for Testing of Chemicals’), and thus carried out under controlled laboratory conditions . For instance, most of toxicity tests are run at a constant temperature which usually represents the optimum temperature for the test species . Yet, toxicity of a contaminant may vary in a temperature-dependent manner, depending on the physiology of the test organism and the chemical properties of the contaminant . Even though an assessment factor of 10 is often used to account for the variability of toxicity data in ERA, no one has investigated the appropriateness of this value to cover the temperature-dependent variability adequately . Here, we compared median lethal concentration (LC50) data of several trace metals and organic pollutants on a range of aquatic species at different temperatures to assess whether an assessment factor of 10 is under- or over-protective for ERA . Our preliminary results indicated a considerable variability of copper toxicity with temperature across different freshwater species . For each species, we used the temperature at which the LC50 is the highest as a reference temperature (i .e ., LC50ref) and the corresponding LC50 value as 1 reference toxic unit (TUref) . The relative difference in the copper toxicity to the same species at a different temperature (T) is expressed as a relative TU value (RTU) which is equal to LC50T/LC50ref . A smaller RTU indicates a higher toxicity . For copper, we detected an average decrease of 0 .139 TU/ºC increase in temperature and a decrease of 0 .076 TU/ºC decrease in temperature . The results imply that an assessment factor of 10 will cover up to 6 .1ºC increase and 11 .8ºC decrease in temperature for copper, translating into a maximum protective range of 17 .9ºC temperature variation . Global surface freshwater temperature was found to have an average variation of 11 .7±1 .4oC . Thus, application of a factor of 10 in this case might be adequately protective to freshwater organisms . In this presentation, we will present more results on other chemicals before drawing a solid conclusion .

TH 265Evaluation of the combined action of natural stressors and chemical pollutants in algae. Assessment of functional, structural and metabolism alterationS . Rodriguez-Mozaz1, M . Ricart1, E . Tornés1, M . Gros1, M . Terrado1, C . Gutierrez1, N . Caceres1, S . Sabater1, D . Barceló2, V . Acuña1

1Catalan Institute for Water Research (ICRA), Girona, Spain2IDAEA-CSIC, Girona, SpainNowadays, there is a growing number of studies exploring the combined effect of different stressors on ecosystems . However, the interactions between natural stressors and toxicants are still poorly understood . Among the stressors that might influence ecosystems, toxicants can directly or indirectly affect all relevant ecosystem processes such as primary production . On the other hand, fast and intense changes in the water temperature is one of the major threats for freshwater ecosystems . The objective of this study was to characterize the physiological responses of cultured algae, Scenedesmus vacuolatus in response to physical (temperature) and chemical (anthropogenic contaminants) stressors . The pollutans selected for this study were the priority pollutant diuron (herbicide, PSII inhibitor) and the emerging compounds propranolol (β-blocker) and fluoxetine (antidepressant) . The experiment followed a factorial design with 2 factors (water temperature and toxicant concentration) and their interaction . A previous experiment determined that the optimal temperature was 20ºC, while 30ºC was used for simulating physical stress . Chemical stress was reproduced at the level of EC30 for each toxicant (5, 850 and 500 μg/L for diuron, propranolol and fluoxetine, respectively) . Changes in photosynthetic efficiency (Yeff), photosynthetic capacity (Ymax), photochemical quenching (qP) and non photochemical quenching (NPQ) were studied in control and exposed algal cultures . The algal toxicity of the three compounds differed considerably . Diuron was the most toxic compound, followed by fluoxetine and propranolol . Effects of diuron occurred immediately after addition, which indicates a very specific toxic action of this compound to algae (inhibition of photosynthesis) . Propanolol and fluoxetine toxicity effects appeared later than those detected with diuron exposure (after 30 minutes) . Most of the endpoints were affected after 12 hours for all the compounds tested, except in the case of diuron, where NPQ mechanisms were inhibited after 12 hours of exposure, suggesting damage in the pigments where the NPQ takes place . The increase of temperature reduced the toxic effect of the herbicide diuron and propanolol at the beginning of the exposure as well as at the final time . In the case of fluoxetine, the temperature increase reduced the toxicity at the initial time, but a synergistic effect was observed at final time with an enhanced toxicity .

TH 266Combined effects of natural (eucalypt leachates) and anthropogenic (copper) stressors on Echinogammarus meridionalis: implications for ecosystem functioning in a global warming climate scenarioM . Gama1, C . Canhoto1, L . Guilhermino2

1Imar-CMA, Coimbra, Portugal2University of Porto, ICBAS & CIIMAR, Porto, PortugalThe combined effects of natural stressors (eucalypt leachates) and a common environmental contaminant (copper), on the crustacean Echinogammarus meridionalis were investigated under a stream water temperature increase scenario . Single and mixtures toxicity bioassays based on mortality and biomarkers were used . In single stressors bioassays, the increase in temperature from 10ºC to 20ºC was found to significantly increment the toxicity of both copper and eucalypt leachates . In the mixtures bioassays, the increase of temperature by itself resulted in a reduction of glutathione S-transferases activity, increased levels of lipid peroxidation (LPO) indicating oxidative damage and inhibition of acetylcholinesterase activity indicating alterations of cholinergic neurotransmission . Furthermore, at 20ºC the toxicity of both stressors was higher, as indicated by a higher mortality and alterations in biomarkers . Toxicological interactions between stressors were also found . These results suggest that the ecological role of E . desmaresti populations can be negatively affected in streams contaminated by eucalypt leachates and/or copper and even more at high temperatures . TH 267Combined effects of temperature and chemical stress on native and exotic species of the genus ArtemiaV .D .F . Almeida1, P .M . Pinto1, M .N .R . Vieira2, L .M .C . Guilhermino3

1CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal2FCUP & CIIMAR - Universidade do Porto, Porto, Portugal3ICBAS & CIIMAR - Universidade do Porto, Porto, PortugalGlobal climate changes may have considerable negative impacts on hypersaline environments of the South of Europe which are also facing other pressures such as chemical

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contamination and biological invasions . Artemia species are key stone species of several hypersaline ecosystems in both marine and continental environments . In the present study, the combined effects of chemical and temperature stress on A . parthenogenetica, a native species in continental and marine hypersaline Portuguese ecosystems, and on the exotic species A . franciscana were investigated . The heavy metal mercury and the organophosphate insecticide fenithrothion were used as chemical stressors and two temperatures were tested 20ºC and 25ºC . Laboratory maintained and field collected organisms were used as test organisms in laboratory bioassays . Both chemicals had distinct effects on different populations and their effects were changed by temperature variation . Furthermore, the sensitivity of native and exotic species were differents . The implications for hypersaline ecosystem functioning and biodiversity in global warming scenarios are discussed .

This study was carried out in the scope of the project “Chemical wars: the role of chemically mediated interactions in the invasiveness potential of non-native Artemia”- Ref . PTDC/MAR/108369/2008 funded by the Portuguese Foundation for the Science and Technology and COMPETE FEDER funds .

TH 268Acute versus chronic temperature dependence of cadmium toxicity in zebrafishL . Vergauwen, D . Knapen, A . Hagenaars, R . BlustUniversiteit Antwerpen, Antwerpen, BelgiumToxicity studies are mainly performed in standardized conditions, most often at the optimal rearing temperature of the test organism . However, environmental temperature has a substantial impact on chemical toxicity . Metal toxicity is generally said to increase with increasing temperature in aquatic organisms, probably due to increased accumulation rates . This study aimed at investigating the temperature effect on cadmium accumulation and toxicity . Adult wild-type zebrafish were acclimated to exposure temperatures for one month prior to experiments . Fish were exposed to nominal concentrations of 0, 38, 52, 77, 115, 183, 284 or 435 µM cadmium at 18, 26, 30 or 34°C for 96 h in an acute experiment . In a chronic experiment zebrafish were exposed to 0 or 5 µM cadmium for 28 days at 12, 18, 26 or 34°C . Mortality was observed and the cadmium total body burden was measured . The temperature dependence of cadmium toxicity differed substantially between the acute and the chronic scenario . The pattern of acute cadmium toxicity was concentration dependent . LC50 values were lower at the intermediate temperature (26°C LC50: 102 .4 µM) when compared to 18 and 34°C (respectively 19 and 12% increased toxicity) . In contrast, the LC10 value at 34°C was significantly lower than those at 18 and 26°C, resulting in exactly the opposite order of toxicity at low exposure concentrations: 34°C > 30°C > 18°C > 26°C . In the chronic scenario cadmium caused mortality to increase with increasing temperature and the observed differences were much more profound (57% mortality at 34°C vs . 2% mortality at 12 and 18°C) when compared to the acute scenario . Furthermore, in the chronic scenario increasing cadmium accumulation with increasing temperature played an important part in increasing toxicity, while in the acute scenario other physiological processes weakened this relationship . Apparently in an acute highly toxic exposure scenario prior acclimation to an altered temperature protected zebrafish against future stress, while in a chronic scenario the continuous heat stress eventually aggrevated cadmium toxicity . More scientific background is necessary to include the temperature effect on chemical toxicity in regulation . Standardized tests are mostly performed at optimum temperature whereas temperatures in the field are often lower . Based on the present results, both in a peak exposure scenario and in a chronic low exposure scenario this could result in overestimation of toxicity .

TH 269The effects of thermal anomalies in mussels exposed to Cd at different seasons: cell and tissue level biomarkersM . Mugica, U . Izagirre, I . MarigomezUPV/EHU, Leioa, SpainMarine organisms subjected to pollution may exhibit altered responses to temperature raise associated to global warming . The consequences of temperature raise for biota may be very different depending on the season . Mussels are able to accumulate high Cd burdens in their tissues and are susceptible to the toxic effects of this metal, both bioaccumulation and biological responsiveness being also seasonal dependent . Presently, the seasonal variability in biomarker responses to temperature elevation in Cd exposed mussels Mytilus galloprovincialis was investigated . Mussels were collected from Gorliz (Biscay Bay) in summer 2009 and winter 2010 and acclimated to local seawater temperature (18ºC summer, 12ºC winter) under laboratory conditions for 5d . A set of mussels was exposed to Cd (0 .1 mg/l) beyond Day 3 acclimation . A control group (not exposed to Cd) was carried out in parallel . At Day 6, mussels were subjected to heat shock (6hr at +10ºC over seasonal local sea water temperature) . Then mussels were maintained at initial temperature for the following 3d . Cell and tissue level biomarkers were recorded in the digestive gland just before the heat shock (0), 1, 2, 3, 4, 5, and 6hr along the heat shock and 2-66hr after returning to initial temperature (8, 10, 24 and 72hr after starting the heat shock): lysosomal enlargement and membrane stability, intracellular neutral lipid accumulation, cell type replacement, atrophy of the digestive diverticula, tissue integrity in digestive gland and intralysosomal metal accumulation . Gamete development was determined on histological sections . In summer Cd exposure provoked significant changes in studied biomarkers, corresponding to a typical general stress response however in winter responses were less marked . In summer studied biomarkers exhibit altered responses after a heat shock, although combined effect of thermal stress and Cd exposure provoked higher changes . After the heat shock biomarkers recovered gradually initial values only in mussels subjected to a temperature elevation . In winter no significant responses were observed after a heat shock . On the other hand, marked alterations were observed with a combined effect of heat shock and cadmium exposure where unlike in summer, biomarkers recovered initial values after the heat shock . Organisms subjected to thermal anomalies exhibit different responses depending on the season . When thermal stress was combined with metal pollution the effect of season was less marked

TH 270Effects of copper on immune performances of Mytilus edulis from different salinitiesN . Höher1, A . Köhler1, M . Nagel2, M . Kriews1, F . Regoli3, K . Broeg1

1Alfred Wegener Institute, Bremerhaven, Germany2Hochschule Bremerhaven, Bremerhaven, Germany3Dipartimento di Scienze della Vita e dell’Ambiente, Ancona, ItalyThe ERANET BONUS+ project BEAST (Biological effects of anthropogenic chemical stress) aimed to test and establish biomarkers in the Baltic Sea, with regard to the predominant salinity gradient . Studies of multiple stressors in this environment are still very scarce, thus the present study tackles the question whether Mytilus edulis adapted to various salinities do respond differently to copper exposure . Individuals of Mytilus edulis were collected in Danish waters at a prevailing salinity of 12 and 20”, respectively, in September 2011 . After an acclimatisation period, mussels were exposed to varying copper concentrations (0, 5 and 15µg/L Cu) for 1, 7 and 13 days . Copper concentrations were chosen based on environmental measurements to ensure ecological relevance . For each copper concentration, salinity and time point 4 glass tanks (9L) were prepared, each of which contained 3 mussels . Water change and copper re-dosing was conducted daily . Copper concentrations were evaluated in water samples as well as in mussel tissue . Assessed biomarkers encompassed cellular, humoral and functional endpoints . In brief, the total and differential haemocyte count, morphological abnormalities in haemocytes, phagocytic (uptake of Neutral-Red stained zymosan particles) and haemolytic activity (lysis of erythrocytes), apoptosis of haemocytes (caspase 3/7 activity) as well as bacterial clearance of haemocytes (competent killing of E .coli) were determined . First results indicate that both environmentally realistic copper concentrations have a negative impact on phagocytic activity of haemocytes in mussels . This decrease was accompanied with a lesser efficiency to kill bacteria . Since the assessment of all data obtained from the experiment is still ongoing, final results including a multivariate statistical analysis will be presented at the meeting . To conclude, phagocytic activity, as an early indicator for changes in molluscan immune responses, is likely to serve as a valuable biomarker in biomonitoring studies on the impact of immunotoxic compounds in brackish water environments . The present study was an attempt to raise awereness to the specific abiotic conditions of the Baltic Sea, with regard to the EU-WFD (2000/60/EC) and the MSFD, as more effort is nedded to improve the understanding of such unique environments as well as the impact of multiple stressors .

TH 271Responses of benthic macroinvertebrate communities to multi-stress conditions in high altitude Andean streamsR .A . Loayza-Muro1, J .K . Marticorena-Ruíz2, E .J . Palomino3, C . Merritt1, M . Asselman1, A . Huiberse1, M .L . De Baat1, M . Van Gemert1, J .F . Duivenvoorden1, M .H .S . Kraak1, W . Admiraal4

1University of Amsterdam - IBED, Amsterdam, Nederland2Universidad Peruana Cayetano Heredia, Lima, Peru3Universidad Nacional Santiago Antúnez de Mayolo, Huaraz, Peru4University of Amsterdam, Amsterdam, NederlandHigh altitude Andes encompass a suite of harsh environmental conditions, including intense ultraviolet radiation (UVR) and acid drainage from metal-rich rocks, which challenge the survival of aquatic biota . The aim of this study was therefore to evaluate the single and combined effects of UVR and metals on benthic macroinvertebrate community composition . Since melanin may play a protective role against both UV and metals, pigments were analysed in field collected animals . Experimental verification of field data was obtained in laboratory experiments exposing mayfly (Cleon simile) and midge (Chironomus riparius) larvae to Cu and UVR . At 4000 m above sea level (m a .s .l .), UV-B was more than two-fold higher than at 3000 m . The concentrations of metals in the polluted sites ranged from 2 to 588 times those at the reference sites . The number of individuals and taxa did not differ between reference sites at both altitudes, but were much lower at the polluted sites than at the reference sites . Canonical Correspondence Analysis indicated a strong influence of UV-B in shaping communities at reference sites, and of metals at polluted sites . Melanin was strongly correlated with UV-B, being two-fold higher in mayflies at 4000 m than 3000 m . Chironomus sp . from polluted sites showed a two-fold higher melanin concentration than those from reference streams at the same altitude . In the laboratory experiments, a decrease in survival in both species was observed in the Cu, UVR and Cu+UVR treatments . Pre-exposure to UVR decreased the sensitivity of only C . riparius to UVR and Cu+UVR, which may explain the persistence of chironomids and the absence of mayflies under extreme metal and UV-B levels in high altitude Andes . A significant increase in melanin in C . riparius but not in C . simile larvae was observed in the Cu toxicity experiment, as seen in chironomids in polluted Andean streams . We conclude that in high altitude Andean streams high UVR and metal leaching create ‘life at the edge’ multi stress conditions which shape macroinvertebrate assemblages . Melanin may be an adaptive strategy of the few persisting specialized species against the effects of intense UV-B radiation and metals, as confirmed by exposure of chironomids to UV and/or Cu .

TH 272Joint effects of carbaryl and stress induced by temperature regimes on Eisenia Andrei (Annelida: Oligochaeta) survival M .P . Lima1, S . Loureiro2, A .M .V .M . Soares2

1Aveiro University, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, Portugal The toxicity of several chemicals in the field can differ depending on the prevailing environmental condition under which species are exposed . Studies have showed that temperature is a key environmental factor governing physiological status, affecting life-trait parameters (survival, growth, reproduction) and can also interact with chemical

2�0 SETAC 6th World Congress/SETAC Europe 22nd Annual Meeting

pollutants in the field . The aim of this study is to compare the toxicity of single exposure of E . andrei to carbaryl and the combined exposure with different air temperature regimes (8º, 12º, 16º, 20º, 22ºC, 24ºC and 28ºC), using survival and biomass loss as endpoints . For this standard acute tests were performed, and adapations for temperature exposure carried out . To evaluate the joint effect of natural and chemical stressor, observed data was compared with the expected effect predicted by the conceptual model of independent action (IA) and deviations for synergistic/antagonistic interactions, dose-level and dose-ratio dependency were used . From the results we can highlight the changes in toxicity to earthworms depending on the temperature regimes, which provide more realistic information for Risk Assessment procedures .

TH 273Exposure of Folsomia candida to carbaryl and natural stressors: effects on reproduction and genotoxicityF .N . Cardoso1, M .P . Lima1, A .M .V .M . Soares2, J . Lourenço1, A . Guimarães1, S . Mendo1, S . Loureiro2

1CESAM/ Aveiro University, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, PortugalTerrestrial organisms can be exposed to a great variety of stressors, such us contaminants and/or other physical or biological stressors that affect their life - and genetic traits . Organisms can experience a large range of environmental fluctuations such as temperature changes, drought and flood conditions or even UV radiation increments . The aim of this work is study the effects of carbaryl to a soil-dwelling collembolan Folsomia candida at different abiotic conditions, evaluating their reproductive effort and genotoxicity . Single and combined exposures were carried out with carbaryl and three different natural stressors: temperature, moisture and UV radiation . The combined effects were compared to carbaryl exposures under standardized condicitons: 20ºC, 60% water holding capacity . We observed that carbaryl induces changes on the survival and offspring production . DNA damage was also detected using the comet assay . Results from the combined exposure showed that fluctuations on environmental conditions such as temperature, soil moisture or radiation can induce changes on chemical toxicity .

TH 274The effects of copper and temperature on interspecific interaction between two collembolan species, Folsomia candida and Sinella curvisetaM .I . Mietelska, D .M . Jevtic, R . LaskowskiInstitute of Environmental Sciences, Jagiellonian University, Krakow, PolandThe effect of copper contamination on interactions between two species of springtails (Collembola) at different temperatures (10 and 20°C) has been examined . The instantaneous population growth rates of Folsomia candida and Sinella curviseta have been compared between monocultures and two-species cultures, influenced by different abiotic factors . Temperature proved to have a more significant impact on interactions between the species than copper contamination . However, the interspecific interactions under suboptimal thermal conditions (10°C) varied depending on copper concentration in soil . The increase in copper concentration caused quantitative changes in the two-species system by increasing the ratio of S. curviseta . Therefore, the presence of interspecific competition pressure may have a significant impact on the toxic effects of chemicals . Due to the complexity of factors influencing toxicity, and their possible effects at population and ecosystem levels, it seems necessary to complement the traditional ecotoxicological studies with more complex tests in order to improve the reliability of ecological risk assessment .

TH 275Combined effects of chlorpyrifos, mancozeb and temperature on the survival and feeding parameters of Porcellionides pruinosusR .G .C . Morgado1, A .M .V .M . Soares2, S . Loureiro2

1CESAM/ University of Aveiro, Aveiro, Portugal2University of Aveiro & CESAM, Aveiro, PortugalEnvironmental contamination and climate changes are two of the most important factors affecting soil ecosystems in agricultural fields . Agriculture regimes are nowadays strongly featured by the use of a wide range of pesticides whose application frequently coincides in time and space . Such mixtures may constitute, per se, a serious problem to soil biota but, in the present scenario of global changes in climate, they assume a particular relevance and unpredictability . The main goal of this work was to evaluate how the terrestrial isopod Porcellionides pruinosus is affected by the combined action of different temperature regimes and two widely used pesticides, the insecticide chlorpyrifos and the fungicide mancozeb . Endpoints measured included survival and two feeding parameters: the consumption ratio and biomass gain/loss . Preliminary tests were undertaken to assess the toxicity of both pesticides individually, as well as their effects in a mixture . The main experiment was then performed based on these results and using a full factorial design with three concentrations of each pesticide (plus an unexposed control) and three temperature regimes, mimicking real scenarios . Results confirmed that both survival and feeding parameters of P. pruinosus seem to be increasingly affected with the interaction of multiple stressors . This highlights the importance of taking climate conditions into account when assessing the impact of pesticides to soil biota, particularly in case of mixtures, and in this particular case that effects on isopods can be transposed to their crucial role in soils as decomposers .

TH 276Toxicity of copper, zinc and nickel mixtures to fathead minnows (Pimephales promelas): implications for Biotic Ligand Model for metal mixturesT .C . Hoang, N .R . LynchLoyola University Chicago, Chicago, United States of AmericaResearch on metal toxicity to aquatic organisms has been conducted extensively . However, most research was conducted with individual metals (e .g ., Cu, Zn, Ni) . This is not always relevant because metals usually present in the natural environment as a mixture of multiple metals . Data on the effects of Cu, Zn, and Ni mixtures to fathead minnows are lacking in the literature . The goals of this research are to characterize the acute toxicity of Cu, Zn, and Ni mixtures to fathead minnows (Pimephales promelas) and to generate data that support the development of a Biotic Ligand Model (BLM) for metal mixtures . 96-h acute toxicity tests were conducted with individual and mixtures of Cu and Zn, Cu and Ni, Zn and Ni, and larval P . Promelas using moderately hard water . For individual metal exposure, the 96-h LC50s for fathead minnows and Cu, Zn and Ni were 207, 873, and 1750 µg/L, respectively . Results of metal mixture exposures indicated that the toxicity of metal mixtures was more than additive (synergistic) . When exposed fathead minnows to either Cu at a concentration of 50 µg/L or to Zn at a concentration of 100 µg/L, no significant mortality was observed . However, 27% mortality were produced when exposed the fathead minnows to a mixture of 50 µg/L Cu and 100 µg/L Zn . When increased exposure concentrations 2 times (100 µg/L Cu and 200 µg/L Zn), total mortality (23%) produced by individual Cu and Zn exposures was less than the total mortality (60%) produced by Cu and Zn mixture exposure . Similarly, mortality (97%) produced by a mixture of 200 µg/L Cu and 400 µg/L Zn was greater the total mortality (40%) produced by individual Cu and Zn . Similar results were also found for Cu and Ni mixtures . At Ni concentrations of ≤ 1000 µg/L, no mortality was observed . However, when exposed fathead minnows to a mixture of 50 µg/L Cu and 500 µg/L Ni, 30% mortality were produced . At 100 µg/L Cu and 1000 µg/L Ni, mortality (97%) produced by mixture exposure were greater than the total mortality produced by individual exposures . For Zn and Ni mixture, at 400 µg/L Zn and 1500 µg/L Ni, 67% mortality were produced by the mixture while total mortality produced by individual Zn and Ni was less than 33% . Using toxic unit (TU) concept, the joint toxicity of Cu and Zn, Cu and Ni, Zn and Ni that produced 50% mortality was 0 .567, 0 .6219, and 1 .154 TU, respectively . Results of the present study are useful for the development of a BLM for metal mixtures .

TH 277Health of and contaminants in fishes from the Slave/Athabasca River System, Canada, relative to oilsands extraction activitiesD . Jones1, E .H . Ohiozebau1, A .H . Hill1, B .T . Tendler1, J .G . Giesy1, P .H . Hodson2, E .K . Kelly3, J .S . Short4, G .C . Codling1

1University of Saskatchewan, Saskatoon, Canada2Queens Uiversity, Kingston, Canada3Government of the Northwest Territories, Yellowknife, Canada4JWS Consulting LLC, Juneau, United States of America The potential environmental impacts of oil extraction activities in the northeastern region of Alberta Canada have recently received significant attention in both the scientific and general media . Chief among the global concerns are the fact that the open pit mining and oil extraction and conversion processes release a variety of environmental contaminants to the local and regional environment . Among the contaminants of concern in these aerial emissions are PAHs and a variety of metals (1) and releases of toxic naphthenic acids to the aquatic environment . The potential for these contaminants to accumulate in fish and wildlife are of particular concern to First Nations consumers of ‘wild foods’ . Consumption of fish and wildlife from the region are of cultural as well as nutritional and economic significance to First Nations communities . An apparent increase in the occurrence of fishes with apparent deformities and lesions has been reported by local communities as remote from the oilsands operations as the lower Slave River . The aim of this study is to investigate the exposure of fish populations in the Athabasca and Slave rivers to environmental contaminants arising from oil sands activities . In addition to investigating the general health condition of the collected fish and we consider contaminant concentrations in their tissues from both fish and human health perspectives .

TH 278Influence of environmental factors on the response of Daphnia magna population to pesticide applicationsI . Dolciotti, K . Foit, A . Herkelrath, M . Kattwinkel, M . LiessUFZ - Helmholtz Centre for Environmental Research, Leipzig, GermanyMost of the tests to assess the risk of pesticides are performed using standard toxicity tests on Daphnia magna cultured individually . However in the environment organisms experience abiotic and biotic interactions like interspecific and intraspecific competition that are likely to be of crucial importance to obtain a response to toxicants’ activity . Moreover input to surface waters from agrochemicals typically occurs in pulses or in repeated exposures due to agricultural runoff and spray drift . However, the literature on the toxicological and physiological responses of populations to fluctuating and repeated pollutant exposure is very limited . In our multispecies test system we investigated how interspecific competition alters the response of Daphnia magna to pulses of pesticide . Populations of Daphnia magna were cultured in the absence and presence of the competitor Culex pipiens for 82 days, and exposed to 2 pulse contaminations (24 hours every 4 weeks) of the insecticide Pirimicarb . Three different concentrations 3, 10, 24 µg/L were tested . Daphnia magna in both setups (we will refer to them “with”and “without”competition) showed an acute sensitivity to the pesticide only at the highest concentration tested . Culex pipiens larvae were insensitive at all concentrations . Multiple contaminations did not increase the sensitivity of Daphnia magna to Pirimicarb . Populations in the setup “without”competition were always able to recover within the 28 days between two pulses . However in the “with competition”system we observed no recovery or extinction . Moreover we investigated the interaction between the two species: at all concentrations of Pirimicarb and in the control, the abundance of daphnia was negatively affected by

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the biomass of Culex larvae . Our experiments revealed how an interspecific interaction can negatively affect the response to pesticide applications of one of the most common test organism in ecotoxicology . We conclude that biotic interactions like competition must be considered when conducting an environmental risk assessment of toxicants .

TH 279Predicting metal and metalloid partitioning in soils using infrared spectroscopic analysisJ . Kirby1, L . Janik1, S . Forrester2, M .J . Mclaughlin3, C . Reimann4

1CSIRO, Adelaide, Australia2Commonwealth Scientific and Industrial Research Organisation (CSIRO), Adelaide, Australia3CSIRO, University of Adelaide, Adelaide, Australia4Geological Survey of Norway, Trondheim, NorwaySolid-solution partitioning of a metal (termed the Kd value) is a key property that determines its environmental fate in both terrestrial and aquatic systems . The Kd value can be used to predict exposure pathways and which biota are likely to be exposed once a metal is released into the environment . The aim of this study was to derive partial least-squares (PLS) regression calibration models from mid-infrared (MIR) spectroscopic analysis plus soil pH data of soils and experimental derived Kd values to predict the solid-solution partitioning (log-Kd) values for selected elements in soils of the EuroGeoSurveys geochemical mapping of agricultural and grazing land soils of Europe (GEMAS) . The strongest spectral correlations for the partitioning of metals in soils were found to be in the regions for carbonate (2500, 1810, and 1350 cm-1), sand (quartz at 1000-1200 cm-1), clay (mostly kaolinite near 3620-3695 cm-1), aluminium oxide (near 3500 cm-1), particle size (quartz near 1000-1200 cm-1), and organic matter (2850-2950 and 1500-1700 cm-1) . The PLS regression plus soil pH models were found to predict experimental log-Kd values for cobalt (Co), lead (Pb), manganese (Mn), nickel (Ni), and zinc (Zn) with good accuracy (R2>0 .8), antimony (Sb(V)), boron (B), molybdenum (Mo(VI)), tellurium (VI) and vanadium (V(V)) with moderate accuracy (R2>0 .6 to 0 .8), and copper (Cu), selenium (Se(VI)), silver (Ag), and tin (Sn(IV)) with poor accuracy (R2<0 .60) . The findings suggest MIR spectroscopy analysis of soils may provide a rapid and cost effective tool to regulators and industry for assessing the partitioning of many elements in soils .

TH 280Levels and trends of PCBs and PBDEs in fish (Rutilus rutilus and Cyprinus carpio) and sediment from South KazakhstanF .B . Botta1, M .N . Mamilov N .2, P .L . Labadie3, F . Alliot4, M .G .E . Moreau Guigon5, K .E . Kozhabaeva2, C .M . Chevreuil5, S .T . Shalakmethova2, H .B . Blanchoud5

1INERIS, Verneuil en halatte, France2University Al-Faraibi, Department of Biology, Almaty, Kazakhstan, Almaty, Kazakhstan3LPTC-UMR 5805 EPOC, CNRS-Université Bordeaux 1, Talence, France4EPHE - UMR Sisyphe, Paris, France5EPHE - UMR SISYPHE, Paris, France

RA21P - Standard vs non-standard methods for hazard and risk assessment

TH 281Evaluation of carcinogenic potential of perfluorinated compounds using in vitro and in silico alternative approachesD . Baderna1, N . Golbamaki2, A . Lombardo2, F . Rotondo3, E . Boriani2, A . Guerrini4, M .G . Mascolo5, M . Lodi2, M . Vaccari5, A . Colacci5, S . Grilli3, E . Benfenati2

1Mario Negri Pharmacological Research Institute, Milano, Italy2Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy3Interdepartmental Center for Cancer Research „G . Prodi„, Bologna, Italy4Department of Experimental Pathology, Cancer Research Section, University of Bol, Bologna, Italy5Center for Environmental Carcinogenesis and Risk Assessment, Environmental Prote, Bologna, ItalyPerfluorinated compounds (PFCs) are a family of fluorine-containing chemicals used in different applications to make materials oil and water resistant . Regarding human exposure, PFCs are used in wide array of consumer products and food packaging as surfactants . PFCs persist in the environment due to the strength of the carbon-fluorine bond which prevents degradation by natural processes . Because of their persistence, toxicity and widespread occurrence in the blood of humans and wildlife, they are considered as emerging environmental pollutants . Toxic effects of PFCs in laboratory animals includes a range of pathological changes, such as liver and kidney damage and reproductive dysfunctions . Despite the global attention of scientific community, the toxicity and the mechanisms of action of PFCs are still partially unknown: only limited evidences are reported about the ability of these compounds to work as promoter in carcinogenic process . The aim of this work is to provide an evaluation of the carcinogenicity of these compounds using alternative methods including in silico methods and in vitro assay . Regarding in silico approach, a selection of dozen software from the list of EU project ANTARES were used to qualitatively evaluate the carcinogenic potential of PFCs . Freely available (i .e . VEGA, Toxtree, Lazar) and commercial software were used to obtain multiple responses in order to make a comparison of the predicted results . In addition to the in silico evaluation, some compounds were tested using the cell transformation assay (CTA) . In vitro cell-transformation systems have been developed to model the in vivo carcinogenesis process and they are regarded as the only possible in vitro alternative to animal testing for the screening of potential genotoxic and non genotoxic carcinogens . Selected compounds were assayed using the CTA based on Balb/c 3T3 clone A31-1-1 cells . When this established cell line is exposed to carcinogens, cells loose the contact-inhibition property and produce foci. Results include the overview of the qualitative prediction from in silico software and the experimental value from in vitro CTA . The evidences from our study underline the need for experimental data for perfluorinated compounds in order to build more suitable in silico models and to confirm results from in vitro assays . Moreover they emphasize the complementarity of the selected alternative methods that could help in reducing the animal testing for this critic endpoint .

TH 282Differences between dossier-based PNECs and EQSs based on all available literature: the case of ImidaclopridL . Van Leeuwen, C .E . SmitRIVM, Bilthoven, Nederland

TH 283Derivation of water quality standards for plant protection products - the importance of open literatureC .E . SmitRIVM, Bilthoven, NederlandThe European and national authorisation of plant protection products (PPP) is based on registration dossiers which in most cases only include reports of GLP-studies according to accepted guidelines . For quality standard (QS) derivation according to the methodology of the Water Framework Directive (WFD), all relevant available information should be considered . This means that dossier data submitted for e .g . the European PPP or biocides authorisation process should be used, but access to registration dossiers for QS-derivation is often restricted due to confidentiality claims . Data from the open literature are included in the QS-derivation, provided that the scientific reliability is sufficient . As a result, the datasets for authorisation and for QS-derivation may substantially differ . There are also methodological differences between the two frameworks, with respect to the treatment of data and the choice of safety factors . During the past years, RIVM has derived water QS for a large number of active PPP substances . For these compounds, we investigated whether the open literature resulted in additional endpoints that were not included in the dossier, and how this influenced the outcome of the QS-derivation as compared to the PPP-authorisation . For 65% of the compounds additional acute data were found, additional data on chronic endpoints were available for 40% of the compounds . Acute or chronic endpoints below the most critical dossier value were retrieved from the open literature for 27 and 21% of the compounds, respectively . In 37% of the cases, the chronic QS was lower than the authorisation value, with differences of a factor of 2 to 30 . This is due to the lower endpoints from the open literature, but also results from the critical evaluation as to whether the potentially sensitive taxa are represented in the dataset . This applies to insecticides and fungicides in particular, since the standard PPP-dossier does not contain chronic data on insects or fungi . Another difference is that transient effects in mesocosms are generally accepted for PPP authorisation if recovery has been demonstrated . It is concluded that an effects assessment based on the dossier data alone may be useful as a first indication of risks . The inclusion of open literature that is foreseen in the new PPP regulation is expected to improve the dataset and yield a more precise and reliable estimate of risk limits that can be used to set regulatory standards .

TH 284How to cope with variability in laboratory aquatic gnotobiotic microcosms?B .J .P . Clément1, H .E . Delhaye1, G .E . Triffault-Bouchet2

1ENTPE, Vaulx en velin, France2Centre d’expertise en analyse environnementale du Québec, Sainte foy, CanadaA protocol of ecotoxicological bioassay in 2-L laboratory microcosms is being developped since 1997 and applied to the study of various pollutants and scenarios of ecotoxicological risk assessment in the field of urban facilities and transport infrastructures (polycyclic aromatic hydrocarbons, municipal solid wastes incineration bottom ashes, road runoff residues, dredged sediments, [3DOTS]) . Effects are assessed on five different organisms (micro-algae, duckweeds, daphnids, amphipods, chironomids) using classical endpoints such as growth, emergence (chironomids), reproduction (daphnids), survival, with a duration exposure of 3-4 weeks . This bioassay can be considered at first glance as a multi-species test which provides ecotoxicity data for each species present in the microcosm, however it is actually more since it takes into account interactions between biota and media (e .g . N and P consumption by primary producers, bioturbation of sediment by chironomid larvae, [3DOTS]) and interactions between populations (e .g . daphnid grazing of micro-algae, competition for nutrients between micro-algae and duckweeds, [3DOTS]) . This type of approach is generally considered as valuable since it states between single-species tests, whick lack ecological relevance and predictivity, and mesocosm assays, which display drawbacks due to their complexity (high variability) . However, even in gnotobiotic systems where the main components are known, variability still exists to some extent and limits the statistical power of the test . In this communication we will illustrate what variability can be in microcosm assays and show improvements brought to the protocol in order to reduce this variability : breedings and cultures of test organisms, microcosm setup, improvement of organisms fitness in control conditions, monitoring and sampling . The best results were obtained when the protocol was changed from a static test (no renewal of water) to a dynamic test (flow-through renewal of water column), which allowed to stabilise physico-chemical parameters of water and thus to improve the development of organisms and the repeatability of the bioassay .

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TH 285Detoxification to Daphnia magna of 4 pharmaceuticals and 7 surfactants by activated sludgeR .G . Dave, G .H . HergerUniversity of Gothenburg, Göteborg, SwedenPharmaceuticals are bioactive compounds generally resistant to biodegradation, which can make them problematic when they are released into nature . The use pattern for pharmaceuticals means that they are discharged into water via sewage treatment plants . Also surfactants are discharged through sewage treatment plants, primarily due to their use in detergents and shampoos and other cleaners . In this study the acute toxicity to Daphnia magna of 4 pharmaceuticals (ciprofloxacin, ibuprofen, paracetamol and zinc pyrithione) and 7 surfactants (C8 alkyl glucoside, C6 alkyl glucoside, sodium caprylimidiopropionate, tallow-trimethyl-ammonium chloride, potassium decylphosphate, propylheptanol ethoxylate and alkylmonoethanolamide ethoxylate) was determined . The abiotic (without activated sludge bacteria) and biotic (with activated sludge bacteria) detoxification was also determined . The 24-h EC50s ranged from 2 µg l-1 for the most toxic substance (zinc pyrithione) to 2 g l-1 for the least toxic compound (C6 alkyl glucoside) . The detoxification rate did not differ between pharmaceuticals and surfactants, but within these groups there were differences in detoxification rate, which should be important in assessments of their environmental hazards and risks . Detoxification rates determined as the ratio between initial EC50 and EC50 after one week in water with activated sludge bacteria ranged from 0 .4 (paracetamol) to 13 (zinc pyrithione) . For most of these chemicals detoxification rate decreased after one week, but for one (alkylmonoethanolamide ethoxylate) it increased from about 2 to 30 times after 2 weeks . Further studies are needed to determine the degradation products that were precipitated (aggregated) for some of the tested chemicals . Altogether, this study has shown that there are large differences in toxicity among chemicals entering sewage treatment plants, but also that the detoxification of them can differ . Therefore, the detoxification should receive more attention in the hazard and risk assessment of chemicals entering sewage treatment plants .

TH 286Toxicity of 9 shampoos to Daphnia magna and their detoxification by activated sludgeR .G . Dave, K .B . BollUniversity of Gothenburg, Göteborg, SwedenShampoos are primarily used for hair cleaning, but also to make hair soft and shiny and to prevent dandruff . To achieve these objectives, shampoos contain, in addition to surfactants, other ingredients like emulsifies, preservatives, plasticizers, perfumes, thickeners and colour . The anti-dandruff shampoos also contain fungicides . Several of these substances are known to be toxic to aquatic organisms, but unlike other cleaning products, eco-labelling is lacking for most shampoos . The aim of this study was to determine if toxicity and detoxification was different for 3 anti-dandruff, 3 ordinary, and 3 eco-labelled shampoos, and to determine their detoxification with and without activated sludge . The results showed that initial 24-h EC50s for Daphnia magna ranged from 0 .54 to 163 mg L-1 (302-fold) . A comparison between labelled ingredients and reported ingredient toxicity using a TU (Toxic Unit) approach could roughly explain the differences among the studied shampoos . The toxicity and also the variation in product toxicity decreased when the shampoos were detoxified (from 302-fold to 5-fold), but still the anti-dandruff shampoos were more toxic than the ordinary and the eco-labelled shampoos . The major finding of this study was that detoxification can be used as a combined determination of toxicity and biodegradation in order to get an estimate of the combined environmental hazard of complex chemical products like shampoos and detergents .

TH 287Sediment toxicity testing for prospective risk assessment - a new framework and how to establish itM .A . Beketov1, N . Cedergreen2, L .Y . Wick1, M . Kattwinkel1, S . Duquesne3, M . Liess1

1UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany2University of Copenhagen, Frederiksberg, Denmark3The Federal Environmental Agency - Umweltbundesamt, Dessau- rosslau, GermanyThe development of ecologically realistic and practical sediment toxicity testing for prospective ecological risk assessment remains a considerable challenge . There is a recognised need to design a new framework for sediment toxicity testing that meets current scientific standards and regulatory requirements, such as reliable assessment of toxicity, which prevents any harmful effects on biodiversity, a strong capability to predict population- and community-level effects, and applicability of the results to decision making . We propose a new framework for prospective sediment toxicity testing, and suggest solutions to the key methodological challenges that hinder establishment of this framework (comparison of sensitivities, design of test batteries, consideration of different exposure routes, extrapolations to population and community levels, use of test results for decision making) . The proposed framework consists of the following three units: test-battery system, higher-tier testing systems and additional ecological models, and a decision support system . The key methodologies proposed to establish this framework are compound- tailored test-battery use approach, relative sensitivity distribution analysis, toxicity tests that combine bacteria and arthropods, micro- and mesocosms studies, population and community models, and model-driven decision support systems . The proposed framework, as well as the key methods mentioned above, has the potential to improve not only prospective toxicity testing for sediments, but also ecological risk assessment in general .

TH 288Improved OECD 105 water solubility test designP .C . Thomas, V . BurosseCEHTRA, Vaulx milieu?, FranceIn this poster a simple modification to the OECD 105 water solubility Guideline is described that can improve fitness of data for use in environmental risk assessment of poorly water soluble substances . The OECD 105 Guideline comprises two methods, one using a shake flask method for water soluble substances (liquids or solids) and the other using a column elution method for poorly soluble solids (despite the fact that liquids are not formally excluded, the column elution is often not technically feasible, due to clogging for example) . Poorly soluble liquids therefore pose a problem as the shake flask method can lead to the formation of emulsions and potential overestimation of the solubility . Furthermore, the solubility of certain substances in pure water may differ, sometimes considerably, from their solubility in the more relevant environmental media potentially leading to misconceptions of the toxicity of the substance in certain cases . The proposed simple modification can be used to improve solubility determinations for poorly soluble liquids and provide reliable indications of true solubility in aqueous media . It was successfully tested in a first experiment, but needs further improvement .

TH 289Intense sweeteners in the aquatic environment - is there a bitter (environmental) aftertaste?K .E . Tollefsen1, L . Nizzetto1, D . Huggett2

1NIVA, Oslo, Norway2University of North Texas, Denton, United States of AmericaPresence of intense (artificial) sweeteners in the aquatic environment have led to concern about toxic effects in non-target species living in areas receiving discharges from anthropogenic activities . The present work presents the current knowledge about sucralose, as a representative of intense sweetener, on environmental occurrence, fate and effects in the aquatic environment to assess whether there is a reason for environmental concern . Sucralose exhibits high chemical stability, high water solubility, is not metabolized by humans and poorly degraded in sewage treatment works, and is therefore discharged largely unaltered to the aquatic environment where it is found in sewage effluents at low ng/L to moderate ug/L concentrations . As sucralose is not efficiently mineralized in the environment, surface water concentrations are typically 1-2 orders of magnitude lower . No acute or chronic toxicity has been demonstrated at environmentally relevant concentrations in standardised regulatory valid ecotoxicological tests with algae, crustaceans and fish . However, reports of sub-lethal responses in aquatic organism behavior, respiration and global gene expression after exposure to sucralose introduce a need for discussing available data in the context of regulatory and non-regulatory risk/hazard assessment .

TH 290Model validation in 96h acute aquatic toxicity testingL .S . MccartyL .S . McCarty Scientific Research & Consulting, Newmarket on, CanadaThe validity of toxicity test results from standard testing protocols is contingent on whether the employed models and assumptions are appropriate and sufficient . A quality control evaluation of conceptual and technical models used in the 96 h acute toxicity test protocol and the ensuing data was carried out . The standard LC50 test design uses exposure concentration as an indirect dose surrogate for the true dose metric - the molar concentration of chemical at site’s of toxic action in the body of the organisms . Thus, three key enabling assumptions need to be routinely examined and validated to ensure that LC50 test results represent consistent, comparable measures of relative toxicity . First, steady-state LC50s must occur . Second, steady-state LC50s must occur at a duration that ensures equivalent exposure . Third, protocol design must ensure that all toxicity modifying factors are controlled . A subset of the U .S . EPA-Duluth fathead minnow 96 h toxicity database was analyzed . Approximately 8% of the tests failed assumption 1 validation and should be considered unusable . Although the time to achieve steady-state varied by a factor of about 2000 in the remaining 92% of the data, these data passed validation of assumption 2 . However, variance from unquantified effects, ostensibly the result of toxicity modifying factors, such as use of inappropriate exposure times (lack of steady-state), metabolic biodegradation, and body size differences, remain embedded in LC50 estimates rendering them inaccurate measures of relative toxicity, thereby failing assumption 3 validation . Improvements in toxicity test design and interpretation, including explicit specification of models, routine validation of assumptions, and collection of additional information for quantifying the influence of metabolic degradation and other currently uncontrolled toxicity modifying factors, as well as more information on mode/mechanism of toxic action, are necessary . A thorough quality control evaluation of 96 h LC50 methodology and related protocols is warranted to ensure that reliable data are being used for hazard and risk assessment and in the development of QSARs .

TH 291Assessment of Cadmium effects using standard single-species and non standard microcosm bioassaysB .J .P . Clément1, H .E . Delhaye1, G .E . Triffault-Bouchet2

1ENTPE, Vaulx en velin, France

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2Centre d’expertise en analyse environnementale du Québec, Sainte foy, CanadaEcotoxicological assessment of chemical substances is mainly based on standard bioassays, most often single-species tests covering the range of acute and chronic toxicity . In our laboratory we have been developping since 1997 a protocol of ecotoxicological bioassay in 2-L laboratory microcosms and applied it to the study of various pollutants and scenarios of ecotoxicological risk assessment in the field of urban facilities and transport infrastructures . Effects are assessed on five different organisms (micro-algae, duckweeds, daphnids, amphipods, chironomids) using endpoints such as growth, emergence (chironomids), reproduction (daphnids), survival, with a duration exposure of 3-4 weeks . The protocol has mainly been used as a static assay, where water was not renewed for the duration of test . A flow-through microcosm assay (dynamic assay) was developped to improve conditions inside the microcosms, resulting in stabilisation of physico-chemical parameters, increase of organisms fitness and reduction of variability . Since toxicity is closely linked to the conditions of bioassay, which influence the chemical speciation of the substance and the dynamic of exposure, we aimed to compare informations produced by standard single-species tests, multi-species tests with an aqueous phase only, and microcosm bioassays (static and dynamic) on five different species used in both types of tests . Cadmium was chosed as a model toxicant and introduced only at the start of test in single- and multi-specific static assays, and continuously in the dynamic microcosm assay . Cadmium concentrations were monitored in several bioassays, especially in the presence of a sediment phase, due to expected sorption of Cd . Effects on the same organisms in various conditions (duration of test, type of medium, presence or absence of sediment, renewal of water or not) were compared and discussed based on these different conditions . The presence of sediment, leading to decline of water cadmium concentrations and to a multiple exposure of some organisms, complicates the establishment of a link between cadmium concentration and effects, and thus the comparison of simple single-species tests and microcosm assays . However, these ones give a better insight of the fate and effects of cadmium in aquatic environment . Compared to the static microcosm test, the dynamic microcosm test, where cadmium concentrations are kept constant in the aqueous phase, provides less variable data and is more sensitive .

TH 292Influence of pH, light cycle, and temperature on ecotoxicity of four sulfonylurea herbicides towards Lemna gibbaR .T . Rosenkrantz1, N . Cedergreen2, A . Baun1, K .O . Kusk1

1Technical University of Denmark, Kgs . lyngby, Denmark2University of Copenhagen, Department of Basic Sciences and Environment, Copenhagen, DenmarkIn chemical risk assessment standard tests are applied to establish the basis for evaluating the hazard of compounds and determination of environmental standards . Using standard tests enables the comparison of the toxicity of different compounds but it may not always describe all aspects of the toxicity of a compound in the environment . The toxicity may be different under different test conditions such as light regime, temperature or pH of the medium . In the environment organisms are rarely exposed to compounds at conditions identical to those of the standard tests, and therefore it is important to study the effect at different test conditions in order to get a more realistic assessment of the potential risk posed by a compound . Therefore, the objective of this study was to investigate what implications changing test conditions may have on the toxicity of a group of model compounds, and thereby obtain a better basis for risk assessment extrapolations . As model compounds were chosen four sulfonylurea herbicides (SUs), which are well-studied compounds in regards to standard tests, although they are somewhat difficult to handle in the lab because they hydrolyse quickly and are in the ionized form depending on pH of the medium . The toxicity of the four SUs towards L . gibba was investigated at three pH levels (6, 7 .5 and 9), at different temperatures (15 and 24 °C) and under different light regimes (continuous and 12:12 h light:dark) . It was shown that test conditions have an effect on the toxicity on the four SUs . Decreasing the pH below the standard test value resulted in a decrease in EC50 values, while increasing the pH caused either no change or an increase in EC50 . In general, the EC50 increased between 2 .2 and 10 times for the four SUs when pH was increased from 6 to 9 . Decreasing the temperature from 24 to 15 °C and introducing a light-cycle did not cause any trends in changes in toxicity . The results indicate that test conditions may have an effect on the toxicity of the compounds studied here and if there is also enough data available to justify a low assessment factor, this should be taken into account when the standard tests are used in determination of environmental standards .

TH 293New short term toxicity test using single algal species and natural phytoplanktonR .T . Rosenkrantz, D . Cupi, A . Baun, K .O . KuskTechnical University of Denmark, Kgs . lyngby, DenmarkStandard algal test using Pseudokirchneriella subcapitata normally have a duration of 72 hours . This method might not be feasible for compounds that have fast kinetics of hydrolysis or photolysis, or compounds that have been shown to exhibit properties of sorption or volatilization . Of special interest are also Engineered Nanoparticles (engNPs) which have been shown to agglomerate or adhere to the surface of test organisms . These physico-chemical characteristics, as well as test exposure/duration can therefore influence test results . An alternative to better control these confounding factors in the experiment is to shorten the duration of exposure .

Currently, there is a need to develop tests using natural algal communities to increase the environmental relevance . Thus, the objective of this study was to develop a short term test with natural community algae and elucidate on advantages and limitations, as well as compare sensitivity with standard algal test . The test principle is to expose the algae to a toxicant gradient in the presence of 14C-labelled bicarbonate for 2-3 hours and measure 14C uptake by scintillation counting . Both natural algae communities from Lake Fure (collected at different times of the year), and a laboratory culture of P . subcapitata were used . The algae were exposed to a range of different toxicants such as photosynthesis inhibiting herbicides, acetolactate synthase (ALS) inhibiting herbicides, titanium dioxide and silver nanoparticles, and standard reference compounds such as potassium dichromate and 3,5-dichlorophenol . Preliminary results show no toxicity of ALS inhibiting herbicides neither for the natural algal community nor the laboratory culture at the concentrations tested .

TH 294Quality evaluation of existing critical body residue data for baseline neutral narcosisL .S . Mccarty1, J .A . Arnot2, D . Mackay3

1L .S . McCarty Scientific Research & Consulting, Newmarket on, Canada2University of Toronto at Scarborough, Toronto, Canada3Trent University, Peterborough, CanadaThe Environmental Residue Effects Database (ERED) developed by the U .S . Army Corps of Engineers and the U .S . Environmental Protection Agency is the most extensive environmental CBR compilation available . ERED was employed for a data quality evaluation of available CBR-effect relationships for baseline neutral narcosis . Evaluation began with an ERED subset consisting of 7574 records for 318 organic chemicals from 651 references for fish, invertebrates, and algae . The first quality screening used 14 different criteria (e .g ., no adverse effects reported, testing of mixtures, difficulties interpreting exposure and responses metrics) to remove inappropriate data . This resulted in a initial working database of 2271 records for 182 chemicals from 294 references that met minimum data quality requirements . A second screening removed data where a specific mode of toxic action was suspected (all or most of the reported CBRs were below about 0 .1 mmol/kg) . A third screening removed data based on a paper by paper review of experimental results . All data removal and screening results and rationales were documented . The penultimate database for baseline neutral narcosis consisted of 278 records for 39 chemicals from 37 references . Three chemical subgroups were noted: neutral organics, polar organics (polar, ionizing, and nitro-containing compounds and substituted phenols), and PAHs . Narcosis quality evaluation proceeded by rating data either as poor (4 rules) or rejected (11 rules) . The rules were based on experimental issues (e .g ., absent, low or difficult to interpret responses, experimental problems, specific toxicity according to the Verhaar classification scheme) . The final evaluation eliminated some of the neutral organics, all of the polar organics group, and 1 PAH . The remaining high quality CBR data was for 27 chemicals in four general classes: substituted benzenes, other organics, PCBs, and PAHs . These chemicals are considered to be baseline narcotics, based on this data quality evaluation . This is not to say that these are the only narcotics in the database, or that they always produce toxicity by the baseline narcosis mode of toxic action, only that these data have passed the specified data quality evaluation process . These evaluated data will form the basis for toxicokinetic model development and evaluation .

TH 295Validity criteria from standard method: the need for assessor common understandingC . Durou1, B . Journel1, P . Adrian2, P .C . Thomas1

1CEHTRA, Sainte-eulalie?, France2CEHTRA Europe SPRL, Brussels, BelgiumStandard data are generated according to recommended and internationally agreed test methods (e .g . OECD Test Guideline, EU Testing Methods) . Test guidelines are well described providing technical information on principle of the test, description of test method and usually validity criteria . For the purpose of the evaluation of data quality under Chemical Regulatory program, scientific study reports are primarily checked by assessors to see whether validity criteria are met or not . Within the context of harmonization of data evaluation worldwide, a common understanding of validity criteria from standard method by assessor is a key component for conducting an appropriate reliability assessment . In this presentation, we describe lack of common interpretation regarding one of the three validity criteria from the recommended guideline on Freshwater Alga and Cyanobacteria, Growth Inhibition Test . We explore the implications for reliability assessment and the possible ways to increase assessor common understanding of validity criteria on Algae toxicity testing .

TH 296Endpoints and dose-response relationships of low-dose and standardized studies of Bisphenol AL . Molander1, A . Beronius2, A . Hanberg2, C . Rudén1

1Royal Institute of Technology (KTH), Stockholm, Sweden2Karolinska Institutet, Stockholm, SwedenBisphenol A (BPA) is used in large volumes to manufacture polycarbonate plastics and epoxy resins, which are materials used in numerous consumer products . Diffuse emissions of endocrine-disrupting chemicals (EDCs), including BPA, from consumer products to both indoor and outdoor environments have been identified as an emerging issue . In risk assessment and risk management of BPA, the evaluation and interpretation of the low-dose studies (where effects are reported below 50 µg/kg/day) has been extensively debated . This study aims to provide an overview of endpoints used in the low-dose toxicity studies of BPA, at what levels the studied effects are reported (NOAEL and/or LOAEL), if the dose-response relationships are monotonic or not, and if exposure is prenatal, postnatal or both . These aspects were compared to the studies of the effects of BPA performed according to standardized test methods . Data were compiled from a comprehensive review of studies concerning the effects of BPA in the open scientific literature . Endpoints were categorized, e .g . into reproductive

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effects, developmental neurotoxic effects and immunological effects, and comparisons were made within and between the endpoint categories . The differences identified between the low-dose studies and the standardized studies are important to consider in the development of appropriate and sensitive criteria for EDC identification and in the refinement of risk assessment methods for BPA, as well as other EDCs .

TH 297Use of public literature and dossier data in WFD EQS derivation compared to risk limit derivation in other regulatory frameworks: the case of silverT .A . Moermond, R . van HerwijnenRIVM, Bilthoven, NederlandRecently in the Netherlands a revised Environmental Quality Standard (EQS) for silver was derived . For EQS derivation according to the methodology of the Water Framework Directive (WFD), all relevant available information should be considered . This means that dossier data submitted under REACH should be used, but also data from open literature should be included, provided that the scientific reliability is sufficient and endpoints are relevant . For the derivation of a chronic EQS for silver in freshwater, 71 unique data sources were found in public literature and the REACH dossier, including reviews . These data sources were scored for reliability using the Klimisch scores (1=reliable; 2=reliable with restrictions; 3=unreliable; 4=unassignable) . Studies with a validity score of 1 or 2 were used for final EQS derivation . In the REACH dossier, for chronic toxicity in freshwater 52 records are available . Within these records, there is a substantial number of duplicates, and the way the records are described varies greatly . Publication sources are often only mentioned as ‘publication’, ‘study report’, or ‘grey literature’ . Thus, public literature is included in the dossier, but it may be difficult to determine the original source . Industry has the obligation to conduct literature searches, thereby disclosing all relevant data, but there is no mechanism in REACH that evaluates this aspect . The only formal requirement, however, is at a minimum fulfilling the basic requirement according to the Annexes VII-X . Within REACH, Klimisch scores may be applied differently than for EQS derivation . For instance, studies in the dossier for silver which were performed using a slightly shorter or longer exposure time than what is described in the OECD guideline, received a reliability score of 3 (‘unreliable’) while in the EQS derivation they could still be seen as reliable . Records in the REACH dossier are divided into key studies and supporting studies . In the silver dossier, there are a number of supporting studies with a reliability of 1 and a number of key studies which refer to ‘grey literature’ without further details . The rationale behind this division is not described in the publicly available part of the dossier . Often, there is no publicly available explanation as to how PNEC values are derived using the available data . This makes a comparison between the PNEC and the EQS difficult .

TH 298Comparison of methods of application on the toxicity of a test substance to the carabid beetle Poecilus cupreusC .A . Jenkins, J . GrayHuntingdon Life Sciences, Suffolk, United KingdomFor soil incorporated substances it is important that the method of application used in evaluation of the effects on the soil dwelling non-target species Poecilus cupreus represents a more realistic situation in regard to exposure and bioavailability than the standard sprayed application in which the carabid beetles, food and substrate are contaminated . Direct spray application of an insecticide product was compared to simulated shank-chisel and drip irrigation applications with the objective of demonstrating that the method of application has a direct influence on the survival, behaviour and predation rate of adult carabid beetles . For the simulated shank-chisel and drip irrigation applications, the test substance was applied at rates equivalent to a field application rate and volume . Beetles were introduced into the test chambers after application . The comparative spray applications were made at the same rate using a compressed air single lance sprayer calibrated to deliver a spray volume equivalent to 400 L/ha, with the beetles in-situ during application (standard test technique) . Control treatments were applied using the same methods . Post application one Musca domestica fly pupa per beetle was introduced into each test chamber to provide a food source . Survival, abnormalities (affected or moribund beetles) and food consumption were assessed and recorded at intervals for 21 days after application . The results showed that there were significant differences in the survival of the adult beetles between the methods of application . Investigation into the relevant application method may be a contributing factor in the design of future studies to ensure the presentation of appropriate data .

TH 299Modification of the OECD 117 Partition coefficient using the HPLC method for high log Kow substancesV . Burosse1, J . Kurtz2, P .C . Thomas1

1CEHTRA, Vaulx milieu?, France2FIRMENICH SA, Geneva, SwitzerlandEstimation of bioaccumulation potential of an organic substance may be obtained through measurement of its partition coefficient . The current OECD/EU method based on HPLC method has been validated up to 6 .5/6 .2, respectively, based on the highest reference substance (DDT) recommended, and extrapolation is expected to lead to high uncertainty . The slow-stir method is not either designed to go higher than log Kow = 8 . However, a low log Kow <4 or extremely high value >9 for Canada or >10 for the EU are considered strong indicators of low potential for bioaccumulation . BCF is expected to increase with log Kow, increasing up to a maximum at approximately 7 .5, but then decreasing thereafter . Substances with very high log Kow (>9) are not expected to bioconcentrate greater than 5000 (for Canada) and with log Kow >10, BCF is expected to be <2000, these values being the thresholds for the B criterion in PBT/vPvB assessment under REACH . Therefore, for test substances beyond the application range of guidelines into force, the limit value does not allow a definitive conclusion on bioaccumulation potential and no method has yet been validated to demonstrate log Kow >9 . This poster illustrates an attempt to select new reference substances for the HPLC method, with higher log Kow values (7 to 10), in order to obtain adequate result for bioaccumulation assessment . The KowWin database was screened for homologous compounds, with linear increase of their log Kow as a function of carbon number, and with structural similarity compared to a given test substance . A total of 18 candidates between 6 .97 and 10 .89 was selected, among which 9 of analytical grade are commercially available . However, the HPLC method may be subject to limitations in this working range, and suitability of this new set of reference substances for high range log Kow determination have to be confirmed by experimentation before any update proposal of the guideline .

TH 300Poorly soluble cosmetic compounds - ecotoxicological assessment and substitution’ (ECOSM)S .K .E . Damme1, H .T . Ratte1, A . Schäffer1, H . Henner1, K . Rettinger2, J . Steber2, A . Coors3, T . Knacker3, C . Schulte4

1RWTH Aachen, Aachen, Germany2German Cosmetic Toiletry, Perfumery and Detergent Association, Frankfurt am main, Germany3ECT Oekotoxikologie GmbH, Flörsheim, Germany4German Federal Environment Agency, Dessau-roβlau, GermanyIn order to meet different product requirements, ‘Personal Care Products’ contain a set of various substances . Each substance is meant to fulfil a specific scope in the final product and therefore the substances may show a great variability in physico-chemical properties . ‘Poorly soluble substances’ are characterised by low water solubility (< 1 mg/l) and represent important ingredients of many cosmetic products . Due to their large use quantities a reliable assessment of their environmental behaviour and their toxicity to aquatic organisms becomes necessary, e .g . as requested by the European REACH-Regulation . However, at the threshold of the water solubility of such substances, ecotoxicity tests are very difficult to conduct . High lipophilicity and thus extensive adsorption of the substance to surfaces, e .g . to test vessels and organisms, complicates the test performance . The majority of existing standard tests provide inadequate test results that can hardly be used for an appropriate risk assessment . The ‘poorly solubles approach’ is one promising method to solve this issue . The basic assumption is that the concentration of highly lipophilic and poorly soluble substances and, thus, their exposure to aquatic organisms is very low in the aqueous phase of water bodies . As a result of an extensive analysis of ecotoxicological data, the so-called aquatic exposure threshold of no ecotoxicological concern (ETNCaqu) could be defined for inert substances with a narcotic mode of action . Neither acute nor long-term adverse effects are anticipated on aquatic organisms when concentration of such substances is below ETNCaqu = 1,9 µg/l . For the screening of potential ecotoxicological substances with PBT-properties (persistent + bioaccumulative + toxic), the ‘poorly solubles approach’ also seems to be pertinent . Due to its potential relevance for assessing the environmental risks of highly lipophilic substances, the scope of the project ECOSM is to develop the ‘poorly solubles approach’ to a pragmatic and reliable evaluation tool . In this context, existing aquatic ecotoxicological test methods have already been adapted to passive dosing techniques and several lipophilic compounds are being tested to validate the ‘poorly solubles approach’ .

TH 301A divergence of paths? Novel endpoints in hazard and risk assessmentE .M . Mihaich1, L .S . Ortego2, C .A . Staples3, N . Caspers4, G .M . Klecka5, S .S . Dimond6, S .G . Hentges7

1Environmental & Regulatory Resources, Durham, nc, United States of America2Bayer CropScience, Research triangle park, nc, United States of America3Assessment Technologies, Keswick, va, United States of America4Currenta, Leverkusen, Germany5The Dow Chemical Company, Midland, mi, United States of America6SABIC Innovative Plastics, Pittsfield, ma, United States of America7American Chemistry Council, Washington, dc, United States of AmericaApical responses on survival, growth, and reproduction have long been the mainstay of ecotoxicology research, integrating effects at the organism level . Yet, as science progresses, and there is increasing pressure to accelerate risk decisions and reduce animal use, biomarker endpoints and in vitro data use in early hazard assessment are taking us down a new path . The challenge is in relating these ‘omic’, cellular, and biochemical markers to adverse effects in a relevant and reproducible manner . Bisphenol A (BPA), is an important high production volume compound used in the production of epoxy resins and polycarbonate plastics . It is also one of the most studied, particularly for potential endocrine interactions . The database of ecotoxicological results for BPA includes traditional guideline acute and chronic data addressing population level effects of survival, growth and reproduction, as well as a variety of studies with secondary, often unique sublethal or biomarker-type endpoints, and a growing library of ‘omic’, cellular,