Polycyclic aromatic hydrocarbon contamination and LUMIStox® solvent extract toxicity of marine...

Polycyclic Aromatic HydrocarbonContamination and Lumistox� Solvent ExtractToxicity of Marine Sediments in the northAegean Sea, Greece

Despina Papadopoulou, Constantini Samara

Environmental Pollution Control Laboratory, Department of Chemistry, Aristotle University ofThessaloniki, G-54124 Thessaloniki, Greece

Received 10 December 2001; revised 10 July 2002; accepted 7 August 2002

Abstract: Organic extracts of surface marine sediment collected from six sites within the bay of Kavala(north Aegean Sea, Greece) were used for determining priority pollutant polycyclic aromatic hydrocarbons(PAHs) and doing toxicity testing. PAH analyses and LUMIStox� acute toxicity measurements wereconducted in two sediment grain-size fractions: silt/clay (� 63 �m) and sand (63–2000 �m). Sixteen PAHconcentrations were found at low- to moderate levels, ranging from 44 to 166 ng/g dry weight in the finefraction and from 45 to 148 ng/g dry weight in the coarse fraction. Molecular indices revealed that PAHsin the bay sediment originate mainly from pyrolytic sources, but some petroleum influence was alsoevident. A comparison of sedimentary PAH levels with sediment quality guidelines (SQGs) indicated anabsence of acutely toxic concentrations. However, all sediment extracts were found to be toxic with theLUMIStox� acute toxicity test, with 15-min EC50s in the ranges of 1.0–4.0 and 1.1–4.5 mg of drysediment/mL for the fine and the coarse fractions, respectively. No significant correlations between EC50sand concentrations of individual or total PAHs was found, suggesting that chemical analysis of PAHsalone cannot be considered a reliable indicator of sediment toxicity. © 2002 Wiley Periodicals, Inc. EnvironToxicol 17: 556–566, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10089

Keywords: marine sediments; PAHs, LUMIStox�; sediment toxicity; SQGs

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitousenvironmental organic pollutants formed during incompletecombustion or in high-temperature pyrolytic processes in-volving fossil fuels or, more generally, materials containingcarbon and hydrogen. Their importance as an environmentalhazard derives from their potential carcinogenic and muta-genic properties (Neff, 1985). The U.S. Environmental Pro-tection Agency (U.S. EPA) has included 16 unsubstitutedPAHs in its priority pollutant list.

The occurrence of PAHs in the marine environment ismainly linked to anthropogenic activities rather than tonatural processes. Some of their most important sourcesinclude urban run-off, atmospheric deposition, accidentaloil leaks, and platform drilling (Manoli and Samara,1999; Witt, 1995). PAHs entering the marine environ-ment tend to adsorb and accumulate in the sedimentcompartment due to their low solubility, hydrophobiccharacter (log Kow � 3– 8) and high persistence (Law etal., 1997). The fundamental problem involved in assess-ing the significance of PAHs in sediments concerns theirbioavailability. Lower-molecular-weight PAHs, whichmay be acutely toxic, are less strongly adsorbed com-pared with the heavier species, which are carcinogenic

Correspondence to: Constantini Samara; e-mail: [email protected].

© 2002 Wiley Periodicals, Inc.

556

when ingested by marine organisms (IARC, 1983; Wood-head et al., 1999).

A variety of attempts have been made to establish asso-ciations between PAH concentrations in sediment and bio-logical effects on various organisms. Amphipodes seem tobe more acutely sensitive to sediment-associated PAHs thanare fish (Alden and Butt, 1987; Lotufo, 1997; Woodhead etal., 1999). Several attempts also have been made to use theavailable ecotoxicity data on PAHs to set numerical sedi-ment quality guidelines (SQGs; Chapman, 1989; Long etal., 1995; Swartz et al., 1995). Despite their limitations,SQGs are frequently used to rank and prioritize both sitesand chemicals of concern.

Comparisons between chemical analyses and sedimenttoxicity bioassays can be complex. Sediment in pollutedenvironments can be contaminated by hundreds of individ-ual compounds able to cause toxicological responses evenbelow analytical detection limits. Interactions among vari-ous contaminants may result in antagonistic or synergisticeffects that cannot be predicted based solely on the sum ofindividual toxicities of identified compounds. However, de-spite these limitations, comparisons between chemical dataand toxicity bioassays can elucidate patterns of anthropo-genic contamination with their resultant biological re-sponses that could not otherwise be provided by either typeof analysis alone (Jacobs et al., 1993). The use of organicsolvent extracts for both chemical analysis of priority or-ganic pollutants and toxicity bioassays provides a goodopportunity to evaluate the relationship between the pres-ence of organic contaminants and the toxicity of the sedi-ment (Tay et al., 1992; Demuth et al., 1993; Salizzato et al.,1997; Johnson and Long, 1998). Fractionation of sedimentextracts into single classes of compounds can provide ad-ditional assessment of the most toxic components (Ho andQuinn, 1993; Salizzato et al., 1998a, 1998b).

In this study surface sediments from the Bay of Kavala inthe north Aegean Sea, Greece, were examined through acombination of chemical analyses for PAHs and TOC andtoxicity testing of organic sediment extracts. Chemical andtoxicity measurements were carried out in organic solventextracts of two grain-size fractions: � 63 �m (silt/clay) and63–2000 �m (sand). The origin of the PAHs in the twosediment fractions was investigated using the molecularindex approach. The ecotoxicological significance of sedi-mentary PAH levels was evaluated by comparison withSQGs. Correlation analysis also was used for investigatingpossible relationships between the chemical and toxicitydata.

MATERIALS AND METHODS

Area Description

The Bay of Kavala in the north Aegean Sea (Fig. 1) has atotal surface area of 220 km2 and the length of its coasts is

about 40 km. Although fluvial discharges are missing, thebay serves as a sink for several contaminants deriving fromvarious local urban and industrial activities. The city ofKavala has about 70 000 inhabitants and is surrounded byseveral smaller residential communities. Domestic effluentsare biologically treated in two wastewater treatment plants;however, discharges of untreated sewage could not be ex-cluded. The city’s port is characterized by relatively inten-sive traffic of ships, ferries, and tankers. A smaller port,used mainly for tourism and as a commercial fishery, is atthe eastern edge of the bay in a leisure area. A medium-sized industrial district that includes two major industries, aphosphate fertilizer–producing plant and an oil refinery, isby the inner part of the bay. Petroleum exploitation isconducted outside the bay approximately 15 km southeastof one of the sampling sites (S6). Six sampling sites withinthe bay (described in Table I) were selected for sedimentcollection.

Sampling and Sample Preparation

Sediment samples were collected from each sampling siteonce a month from February through April 1999. Sampleswere taken from the top 5-cm layer of bottom sedimentusing a stainless steel Van-Veen grab. Several grabs (about10) from each sediment site were put into 2-L solvent-rinsedamber glass jars with minimal headspace. Samples weretransported on ice to the laboratory within 8 h of collection.On receipt, they were wrapped in aluminum foil and frozenat about 20°C until processing (storage time � � 1 week;Jacobs et al., 1993). Before extraction, frozen samples werethawed and homogenized until textural and color homoge-neity was achieved. Homogenized sediments were mechan-ically sieved through 63-�m and 2000-�m sieves. Fractionsof � 63 �m (silt/clay) and 63–2000 �m (sand) were freeze-dried before used for chemical analysis and toxicologicaltesting. All stainless steel and glass equipment used forsediment treatment and storage was precleaned with acetoneand hexane.

Fig. 1. Map of Kavala Bay with sampling sites.

PAH CONTAMINATION AND SOLVENT TOXICITY OF SEDIMENT IN NORTH AEGEAN 557

Sample processing for PAH analysis and toxicity testingis schematically described in Figure 2. Briefly, PAHs wereextracted from freeze-dried sediment samples using ultra-sonication with acetonitrile (HPLC grade, T. J. Baker). Thepurification of the extracts was tested using solid-phaseextraction cartridges filled with octadecyl-bonded silica(C18). However, because only insubstantial improvement ofthe chromatographic determination of PAHs was observed(given the high selectivity of the fluorescence detection;Manoli and Samara, 1996), sediment extracts were notcleaned up before HPLC analysis.

All sediment extracts used for toxicity measurementwere first solvent exchanged to dimethyl sulfoxide (UVSpectrophotometric quality, T. J. Baker). DMSO is fre-quently used as carrier solvent because of its low toxicity,low volatility, and low freezing point (Cambell et al., 1992,Jacobs et al., 1992, Jacobs et al., 1993; Johnson and Long,1998,). The final concentration of all test samples was 1% in

DMSO extract. Extraction blanks were prepared in an iden-tical manner but without sediment. The DMSO solubiliza-tion efficiency was improved by a 10-min sonication. Afterchanging the solvent, PAH analysis was again performed tomeasure the actual amount of contaminants redissolved inDMSO. The mean recovery after redissolution was calcu-lated as � 80% for all PAHs.

Determination of PAHs

HPLC coupled with programmable fluorescence detectionwas employed for the determination of PAHs in sedimentextracts using a Hypersil Green PAH column (100 � 4.6mm, 5 �m particle size, carbon loading of 13.5% � 0.5%)and a guard column packed with the same material. Themobile phase was a CH3CNOH2O gradient starting with a50% (v/v) CH3CN concentration during the first 5 min andgradually being raised to 100% CH3CN in between 5 and 20min. The final composition was maintained for an additional10 min. An equilibrium time of 10 min was applied betweensuccessive injections. The mobile-phase flow rate was 1.5mL/min. Solvents were degassed in an ultrasonic apparatusbefore chromatography. In addition, helium was continu-ously passed through the mobile phase during chromatog-raphy. The column temperature was 30°C and the injectionvolume 20 �L. The fluorescence detector program included5 excitation/emission wavelength changes programmed for0 min (250 nm/245 nm), 10.4 min (240 nm/425 nm), 12.6min (265 nm/380 nm), 18 min (290 nm/430 nm), and 24.4min (300 nm/500 nm). The NIST SRM 1647c containing 16PAHs included in the EPA’s priority pollutants list was usedas a calibration mixture. Acenaphthylene, although con-tained in the standard, is only weakly fluorescent, and thusnot determined. Instead, benzo[e]pyrene was added becauseit is frequently used as a reference PAH.

Recoveries of PAHs from spiked sediment samples werein the range of 78% (indeno[1,2,3-cd]pyrene) and 130%(pyrene). Only naphthalene showed a relatively lower re-covery (59%). Procedural blanks analyzed in the samemanner as real samples showed concentrations lower thandetection limits for all PAHs. The accuracy of the experi-

TABLE I. General description of sampling sites and sediment samples

SamplingPoint Main Activities in the Area

WaterDeptha

(m)Silt/Claya

(%) Sanda (%)Gravela

(%)

S1 Commercial shipment, slightly urbanized and leisure area 11.7 � 0.6 29.0 � 2.5 31.8 � 2.6 39.3 � 3.1S2 Sewage treatment plant outfall 25.8 � 2.1 18.6 � 3.1 47.6 � 2.2 33.9 � 2.2S3 Harbor activities, urbanized area 11.6 � 0.7 14.8 � 2.9 51.6 � 3.8 33.6 � 4.3S4 Sewage treatment plant outfall 18.7 � 1.3 20.3 � 3.2 45.3 � 2.2 39.0 � 6.3S5 Phosphoric fertilizer production 12.6 � 0.2 30.9 � 8.3 43.7 � 3.2 30.5 � 4.2S6 Petroleum refining terminal 13.4 � 1.9 47.3 � 5.4 40.6 � 6.7 11.9 � 2.8

a Mean � SD of three samples collected once a month in the period February–April 1999.

Fig. 2. Procedural scheme for PAH analysis and sedimenttoxicity measurement.

558 PAPADOPOULOU AND SAMARA

mental procedure was tested by analyzing two certifiedreference materials (CRMs), both obtained from BCR:the BCR-088 (sewage sludge) and the BCR-524 (contami-nated industrial soil) containing seven of the targeted com-pounds: pyrene, benzo[a]anthracene, benzo[e]pyrene, ben-zo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene,and indeno[1,2,3-cd]pyrene. Replicate analyses of theCRMs yielded similar errors � 5%–15% for individualanalytes.

Toxicity Measurements

A LUMIStox 300 luminometer, a LUMIStherm incubator,and the nonpathogenic bacteria Vibio fischeri LCK 486, allobtained from Dr. Lange, GmbH, Germany, were used fortoxicity measurements. The LUMIStox� acute toxicity testwas performed according to the standard procedure outlinedin Lange (1998). Briefly, the procedure included reconsti-tution of freeze-dried bacteria, preparation of a homogenousbacterial suspension, and measurement of the initial biolu-minescence. Control solutions (8.0 mg of K2CrO7/L and 7%NaCl) were used with each batch of bacteria to verifybacteria and reagent quality. Bacterial suspensions werethen exposed to 9 dilutions of each DMSO extract, osmot-ically adjusted to 2% NaCl, and were incubated for specifictime intervals (5, 15, and 30 min) under a constant temper-ature (15°C � 1°C). After each incubation time the changein bioluminescence was recorded. Light readings were nor-malized using blank solutions containing the same NaCl andDMSO concentration as test samples. The EC50s of pureDMSO and solvent blanks were 5.81% and 5.24%, respec-tively, consistent with reported values (Schiewe et al., 1985;Jacobs et al., 1992). Gamma values were adjusted for thecontribution of solvent vehicle. The effective concentrationpercentages of tested solutions resulting in a 50% decreasein bioluminescence (EC50) were calculated following theprocedures outlined in Lange (1998). EC50s (%) were sub-sequently transformed to dry weight equivalents, represent-ing the mass of dry sediment that, if extracted and solventexchanged, would result in a 50% decrease in biolumines-cence. The use of dry weight equivalents normalizes sam-ples for the amount of solid material present in bulk sedi-ment (Jacobs et al., 1992).

TOC Analysis

The organic carbon content (TOC) of sediments was deter-mined in freeze-dried subsamples by the Wakley–Blackmethod, adopted and modified by Jackson (1958). Theprecision of analysis was �5% based on replicate measure-ments.

RESULTS

PAH and TOC Concentrations in Sediments

The concentrations of PAHs found in the two grain-sizefractions of the examined sediments are summarized inTable II. All 16 EPA PAHs (except acenaphthylene) plusB[e]Py could be identified and quantified in tested sedi-ments. The distribution of PAHs showed similar patterns inthe two sediment fractions. Except from Np, highest abun-dances were exhibited by the 3–4 ringed PAHs Ph, Fl, andPy, as well as by the 5–6 ringed species B[e]P, B[b]F andIPy. The least abundant PAHs were dB[a,h]A and An.One-way analysis of variance followed by the Tukey’s posthoc test were applied to sedimentary PAH data to identifysignificant differences among sites. The concentrations oftotal PAHs (�PAH) in the silt/clay fraction (44–166 ng/gdry wt) exhibited significant differences (at the 0.05 level)only between S1 and S3/S4, whereas strong similarity wasobserved for the pairs S1–S6 and S2–S5. Concerning thesand-associated �PAH content (45–148 ng/g dry wt), dif-ferences were significant only between S5 and S6, whereasstrong similarities were found among S1, S2, S3, and S6. Thesum of carcinogenic PAHs (�PAHcarc) exhibited a distri-bution pattern similar to total PAHs.

The mean TOC content ranged between 0.62% and1.89% in the silt/clay fraction and between 0.88% and1.96% in the sand fraction (Table II). The observed valueswere consistent with those reported for other locations in theAegean Sea with limited anthropogenic influence (An-gelides and Aloupi, 2000; Gogou et al., 2000; Zabetoglou etal., 2002).

Sediment Toxicity

The DMSO extracts of all sediment samples indicated thepresence of acutely toxic substances. No significant differ-ence between 15 and 30 min of bacteria exposure times wasobserved, suggesting that the toxic effect was complete after15 min (Guzzella, 1998). In Table III, 15-min EC50s aregiven as percent concentrations of tested solutions and asdry weight equivalents (mg dry sediment/mL). As shown,toxicity did not display substantial variation among sites.Furthermore, it was not consistently associated with a par-ticular sediment fraction. The most toxic samples were thesilt/clay fraction from S4 and the sand fraction from S5 andS6. The least toxic sample was the sand fraction from S2.

DISCUSSION

Distribution of Sedimentary PAHs—The Roleof Organic Matter

In general, the distribution of sediment contaminants indifferent grain sizes can provide useful information for the


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assessment of the ecological risk imposed on benthic organ-isms whose activity is connected with certain grain fractions(Foster and Wright, 1988).

The distribution of PAHs in the sediments examined inthis study was not consistently associated with a specificgrain-size fraction. The silt/clay–to–sand �PAH ratiosranged from 0.8 at S1 to 2.5 at S3. The distribution of PAHsin different grain-size sediment fractions depends on phys-icochemical factors, such as the organic carbon content andthe binding capacity of particles (Evans et al., 1990; Guinanet al., 2001). Literature data on the grain-size distribution ofPAHs in sediments are conflicting. In some studies it wasfound that PAHs were mainly associated with silt and clayfractions (Di Toro et al., 1991; Maruya et al., 1996),whereas in others, PAHs were associated with large-sizefractions that contained high particulate organic matter ofcharcoal, plant detritus, or fecal pellets (Simpson et al.,1998; Wang et al., 2001). Organic carbon is the predomi-nant controlling factor in determining the partitioning be-havior of PAHs in the different particle-size fractions of asediment (Wang et al., 2001). Despite some exceptions(Zhou et al., 2000), a positive relationship between sedi-mentary organic matter and PAH contents has been reported(Simpson et al., 1998; Kim et al., 1999; Gogou et al., 2000;Wang et al., 2001). In the present study a strong linearrelationship between total PAH and TOC concentrationscould be observed in both sediment fractions (Fig. 3). All

PAH species showed significant (at the 0.05 level) positivecorrelation with TOC, except naphthalene and fluorene.

Comparison with Literature PAH Data

There is a relatively large body of sedimentary PAH data forthe Mediterranean Sea, particularly for its northwestern part(Lipiatou and Saliot, 1991; Domine et al., 1994; Guzzellaand De Paolis, 1994; Benlachen et al., 1997; Lipiatou et al.,1997; Salizzato et al., 1997; Baumard et al., 1998a; Eljarratet al., 2001; Notar et al., 2001). Significantly fewer data areavailable for the eastern Mediterranean (summarized inTable IV). A quantitative comparison across reported PAHdata is difficult because of variances in the number and typeof individual species determined in each study, the sedimentfraction analyzed, and the analytical methods used. Briefly,total PAH concentrations range from very low in offshoreareas and unaffected coasts to very high in the vicinity ofurban centers, industrial sources, or river outflows. ThePAH levels found in the present study are consistent with

TABLE III. Toxicity of the organic extractsof sediment fractions

SamplingSite

EC50 (%)EC50 (mg drysediment/mL)

Silt/Clay Sand Silt/Clay Sand

S1 7.0 � 2.8 3.0 � 1.4 3.5 � 1.4 2.3 � 1.1S2 8.0 � 2.8 2.5 � 0.7 4.0 � 1.4 1.9 � 0.5S3 4.0 � 2.8 6.0 � 4.2 2.0 � 1.4 4.5 � 3.2S4 2.0 � 0.1 4.0 � 2.8 1.0 � 0.0 3.0 � 2.1S5 2.5 � 0.7 1.5 � 0.7 1.3 � 0.4 1.1 � 0.5S6 2.5 � 0.7 1.5 � 0.7 1.3 � 0.4 1.1 � 0.5

Mean � SD of three samples collected once a month in the periodFebruary–April 1999.

Fig. 3. PAH versus TOC sediment contents.

TABLE IV. Total PAH concentrations in sediments from eastern Mediterranean Seaa

Location �PAH (ng/g dry wt) Grain Size Fraction Pollution Level Reference

Ismit Bay, Turkey 2500–25000 whole sediment high to very high Tolun et al., 2001Thermaikos Gulf, Greece 580–930 whole sediment moderate Kilikidis et al., 1994Aegean Sea, Greece—open sea sites 15.3–145 whole sediment low to moderate Hatzianestis and Sklivagou, 2001—estuarine sites 185–654Cretan Sea, Greece 14.6–158 whole sediment low to moderate Gogou et al., 2000Bay of Kavala, Greece 44–166 �63 �m low to moderate This studyBay of Kavala, Greece 45–148 63–2000 �m low to moderate This study

a Pollution levels are assigned as low: 0–100 ng/g, moderate: 100–1000 ng/g, high: 1000–5000 ng/g, very high: �5000 ng/g.


those reported for open sea sediments but lower than thePAH levels found in estuarine sediments of the north Ae-gean Sea (Hatzianestis and Sklivagou, 2000) According toBaumard et al. (1998a), pollution levels with PAHs can becharacterized as low, moderate, high, and very high whentotal PAH concentrations are 0–100 ng/g, 100–1000 ng/g,1000–5000 ng/g, and � 5000 ng/g, respectively. Based onthis classification, the sediments from the Bay of Kavala canbe considered slightly to moderately polluted with PAHs.

Identifying Sedimentary PAH Origin

Molecular indices of isomeric PAH compounds are fre-quently used to assess the sources of PAHs in sediments(Baumard et al., 1998a, 1998b; Woodhead et al., 1999;Soclo et al., 2000). The relative concentrations of isomericcompounds are temperature dependent, thus indicating theprocess (pyrolytic-high temperature process or petrogenic-low temperature process) through which PAHs were gener-ated. Often, the pyrolytic fingerprint is so strong that thepetrogenic or diagenetic profiles cannot be observed insediments.

Molecular ratios applicable to this study are shown TableV. The one-way analysis of variance followed by theTukey’s post hoc test indicated similar ratio values amongsites, suggesting a similar PAH origin. Statistically signifi-cant differences (at the 0.05 level) were only observedbetween S1 and S4 for the Fl/Py ratio in the silt/clay fractionand between S1 and S5 for the Ph/An ratio in the sandfraction. When the sedimentary PAH ratio values are com-pared with the corresponding values in specific PAHsources (Baumard et al., 1998a, 1998b; Woodhead et al.,1999; Soclo et al., 2000; Notar et al., 2001) a major pyro-lytic origin of sedimentary PAHs throughout the Bay ofKavala might be suggested. However, for some sites, suchas S5 and S6, inputs of PAHs from petroleum sources couldnot be excluded.

Investigations have also shown that PAH profiles maychange with distance from discharge because of the varyingtransport behavior and incorporation mechanisms of indi-vidual PAHs in sediments (Naes et al., 1995). Becauseisomeric PAHs have different reactivities, their ratios canenable the recognition of major transport pathways. Thuslow values of the Ph/An or the B[a]P/B[e]P�B[a]P ratiosmight indicate the occurrence of less “reworked” PAHinputs deriving from local sources (Woodhead et al., 1999;Gogou et al., 2000). In the present study the lowest Ph/Anratio value was observed at S4, which is in the immediatevicinity of the wastewater treatment plant outflow.

Ecotoxicological Significance of PAHConcentrations in Sediments

Sediment quality guidelines (SQGs) have been developedfor several sediment contaminants, including PAHs (Wood- T

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head et al., 1999). Although many of these values areconsidered site-specific and are not intended for use asregulatory guidelines, they provide useful benchmarks forassessing the potential toxicity of contaminated sediments.In essence, SQGs involve either weight-of-evidence ap-proaches based on field and laboratory observations ofbiological effects (e.g., Long et al., 1995; MacDonald et al.,1996) or methods based on variations of the equilibriumpartitioning (EqP) approach, which calculate the interstitialwater concentrations and compare them with “safe” or“effect” concentrations for the relevant chemicals in water(e.g., Di Toro et al., 1991; Swartz et al., 1995). ExistingSQGs for PAHs in marine sediments are presented in TableVI. The comparison of sedimentary PAH data with thesecriteria indicates that there are substantial margins of safety.Thus, PAHs would not be expected to be a primary causefor biological/ecological degradation in Kavala Bay.

Toxicity Versus Chemistry Data

Sediment toxicities measured in the present study (1.0–4.5mg ds/mL) in general are consistent with the toxicitiesfound by other investigators in sediment extracts containingall classes of pollutants (i.e., extracts not purified fromsulfur or other contaminants before toxicity measurement).Guzzella et al., (1998), employing the LUMIStox� bioassayin DMSO extracts of silt/clay sediment fractions from thePo River found 15-min EC50s in the range of 0.56–1.96 mgds/mL. Demuth et al. (1993), applying the Microtox� bio-assay on whole sediment extracts in absolute ethanol, found

15-min EC50s between 0.42 and 1.3 mg ds/mL for BostonHarbor sediments and between 1.4 and 8.9 mg ds/mL for theHudson–Raritan Bay estuary. It is worth noting that sedi-ments tested by Demuth et al. (1993) contained 3–100 timeshigher PAH concentrations than the sediments tested in thepresent study, as well as considerable concentrations ofPCBs and pesticides.

Sedimentary sulfur has been reported to be a significantcontributor to the toxicity of sediment extracts (Jacobs et al.,1992; Salizzato et al., 1997). Salizzato et al. (1998a) ob-served that, although tests with untreated sediment extractsshowed a total loss of bioluminescence, the toxic effect waslower after sulfur elimination, and no toxicity was found infractions containing separated mixtures of PAHs or PCBs.Sulfur was found to be the most toxic component of thesediments tested in that study, with an EC50 of 0.022 �g/mLcompared with 0.5 and 1.4 �g/mL, respectively, for thePAH- and PCB-containing fractions (Salizzato et al.,1998a).

The EC50s of individual PAHs in DMSO solutions asmeasured by the Microtox� bioassay have been found in therange of 0.26 (B[a]A)–33.4 (An) mg/mL (Jacobs et al.,1993). The EC50s measured by Johnson and Long (1999)using the same test ranged between 0.34 (acenaphthylene)and 10.7 (B[a]Py) mg/mL. The two-ringed PAHs weremore toxic than the five-ringed species; however, the lattertended to be more acutely toxic in complex mixtures withother PAHs, pesticides, or PCBs. In any case, in the litera-ture the EC50s of PAH solutions in DMSO are at least 2orders of magnitude higher than the total PAH concentra-

TABLE VI. Comparison of sedimentary PAH concentrations with SQCs (ng/g dry wt)

PAHsConcentration

Range TELa PELa ER-Lb ER-Mb AETc WSSQd

Np 2.8–3.0 34.6 391 160 2100 500 2000Ace 1.4–7.1 6.71 88.9 16 500 150 320F 1.1–2.6 21.2 144 19 540 350 460Ph 1.8–14 86.7 54.4 240 1500 260 2000An 0.19–2.6 46.9 245 85 1100 300 4400Fl 2.0–30 113 1494 600 5100 1000 3200Py 1.7–26 153 1398 670 2600 1000 20000B[�]A 0.83–16 74.8 693 260 1600 550 2200Chr 0.49–19 108 846 380 2800 900 2200B[e]P 1.1–40 — — — — — —B[b]F 1.5–23 — — — — — —B[k]F 0.72–9.8 — — — — — —B[�]P 1.4–27 88.8 763 430 1600 550 2200dB[�,h]A 0.15–4.6 6.22 135 63 260 100 240B[ghi]Pe 0.98–18 — — — — — —IPy 3.2–32 — — — — — —�PAHs 44–166 1684 16770 4022 44792 22000 —

a Threshold effect levels and probable effect levels (MacDonald et al., 1996).b Effects range–low and effects range–median values (Long et al., 1995).c Apparent effects threshold (Long and Morgan, 1990).d Washington State Sediment Quality Criteria (1991).


tion corresponding to the EC50s of the sediment extractstested in the present study (0.46–3.18 ng/mL). As a con-clusion, the toxicity measured in our sediment extractsmight be attributed to other coextracted toxicants (e.g.,sulfur, PCBs, aliphatic hydrocarbons, pesticides, etc.) ratherthan to sedimentary PAHs. Although PAHs are reported asthe major semivolatile organic priority pollutants in sedi-ments, organic priority pollutants represent only a fractionof the total sediment contaminants (Jacobs et al., 1993).

There has been some evidence supporting a relationshipbetween solvent-extracted sediment toxicity with field tox-icity data. A significant association between Microtox�toxicity of solvent sediment extracts and sums of aromatichydrocarbons, naphthalenes, and chlorinated hydrocarbonswas found in some studies (Schiewe et al., 1985). However,in other studies individual or total PAH concentrations werepoor indicators of sediment extract toxicity (True and Hey-ward, 1990; Ho and Quinn, 1993; Jacobs et al., 1993). Thisinconsistency may be a result of the different sedimentsexamined in each study having varying types and degrees ofcontamination. The statistical procedure also seems to havea significant impact on the subsequent interpretation be-cause in some instances PAHs were found to be signifi-cantly associated with toxicity in the Spearman correlationprocedure but not in the linear correlation procedure (Jacobset al., 1993; Schiewe et al., 1985).

In this study both Spearman rank order correlation andlinear correlation analyses were applied to make compari-sons. Both procedures yielded correlation coefficients thatwere not significant at the 0.05 confidence level. Althoughthe negative Spearman correlation coefficients between theEC50s and certain PAHs in the sand fraction (0.37 withNp, 0.29 with Ace, 0.26 with Fl and Py, 0.25 withB[b]F) might indicate some relationship, it is clear that PAHconcentrations alone cannot explain the variation in toxicityof tested sediments. Further investigation is needed to beable to attribute the sediment toxicity measured to specificsediment contaminants.

CONCLUSIONS

Surficial sediments from the Bay of Kavala in the northAegean Sea were examined chemically and toxicologicallyusing organic extracts of silt/clay and sand fractions forPAH analysis and LUMIStox� acute toxicity testing. Chem-ical analysis indicated low to moderate pollution of sedi-ments with PAHs. Comparison with numerical SQGs indi-cated the absence of acutely toxic concentrations ofsedimentary PAHs, suggesting that PAHs are not likely tobe the primary cause of the biological/ecological degrada-tion in Kavala Bay. Toxicity was exhibited by all sedimentextracts; however, that toxicity should be attributed to alltoxicants present as well as to antagonistic or synergisticeffects. Toxicity values were not significantly correlated

with PAH concentrations, thus suggesting that chemicalanalysis for PAHs alone is not a reliable indicator of sedi-ment toxicity.

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