Pedosphere 24(2): 280–284, 2014

ISSN 1002-0160/CN 32-1315/P

c© 2014 Soil Science Society of China

Published by Elsevier B.V. and Science Press

Ecotoxicological Tools for Landfarming Soil Evaluation

in a Petrochemical Complex Area∗1

F. M. R. DA SILVA JUNIOR1,∗2, P. F. SILVA1, F. S. GUIMARAES1, K. A. DE ALMEIDA1, P. R. M. BAISCH2

and A. L. MUCCILLO-BAISCH1,3

1Laboratorio de Ensaios Farmacologicos e Toxicologicos, Instituto de Ciencias Biologicas, Universidade Federal do Rio Grande do

Sul (FURG), Av. Italia, km 8, Campus Carreiros, CEP 96201-900, Rio Grande-RS (Brazil)2Laboratorio de Oceanografia Geologica, Instituto de Oceanografia, Universidade Federal do Rio Grande do Sul (FURG), Av. Italia,

km 8, Campus Carreiros, CEP 96201-900, Rio Grande-RS (Brazil)3Programa de Pos graduacao em Ciencias Fisiologicas, Fisiologia Animal Comparada, Instituto de Ciencias Biologicas, Universidade

Federal do Rio Grande do Sul (FURG), Av. Italia, km 8, Campus Carreiros, CEP 96201-900, Rio Grande-RS (Brazil)

(Received July 28, 2013; revised January 9, 2014)

ABSTRACTThe demand for simple and rapid bioassays in ecotoxicological evaluations is of paramount importance in order to speed up

environmental monitoring programs. In this study we performed bioassays with lettuce seeds and two species of terrestrial isopods

(Armadillidium vulgare and Porcellio dilatatus) for the ecotoxicological assessment of a landfarming soil from a petrochemical complex

area. The solubilized content of test soil demonstrated a concentration-response type toxic effect on seed germination rate, and a delay

on germination, but showed toxic effect on seedlings wet weight only at the highest concentration. Toxic effects were also observed

in mortality rate and avoidance behavior of the two woodlice species. These results demonstrated the sensitiveness of the organisms

studied, and highlighted the possibility to use these bioassays in environmental monitoring programs in areas contaminated with fossil

fuels.

Key Words: Armadillidium vulgare, lettuce, Porcellio dilatatus, terrestrial woodlice

Citation: Da Silva Junior, F. M. R., Silva, P. F., Guimaraes, F. S., De Almeida, K. A., Baisch, P. R. M. and Muccillo-Baisch, A. L.

2014. Ecotoxicological tools for landfarming soil evaluation in a petrochemical complex area. Pedosphere. 24(2): 280–284.

The use of bioassays to detect alterations causedby toxic agents has become an important complementto the simple chemical analysis. Ideal bioassays musthave some particular properties, such as being robust,standardized, cheap and simple. They must also at-tempt to include different ecological/biological func-tions, to have a clear endpoint and to allow compari-sons between different sites, besides responding to en-vironmental stress and changes in such stress (Patonet al., 2005).

Ecotoxicological evaluations on soils have beenperformed mainly with aqueous fraction and usingaquatic organisms. Studies with terrestrial species havebeen focused on plants and invertebrates (earthwormsand springtails) (Sverdrup et al., 2003; Paton et al.,2005). These organisms are chosen based on the sen-sitiveness of their response. Such choice of organismsis normally related to the benefits of the soil and toecosystem functions such as water storage, decomposi-tion and nutrient cycling (Doran and Zeiss, 2000).

In this study, we performed bioassays with a ve-getable species (Lactuca sativa, lettuce) and with twospecies of terrestrial invertebrates which are impor-tant to the decomposition process in the soil (Ar-madillidium vulgare and Porcellio dilatatus, two com-mon Isopoda species). Germination, growth and dry orwet biomass are the parameters used in phytotoxicityevaluation of contaminated soils, which can be ana-lyzed by planting the seeds directly in the soil or usingaqueous fractions for watering the seeds (Wang andFreemark, 1995; Gong et al., 1999; Henner et al., 1999;P�laza et al., 2005; Smith et al., 2006). Lettuce is one ofthe most used vegetables in phytotoxicity assessment(Robidoux et al., 2004; Eom et al., 2007; Martı et al.,2007; Valerio et al., 2007).

Terrestrial invertebrates have become importantorganisms for monitoring polluted sites because theyplay important roles in decomposing organic matterand recycling nutrients. Among such invertebrates, te-rrestrial woodlice are particularly interesting because

∗1Supported by Brazil National Petroleum Agency and Petrobras (No. PRH-ANP/MME/MCT 27).∗2Corresponding author. E-mail: f.m.r.silvajunior@gmail.com.

ECOTOXICOLOGICAL TOOLS FOR SOIL EVALUATION 281

they are detritivore and accumulate heavy metals.Also, it is a group of organisms composed of speciesthat can be very resistant or even highly sensitive(Cortet et al., 2000). Mortality, growth and reproduc-tion rates and also their avoidance behavior have beenthe most important endpoints used in ecotoxicologi-cal studies with woodlice (Paoletti and Hassall, 1999;Jansch et al., 2005; Loureiro et al., 2005).

The objectives of the present study were to inves-tigate the responses of different terrestrial organismsto a soil from a petrochemical area treated by land-farming with the lettuce and woodlice bioassays andto indicate the recovery status of the site.

MATERIALS AND METHODS

Study area and measurements of polycyclic aromatichydrocarbons and heavy metals

The soil was collected from a waste treatmentsystem SICECORS in an area under landfarming bi-oremediation process at Polo Petroquımico do Sul, Tri-unfo, Rio Grande do Sul, Brazil, and was supplied byCompanhia Petroquımica do Sul (COPESUL). Thissoil was at initial treatment stage (less than one year),and was contaminated with many types of pollutants,including polycyclic aromatic hydrocarbons (PAHs)and heavy metals, listed in Table I. The landfarmingsoil had sand 65.4%, silt 13.7%, and clay 20.9%. Acontrol artificial soil with the same granulometry wasprepared to mix with landfarming soil.

PAH analysis was performed using gas chroma-tography coupled to mass spectrometry (GCMS) byinjecting 1 μL aliquot of the extracts, with a split/splitless injector (1:50) and an HP-5 fused silica capi-

llary column (60 m × 0.25 mm × 0.25 μm). Electronimpact mass spectra were obtained at 1 keV of ioni-zation energy. Helium was used as the carrier gas ata flow of 1 mL min−1. Temperature was programmedfrom 120 to 220 ◦C at 5 ◦C min−1, followed by a 10◦C min−1 increasing rate until it reached 280 ◦C. Theinterface temperature was 280 ◦C.

Copper and zinc in the soil samples were ana-lyzed by flame atomic absorption spectrophotometry(AASPerkin-Elmer 800, Uberlingen, Germany), whileelectrochemical atomization mode with Zeeman cor-rection was used in chromium, nickel, lead and arsenicanalysis in the soil samples.

Organisms test

Lettuce seeds from TopSeed Garden, Petropolis,Brazil, were submerged in diluted hypochlorite (10 gL−1), and rinsed in tap water before use. All lettuceexperiments were initialized on the same day and theywere performed with the seeds from the same batch.The woodlice A. vulgare and P. dilatatus were collec-ted in areas of organic farming and maintained in la-boratory conditions (temperature 28 ◦C, in the dark,and frequently moistened using water sprinklers). Onlyantenna-bearing adults of undistinguished sex were se-lected for the tests. No gravid females were used. Theanimals were kept in the laboratory in earthworm hu-mus and were fed with jambul tree leaves (Syzygiumjambolanum).

Acute toxicity test with lettuce

The soil was solubilized at six different concentra-tions (0, 5, 15, 50, 150 and 500 mg L−1) using mineralwater as solvent. The stock solution (500 g L−1) was

TABLE I

Contents of polycyclic aromatic hydrocarbons (PAHs) and heavy metals in the landfarming soil from a petrochemical industrial

complex area

PAH Concentration PAH Concentration Metal Concentration

μg kg−1 dry soil μg kg−1 dry soil mg kg−1 dry soil

Naphthalene 62.60 Benzo(a)anthracene 2 940.08 Nickel 19.49

2-Methyl naphthalene 71.87 Chrysene 5 348.95 Zinc 97.78

1-Methyl naphthalene 70.56 Benzo(b)fluoranthene 930.58 Lead 24.37

2,6-Dimethyl naphthalene 83.77 Benzo(k)fluoranthene 205.93 Copper 21.23

1,7-Dimethyl naphthalene < 1.66 Benzo(e)pyrene 860.07 Chromium 27.95

Biphenyl 115.48 Benzo(a)pyrene 838.45 Arsenic 3.61

Acenaphthylene 5 442.47 Perylene 226.40

Acenaphthene 118.56 Indeno(1,2,3-cd)pyrene 359.68

Fluorene 1 056.52 Dibenzo(a,h)anthracene 188.00

Dibenzothiophene 21.04 Benzo(g,h,i)perylene 343.62

Phenanthrene 3 422.89 ΣTotal PAHs 29 177.97

Anthracene 3 630.22 Σ2–3 rings PAHs 14 095.99

Fluoranthene 1 540.49 Σ4–6 rings PAHs 13 541.49

Pyrene 1 299.72

282 F. M. R. DA SILVA JUNIOR et al.

prepared in a soil:solvent proportion of 1:2 (w:v) ac-cording to Da Silva Junior et al. (2009) and it wasmechanically shaken for 24 h. All concentrations usedin the bioassays were obtained from this stock solution.Solutions of 0.02 mol L−1 CuSO4 were used as a posi-tive control. Every concentration was tested in 5 repli-cates using 25 lettuce seeds distributed in a Petri platecontaining a paper filter, to which was added 3 mL ofthe corresponding concentration solution. At the endof 5-d exposure, the rate of germination, daily germi-nation, and wet weight of the plants were recorded.

Acute toxicity test with woodlice

The test-soil was diluted with the control-soil tofive concentrations of 0, 30, 100, 300 and 1 000 gkg−1. Fifty grams of soil were placed into jars (8 cmheight, 7 cm diameter). Every concentration was testedin 6 replicates with 5 animals each. The animals wereexposed for 14 d with access to food (jambul treeleaves), at a temperature of 28 ◦C, in the dark and inconditions of high humidity. At the end of the exposureperiod, the number of dead organisms was counted.

Avoidance behavior response test

This test was divided in two sections: group andindividual. The animals were put in plastic boxes (18cm × 13 cm × 12 cm) divided in two compartmentswith the same dimensions, one containing the test-soiland the other the control-soil. For the group avoidancetest ten animals of each species were put in each box infive replicates. The individual avoidance test was madewith a single animal in each box in 10 replicates due tothe fact that woodlice present an aggregative behavior(Loureiro et al., 2009). The animals were kept in theboxes for 48 h, without food, at a temperature of 28◦C, in the dark and in conditions of high humidity. Af-ter the exposure period, a wall was put in the center ofthe box, and the number of animals in both test- andcontrol-soil was counted.

Data analysis

Parametric data in wet weight of seedling as well indaily and final germination in different treatments wereanalyzed by Dunnett’s test. The median effective con-centration (EC50) of the assay with lettuce seeds andthe median lethal concentration (LC50) of the assaywith terrestrial isopods were obtained using the Curve-Expert 1.4 software (Daniel Hyams, Hixson, USA)(Meng et al., 2010, Silva et al., 2012).

For the analysis of group and individual avoidancebehavior tests, the Guideline for Earthworm Avoidance

Test (ISO 17512) was used, in which the habitat func-tion of the soil is considered limited when more than80% of specimens are found in the control-soil. Statis-tical analysis was conducted using STATISTICA 8.0.

RESULTS

Acute toxicity test with lettuce

The EC50 value for lettuce seeds after 5 d was 309mg L−1 of aqueous extract of soil. The germinationrate of the positive control was null after 5 d of experi-ment (data not shown). The increase in concentrationof landfarming soil caused a diminished germinationrate and a delay on lettuce seed germination. Seedlingwet weight decreased only at 500 mg L−1, the highestconcentration tested (Table II).

TABLE II

Cumulative average percentages (n = 5) of germinated seeds ex-

posed for 5 d and wet weights of 5-d-old seedlings under different

concentrations of solubilized landfarming soil, using mineral wa-

ter as solvent, collected from a petrochemical industrial complex

area

Soil Seed germination Wet

concentration weight1 d 2 d 3 d 4 d 5 d

mg L−1 % g plant−1

0 70.4 76.8 84.0 85.6 88.0 0.170

5 59.2 66.4* 69.6 78.4 85.6 0.173

15 48.8 60.0* 68.8 78.4 85.6 0.170

50 49.6 60.0* 63.2 69.6* 73.6 0.183

150 41.6* 50.4* 53.6* 60.0* 61.6* 0.174

500 13.6* 26.4* 28.0* 36.8* 40.8* 0.086*

*Significant at P < 0.05 when compared to the control (0 mg

L−1) within each column.

Acute toxicity test with woodlice

The experiments with the two woodlice species wasconducted with a mixture of landfarming soil and con-trol soil at different concentrations. The LC50 valuesfor the two species were 74% (A. vulgare) and 27% (P.dilatatus), respectively. The landfarming soil (concen-tration 100%) caused a mortality rate of 70% for A.vulgare and 100% for P. dilatatus.

Avoidance behavior response test

There was no mortality during the avoidance be-havior response test. Both woodlice species escapedfrom the tested landfarming soil, in which less than20% of the animals were found in the contaminatedpart in the group test, whereas 20% of the animalswere found in the contaminated portion in the indi-vidual test. The results of these tests were similar forboth species tested (Fig. 1).

ECOTOXICOLOGICAL TOOLS FOR SOIL EVALUATION 283

Fig. 1 Percentages of the exposed test organisms Armadilli-

dium vulgare and Porcellio dilatatus in the landfarming soil from

a petrochemical industrial complex (test soil). The dash line

(20%) indicates the habitat function limit. Vertical bars re-

present the standard deviations of the means (n = 50).

DISCUSSION

Several studies have demonstrated the need for us-ing different organisms to evaluate environmental risksor remediation processes (Eom et al., 2007; Leitgib etal., 2007; Lors et al., 2010). In this study an acute toxi-city test using lettuce seeds and an avoidance behaviortest using two species of terrestrial woodlice were con-ducted to evaluate the bioremediation quality of a soilcontaminated with PAHs from a petrochemical com-plex area in the South of Brazil.

The evaluation of soil quality using phytotoxi-city is a common strategy (Keddy et al., 1995; Wangand Freemark, 1995), and bioassays with lettuce seedsare commonly used for the evaluation of contami-nated sites (USEPA, 1989). Bioassays with plants havedemonstrated sensitiveness while measuring the effi-ciency of bioremediation processes (Hamdi et al., 2006,2007). P�laza et al. (2005) studied 6 higher plants asbioremediation indicators of a soil contaminated by oil.They found that L. sativa growth was a more sensitivetoxicity indicator than germination itself. Eom et al.(2007) also highlighted the low sensitivity of lettuceseed germination rates as a phytotoxicity parameter,but they indicated that dry and wet weights are valu-able parameters, mainly the latter.

In our study the seedling wet weight was efficient todemonstrate phytotoxicity of the studied soil, but onlyat the highest concentration tested (500 mg L−1). Onthe other hand, germination after 5 d of incubationwas affected even at low concentration of the studiedsoil (≥ 100 g kg−1).

The strategy of evaluating daily germination in-creased the sensitiveness of the bioassay with lettuceseeds. For instance, after only 1 d of incubation, con-centrations of the landfarming soil above 30 g kg−1

were enough to reduce germination. Such evaluationhas already demonstrated to be an invaluable tool instudies of soils with moderate contamination (unpub-lished data). Thus, in the present study it shows to bea useful tool for the assessment of the process of reme-diation.

Similarly to the lettuce seed bioassay, bioassaysperformed with the two terrestrial woodlice speciesdemonstrated sensitiveness while detecting lethal andsub-lethal toxic effects. Both species of woodlice stu-died (A. vulgare and P. dilatatus) are found world-wide and can live in “aggregation” (Loyola e Silva andCoraiola, 1999). From the morphological point of view,they are part of two distinct groups: A. vulgare is aroller and P. dilatatus is a runner. In the avoidancebehavior test the responses of the two species were si-milar (escaping from the landfarming soil) and the soilprovoked a high rate of mortality in both species af-ter 14 d of exposure, which indicates an early stage ofbioremediation of the soil. Lethal and sub-lethal pa-rameters were demonstrated to be useful for determi-ning soil toxicity, in disagreement to the propositionof Geissen et al. (2008), who defended the use of sub-lethal parameters to estimate the efficiency of restora-tion techniques.

Even though terrestrial woodlice have been targe-ted in various studies of toxicity related to metals(Kohler and Eckwert, 1997; Jones and Hopkin, 1998;Odendaal and Reinecke, 2003; Godet et al., 2011) andpesticides (Ribeiro et al., 2001; Jansch et al., 2005),the use of these animals in studies of organic com-pounds derived from petroleum is restricted to bioaccu-mulation via feeding (Van Brummelen et al., 1996). Inour study, these organisms have demonstrated to besensitive enough to indicate the presence of toxiccompounds in the soil, among which are metals andpetroleum derived-hydrocarbons, and to reveal theearly stage of remediation in the soil studied.

CONCLUSIONS

The bioassays (lettuce seed germination, woodlicemortality and woodlice avoidance behavior) used inthis study highlighted the sensibility of the test or-ganisms for the assessment of soils contaminated byfossil fuels. They are suitable for environmental moni-toring programs that aim at following-up bioremedia-tion processes and environmental restoration.

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