Ecological risk assessment of contaminated soils through direct toxicity assessment

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Ecotoxicology and Environmental Safety 62 (2005) 174–184 www.elsevier.com/locate/ecoenv

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Ecological risk assessment of contaminated soils through direct toxicity assessment Marı´ a Dolores Ferna´ndeza,, Ekain Cagigalb, Marı´ a Milagrosa Vegac, Arantzazu Urzelaib, Mar Babı´ na, Javier Proa, Jose´ Vicente Tarazonaa a

Laboratory for Ecotoxicology, Department of the Environment, INIA, Ctra. A Corun˜a, km. 7.5, 28040 Madrid, Spain b LABEIN, Unidad de Construccio´n y Medio Ambiente, Cuesta de Olabeaga 16, 48013 Bilbao, Spain c ERA-Consult., P.O. Box 3350, 28080 Madrid, Spain Received 11 March 2004; received in revised form 6 October 2004; accepted 29 November 2004 Available online 29 January 2005

Abstract A microcosm (MS-3) with a multispecies soil system is introduced as an experimental tool for direct toxicity assessment of contaminated soils. The capacity of MS-3 to determine soil ecotoxicity potential was evaluated using samples from three sites contaminated with organic and/or inorganic compounds. Soils were toxic to soil-dwelling organisms (earthworm, plants, and microorganisms) and to aquatic organisms (algae and RTG-2 cell fish). As expected, responses varied substantially among different soils and organisms. The application of this evaluation system provided complementary information to the chemical characterization. For soils containing metals the toxic response was lower than predicted from total metal concentrations. For hydrocarbons, the toxicity response agreed with estimated values. The induction of EROD activity suggested the presence of dioxinlike compounds, which had not been addressed in the chemical characterization. The proposed multispecies system affords the measurement of 11 endpoints covering three soil and three aquatic taxonomic groups, reproduces soil conditions and gradients, and appears as an excellent complementary tool to chemical analysis for characterization of contaminated sites. r 2005 Elsevier Inc. All rights reserved. Keywords: Soil characterization; Bioassays; Microcosm; Contaminated soil; Soil toxicity

1. Introduction Problems of contaminated soils are currently an important issue that can potentially affect terrestrial and aquatic communities, owing to the drainage and surface runoff of toxic substances in water from contaminated sites. Actual environmental hazard assessment of soils is usually performed exclusively by chemical analysis (Bundesanzeiger, 1999; Ha¨mmann et al., 1998; Ministry of Housing, Spatial Planning and the Environment, 2001). The spectrum of the analyzed substances is selected based on a historical background of the area. However, not all the present contaminants Corresponding author. Fax: +34913572293.

E-mail address: [email protected] (M.D. Ferna´ndez). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.11.013

in the soil, including metabolites or transformation byproducts, may be considered during the analysis, resulting in an underestimation of the real environmental risk. Furthermore, the chemical analysis does not allow an integration of the combined effects produced by the chemical mixture present at a polluted site. In addition, total concentrations can overestimate the real risk, as aging processes can strongly reduce the bioavailability and, subsequently, the toxicity of pollutants. Bioassays try to integrate these effects and help in evaluating the risk associated with exposure to bioavailable substances present in a soil. Ecotoxicological analyses are recommended for estimating the risk to ecological receptors associated with contaminants in soils (Loibner et al., 2003; Stephenson et al., 2002).

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As a result, there is an increasing need to incorporate toxicity tests in risk assessments and hazard identification. The toxicity tests available for assessing polluted sites were first described by the US Environmental Protection Agency (US EPA) (1989, 1992) with the European perspective summarized by Van Straalen and Løkke (1997). In contrast to aquatic toxicity testing where test protocols have been standardized, testing protocols for soils still lack harmonization. During the early 1980s, acute toxicity tests for plants and earthworm were developed jointly by the European Union (EU) and by the Organization for Economic Cooperation and Development (OECD), mainly to satisfy the regulatory requirements for registration of new chemicals. Later, Greene et al. (1989) recommended a battery of tests to assess soils at contaminated sites. Additionally, aquatic toxicity tests were recommended for assessing the toxicity of soil leachates. A review of the toxicity tests available for ecotoxicity assessment of contaminated sites was compiled by Stephenson et al. (2002). The alternative to a battery of independent toxicity test is the use of multispecies systems. Among soil microcosms two options are available: intact soil cores with autochthonous soil organisms (US Environmental Protection Agency (US EPA), 1996; Sheppard, 1997; Knacker et al., 2004) or artificial assemblages adding soil organisms on sieved soil columns (Salminen and Haimi, 1997; Chen and Edwards, 2001; Ferna´ndez et al., 2004). These systems have been used for testing potentially harmful substances. In this paper, we evaluated the potential of a multispecies soil system as an experimental tool for developing an assessment strategy through direct toxicity assessment. This soil microcosm system, known as ‘‘Multispecies Soil System’’ (MS-3), enables the evaluation of toxic effects of contaminated soils to organisms living in soils and the potential for leaching of toxic substances through the soil column. The soil microcosms system was initially designed in our laboratory for testing pure chemicals and complex mixtures (Carbonell et al., 1998; Ferna´ndez et al., 2004). This paper presents the applications of the

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experimental design for testing contaminated soils using samples collected from hot spots contaminated with organic and/or inorganic compounds.

2. Materials and methods Soil ecotoxicity was assessed by the multispecies soil system (MS-3). This system consists of soil columns coupled to a leachate collector system. Representative soil macroorganisms (plants and terrestrial invertebrates) are introduced into these soil columns and exposed to chemicals under simulated realistic environmental conditions. To ensure conservation of the microbial community, soils are manipulated following procedures similar to those recommended for soil degradation studies. During the exposure period, the system is watered to simulate rainfall events, and artificial sunlight is provided for plant growth and to provide a photoperiod (16 h daylight and 8 h darkness) and temperature gradient approaching natural conditions. Leachates are collected and tested for toxicity using representative aquatic organisms (i.e., algae, daphnia, and fish cell lines) for which test procedures have been standardized. 2.1. Soil samples Soil samples were collected in a highly polluted industrial area of Vizcaya (Spain). Soils were air-dried and sieved (2-mm mesh). Table 1 details the physicochemical characteristics and chemical concentrations of these soils. Soil 1 and soil 2 were contaminated with several metals; soil 1 showed relatively high metal concentrations compared to soil 2 where metal concentrations were lower. Soil 2 also presented levels of polychlorinated byphenyls (PCB) and hexachlorocyclohexanes (HCH). Soil 3 contained low concentrations of metals but in addition, it has a high concentration of petroleum hydrocarbons derived from mineral oil. Control soil was collected from a surface layer of a field located near Madrid (Spain) and used as control

Table 1 Main physicochemical characteristics and soil chemical concentrations in the control and test soils Texture

Control soil Soil 1 Soil 2 Soil 3

Metals (mg/kg)

Clay (%)

Silt (%)

Sand (%)

pHw (1–2.5)

Organic matter (%)

As

Cd

Cu

Pb

Zn

7.8 17.4 12.3 5.3

18.8 19.7 29.4 6.9

73.4 62.9 58.3 87.8

7.27 5.56 6.9 7.94

1.9 17.5 10.3 14.6

nm 2492 484 nm

0.2 209 44 1

27 13547 372 1376

31.3 24127 4084 151

62 7458 1056 872

nm, not measured; nd, not detected.

PCB (mg/kg)

HCH (mg/kg)

TPH (mg/kg)

nm nm 189 nm

nm nm 43 nm

nd nm nm 65335

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treatment and for the dilution series. This soil fulfilled the conditions outlined by the OECD (OECD (Organisation for Economic Cooperation and Development), 2000) for use as control soil in microorganism assays (sand 470%, pH 5.5–7.5 and organic carbon content ranging from 0.5% to 1.5%). A reference soil was prepared to use as an additional control in the toxicity assays with nondiluted samples of soil 2. In order to obtain this soil, kaolin clay (50 g/kg dry soil), peat (79 g/ kg dry soil), mold (173 g/kg dry soil), and control soil were mixed thoroughly. A mixture of peat and mold was used to achieve a soil pH similar to soil 2 (pH 6.9). Properties of the reference soil (pH, soil organic matter, and clay contents) were those described to soil 2. 2.2. Chemical analysis Owing to the historical use of the site, soils were analyzed for heavy metals, mineral oil, polychlorinated byphenyls and hexachlorocyclohexanes. For the determination of metal concentrations, soil samples were digested in aqua regia by means of MAE (microwaveassisted extraction) using a microwave oven. After filtration, metals in the solution were analyzed by inductively coupled plasma-atomic emission spectrophotometry (ICP) using a model Plasma 400 (PerkinElmer) ICP system. Total petroleum hydrocarbons (TPH) were measured by first extracting the soil with dichloromethane, following a cleanup with Fluorisil based on the standard method (e.g., ISO/TR 11046, 1994) and analyzed by gas chromatography-flame ionization detector GC-FID Model Autosystem (Perkin-Elmer). The PCB analysis was performed on a gas chromatograph (Autosystem, Perkin-Elmer) equipped with an electron capture detector (ECD), after petroleum ether extraction and adequate cleanup based on a standard method (ISO 10382: 2002). Calibration was done with a standard mixture (PCB MIX3, 6 congener) 10 mg/mL supplied by Dr. Ehrenstorfer GmbH. HCHs were extracted with petroleum ether. After cleanup, the chromatographs of the extracts were generated using gas chromatography equipped with an ECD (Autosystem, Perkin-Elmer). Calibration was done with pesticide MIX 8, 10 mg/mL supplied by Dr. Ehrenstorfer GmbH. 2.3. Multispecies soil system (MS-3) 2.3.1. Management of soil columns Soil samples were assessed at four dilutions of contaminated soil: 0.3, 0.6, 1.2, and 2.5% (w/w). Higher exposure levels, 25 and then 100%, were tested only when no toxicity above 50% was observed in the diluted soils. Dilutions of polluted soil with control soil were prepared on a dry-weight basis and were produced by hand mixing the soils. Soils were placed in 15 cm height
15 cm diameter methacrylate columns

(1.7–2.2 kg soil dry wt per column, depending on soil bulk density) and ten adult Eisenia fetida (Oligochaeta: Lumbricidae) from our laboratory cultures were added on Day 0 to each soil microcosm and used to represent soil invertebrates. Seven seeds of three plant species (i.e., wheat, Triticum aestivum; rape, Brassica napus, and red clover, Trifolium pratense) were sown on to the soil in each microcosm. Seeds were obtained from the Spanish National Center for Seeds and Vegetal Varieties (Madrid). Three replicates for each treatment were carried out. Columns were incubated in a climate room at a controlled temperature (2072 1C) and illuminated with fluorescent bulbs (18 W) with a photoperiod of 16 h daylight and 8 h darkness; the light intensity was 1600–1900 lx. A volume of water was added to the soil in order to reach its water-holding capacity. Columns were watered 5 days a week with 50 ml of dechlorinated water simulating 1000 mm rainfall/year, allowing the soils to drain to field capacity. Leachates were collected during 21 days, in association with watering events. Successive leachate fractions of each microcosm were mixed and kept refrigerated at 4 1C for further chemical analysis and used in toxicity tests with aquatic organisms. 2.3.2. Ecotoxicological endpoints Tests were performed to assess the direct toxicity of soil and soil leachate to soil and aquatic organisms, respectively (Table 2). In order to determine soil toxicity to soil organisms, after 21 days MS-3 columns were opened and the numbers of adult earthworms surviving as well as emergence of seedlings and above-ground biomass production, measured as wet mass of shoots, were recorded. Effects to microorganisms were determined using the soil respiration test following the principles of standardized methods (OECD, 2000). Microbial activities were measured in the experimental soils using subsamples of soil colleted from the surface layer of the soil columns in the microcosms. These subsamples were dried at room temperature until a soil moisture content between 40% and 60% of the maximum water-holding soil capacity was achieved. Soils were amended with 4 mg glucose/g soil (dry weight) and carbon dioxide release was measured in a Microbiological Analyzers, BacTrac 4000 SY-Lab. Toxicity of leachate to aquatic organisms was determined for the leachate samples obtained from the soils in the microcosms using acute aquatic tests that included an algal test (OECD (Organization for Economic Cooperation and Development), 1984b; Ramos et al., 1996), the daphnid test (OECD (Organization for Economic Cooperation and Development), 1984c), and the RTG-2 fish cell line test (Babı´ n et al., 2001), to measure growth, immobilization, and cytotoxicity of leachate, respectively. Cytotoxicity was determined to the RTG-2 fish cell line (ATCC, CL

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Table 2 Summary of test selected to assess the toxicity of soil and soil leachates to soil and aquatic organisms, respectively, exposed to soil in a microcosm system (MS-3) Test organisms

Tests

Measured parameters

Reference

Microorganisms Invertebrates

Soil respiration test Earthworm acute test

Carbon transformation Survival

Plants

Plants growth test

Emergence and growth

Modified OECD (2000) Modified OECD (Organization for Economic Cooperation and Development), (1984a) Modified OECD (Organization for Economic Cooperation and Development) (2004)

Algae

Algae, growth inhibition test

Growth inhibition

Invertebrates

Daphnid acute test

Immobilization

EROD activity b-Galactosidase act Neutral red Proteins

Induction or inhibition Inhibition Cellular viability Cellular mortality

RTG-2

No. 55) using four different endpoints parameters: enzymatic activities as 7-ethoxyresorufin O-deethylase (EROD) (Bols et al., 1999) and b-galactosidase (Pablos et al., 1998), uptake of neutral red stain (NR) to evaluate cell viability (Borenfreund and Puerner, 1985), and FRAME KB protein assay to evaluate cell detachment from a substrate (Knox et al., 1986). In these assays, two controls were used: the test medium prescribed by OECD and the leachates obtained from the control soil in the assay conditions. 2.4. Statistical analysis Toxicity effects were calculated as percentages of inhibition at the given concentration or as L(E)Cx values. Percentages of inhibition were determined with respect to the control soil in the diluted soils and with respect to reference soil in the assays performed with nondiluted samples of soil 2. In the earthworm acute test and daphnia test, percentage of mortality or immobilization, respectively, was determined with respect to the number of test organisms (10 organisms in each microcosm). L(E)Cxs were calculated using log-probit methods (Litchfield and Wilcoxon, 1949). L(E)C20 is used instead of (L)EC50 in those cases where the determined toxicity was too low for calculating L(E)C50. Toxicity responses obtained in test soils were compared with those in the control soil by one-way analysis of variance (ANOVA), with Fisher’s leastsignificant difference procedure (LSD, Po0:05), in the software program STATGRAPHICS.

3. Results According to the proposed criteria, soil 1 was tested at 0.3%, 0.6%, 1.2%, and 2.5%, soil 2 at 0.3%, 0.6%,

OECD (Organization for Economic Cooperation and Development) (1984b); Ramos et al. (1996) OECD (Organization for Economic Cooperation and Development) (1984c) Bols et al. (1999) Pablos et al. (1998) Borenfreund and Puerner (1985) Knox et al. (1986)

1.2%, 2.5%, 25%, and 100%; and soil 3 at 0.3%, 0.6%, 1.2%, 2.5%, and 25% (w/w) concentrations of contaminated soil. Table 3 summarizes the toxicity results of the microcosm study for the three soils tested. Only those data significantly different from control soil by analysis of variance/least-significant difference (Po0:05) are shown. Mean values of variation coefficients for the control soil in the three assays ranged from 2% to 30% for soil organisms and from 2% to 16% for aquatic organisms, respectively. For soil organisms maximum and minimum variability were obtained for emergence of B. napus (31%) and for the earthworm test (11%), respectively. For aquatic organisms the maximum variability was obtained for the citotoxicity assay with RTG-2 cell when the measured parameter was EROD activity (16%) and minimum variability when the measured parameters were neutral red and proteins (2%). Variation coefficients were in the range of the results obtained by others authors using a single-species test (e.g., van Gestel et al., 2001). The three soils tested produced different toxicological responses in soil organisms for the different toxicity endpoints (Table 3). Exposure–concentration response relationships were described for the earthworms. Effects to plants and soil microorganisms were mostly observed at the highest concentrations. In the plant test, the aerial biomass production appeared to be the most sensitive endpoint and showed less variation than emergence of seedlings. Soil 1 presented the highest toxicity among the three soils tested (Table 3). This soil showed a concentrationdependent effect on earthworm survival (Fig. 1) with an LC50 ¼ 1.44% (w/w) soil concentration (r ¼ 0:97). Statistical analyses of wet mass of shoots revealed significant differences between treatment and control soil, but not on seedling emergence. Dry weight was significantly reduced with respect to control soil in

178

Soil

Concentration of contaminated soil (%, w/w)

Earthwormsa (Mortality)

Percentage of inhibition compared with control soil (%)

Plants (Wet weight plant)

Control soil Soil 2

Control soil Soil 3

2.5

2.5 25 100

2.5 25

Algae (Growth rate)

Triticum aesivum

Brassica napus

Trifolium pratense

0717

0710

0730

0721

0735

075

53711

3174

ns

4779

ns

0710

0729

0736

0719

ns ns nsb

ns ns nsb

ns ns nsb

070

0712

2072 3073

ns 6078

Daphniaa (Immobilization)

RTG-2 cell (ATCC, CL No. 55)

EROD

Neutral red

Protein

075

0711

070.5

070.43

16.270.8

ns

3173

12.470.3

7.5570.07

077

077

—

0720

073

071

ns 2575 4779b

ns 3072 ns

ns ns 2073

— — —

ns ns 12977

ns ns ns

ns ns ns

076

0736

077

0716

070

0717

073

073

ns 2575

ns 51718

ns 4176

1973 ns

ns ns

ns 72717

ns ns

ns ns

Shown data were significantly different from control (Po0:05) by the LSD procedure; ns, not significantly different from control (Po0:05) by the LSD procedure; italics indicate increase effects compared with the control; —data of daphnid test with soil 2 are not included because high daphnia mortality in assays with leachates from reference soil were observed. a In earthworm and daphnid tests, percentages of mortality or immobilization, respectively, were determined with respect to the number of organisms in the test (10 organisms in each microcosm). b At 100% soil 2, responses are expressed as percentage of effect compared to the reference soil.

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Control soil Soil 1

Microorganisms (C. transformation)

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Table 3 Toxicity data to soil organisms exposed to soils 1, 2, and 3, and to aquatic organisms exposed to leachates from these soils in the microcosm system MS-3

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c*

Mortality (%)

100

Soil 1

80

Soil 2

60

Soil 3

40

b

a

a ab´

b´

ab

ab

c´

ab ab

20

a

ab´

ab´

ab´

a´´

ab´

bc´

a´

0 Control soil

0.3

0.6

1.25

2.5

25

100

Concentration of contaminated soil (%, w/w) Fig. 1. Adult mortality (%) of earthworms exposed for 21 days to soils 1,2, and 3 in microcosms. Note: Different letters indicate significantly different values using one-way ANOVA (LSD; Po0:05). *Value measured in glass containers at 20 1C, in parallel to the MS-3.

Table 4 Carbon dioxide-released rates in the carbon transformation test for soils 1,2, and 3 assessed in the microcosm system MS-3 (Tests were performed according to OECD 2000) Concentration of contaminated soil (%, w/w)

Soil 1 (mg CO2/kg day wt soil/h)

Soil 2 (mg CO2/kg day wt soil/h)

Soil 3 (mg CO2/kg day wt soil/h)

Control soil 0.3 0.6 1.25 2.5 25 100

73726a 59723a 65711a 78713a 53716a — —

6873a 6773ab 6573ab 5577ab 5978ab 4773b 68716ab

6278a 6171ab 5075bc 6677a 6276a 3770c —

—, Soils were not assessed at this concentration; values with different letters within a column indicate significantly different values using one-way ANOVA (LSD; Po0:05).

T. aestivum (3174) and in T. pratense (4779%) at 2.5% soil concentration, whereas B. napus was not affected (Table 3). No inhibition of carbon mineralization was observed for soil 1, indicating that the microbial communities were not adversely affected in this soil. Additionally, ecotoxicity of aqueous leachates obtained from soil 1 at concentrations of 0.3%, 0.6%, 1.25%, and 2.5% was tested. Algal cell numbers increased when organisms were exposed to leachates from control and test soils with respect to those exposed to standard OECD medium. This phenomenon probably reflects the growth-stimulating effect attributable to nutrients in the soil. In order to avoid this phenomenon, in bioassays with aquatic organisms, percentages of inhibition (Table 3) were calculated relative to the leachates from control soil despite the respective standard blanks normally used in these tests. Thus, soil 1 produced a slight, but significant, inhibition of algae growth rate at all concentrations tested (mean value ¼ 11.670.6%), which cannot be explained from the soil concentration. Effect of leachates in fish was assessed with the RTG-2 cell line, using four different

endpoints: EROD activity, b-galactosidase activity, unspecific lysosomal activity by neutral red, and cellular protein contents. Leachates from soil 1 at the highest concentration tested (2.5% soil concentration) caused an induction in the EROD activity (3173%) and a reduction of neutral red activity (12.470.3%) and protein content (7.5570.07%). No significant effect was observed when the enzymatic activity of bgalactosidase was measured. In the daphnids test, no significant differences were observed for Daphnia magna exposed to the leachates from control soil and soil 1 (Table 4). Soil 2 was the least toxic presenting toxicity only for 4 among the 11 endpoints measured. Moreover, L(E)C50 s were greater than 100% in all organisms tested, except in the citotoxicity test when EROD activity was measured. At low exposure levels (0.3–1.25% soil concentration), the only adverse effect observed was an inhibition of carbon mineralization with EC20 ¼ 3.99% (w/w) soil concentration (r ¼ 0:94) (Table 4). Survival of the earthworm was not affected at any soil 2 concentration (Fig. 1) but in the plant test, a

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decrease on T. pratense weight of 2575 and 4779%, compared to the control soil, was observed at 25% and 100% soil concentration, respectively. Leachates from soil 2 caused a 2073% inhibition of average specific growth rate of algae relative to that in the leachates from control soil. The assay RTG-2 cells indicated a positive response only when EROD activity was measured. Thus, fish cell exposed to leachates obtained from soil 2 (100% soil concentration) were characterized by values of EROD activity that were 2.3 times higher than the corresponding value in the control soil; this corresponds to a percentage of effect equal to 12977% (Table 3). Results of daphnia assays for this soil were not considered due to high mortality observed in the assay with leachates from reference soil. For soil 3, dilutions ranging from 0.3% to 2.5% were toxic to earthworm only. Adult earthworms survival was adversely affected (Fig. 1) with an LC20 of 7.0% (w/w) soil concentration (r ¼ 0:94). At 25% of soil concentration, soil 3 produced toxic effects on all taxonomic groups. Soil 3 adversely affected plant growth but not seedling emergence for the three species tested. Microorganisms were also adversely affected with a 4176% inhibition of carbon mineralization occurring at 25% soil concentration (Table 4). Regarding aquatic organisms, the most sensitive test was the RTG-2 cell line. Leachates from soil 3 did not adversely affect algal growth or daphnid immobilization. An increase in algal growth was observed for organisms exposed to leachates from soil concentrations ranging from 0.3% to 2.5%. Fish cells exposed to leachates obtained from soil 3% at 25% soil concentration presented values of EROD activity 1.7 higher than the corresponding value in control soil; this corresponds to a percentage of effect equal to 72717% (Table 3). The other parameters measured in this test were not affected.

4. Discussion Addressing contaminated soils is always a challenge as soil characteristics and the aging process modify the toxicity of the pollutants. A proper assessment requires the combined use of chemical and toxicological analysis. Our toxicity testing protocol is based on a soil microcosm, using dilution series whereby the contaminated soil is diluted with a clean control soil, as suggested previously (Greene et al., 1989). Toxicity of contaminants in the soil may decrease with time due to adsorption and aging processes. However, some of these processes may be reversible by changes in physicochemical soil properties. As a consequence, bioavailable pollutants may be released in the environment due to significant change in soil properties. This fact is particularly important during soil

remediation. Dilution processes modify the bioavailability of chemicals in the soil. The proposed testing strategy starting with large dilution factors covers the potential for these reversible processes. In addition, assays at low exposure levels allow determination of the risk for spreading the contamination to other areas due to surface movement of contaminated soil caused by erosion, flooding, etc. On the other hand, in diluted soils, especially at concentrations ranging from 0.3% to 2.5%, the influence of the tested soil on the overall soil characteristics is negligible and therefore the soil characterization reflects those of the control soil. In this way, effects of the test soil will not be confused with the effect caused by the contaminants, allowing comparisons among different soils. As expected, the toxic effects varied substantially depending on soil and test species. Microorganisms and plants were the most sensitive groups for soil 2 containing metals and soil 3 containing petroleum hydrocarbons, respectively. Daphnids were the least sensitive organisms, since no response was observed in the leachates despite of elevated metal or oil soil concentrations. These differences in species sensitivity stress that a valid assessment of the environmental hazard of contaminated soils can only be obtained on the basis of test systems covering different key organisms. Table 1 shows the characteristics and pollutant levels in the soils. Both soil 1 and soil 2 are contaminated with the same metals at different concentration. Soil levels of As, Cd, Pb, and Zn were 5–7 times higher in soil 1 than in soil 2, whereas, Cu in soil 1 was 38 times higher than in soil 2. Soil 2 also contained organic contaminants, specifically, PCB and HCH. Bioassay responses were in agreement with these measured values. Soil 1 was the most toxic to different soil and aquatic organisms. Toxicity of soil 1 to plants and earthworms could be explained by soil metal content, which was higher than EC50 values for all metals (Table 5). However, toxicity was lower than the expected from total metal concentrations, suggesting a low bioavailability of metals in this soil. At 2.5% soil concentration, soil lead level (600 mg/kg soil approximately) was about 6 times higher than described EC50 for plants (EC50 ¼ 50–100 mg/kg soil dry wt) and 60 times above the reported LC50 values for earthworm (LC50 ¼ 3.7–10 mg/kg soil dry wt). Even at 0.3% soil concentration, lead level (110 mg/ kg approximately) was 10 times higher than the described LC50 value of this metal for earthworms. However, earthworm mortality caused by soil 1 was less or equal to 50% at all concentrations tested, whereas earthworm mortality at 0.3% was only 2371% (Fig. 1). Soil 2 did not present toxicity to earthworm or plants at any concentration tested, except to T. pratense (Fig. 1). However, toxicity to plants and earthworms should be expected according to high levels of lead (4084 mg/kg

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Table 5 Data of EC50 ecotoxicity values to soil and aquatic organisms for metals as described in the literature

Soil organisms (mg/kg) Plantsa Earthworma

As

Cd

Cu

Pb

Zn

77–325

350 300

364–7500 1500–4000

50–100 3.7–10

600–1000 68–1600

0.004–0.036 50 0.002–0.038

0.03–0.06 0.02 0.03–2.3

0.15–9.5 0.53–1.83 0.48–2.8

0.15–4 0.7–2.3 4–80

6.2 (4.9–7.8) 7.7 (6.1–9.8)

9.5 (7.7–11.7) 12.7 (9.7–16.7)

Aquatic organisms (mg/L) 44.6 Daphniaa Algaea 0.16 22–105 Fisha RTG-2 cell (ATCC, CL No. 55) Neutral redb Proteinb a

11.0 (6.9–17.8) 15.1 (7.5–30.3)

Toxicity data obtained from ECOTOX Database, 2004. Data reported by Castan˜o et al. (1995).

b

soil) in this soil. Moreover, the increased level of arsenic, cadmium, copper, and zinc should also contribute to the toxicity of this soil to plants. Toxicity not only depends on metal concentration but also on the solubility, mobility, and chemical speciation (Bell et al., 1991; Lorenz et al., 1997). All of these factors can influence bioavailability of metals, which will differ among taxonomic groups and will depend on the exposure route. Both soils had a high content of organic matter that favors soil adsorption and restricts metal bioavailability. However, soil 1 was slightly acid (pH 5.6), which increased metal solubility and consequently increased metal bioavailability and hence the toxicity. The lack of a toxic response on the earthworm test for soil 2 and the low response observed on plants suggest that the contaminants in this soil could be bound and not biologically available. Although the information is scarce, available data suggest that HCH (43 mg/kg soil) and PCB (189 mg/kg) concentrations in soil 2 are too low to produce measurable responses in the toxicity assays. The toxicity of g-HCH to earthworms and plants has been evaluated and an LC50 value of 50–170 mg/kg soil dry wt to earthworms (Haque and Ebbing, 1983; Heimbach, 1985) and an EC50 value of 100–1000 mg/kg soil dry wt to plants (Pestemer and Auspurg, 1989; Hulzebos et al., 1989) have been reported. Moreover, studies performed by Das and Mukherjee (2000) demonstrated that HCH soil concentrations ranging from 0.46 to 10 mg/kg were not toxic and resulted in an increase in the population of the soil microorganisms. For PCB, Luepromchai et al. (2002) found that soils spiked with Aroclor 1242 (Technical PCB preparation) at 100 ppm did not cause toxicity to earthworm. Regarding soil 3 with its high content of mineral oil and low metal content, plants were the most sensitive organisms for this soil. At 25% of soil concentration (16,000 mg of TPH/kg approximately), this soil caused effects in the three species tested. T. aestivum and T.

pratense were adversely affected at concentrations higher than 50%. At 2.5% of soil concentration, corresponding to a level of total hydrocarbon about 1600 mg/ kg soil, plants were not adversely affected. Mortality to earthworm was observed at 2.5% and 25%, although effects were lower than 50%. Low toxicity observed to plants and earthworm at 2.5% of soil concentration is in agreement with previous data in the literature (Saterbak et al., 1999; Wong et al., 1999; Dorn and Salanitro, 2000) where oil levelso4000 mg/kg dry soil were described to have little effect on plants. Soil 3 was also contaminated with metals. At 25% of soil concentration, levels of the major metals, Cu and Zn, were approximately 340 and 220 mg/kg soil, respectively, which could contribute to the toxicity observed on plants and earthworm, respectively. In the microorganism assay, a significant reduction in the activity of microbial population was observed when tested at 25% for soils 2 and 3. On the other hand, for nondiluted samples of soil 2 (100% soil concentration), carbon mineralization rates were similar to those observed for the control and reference soils, indicating an active microbial population. These results may be explained by the adaptation of soil microbial populations to the contaminants (Bruins et al., 2000). Nonadapted populations of the control soil are affected as observed at the 25% dilution. These results confirm the added value of testing samples of contaminated soil diluted with a control soil. Toxicity to aquatic organisms is usually determined assessing soil extracts. A common procedure is to shake soil with water (1:10) for 24 h (DIN 38414–S4, 1984). However, several authors question the environmental relevance of these extracts since the extraction is performed with a high content of water and therefore the ionic strength is lower than in the pore water of the soil (Friege, 1990). In our experiments, leachates are obtained from the soil microcosms, such that the equilibrium between the pollutant(s) in the soil and in

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the soil pore water is expected to be reestablished between watering events. Moreover, this procedure simulates processes occurring during rainfall periods. Low variation coefficients were obtained in the aquatic bioassays applied to leachates samples. Therefore, this method seems to be reproducible and offers an initial estimation of the potential for groundwater contamination. Toxicity of soil leachates to aquatic organisms depends, in part, on the nature of the contaminants (Table 5), as well as on the transfer of pollutants from soil to leachates. Mobility patterns of pollutants in soils differed for different metals and they are strongly influenced by soil parameters such as soil pH, organic matter content, clay mineralogy, and the concentration and combination of metals present in soil (Adriano, 2001). Toxicity to aquatic organisms (algae and RTG-2 cells) was higher in soil 1 than in soil 2 leachates as expected according to the higher metal levels in soil 1 than in soil 2 as well as the acid pH in soil 1 that favors the metal mobility. Specifically for soil 2, these metals did not affect aquatic organisms at any of the concentrations tested except at the highest dose (100% soil concentration), indicating that heavy metals are released to the water in trace amounts. From these results it seems that the extraction method gives an indication of the bioavailability of metals in the soil tested. The results obtained in the cell fish assay show an induction of EROD activity in the leachates of all soils. In soil 2 and soil 3, induction of EROD activity might be related to the presence in these soils of PCBs and PAH, respectively. It is known that some PCB congeners (Van Schanke et al., 2000) and PAHs (Villeneuve et al., 2001) act as dioxin-like compounds that can induce EROD activity. However, an exhaustive analysis of these compounds was not performed due to analytical difficulties, as well as the expense associated with the measure of all possible congeners. Soil 1 is contaminated by metals, which cannot elicit this effect (Bruschweiler et al., 1996; Bozcaarmutlu and Arinc, 2004). Consequently, in this case, the increase of EROD activity suggests the presence of toxic products in soil 1 that remained undetected by chemical characterization based on the historical use of the site. Metals decrease cellular viability (Castan˜o et al., 1995) according to inhibition of neutral red and proteins observed in soil 1, containing the highest concentration of metals. Moreover, protein measurement was less sensitive than neutral red, which agrees with data usually observed in cytotoxicity test on RTG-2 (Castan˜o et al., 1996).

5. Conclusions As demonstrated in this study, the application of bioassays provides information that cannot be obtained

from chemical analyses. The toxicity of metals in soils was less than that expected to correspond to metal soil concentration, indicating the need for considering bioassays to assess the bioavailable fraction of the contaminants as well as synergistic and antagonistic interactions. In addition, bioassays have the potential to elucidate adverse effects or responses that are not directly attributable to known contaminants in soils and can trigger further investigations into causal agents of toxicity. In this study, analyses of leachate samples with the RTG-2 bioassay detected the presence in the soil of chemicals that increased EROD activity in fish cells; however, these chemicals were not detected in the chemical characterization. Biological tests and conventional chemical analyses of soils complement each other in the assessment of soil pollution. Moreover, toxicity tests may be used as screening tools in order to identify polluted soil or water samples and to reduce the number of samples that require full chemical and toxicological analysis. A Multispecies Soil System (MS-3) has proven to be a useful tool for characterizing polluted soils. In this system, organisms were selected from different trophic levels and included taxonomic groups that cover essential ecological roles for the sustainability of soil use. Therefore, the present microcosm system allows for a good soil characterization in terms of ecotoxicological effects and provides information on the potential fate of chemical substances in soils. Leachates are obtained following a procedure that simulates real conditions. Moreover, by contrast to test batteries with single species, this multispecies system accounts for species interactions and permits simultaneous measurement of 11 endpoints. In addition, the use of a testing strategy covering different dilutions at low and high exposure levels offers the possibility of obtaining a clear view of the (eco)toxicological profile of the soil tested.

Acknowledgments The authors are grateful to Pilar Garcı´ a, Azucena Lo´pez, and Marı´ a Jose´ Gallardo for technical assistance. This work has been funded by the Plan Nacional de Medio Ambiente, Project No. 2000/215 and by the Comunidad de Madrid, Project No. 07M/0036/2002.

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