Environ Monit Assess (2008) 139:107118 DOI 10.

1007/s10661-007-9819-7

Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay
B. Lah & T. Vidic & E. Glasencnik & T. Cepeljnik & G. Gorjanc & Romana Marinsek-Logar

Received: 10 October 2006 / Accepted: 11 May 2007 / Published online: 14 June 2007 # Springer Science + Business Media B.V. 2007

Abstract Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO2) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil

B. Lah : T. Cepeljnik : G. Gorjanc : R. Marinsek-Logar (*) Zootechnical Department, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domzale, Slovenia e-mail: romana.marinsek@bfro.uni-lj.si T. Vidic Biotechnical Faculty, Department of Biology, University of Ljubljana, Vena pot 111, 1000 Ljubljana, Slovenia E. Glasencnik ERICo Velenje, Environmental Research & Industrial Co-operation Institute, Koroka 58, 3320 Velenje, Slovenia

leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated. Keywords Soil . In vitro bioassays . Water soil leachates . Genotoxicity . Ames test . Comet assay . Tradescantia micronucleus test

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Introduction Genotoxic compounds in soil may have an effect on human health through inhalation of dust, ingestion of plants that absorbed the compounds from the soil, and the leaching of the compounds from soil to ground and surface water used for drinking (Watanabe and Hirayama 2001). The physical and chemical nature of soil is very complex and standard chemical and pedological analyses are limited in their ability to characterize the chemical composition of genotoxicants in soil. On the other hand, genotoxicological and mutagenicity bioassays provide a means for assessing the (geno)toxicity of complex mixtures without the need of precise chemical characterization. The genotoxicity of unknown mixtures is usually evaluated by exposing such samples to living organisms which are further examined for genetic damage (Chenon et al. 2003; Martin et al. 2005; Mouchet et al. 2006). A number of tests have been developed using aquatic and soil animals, amphibians, protozoa and prokaryotic microorganisms which can potentially assess the genotoxic potential of the environmental sample (Gauthier et al. 1993; Sauvant et al. 1995; Bkaert et al. 2002; Kim and Hyun 2006). Most of the studies looking at the genotoxicity evaluation of soil samples focused on preparing the leachates from soil samples prior to performing the bioassays (Bkaert et al. 1999, 2002; White and Claxton 2004). The bioavailability of contaminants is a key factor for ecotoxicological effects therefore water extractable eluates were prepared and tested for genotoxicity in this study. Even though water soil leachates derived from polluted soil often appeared to contain few toxic or genotoxic pollutants the impact of these complex matrices was confirmed (Knasmller et al. 1998). To examine the genotoxicity of soil, the bacterial Salmonella/Ames test is most commonly used (Monarca et al. 2002; White and Claxton 2004). This test can be carried out rapidly and cheaply, but the main drawback is the lack of eukaryotic metabolic enzyme systems. Soil genotoxicity has also been frequently evaluated by plant bioassays like Tradescantia micronucleus test, Tradescantia stamen hair mutation test, Allium cepa test, and Vicia faba test, which demonstrate higher sensitivity and allow in situ monitoring of soil pollution (Ma et al. 1994; Cottele et al. 1999; Gichner and Veleminsky 1999; Knasmller et al. 2003; Liu et al. 2003). Primary DNA damage caused

by the potential presence of genotoxic substances in soil samples has been verified by the alkaline version of the comet assay in water soil leachates in few cases, too. The aim of our study was to evaluate the genotoxicity of soil in aleka valley (Slovenia) by applying an integrated physico-chemical-biological approach and to evaluate the relevance of the test battery, using several biotests with different levels of sensitivity and specificity and using different cells or organisms from more than one trophic level. The environment in aleka valley is contaminated by the emissions of the Thermal power plant otanj, releasing aerosols of trace elements, heavy metals, nitrogen and sulphur compounds which settle into the upper layers of soils and vegetation, by municipal wastes and intensive agricultural activities. Genotoxicity evaluations were performed by a short-term bacterial mutagenicity test (Ames test); by comet assay with two human cell lines (Caco-2 and HepG2) and a plant genotoxicity test (Tradescantia micronucleus test). In addition, pedological and standard physico-chemical analyses of soil samples were conducted, too.

Materials and methods Soil samples, pedological- and physico-chemical analysis Coal and lignite-mining, coal burning, industry, a nearby thermal power plant (TPP), traffic, and farming left ecotoxicological burdens in a Slovenian industrial-rural region called aleka dolina (aleka valley) and the surrounding regions. Six soil samples (Velenje sample 1 was taken 5 km from the TPP; Graka gora sample 2 is 6.9 km from the TPP; Zavodnje sample 3 is 7.2 km from the TPP; otanj sample 4 is 0.4 km from the TPP; Veliki vrh sample 5 is 2.7 km from the TPP; Zgornja Savinjska vally sample 6, the reference location, which is situated in the neighboring valley) were taken by standard procedure (ISO 11464 1994; ISO 10381 1996) on December 2003. The soil samples were dried and sieved according to standard procedure (ISO 11464 1994) and afterwards stored at 20C for further analyses. Since heavy metals (lead, cadmium, zinc and mercury), mainly from the thermal power plant, are released into the environment as dust particles

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pedological and physico-chemical analyses were performed according to ISO standards (SIST ISO: 5664 1996, 5666 1996, 10523 1996, 11083 1996; SIST EN ISO 10304-2 1998; SIST ISO 8245 2000; SIST DIN 38406-29 2000). The term heavy metals in this study refers to: lead (Pb), zinc (Zn), cadmium (Cd), arsenic (As), mercury (Hg), nickel (Ni), molybdenum (Mo), chromium (Cr), cobalt (Co) and copper (Cu). SO2 content in the air 15 m above the soil sampling locations were measured by automatic measuring stations (1 h measuring interval), except for the sampling location T6. For biological testing, soil samples were leached with water (the soil/water ratio was 1:5) as described by Bkaert et al. (1999). The supernatant was then stored at 4C and used for biological testing next day. In order to eliminate possible bacterial contamination leachates were sterilized by membrane filtration (0.22 m) prior to genotoxicity testing (Bkaert et al. 1999). Ames/Salmonella typhimurium assay Ames test was performed as a standard plate incorporation assay with Salmonella typhimurium strains TA97a, TA98 and TA100 with (+S9) and without (S9) in vitro extracellular microsomal activation (by S9 rat liver enzyme homogenate; Maron and Ames 1983). Strain specific genetic markers were verified prior to use. For each tester strain a specific positive control was used with/ without metabolic bioactivation: for strains TA97aICR-acridine; TA98-4NQNO and TA100-sodium azide, without metabolic bioactivation and 2AF for the strains TA98 and TA100; and B(a)P for TA97a strain with metabolic bioactivation. Following 48 h incubation at 37C genotoxic activities were expressed as induction factors (induction factor of reversions) i.e. as multiples of the background levels. Statistical significance was demonstrated with the KruskalWallis test (non-parametric ANOVA) for differences between treatment groups and Dunnetts multiple comparison for differences to the negative control. The interpretation of the Ames test results followed OECD 471 guidelines (Organisation for Economic Cooperation and Development 1997) and EPA Health Effects Tests Guidelines (OPPTS 870.5265; United States Environmental Protection Agency 1996) for genotoxicity testing of chemicals. According to EPA and Organisation for Economic

Cooperation and Development (1997) guidelines, a mutagenic potential is assumed, if the mutant frequency is 2.0 or higher. A dose effect relationship could underline this conclusion. A possible mutagenic potential is assumed if the mutant frequency is from 1.7 to 1.9, in combination with a dose effect relationship. No mutagenic potential for a tested item is assumed if all mutant frequencies range between 1.0 (and lower) and 1.6. In vitro comet assay with Caco-2 and HepG2 cell lines Epithelial colon cancer cells (Caco-2) were obtained from Instituto Zooprofilatico Sperimentale, Brescia, Italy, and the human hepatoma cell lines (HepG2 cells) were obtained from the Institute of Cancer Research at The University of Vienna, Austria. Both cell lines were grown in a monolayer culture at 37C in a humidified atmosphere of 5% CO2 in Dulbeccos Modified Eagles Medium (DMEM), supplemented with 10% fetal calf serum (FCS) for HepG2 cells and 15% FCS for Caco-2 cells. In both cases, gentamycin (40 g/ml) was added to the medium. The cells were seeded at a density of 40,000 cells per cm2 in 24 well tissue plates and cultured for 8 days. The medium was changed three times a week. The comet assay was performed as described by Lah et al. (2005a,b) with minor modifications. Cells in wells were exposed to sterile water soil leachates with the added components of DMEM medium (test samples T1T6) and sterile DMEM medium (negative control) for 24 h in triplicates. Nine independent replicates on water soil leachates and negative controls were carried out on each cell line. Single cell suspensions for each treatment were prepared by adding 0.25% trypsinEDTA solution, resuspending in DMEM medium and passed through pipette tips several times. The dye-exclusion test with Trypan blue (Duthie and Collins 1997) was used to examine the viability of cells before the comet assay was performed. The viability of cells was always higher than 90%. Briefly, the collected Caco-2 or HepG2 cells were immobilized in the third agarose layer of four layered microgels on microscope slides at a concentration of approximately 12 105 cells/ml. For a positive control, the four layered microgels with cells grown in sterile DMEM were treated with 500 M hydrogen peroxide for 5 min before lysis. The microgels with

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incorporated cells were submerged afterwards into an alkaline lysis buffer for 1 h and followed by electrophoretic buffer (pH>13) for 40 min. The electrophoresis was carried out at 2 V/cm and 300 mA for 30 min, followed by neutralisation in 400 mM Tris-HCl pH 7.5 for 15 min. The damaged DNA traveled toward the anode and formed an image of a comet tail. The comets were visualized by staining with ethidium bromide (20 g/ml). Quantitative analysis of nuclear DNA damage in Caco-2 and HepG2 cells was done with an epifluorescence microscope (Olympus BX 50) using 20 objective magnification; using a BP 515 560 nm filter and BA 590 nm barrier filter and digital camera (Hammamatsu Orca 2) connected to a computer. The comets were scored using Komet 5.0 Computer Software (Kinetic Imaging 2001). Olive tail moment (OTM) was chosen as the most relevant measure of DNA damage (Olive et al. 1996). Images of 100 comets were collected from each of the two replicate slides per test sample T1T6 and OTMs were collected for further statistical analyses. Full Bayesian approach (Gelman et al. 2004) was used for the statistical analysis of comet assay data. Analysis was performed in two stages. In the first stage, following Lovell et al. (1999), a quantitative measure for each sample of cells was calculated. We did not only calculate a quantitative measure such as mean or mean of log transformed data, but we built a probability model and calculated a quantitative measure based on the fit of the probability model. The distribution of OTM data was substantially skewed and we assumed that olive tail moments Yij of scored cells (index j) follow Weibull distribution Eq. 1 as has been proposed by Ejchart and SadlejSosnowska (2003) and to vary between cell samples (index i) according to distribution parameters i and li. We used noninformative distributions for both parameters i.e. exponential for i, which is decreasing slowly for positive values, and uniform with wide range for i. Based on fit of this model a quantitative measure and its standard error for each sample of cells were calculated separately. We choose median value q (mediani log 2l1 i ).

Altogether, the medians and standard errors for 57 samples of cells were calculated for CaCo-2 line of cells and 49 medians for HepG2 line of cells. Among each of these medians, three medians represented DNA damage in negative controls. To express DNA damage compared to the negative control, corresponding medians and their standard errors were subtracted i.e. we performed a background correction to the negative control. In the second stage, background corrected medians (Yijk) were modeled Eq. 2 including intercept (), effects of water soil leachates (Ti), day (Di) and replicate within the day. Noninformative prior distributions (Gelman et al. 2004) were assigned for all parameters of the model.
  yijk ~ Normal ijk ; 2 k ijk Ti Dj eijk ; Ti ; Dj ~ Normal 0; 1002 ; eijk ~ Normal 0; 2 ; e ~ Uniform0; 10 e

For each stage of analysis, a Markov chain Monte Carlo method with three chains of 5,000 samples was used. Length of burn-in period was assessed visually by chain inspection and BGR (Brooks-Gelman-Rubin) statistic (Gelman et al. 2004). Chains appeared to converge to stationary distribution very fast (in only few iterations) and we discarded half of the samples (2,500) in each chain as a conservative choice. All calculations were performed with R (R Development Core Team 2005; Sturtz et al. 2005) and BUGS (Bayesian inference using Gibbs sampling; Spiegelhalter et al. 2003) programs. Data and program codes for analysis are available from the authors upon request. Results in each stage of the analysis are graphically represented with posterior medians and 95% credible intervals. The preliminary Tradescantia micronucleus (MN) assay The Tradescantia MN test was performed as described by Knasmller et al. (2003) with some minor modifications. The cuttings were exposed to water soil leachates (T1T6), to tap water (as negative control) for 48 h, and to 5 mM maleic hydrazide C4H4N2O2 (as positive control) for 6 h, followed by 24 h recovery before fixation. The inflorescences were fixed in 1:3 acetic acid-ethanol (Carnoys) solution and transferred into 70% ethanol.

yij ~ Weibull i ; 1i i ~ Exponential 0:001; log 1i ~ Uniform100; 100

Environ Monit Assess (2008) 139:107118 Table 1 Pedological parameters of the six soil samples (T1T6) Parameter Fine sand Rough sand Clay Sand Texture class Unit % % % % / T1 T2 T3 T4 T5 T6 15.50 19.30 17.90 26.10 26.90 28.20 15.50 12.00 7.70 9.90 7.10 8.10 10.90 19.10 13.10 14.00 19.10 21.00 58.10 49.60 61.30 50.00 46.90 42.70 S-C C S-C C C C

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in terms of the number of micronuclei/100 tetrads (MN/100 tetrads).

Results Pedological and chemical analyses of soil samples According to pedological (Table 1) and physicochemical analyses (Table 2) of the six soil samples, soil from sampling point T1 is listed as a sandy-clay (S-C) that is weakly alkaline and has low concentrations of phosphorous and potassium. T2 soil is listed as a medium-heavy clay soil (C) that is weakly acidic with a high concentration of potassium but a low concentration of phosphorous, both T1 and T2 soils are low in organic carbon and Kjeldahl nitrogen compared to the rest four samples. T3 soil is a light sandy-clay soil (S-C) that is weakly acidic with high concentrations of potassium and phosphorous. T4 and T5 soils are listed as medium to heavy clay soils (C). T4 soil is weakly alkaline and has low concentrations of potassium and phosphorous. T5 on the other hand is weakly acidic and has high concentrations of potassium and low concentrations of phosphorous. Heavy metal concentrations of six soil samples (Table 3) were compared to permitted immission limit values (PIL values) according to Slovenian Regulations for the Amounts of hazardous compounds, heavy metals and fertilizers in soils (Official Gazette 1996). According to the immission limit values for the following metals: Pb, Hg, Zn, Cu, Co, Mo and Ni, none of the chosen sampling locations could be declared as polluted. PIL values and even warning immission values for Cr however were exceeded in all soil samples. Immission limit values for Cd in

Measurements of the determined parameters were performed according to the ISO standard methods described in Materials and methods; T1T6=soil samples S-C Sandy-clay soil particles; C clay soil particles

The MN assay is based upon the fact that DNA damage in pollen mother cells leads to formation of micronuclei in the early tetrad stage. Micronuclei are small masses of chromatin which appear beside the main nuclei when the plant is in contact with aneugen agents (fusorial poisons) or clastogen agents (inducing chromosome breaks). Inflorescences containing early tetrad cells were found in buds with a length of 3 4 mm and afterwards slides were prepared and stained with aceto-carmine as described by Knasmller et al. (2003). Usually, 1,500 tetrads are scored under the microscope from five slides per sample and controls. Due to the lack of available plant material at the time of the experiment, not enough flower buds containing tetrads could be obtained. Consequently, only 300 500 tetrads per sample were scored and the results are therefore only qualitatively informative and regarded as preliminary. The micronuclei frequencies were calculated by dividing the total number of detected micronuclei with the total number of tetrads scored and expressed

Table 2 Physico-chemical parameters of the six soil samples (T1T6)

Parameter Unit

pH (H2O) / 7.42 6.16 6.40 6.91 5.53 6.27

pH (KCl) / 6.90 5.29 5.74 6.34 4.22 5.62

Organic carbon g/kg d.w. 28.70 28.50 96.20 44.00 61.70 45.10

Kjeldahl nitrogen % 0.33 0.35 0.97 0.44 0.58 0.56

Easy accessible potassium (K) mg K2O/100 g 9.30 27.20 22.30 9.24 53.00 25.90

Easy accessible phosphorus (P) mg P2O2/100 g 1.95 4.94 12.40 8.11 4.77 1.56

Measurements of the determined parameters were performed according to the ISO standard methods described in Materials and methods; T1T6=soil samples d.w Dry weight

T1 T2 T3 T4 T5 T6

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Table 3 Heavy metals and nitrates concentration in six soil samples and in water extracts Parameter (unit) Samples T1 Pb (mg/kg) Zn (mg/kg) Cd (mg/kg) As (mg/kg) Hg (mg/kg) Ni (mg/kg) Mo (mg/kg) Cr (mg/kg) Co (mg/kg) Cu (mg/kg) SO2 (g/m3)a Nitrates (mg/kg) 38.00 119.00 0.59 13.70 0.11 43.60 2.61 193.00 16.30 31.90 54.00 6.90 T2 37.20 90.10 0.64 10.70 <0.10 43.20 1.90 193.00 10.00 17.90 126.00 <4.00 T3 72.00 144.00 1.74 6.66 0.24 16.60 2.31 187.00 11.70 16.90 138.00 4.00 T4 44.00 131.00 0.62 11.60 0.15 40.90 2.55 216.00 15.70 33.50 525.00 4.60 T5 33.00 126.00 1.11 8.86 0.42 31.30 3.22 169.00 8.78 23.10 266.00 6.40 T6 46.20 123.00 0.98 21.20 0.15 42.30 4.66 191.00 14.40 35.10 <4.00 85 200 1 20 0.8 50 10 100 20 60 350 PIL values

Measurements of the determined parameters were performed according to the ISO standard methods described in Materials and methods; T1T6=soil samples PIL values Permitted immission limit values according to Slovenian regulations
a

SO2 was measured in the air 15 m above soil sampling locations by automatic measuring system.

Fig. 1 Degree of CaCo-2 (left two graphs) and HepG2 (right two graphs) nuclear DNA damage presented as Olive Tail Moment (OTM) detected in six water soil leachates after 24 h cell treatment. The upper two graphs represent the posterior medians and 95% credible intervals for each sample of cells according to first stage of the analysis Eq. 1, while the lower two graphs represent the posterior medians and 95% credible intervals for each water soil leachates according to second stage of the analysis Eq. 2. Genotoxicity is indicated in the cases where the credible intervals do not overlap zero value

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Fig. 2 Induction of micronuclei (MN) in Tradescantia micronucleus assay after 48 h exposure to water soil leachates. PC positive control (5 mM C4H4N2O2), NC negative control (tap water)

samples T3 and T5 and for As in sample T6 were exceeded, too. The proportions of the metals extracted into water samples which were further applied for biotests ranged from 0.02 to 0.1% of the total heavy metal amounts present in soils. The highest concentrations of sulphur dioxide in the air were detected above the sampling locations T4 and T5 (Table 3), the PIL value was exceeded only at T4. There might be a correlation between SO2 immissions and low pH value of sample T5. These two locations are the closest to the thermal power plant and are directly exposed to its emissions. Results of the bioassays Ames test/Salmonella typhimurium assay usually represents the first step in genotoxicity/mutagenicity testing of various pure chemicals and environmental samples (Bkaert et al. 1999; Bispo et al. 1999; Ehrlichmann et al. 2000; Monarca et al. 2002). In our study, the plate incorporation method of the Ames test was performed. In our study, the Ames test (+S9 and S9) failed to detect the genotoxic potential in any of water soil leachates as the results never exceeded the critical value of 2.0 and all the quotients ranged below 1.6. As recommended by Maron and Ames (1983), primary tester strains TA97a, TA98 and TA100 were used. Regarding the comet assay on CaCo-2 and HepG2 cells, calculated medians and corresponding credible intervals for each cell sample (water soil leachates and negative controls) after first stage of the analysis are presented in Fig. 1 (upper two graphs). All credible intervals are above zero and indicate genotoxicity for all samples. Additionally, substantial variability can be observed. In the second stage of the analysis

genotoxicity of the water soil leachates in comparison to the negative controls was assessed. Results are presented in Fig. 1 (lower two graphs) and indicate clear genotoxicity of all six water soil leachates on CaCo-2 cells, with the highest genotoxicity found in sample T3. On the other hand, the results for the HepG2 cells do not clearly indicate genotoxicity for samples T1 and T4 and demonstrated the highest genotoxicity of samples T3 and T5. Due to the lack of plant material only one exposure of plants to water soil leachates and controls was possible at the time and in some cases not enough flower buds containing tetrads could be obtained. Consequently, fewer than 1,500 tetrads could be enumerated, precisely 300500 tetrads per sample. It is important to stress that due to the lack of plant material, these results are therefore only qualitatively informative. However, it is interesting to note that an increasing trend in the number of micronuclei for samples T1, T2 and T4 could be seen, indicating genotoxicity (Fig. 2).

Discussion Extensive previous studies of soil contamination in aleka valley indicated the heavy metals as the main contaminating agents in this region. The levels of the total concentration of PCBs and PAHs have not exceeded 0.01 and 0.02 mg/kg, respectively, what is well below the permitted immission limit values. The concentrations of the analysed pesticides and AOX have not exceeded the limit values, either (Kugoni and Stropnik 2004; Kugoni et al. 2004). Most metallic salts are effective poisons at particular concentrations, because they are able to bind to thiol groups and induce spindle disturbances in the cells (Patra et al. 2004). When considering heavy metals in soil, the bioavailability is a very important factor in the ecotoxicological evaluations of the soil. Bioavailable compounds are usually water soluble mobile compounds, on the contrary, biounavailable compounds are complex and not mobile; however, bioavailability can be increased in fluid media (Giller et al. 1998; Bkaert et al. 1999; Fent 2004; Loska et al. 2004). By now, the majority of metal (geno)toxicity studies in aleka valley have been focused on soilplant interactions (Glasennik et al. 2002; Kugoni et al. 2004). The presented results of the chemical analyses of six soil samples (T1T6) indicated that the tested soils are

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in general moderately contaminated according to Slovenian Regulations (Official Gazette 1996). For all soil samples collected in the aleka valley, the chromium content in soil samples exceeds the warning and the permitted limit immission value, for Cd permitted limit immission value is exceeded in samples T3 and T5, for As in T6 and for Ni slightly exceeded in samples T1 and T2. Heavy metals and/or their compounds are classified as mutagenic, carcinogenic and clastogenic substances (White and Claxton 2004). Anyhow, the negative results of the Ames test in present study fail to prove the presence of mutagens in soil leachates, causing frameshift or basepair substitutions. There are several reports on lower sensitivity of this point mutation test and disability in detection of genotoxic effects caused by heavy metals (Knasmller et al. 1998; Monarca et al. 2002; Radetski et al. 2004). At the same time these negative results could confirm the absence of PAHs in soil samples, which account in many cases of genotoxic effects of soils and sediments (White 2002; White and Claxton 2004). The preparation of the samples can be connected to and could partly explain the lower sensitivity of the Ames test (Bkaert et al. 1999). The filtration step (0.2 m) used in the Ames test procedure may be one of the causes of this bioassays lower sensitivity as compared to the other tests. Some of the components in leachates could attach to the filters and are consequently lost for further testing. Filtration of the samples however partly eliminates the contaminant fraction potentially present in the sample which is otherwise available to living organisms in other in vivo tests or in plant micronucleus assay (Mouchet et al. 2006). The negative results recorded on water soil leachates by the Ames test can also be explained by the low water extractability of metals from soil samples. Very low concentrations (g/l) of heavy metals are actually extracted from the soil into the water as it was reported by Ivask et al. (2004). Knasmller et al. (1998) reported that the metal amounts in the aqueous leachates were five- to 10-fold lower than the concentrations required to cause detectable effects on salmonella DNA. The proportions of the metals extracted into water samples which were further applied for biotests in the present study were low, too, and ranged from 0.02 to 0.1% of the total heavy metal amounts present in soils. Due to the lower sensitivity of Ames test for heavy metals, further work must be directed to the develop-

ment of bioassays with higher organisms/cells (Gatehouse et al. 1990). This is also one of the reasons why short-term tests with cultivated mammalian cells are widely used for the detection of potential environmental mutagens and carcinogens. Since numerous compounds exert their genotoxic and carcinogenic effects only following metabolic activation, it is important that indicator cell lines are metabolically competent (Glatt et al. 1990). According to the findings of Knasmller et al. (2004), HepG2 cells represent an excellent tool for the detection of environmental and dietary genotoxicants. The comet assay results on HepG2 cells clearly reveal the genotoxic effects on the level of DNA damage by DNA breaks (which might potentially lead to carcinogenicity) in water soil leachates for samples T2, T3, T5 and T6 (Fig. 1). These effects are not as clear for samples T1 and T4. At the same time, the highest genotoxic potential was proven for samples T3 and T5, which might correlate with the highest concentrations of Pb, Zn and especially Cd for sample T3, with the highest concentrations of Hg for sample T5 and with the highest concentration of organic carbon and nitrogen for both (Tables 2 and 3). It is important to stress, that DNA damage is also suspected to be due to increased chromium contents in all six soil samples The possible interactions between the sample compounds present must also be taken into account. HepG2 cells have been several times successfully used to study genotoxic and cytotoxic effects and mechanisms of action of heavy metals. Tchounwou et al. (2004) clearly demonstrated on HepG2 cells the potential of lead nitrate to undergo biotransformation in the liver, to cause the proliferation, protein damage, metabolic perturbation, and growth arrest and DNA strand breaks. Fatur et al. (2002) used the comet assay to demonstrate the DNA damage caused by Cd in HepG2 cells and supported the hypothesis that direct and indirect mechanisms are involved in Cd-induced DNA damage. The second chosen indicator cell line for DNA damage was Caco-2. Even though this cell line appears not to posses all the desirable xenobiotic metabolizing enzymes of phase I and phase II, it exhibits different degree of specialization and enterocyte like functions. The intestinal epithelium permeability, which Caco-2 cell line resembles, is a critical characteristic that determines the rate and extent of human absorption and ultimately the bioavailability of

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a xenobiotic compound (Duthie et al. 1997). The detected genotoxicity in all water soil leachates (Fig. 1, the left two graphs) could be partially, but not specifically ascribed, to increased Cr contents in all six soil samples, to exceeded limit immission values for Cd in sample T3 and T5 and to the exceeded limited immission value for As in sample T6 (Table 3) like in HepG2 cells, although the two cell lines showed a bit different pattern of responses. A lot of research work has been done regarding the genotoxicity of heavy metals, especially of Cr and As. Hartmann and Speit (1994) and Basu et al. (2001) agree about the possible mechanism of the genotoxic action of As, where arsenic indirectly inhibits the enzymes for DNA repair. Several studies report the genotoxic, cytotoxic and carcinogenic effects of Cr (VI) and its reduced form (III), which is a reactive intermediate and which in combination with oxidative stress in cells causes oxidative DNA damage (Singh et al. 1998; Cavas and Ergene-Gzkara 2005; Levina and Lay 2005; Rudolf et al. 2005; Sargeant and Goswami 2006). However, Majer et al. (2002) reported that the genotoxic effects of soil are to a large extent connected to the physico-chemical properties of the soil and influence profoundly the effects of heavy metal contamination. The interactions among the metals and among the metals and other soil components should be taken into consideration when explaining the genotoxic potential of contaminated soils (White and Claxton 2004). Water soil leachates T1, T2 and T4 show a trend toward increasing numbers of micronuclei formation with the Tradescantia MN test (Fig. 2), indicating clastogenicity, which can be explained by elevated levels of Cr and relatively high Pb, Ni and Hg contents in soil samples. Tradescantia MN assay, based on the formation of micronuclei resulting from chromosomal breakage in the meiotic pollen mother cells of Tradescantia ssp. Inflorescences. has been one of the most frequently applied genotoxicity assays for detecting clastogenicity of contaminated soils (White and Claxton 2004). The extensive study of Majer et al. (2002) of soils contaminated with varying amounts of heavy metals (As, Cd, Cr, Cu, Mn, Ni, Pb, Zn...) proved numerous significant positive responses, even threefold above the mean control value by Tradescantia MN assay. Beside micronuclei induction in plants, the induction of chromosomal aberrations and sister chromatide ex-

change by heavy metals has been detected by different plant bioassays (White and Claxton 2004). In this study, a battery of bioassays detecting different genetic end-points were used: point mutations due to base pair substitution or base insertion or deletion, detected by the Ames test; DNA breaks/ primary DNA damage detected by the comet assay and cytogenetic damages due to an alteration in the plant chromosomal integrity expressed as micronuclei and detected by the Tradescantia micronucleus test. According to several scientific reports (Maron and Ames 1983; Knasmller et al. 1998, 2004; Majer et al. 2002; Crebelli et al. 2005; Mouchet et al. 2006) describing the involvement of genetic end-points in pathological processes, all these end-points should be taken into consideration while monitoring the environment for genotoxic hazards. Crebelli et al. (2005) explain that chemical mutagens usually do not affect different genetic end-points with the same degree of efficiency. These conclusions are in agreement with the results of our study, where we are mostly dealing with heavy metal soil contamination. Several different bioassays used in our study complement each other for the genotoxic evaluation of soil samples. This study is completed by the use of physico-chemical analyses which alone are insufficient for ecotoxicity monitoring, but are very useful in investigations of genotoxicity causing factors and genotoxicity mechanisms.

Conclusions Most of the mutagenic/genotoxic substances are poorly water soluble, and the standard test systems are not sensitive enough to detect these low concentrations (Ehrlichmann et al. 2000). According to the results of this study, it can be estimated that the comet assay and the micronucleus test are sensitive enough for the effective evaluation of genotoxicity in aqueous soil leachates. In spite of the low extractability of heavy metals and other xenobiotic compounds by water, genotoxic effects were detected in two of the selected bioassays. To summarize, the so-called poliphase approach, combining physico-chemical, pedological and (geno)toxicological approaches in the studies and monitoring of genotoxins in soil samples, improves the evaluation of the ecotoxicological effects of environmental samples, due to many different classes of xenobiotic compounds being

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Environ Monit Assess (2008) 139:107118 of soil samples from contaminated sites. Ecotoxicology and Environmental Safety, 46, 7380. Ejchart, A., & Sadlej-Sosnowska, N. (2003). Statistical evaluation and comparison of comet assay results. Mutation Research. Genetic Toxicology, 534, 8592. Duthie, S. J., & Collins, A. (1997). The influence of cell growth detoxifying enzymes and DNA repair on hydrogen peroxidemediated DNA damage (measured using the comet assay) in human cells. Free Radical Biology & Medicine, 4, 717724. Fatur, T., Tusek, M., Falnoga, I., Scancar, J., Lah, T. T., & Filipic, M. (2002). DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium. Food & Chemical Toxicology, 40, 10691076. Fent, K. (2004). Ecotoxicological effects at contaminated sites. Toxicology, 20, 22240. Gatehouse, D. G., Wilcox, P., Forster, R., Rowland, I. R., & Callander, R. D. (1990). In D. J. Kirkland (Ed.), Bacterial mutation assays UKEM recommended procedures guidelines for mutagenicity testing (pp. 1359). Massachusetts: Cambridge University Press. Gauthier, L., Van der Gaag, M. A., Haridon, J. L., Ferrier, V., & Fernandez, M. (1993). In vivo detection of waste water and industrial effluent genotoxicity: Use of the Newt Micronucleus test (Jaylet test). Science of the Total Environment, 138, 249269. Gelman, A., Carlin, J. C., Stern, H., & Rubin, D. B. (2004). Bayesian data analysis (2nd ed., pp. 58488388). New York: Chapman & Hall. Giller, K. E., Witter, E., & McGrath, S. P. (1998). Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review. Soil Biology & Biochemistry, 30, 13891414. Gichner, T., & Velemnsk, J. (1999). Monitoring the genotoxicity of soil extracts from two heavily polluted sites in Prague using the Tradescantia stamen hair and micronucleus (MNC) assays. Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis, 426, 163166. Glasennik, E., Ribari Lasnik, C., Mller, M., Grill. D., & Bati, F. (2002). Impact of air pollution on genetic material of the shallot (Allium cepa L var ascalanicum) exposed in the vicinity of major Slovene local emission sources in 1999. Phyton, 42, 237250. Glatt, H., Gemperlein, I., Setiabudi, F., Platt, K. L., & Oesch, F. (1990). Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds. Mutagenesis, 5, 241249. Organisation for Economic Cooperation and Development (OECD) (1997). Guidelines for the Testing of Chemicals. Bacteria reverse mutation test guideline 471. Paris, France: OECD Hartmann, A., & Speit, G. (1994). Comparative investigations of the genotoxic effects of metals in the single cells gel (SCGE) assay and their sister chromatide (SCE) test. Environmental and Molecular Mutagenesis, 23, 299305. Ivask, A., Franois, M., Kahru, A., Dubourguier, H.-C., Virta, M., & Douay, F. (2004). Recombinant luminiscent bacterial sensors for the measurement of bioavailability of cadmium and lead in soils polluted by metal smelter. Chemosphere, 55, 147156.

present in the environment. It might also be important to consider the use of in vivo tests for genotoxicity, which would complement the in vitro results and give an even clearer picture of the genotoxic potential of environmental samples.
Acknowledgements This study was supported by the Slovenian Ministry of Education, Science and Sports; Ministry of Agriculture, Forestry and Food and the Coal mining corporation Rudnik Velenje. The authors are thankful to Mr Darryl Glover for reviewing the English language in the article.

References
Basu, A., Mahat, J., Gupta, S., & Giri, A. K. (2001). Genetic toxicology of a paradoxical human carcinogen arsenic: A review. Mutation Research, 488, 17194. Bkaert, C., Ferrier, V., Marty, J., Pfohl-Leszkowicz, A., Bispo, A., Jourdain, M. J., et al. (2002). Evaluation of toxic and genotoxic potential of stabilized industrial waste and contaminated soils. Waste Management, 22, 241247. Bkaert, C., Rast, C., Ferrier, V., Bispo, A., Jourdain, M. J., & Vasseur, P. (1999). Use of in vitro (Ames and Mutatox tests) assays to assess the genotoxicity of leachates from contaminated soil. Organic Geochemistry, 30, 953962. Bispo, A., Jourdain, M. J., & Jauzein, M. (1999). Toxicity and genotoxicity of industrial soils polluted by polycyclic aromatic hydrocarbons (PAHs). Organic Geochemistry, 30, 947952. Cavas, T., & Ergene-Gzkara, S. (2005). Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents. Aquatic Toxicology, 74, 264 271. Chenon, P., Gauthier, L., Loubieres, P., Severac, A., & Delpoux, M. (2003). Evaluation of the genotoxic and teratogenic potential of a municipal sludge and sludgeamended soil using the amphibian Xenopus laevis and the tobacco: Nicotina tabacum L. var xanthi Dulieu. Science of the Total Environment, 301, 139150. Cottele, S., Masfaraud, J.-F., & Ferard, J.-F. (1999). Assessment of the genotoxicity of contaminated soil with the Allium/ Vicia-micronucleus and the Tradescantia-micronucleus assays. Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis, 426, 167171. Crebelli, R., Conti, L., Monarca, S., Feretti, D., Zerbini, I., Zani, C., et al. (2005). Genotoxicity of the disinfection by-products resulting from paracetic acid- or hypochloritedisinfected sewage wastewater. Water Research, 39, 11051113. Duthie, S. J., Johnson, W., & Dobson, V. L. (1997). The effect of dietary flavonoids on DNA damage (strand breaks and oxidised pyrimidines) and growth in human cells. Mutation Research, 340, 141151. Ehrlichmann, H., Dott, W., & Eisentraeger, A. (2000). Assessment of the water-extractable genotoxic potential

Environ Monit Assess (2008) 139:107118 Kim, I.-Y., & Hyun, C.-K. (2006). Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms. Ecotoxicology and Environmental Safety, 64, 288297. Knasmller, S., Gottmann, E., Steinkellner, H., Fomin, A., Pickl, C., Paschke, A., et al. (1998). Detection of genotoxic effects of heavy metal contaminated soils with plant bioassays. Mutation Research, 420, 3748. Knasmller, S., Mersch-Sundermann, V., Kevekordes, S., Darroudi, F., Huber, W. W., Hoelzl, C., Bichler, J., & Majer, B. J. (2004). Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants: Current state of knowledge. Toxicology, 198, 315328. Knasmller, S., Uhl, M., Gottmann, E., Hlzl, C., & Majer, B. J. (2003). The Tradescantia micronucleus bioassay. In J. Maluszynska & M. Plewa (Eds.), Bioassays in Plant cells for improvement of Ecosystem and Human Health (pp. 95106). Katowice: Wydawnictwo Uniwersytetu lskiego Kugoni, N., Pokorny, B., & eerko, M. (2004). Pollution of garden soils and vegetables in the alek Valley. Acta Biologica Slovenica, 1, 2125. Kugoni N., & Stropnik M. (2004). Heavy metal contents in agricultural soil and plants in aleka valley (p. 165). Final Research Report, Erico Velenje, Slovenija. Lah, B., Zinko, B., Narat, M., & Marinsek-Logar, R. (2005a). Monitoring of genotoxicity in drinking water using in vitro comet assay and Ames test. Food Technology and Biotechnology, 432, 139146. Lah, B., Zinko, B., Tisler, T., & Marinsek-Logar, R. (2005b). Genotoxicity detection in drinking water by Ames test Zimmermann test and Comet assay. Acta Chimica Slovenica, 523, 341348. Levina, A., & Lay, P. A. (2005). Mechanistic studies of relevance to the biological activities of chromium. Coordination Chemistry Reviews, 249, 281298. Liu, W., Zhou, Q.-X., Li, P.-J., Sun, T.-H., Yang, Y.-S., & Xiong, X.-Z. (2003). 124-trichlorobenzene induction of chromosomal aberrations and cell division of root-tip cells in Vicia faba seedlings. Bulletin of Environmental Contamination and Toxicology, 71, 689697. Loska, K., Weichula, D., & Korus, I. (2004). Metal contamination of farming soils affected by industry. Environment International, 30, 59165. Lovell, D. P., Thomas, G., & Dubow, R. (1999). Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies. Teratogenesis, Carcinogenesis, and Mutagenesis, 19, 109119. Majer, B. J., Tscherko, D., Paschke, A., Wennrich, R., Kundi, M., Kandeler, E., et al. (2002). Effects of heavy metal contamination of soils on micronucleus induction in Tradescantia and on microbial enzyme activities: A comparative investigation. Mutation Research, 515, 111124. Ma, T. H., Cabrera, G. L., Cebulska-Wasilewska, A., Chen, R., Loarca, F., Vandenberg, A. L., et al. (1994). Tradescantia stamen hair mutation bioassay. Mutation Research, 310, 211220. Monarca, S., Donatella, F., Zerbini, I., Alberti, A., Zani, C., Sergio, R., et al. (2002). Soil contamination detected using

117 bacterial and plant mutagenicity tests and chemical analyses. Environmental Research Agenda, 88, 6469. Maron, D. M., & Ames, B. N. (1983). Revised methods for the salmonella mutagenicity test. Mutation Research, 113, 173215. Martin, F. L., Piearce, T. G., Hewer, A., Phillips, D. H., & Semple, K. T. (2005). A biomarker model of sublethal genotoxicity (DNA single-strand breaks and adducts) using the sentinel organism Aporrectodea longa in spiked soil. Environmental Pollution, 138, 307315. Mouchet, F., Gauthier, L., Mailhes, C., Jourdain, M. J., Ferrier, V., Triffault, G., et al. (2006). Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration using the comet and micronucleus tests on amphibian (Xenopus laevis) larave and bacterial assays (Mutatox and Ames tests). Science of the Total Environment, 355, 232246. Official Gazette (1996). Uredba o vnosu nevarnih snovi in rastlinskih hranil v tla. Uradni list Republike Slovenije, 68, 5769. Olive, P. L., Banath, P. J., & Durand, R. E. (1996). Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the comet assay. Radiation Research, 112, 8694. Patra, M., Bhowmik, N., Bandopadhyay, B., & Sharma, A. (2004). Comparison of mercury lead and arsenic with respect to genotoxic effects on plant systems and the effects on plant systems and the development of genetic tolerance. Environmental and Experimental Botany, 52, 199223. R Development Core Team (2005). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. ISBN 3-900051-070, URL http://www.R-project.org. Radetski, C. M., Ferrari, B., Cotelle, S., Masfaraud, J.-F., & Ferard, J. F. (2004). Evaluation of the genotoxic mutagenic and oxidant stress potentials of municipal solid waste incinerator bottom ash leachates. Science of the Total Environment, 333, 209216. Rudolf, E., Cervinka, M., Cerman, J., & Schroterova, L. (2005). Hexavalent chromium disrupts the actin cytoskeleton and induces mitochondria-dependent apoptosis in human fibroblasts. Toxicology In Vitro, 19, 713723. Sauvant, M. P., Pepin, D., Bohatier, J., & Grolierere, C. A. (1995). Comparison of six bioassays for the assessing in vitro acute toxicity and structure-activity relationships for vinyl chloride monomer its main metabolites and derivatives. Science of the Total Environment, 172, 7992. Sargeant, A., & Goswami, T. (2006). Hip implants-Paper VIIon concentrations. Materials & Design, 27, 287307. Singh, J., Carlisle, D. L., Pritchard, D. E., & Patierno, S. R. (1998). Chromium-induced genotoxicity and apoptosis: Relationship to chromium carcinogenesis (review). Oncology Reports, 5, 13071318. SIST ISO 11464 (1994). Soil quality pre-treatment of samples for physico-chemical analyses. Geneve, Switzerland: International Organization for Standardization. SIST ISO 5666 (1996a). Water quality Determination of mercury. Geneve, Switzerland: International Organization for Standardization.

118 SIST ISO 10523 (1996). Water quality Determination of pH. Geneve, Switzerland: International Organization for Standardization. SIST ISO 11083 (1996). Water quality Determination of chromium (VI)-spectrometric method using 15diphenylcarbazide. Geneve, Switzerland: International Organization for Standardization. SIST ISO 10381 (1996). Soil quality-sampling Part 6. Geneve, Switzerland: International Organization for Standardization. SIST EN ISO 10304-2 (1998). Water quality Determination of dissolved anions by liquid chromatography of ions Part 2: Determination of bromide chloride nitrate nitrite orthophosphate and sulphate in waste water. Geneve, Switzerland: International Organization for Standardization. SIST DIN 38406-29 (2000). German standard methods for the examination of water wastewater and sludge-Cations (group E) Part 29: Determination of highly volatile halogenated hydrocarbons Gas-chromatographic methods. Geneve, Switzerland: International Organization for Standardization. SIST ISO 8245 (2000). Water quality Guidelines for the determination of total organic carbon TOC and dissolved organic carbon DOC. Geneve, Switzerland: International organization for standardization.

Environ Monit Assess (2008) 139:107118 Spiegelhalter, D. J., Thomas, A., Best, N. G., Gilks, W. R., & Lunn, D. (2003). BUGS: Bayesian inference using Gibbs sampling. Cambridge, England: MRC Biostatistics Unit. http://www.mrc-bsu.cam.ac.uk/bugs/. Sturtz, S., Ligges, U., & Gelman, A. (2005). R2WinBUGS: A package for running WinBUGS from R. Journal of Statistical Software, 12, 3, 116. http://www.jstatsoft.org/v12/i03. Tchounwou, P. B., Yedjou, C. G., Foxx, D. N., Ishaque, A. B., & Shen, E. (2004). Lead-induced cytotoxicity and transcriptional activation of stress genes in human liver carcinoma Hep-G2) cells. Molecular and Cellular Biochemistry, 255, 161170. United States Environmental Protection Agency (1996). Health effects tests guidelines: OPPTS 8705265. The Salmonella typhimurium reverse mutation assay. EPA712-C-96219. Washington: United States Environmental Protection Agency. Watanabe, T., & Hirayama, T. (2001). Genotoxicity of soil. Journal of Health Science, 47, 433438. White, P. (2002). The genotoxicity of priority polycyclic aromatic hydrocarbons in complex mixtures. Mutation Research, 515, 8598. White, P. A., & Claxton, L. D. (2004). Mutagens in contaminated soil: A review. Mutation Research, 567, 227345.