genotoxicity evaluation of water soil leachates by ames test, comet assay, and preliminary...

Genotoxicity evaluation of water soil leachates by Ames test,comet assay, and preliminary Tradescantiamicronucleus 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 testsand physico-chemical methodologies can be usefulfor determining the potential genotoxic contaminantsin soil samples. The aim of our study was to evaluatethe genotoxicity of soil by applying an integratedphysico-chemical-biological approach. Soil sampleswere collected at six sampling points in a Slovenianindustrial and agricultural region where contami-nation by heavy metals and sulphur dioxide (SO2) areprimarily caused by a nearby power plant. The in vitroalkaline version of the comet assay on water soil

leachates was performed with Caco-2 and HepG2cells. A parallel genotoxicity evaluation of the sampleswas performed by Ames test using Salmonellatyphimurium and the Tradescantia micronucleus test.Pedological analyses, heavy metal content determina-tion, and different physico-chemical analyses, werealso performed utilizing standard methodology. Waterleachates of soil samples were prepared according tostandard methods. Since only a battery of biotests withprokaryotic and eukaryotic organisms or cells canaccurately estimate the effects of (geno)toxicants insoil samples and water soil leachates, a combination ofthree bioassays, with cells or organisms belonging todifferent trophic levels, was used. Genotoxicity of allsix water soil leachates was proven by the comet assayon both human cell lines, however no positive resultswere detected by bacterial assay, Ames test. TheTradescantia micronucleus assay showed increase inmicronuclei formation for three samples. Accordingto these results we can assume that the comet assaywas the most sensitive assay, followed by themicronucleus test. The Ames test does not appear tobe sensitive enough for water soil leachates genotox-icity evaluations where heavy metal contamination isanticipated.

Keywords Soil . In vitro bioassays .

Water soil leachates . Genotoxicity . Ames test .

Comet assay . Tradescantia micronucleus test

Environ Monit Assess (2008) 139:107–118DOI 10.1007/s10661-007-9819-7

B. Lah : T. Cepeljnik :G. Gorjanc :R. Marinsek-Logar (*)Zootechnical Department, Biotechnical Faculty,University of Ljubljana,Groblje 3,1230 Domzale, Sloveniae-mail: [email protected]

T. VidicBiotechnical Faculty, Department of Biology,University of Ljubljana,Večna pot 111,1000 Ljubljana, Slovenia

E. GlasencnikERICo Velenje, Environmental Research & IndustrialCo-operation Institute,Koroška 58,3320 Velenje, Slovenia

Introduction

Genotoxic compounds in soil may have an effect onhuman health through inhalation of dust, ingestion ofplants that absorbed the compounds from the soil, andthe leaching of the compounds from soil to groundand surface water used for drinking (Watanabe andHirayama 2001). The physical and chemical nature ofsoil is very complex and standard chemical andpedological analyses are limited in their ability tocharacterize the chemical composition of genotoxi-cants in soil. On the other hand, genotoxicological andmutagenicity bioassays provide a means for assessingthe (geno)toxicity of complex mixtures without theneed of precise chemical characterization. The geno-toxicity of unknown mixtures is usually evaluated byexposing such samples to living organisms which arefurther examined for genetic damage (Chenon et al.2003; Martin et al. 2005; Mouchet et al. 2006). Anumber of tests have been developed using aquaticand soil animals, amphibians, protozoa and prokary-otic microorganisms which can potentially assess thegenotoxic potential of the environmental sample(Gauthier et al. 1993; Sauvant et al. 1995; Békaertet al. 2002; Kim and Hyun 2006). Most of the studieslooking at the genotoxicity evaluation of soil samplesfocused on preparing the leachates from soil samplesprior to performing the bioassays (Békaert et al. 1999,2002; White and Claxton 2004). The bioavailability ofcontaminants is a key factor for ecotoxicologicaleffects therefore water extractable eluates were pre-pared and tested for genotoxicity in this study. Eventhough water soil leachates derived from polluted soiloften appeared to contain few toxic or genotoxic pol-lutants the impact of these complex matrices was con-firmed (Knasmüller et al. 1998).

To examine the genotoxicity of soil, the bacterialSalmonella/Ames test is most commonly used (Mon-arca et al. 2002; White and Claxton 2004). This testcan be carried out rapidly and cheaply, but the maindrawback is the lack of eukaryotic metabolic enzymesystems. Soil genotoxicity has also been frequentlyevaluated by plant bioassays like Tradescantia micro-nucleus test, Tradescantia stamen hair mutation test,Allium cepa test, and Vicia faba test, which demon-strate higher sensitivity and allow in situ monitoring ofsoil pollution (Ma et al. 1994; Cottele et al. 1999;Gichner and Veleminsky 1999; Knasmüller et al.2003; Liu et al. 2003). Primary DNA damage caused

by the potential presence of genotoxic substances insoil samples has been verified by the alkaline versionof the comet assay in water soil leachates in fewcases, too.

The aim of our study was to evaluate the genotox-icity of soil in Šaleška valley (Slovenia) by applying anintegrated physico-chemical-biological approach andto evaluate the relevance of the test battery, usingseveral biotests with different levels of sensitivity andspecificity and using different cells or organisms frommore than one trophic level. The environment inŠaleška valley is contaminated by the emissions ofthe Thermal power plant Šoštanj, releasing aerosols oftrace elements, heavy metals, nitrogen and sulphurcompounds which settle into the upper layers of soilsand vegetation, by municipal wastes and intensiveagricultural activities. Genotoxicity evaluations wereperformed 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, pedo-logical and standard physico-chemical analyses of soilsamples were conducted, too.

Materials and methods

Soil samples, pedological- and physico-chemicalanalysis

Coal and lignite-mining, coal burning, industry, anearby thermal power plant (TPP), traffic, andfarming left ecotoxicological burdens in a Slovenianindustrial-rural region called Šaleška dolina (Šaleškavalley) and the surrounding regions. Six soil samples(Velenje – sample 1 was taken 5 km from the TPP;Graška gora – sample 2 is 6.9 km from the TPP;Zavodnje – sample 3 is 7.2 km from the TPP; Šoštanj –sample 4 is 0.4 km from the TPP; Veliki vrh – sample 5is 2.7 km from the TPP; Zgornja Savinjska vally –sample 6, the reference location, which is situated inthe neighboring valley) were taken by standardprocedure (ISO 11464 1994; ISO 10381 1996) onDecember 2003. The soil samples were dried andsieved according to standard procedure (ISO 114641994) and afterwards stored at −20°C for furtheranalyses. Since heavy metals (lead, cadmium, zincand mercury), mainly from the thermal power plant,are released into the environment as dust particles

108 Environ Monit Assess (2008) 139:107–118

pedological and physico-chemical analyses wereperformed 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 inthis study refers to: lead (Pb), zinc (Zn), cadmium(Cd), arsenic (As), mercury (Hg), nickel (Ni), molyb-denum (Mo), chromium (Cr), cobalt (Co) and copper(Cu). SO2 content in the air 15 m above the soilsampling locations were measured by automaticmeasuring stations (1 h measuring interval), exceptfor the sampling location T6. For biological testing,soil samples were leached with water (the soil/waterratio was 1:5) as described by Békaert et al. (1999).The supernatant was then stored at 4°C and used forbiological testing next day. In order to eliminatepossible bacterial contamination leachates were ster-ilized by membrane filtration (0.22 μm) prior togenotoxicity testing (Békaert et al. 1999).

Ames/Salmonella typhimurium assay

Ames test was performed as a standard plateincorporation assay with Salmonella typhimuriumstrains TA97a, TA98 and TA100 with (+S9) andwithout (−S9) in vitro extracellular microsomalactivation (by S9 rat liver enzyme homogenate;Maron and Ames 1983). Strain specific geneticmarkers were verified prior to use. For each testerstrain a specific positive control was used with/without metabolic bioactivation: for strains TA97a-ICR-acridine; TA98-4NQNO and TA100-sodiumazide, without metabolic bioactivation and 2AF forthe strains TA98 and TA100; and B(a)P for TA97astrain with metabolic bioactivation.

Following 48 h incubation at 37°C genotoxicactivities were expressed as induction factors (inductionfactor of reversions) i.e. as multiples of the backgroundlevels. Statistical significance was demonstrated withthe Kruskal–Wallis test (non-parametric ANOVA) fordifferences between treatment groups and Dunnett’smultiple comparison for differences to the negativecontrol. The interpretation of the Ames test resultsfollowed OECD 471 guidelines (Organisation forEconomic Cooperation and Development 1997) andEPA Health Effects Tests Guidelines (OPPTS870.5265; United States Environmental ProtectionAgency 1996) for genotoxicity testing of chemicals.According to EPA and Organisation for Economic

Cooperation and Development (1997) guidelines, amutagenic potential is assumed, if the mutant frequen-cy is 2.0 or higher. A dose effect relationship couldunderline this conclusion. A possible mutagenicpotential is assumed if the mutant frequency is from1.7 to 1.9, in combination with a dose effectrelationship. No mutagenic potential for a tested itemis assumed if all mutant frequencies range between 1.0(and lower) and 1.6.

In vitro comet assay with Caco-2 and HepG2 celllines

Epithelial colon cancer cells (Caco-2) were obtainedfrom Instituto Zooprofilatico Sperimentale, Brescia,Italy, and the human hepatoma cell lines (HepG2 cells)were obtained from the Institute of Cancer Research atThe University of Vienna, Austria. Both cell lines weregrown in a monolayer culture at 37°C in a humidifiedatmosphere of 5% CO2 in Dulbecco’s ModifiedEagle’s Medium (DMEM), supplemented with 10%fetal calf serum (FCS) for HepG2 cells and 15% FCSfor Caco-2 cells. In both cases, gentamycin (40 μg/ml)was added to the medium. The cells were seeded at adensity of 40,000 cells per cm2 in 24 well tissue platesand cultured for 8 days. The medium was changedthree times a week. The comet assay was performed asdescribed by Lah et al. (2005a,b) with minor mod-ifications. Cells in wells were exposed to sterile watersoil leachates with the added components of DMEMmedium (test samples T1–T6) and sterile DMEMmedium (negative control) for 24 h in triplicates. Nineindependent replicates on water soil leachates andnegative controls were carried out on each cell line.

Single cell suspensions for each treatment wereprepared by adding 0.25% trypsin–EDTA solution,resuspending in DMEM medium and passed throughpipette tips several times. The dye-exclusion test withTrypan blue (Duthie and Collins 1997) was used toexamine the viability of cells before the comet assaywas performed. The viability of cells was alwayshigher than 90%.

Briefly, the collected Caco-2 or HepG2 cells wereimmobilized in the third agarose layer of four layeredmicrogels on microscope slides at a concentration ofapproximately 1–2×105 cells/ml. For a positivecontrol, the four layered microgels with cells grownin sterile DMEM were treated with 500 μM hydrogenperoxide for 5 min before lysis. The microgels with

Environ Monit Assess (2008) 139:107–118 109

incorporated cells were submerged afterwards into analkaline lysis buffer for 1 h and followed by electro-phoretic buffer (pH>13) for 40 min. The electropho-resis was carried out at 2 V/cm and 300 mA for 30 min,followed by neutralisation in 400 mM Tris-HCl pH 7.5for 15 min. The damaged DNA traveled toward theanode and formed an image of a “comet” tail. Thecomets were visualized by staining with ethidiumbromide (20 μg/ml). Quantitative analysis of nuclearDNA damage in Caco-2 and HepG2 cells was donewith an epifluorescence microscope (Olympus BX 50)using 20× objective magnification; using a BP 515–560 nm filter and BA 590 nm barrier filter and digitalcamera (Hammamatsu Orca 2) connected to a com-puter. The comets were scored using Komet 5.0Computer Software (Kinetic Imaging 2001). Olive tailmoment (OTM) was chosen as the most relevantmeasure of DNA damage (Olive et al. 1996). Imagesof 100 comets were collected from each of the tworeplicate slides per test sample T1–T6 and OTMswere collected for further statistical analyses.

Full Bayesian approach (Gelman et al. 2004) wasused for the statistical analysis of comet assay data.Analysis was performed in two stages. In the firststage, following Lovell et al. (1999), a quantitativemeasure for each sample of cells was calculated. Wedid not only calculate a quantitative measure such asmean or mean of log transformed data, but we built aprobability model and calculated a quantitativemeasure based on the fit of the probability model.The distribution of OTM data was substantiallyskewed and we assumed that olive tail moments Yijof scored cells (index j) follow Weibull distributionEq. 1 as has been proposed by Ejchart and Sadlej-Sosnowska (2003) and to vary between cell samples(index i) according to distribution parameters θi andli. We used “noninformative” distributions for bothparameters i.e. exponential for θi, which is decreasingslowly for positive values, and uniform with widerange for θi. Based on fit of this model a quantitativemeasure and its standard error for each sample of cellswere calculated separately. We choose median value(mediani ¼ log 2ð Þl�1

� �qi ).

yij ~Weibull θi; 1ið Þθi ~ Exponential 0:001ð Þ; log 1ið Þ ~ Uniform �100; 100ð Þ

ð1Þ

Altogether, the medians and standard errors for 57samples of cells were calculated for CaCo-2 line ofcells and 49 medians for HepG2 line of cells. Amongeach of these medians, three medians representedDNA damage in negative controls. To express DNAdamage compared to the negative control,corresponding medians and their standard errors weresubtracted i.e. we performed a background correctionto the negative control.

In the second stage, background corrected medians(Yijk) were modeled Eq. 2 including intercept (α),effects of water soil leachates (Ti), day (Di) andreplicate within the day. “Noninformative” priordistributions (Gelman et al. 2004) were assigned forall parameters of the model.

yijk ~ Normal μijk ; σ2k

� �

μijk ¼ αþ Ti þ Dj þ eijkα;Ti;Dj ~ Normal 0; 1002ð Þ;eijk ~ Normal 0; σ2e

� �; σe ~ Uniform 0; 10ð Þ

ð2Þ

For each stage of analysis, a Markov chain MonteCarlo method with three chains of 5,000 samples wasused. Length of burn-in period was assessed visuallyby chain inspection and BGR (Brooks-Gelman-Rubin)statistic (Gelman et al. 2004). Chains appeared toconverge to stationary distribution very fast (in only fewiterations) and we discarded half of the samples (2,500)in each chain as a conservative choice. All calculationswere performed with R (R Development Core Team2005; Sturtz et al. 2005) and BUGS (Bayesianinference using Gibbs sampling; Spiegelhalter et al.2003) programs. Data and program codes for analysisare available from the authors upon request. Results ineach stage of the analysis are graphically representedwith posterior medians and 95% credible intervals.

The preliminary Tradescantia micronucleus (MN)assay

The Tradescantia MN test was performed as de-scribed by Knasmüller et al. (2003) with some minormodifications. The cuttings were exposed to watersoil leachates (T1–T6), to tap water (as negativecontrol) for 48 h, and to 5 mM maleic hydrazide –C4H4N2O2 (as positive control) for 6 h, followed by24 h recovery before fixation. The inflorescenceswere fixed in 1:3 acetic acid-ethanol (Carnoy’s)solution and transferred into 70% ethanol.


The MN assay is based upon the fact that DNAdamage in pollen mother cells leads to formation ofmicronuclei in the early tetrad stage. Micronuclei aresmall masses of chromatin which appear beside themain nuclei when the plant is in contact with aneugenagents (fusorial poisons) or clastogen agents (induc-ing chromosome breaks). Inflorescences containingearly tetrad cells were found in buds with a length of 3–4 mm and afterwards slides were prepared and stainedwith aceto-carmine as described by Knasmüller et al.(2003). Usually, 1,500 tetrads are scored under themicroscope from five slides per sample and controls.Due to the lack of available plant material at the timeof the experiment, not enough flower buds containingtetrads could be obtained. Consequently, only 300–500 tetrads per sample were scored and the results aretherefore only qualitatively informative and regardedas preliminary.

The micronuclei frequencies were calculated bydividing the total number of detected micronucleiwith the total number of tetrads scored and expressed

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 physico-chemical 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 concen-trations of phosphorous and potassium. T2 soil islisted as a medium-heavy clay soil (C) that is weaklyacidic with a high concentration of potassium but alow concentration of phosphorous, both T1 and T2soils are low in organic carbon and Kjeldahl nitrogencompared to the rest four samples. T3 soil is a lightsandy-clay soil (S-C) that is weakly acidic with highconcentrations of potassium and phosphorous. T4 andT5 soils are listed as medium to heavy clay soils (C).T4 soil is weakly alkaline and has low concentrationsof potassium and phosphorous. T5 on the other handis weakly acidic and has high concentrations ofpotassium and low concentrations of phosphorous.

Heavy metal concentrations of six soil samples(Table 3) were compared to permitted immission limitvalues (PIL values) according to Slovenian Regula-tions for the Amounts of hazardous compounds,heavy metals and fertilizers in soils (Official Gazette1996). According to the immission limit values forthe following metals: Pb, Hg, Zn, Cu, Co, Mo and Ni,none of the chosen sampling locations could bedeclared as polluted. PIL values and even warningimmission values for Cr however were exceeded in allsoil samples. Immission limit values for Cd in

Table 1 Pedological parameters of the six soil samples (T1–T6)

Parameter Fine sand Rough sand Clay Sand Texture classUnit % % % % /

T1 15.50 15.50 10.90 58.10 S-CT2 19.30 12.00 19.10 49.60 CT3 17.90 7.70 13.10 61.30 S-CT4 26.10 9.90 14.00 50.00 CT5 26.90 7.10 19.10 46.90 CT6 28.20 8.10 21.00 42.70 C

Measurements of the determined parameters were performedaccording to the ISO standard methods described in “Materialsand methods”; T1–T6=soil samples

S-C Sandy-clay soil particles; C clay soil particles

Parameter pH(H2O)

pH(KCl)

Organiccarbon

Kjeldahlnitrogen

Easy accessiblepotassium (K)

Easy accessiblephosphorus (P)

Unit / / g/kg d.w. % mg K2O/100 g mg P2O2/100 g

T1 7.42 6.90 28.70 0.33 9.30 1.95T2 6.16 5.29 28.50 0.35 27.20 4.94T3 6.40 5.74 96.20 0.97 22.30 12.40T4 6.91 6.34 44.00 0.44 9.24 8.11T5 5.53 4.22 61.70 0.58 53.00 4.77T6 6.27 5.62 45.10 0.56 25.90 1.56

Table 2 Physico-chemicalparameters of the six soilsamples (T1–T6)

Measurements of the deter-mined parameters were per-formed according to the ISOstandard methods describedin “Materials and methods”;T1–T6=soil samples

d.w Dry weight


Table 3 Heavy metals and nitrates concentration in six soil samples and in water extracts

Parameter (unit) Samples PIL values

T1 T2 T3 T4 T5 T6

Pb (mg/kg) 38.00 37.20 72.00 44.00 33.00 46.20 85Zn (mg/kg) 119.00 90.10 144.00 131.00 126.00 123.00 200Cd (mg/kg) 0.59 0.64 1.74 0.62 1.11 0.98 1As (mg/kg) 13.70 10.70 6.66 11.60 8.86 21.20 20Hg (mg/kg) 0.11 <0.10 0.24 0.15 0.42 0.15 0.8Ni (mg/kg) 43.60 43.20 16.60 40.90 31.30 42.30 50Mo (mg/kg) 2.61 1.90 2.31 2.55 3.22 4.66 10Cr (mg/kg) 193.00 193.00 187.00 216.00 169.00 191.00 100Co (mg/kg) 16.30 10.00 11.70 15.70 8.78 14.40 20Cu (mg/kg) 31.90 17.90 16.90 33.50 23.10 35.10 60SO2 (μg/m

3)a 54.00 126.00 138.00 525.00 266.00 – 350Nitrates (mg/kg) 6.90 <4.00 4.00 4.60 6.40 <4.00 –

Measurements of the determined parameters were performed according to the ISO standard methods described in “Materials andmethods”; T1–T6=soil samples

PIL values Permitted immission limit values according to Slovenian regulationsa 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) nuclearDNA damage presented asOlive Tail Moment (OTM)detected in six water soilleachates after 24 h celltreatment. The upper twographs represent the poste-rior medians and 95% cred-ible intervals for eachsample of cells according tofirst stage of the analysisEq. 1, while the lower twographs represent the poste-rior medians and 95% cred-ible intervals for each watersoil leachates according tosecond stage of the analysisEq. 2. Genotoxicity is indi-cated in the cases where thecredible intervals do notoverlap zero value


samples T3 and T5 and for As in sample T6 wereexceeded, too. The proportions of the metals extractedinto water samples which were further applied forbiotests ranged from 0.02 to 0.1% of the total heavymetal amounts present in soils. The highest concen-trations of sulphur dioxide in the air were detectedabove the sampling locations T4 and T5 (Table 3), thePIL value was exceeded only at T4. There might be acorrelation between SO2 immissions and low pHvalue of sample T5. These two locations are theclosest to the thermal power plant and are directlyexposed to its emissions.

Results of the bioassays

Ames test/Salmonella typhimurium assay usuallyrepresents the first step in genotoxicity/mutagenicitytesting of various pure chemicals and environmentalsamples (Békaert et al. 1999; Bispo et al. 1999;Ehrlichmann et al. 2000; Monarca et al. 2002). In ourstudy, the plate incorporation method of the Ames testwas performed.

In our study, the Ames test (+S9 and −S9) failed todetect the genotoxic potential in any of water soilleachates as the results never exceeded the criticalvalue of 2.0 and all the quotients ranged below 1.6.As recommended by Maron and Ames (1983), primarytester strains TA97a, TA98 and TA100 were used.

Regarding the comet assay on CaCo-2 and HepG2cells, calculated medians and corresponding credibleintervals for each cell sample (water soil leachates andnegative controls) after first stage of the analysis arepresented in Fig. 1 (upper two graphs). All credibleintervals are above zero and indicate genotoxicity forall samples. Additionally, substantial variability canbe observed. In the second stage of the analysis

genotoxicity of the water soil leachates in comparisonto the negative controls was assessed. Results arepresented in Fig. 1 (lower two graphs) and indicateclear genotoxicity of all six water soil leachates onCaCo-2 cells, with the highest genotoxicity found insample T3. On the other hand, the results for theHepG2 cells do not clearly indicate genotoxicity forsamples T1 and T4 and demonstrated the highestgenotoxicity of samples T3 and T5.

Due to the lack of plant material only one exposure ofplants to water soil leachates and controls was possibleat the time and in some cases not enough flower budscontaining tetrads could be obtained. Consequently,fewer than 1,500 tetrads could be enumerated, precisely300–500 tetrads per sample. It is important to stress thatdue to the lack of plant material, these results aretherefore only qualitatively informative. However, it isinteresting to note that an increasing trend in the numberof micronuclei for samples T1, T2 and T4 could beseen, indicating genotoxicity (Fig. 2).

Discussion

Extensive previous studies of soil contamination inŠaleška valley indicated the heavy metals as the maincontaminating agents in this region. The levels of thetotal concentration of PCBs and PAHs have notexceeded 0.01 and 0.02 mg/kg, respectively, what iswell below the permitted immission limit values. Theconcentrations of the analysed pesticides and AOX havenot exceeded the limit values, either (Kugonič andStropnik 2004; Kugonič et al. 2004). Most metallicsalts are effective poisons at particular concentrations,because they are able to bind to thiol groups andinduce spindle disturbances in the cells (Patra et al.2004). When considering heavy metals in soil, thebioavailability is a very important factor in the ecotox-icological evaluations of the soil. Bioavailable com-pounds are usually water soluble mobile compounds, onthe contrary, biounavailable compounds are complexand not mobile; however, bioavailability can beincreased in fluid media (Giller et al. 1998; Békaertet al. 1999; Fent 2004; Loska et al. 2004). By now, themajority of metal (geno)toxicity studies in Šaleškavalley have been focused on soil–plant interactions(Glasenčnik et al. 2002; Kugonič et al. 2004).

The presented results of the chemical analyses of sixsoil samples (T1–T6) indicated that the tested soils are

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


in general moderately contaminated according toSlovenian Regulations (Official Gazette 1996). For allsoil samples collected in the Šaleška valley, thechromium content in soil samples exceeds the warningand the permitted limit immission value, for Cdpermitted limit immission value is exceeded in samplesT3 and T5, for As in T6 and for Ni slightly exceededin samples T1 and T2.

Heavy metals and/or their compounds are classi-fied as mutagenic, carcinogenic and clastogenicsubstances (White and Claxton 2004). Anyhow, thenegative results of the Ames test in present study failto prove the presence of mutagens in soil leachates,causing frameshift or basepair substitutions. There areseveral reports on lower sensitivity of this pointmutation test and disability in detection of genotoxiceffects caused by heavy metals (Knasmüller et al.1998; Monarca et al. 2002; Radetski et al. 2004). Atthe same time these negative results could confirm theabsence of PAHs in soil samples, which account inmany cases of genotoxic effects of soils and sedi-ments (White 2002; White and Claxton 2004).

The preparation of the samples can be connected toand could partly explain the lower sensitivity of theAmes test (Békaert et al. 1999). The filtration step(0.2 μm) used in the Ames test procedure may be oneof the causes of this bioassay’s lower sensitivity ascompared to the other tests. Some of the componentsin leachates could attach to the filters and areconsequently lost for further testing. Filtration of thesamples however partly eliminates the contaminantfraction potentially present in the sample which isotherwise available to living organisms in other in vivotests or in plant micronucleus assay (Mouchet et al.2006). The negative results recorded on water soilleachates by the Ames test can also be explained by thelow water extractability of metals from soil samples.Very low concentrations (μg/l) of heavy metals areactually extracted from the soil into the water as it wasreported by Ivask et al. (2004). Knasmüller et al. (1998)reported that the metal amounts in the aqueous leachateswere five- to 10-fold lower than the concentrationsrequired to cause detectable effects on salmonella DNA.The proportions of the metals extracted into watersamples which were further applied for biotests in thepresent study were low, too, and ranged from 0.02 to0.1% of the total heavy metal amounts present in soils.

Due to the lower sensitivity of Ames test for heavymetals, further work must be directed to the develop-

ment of bioassays with higher organisms/cells(Gatehouse et al. 1990). This is also one of thereasons why short-term tests with cultivated mam-malian cells are widely used for the detection ofpotential environmental mutagens and carcinogens.Since numerous compounds exert their genotoxic andcarcinogenic effects only following metabolic activa-tion, it is important that indicator cell lines aremetabolically competent (Glatt et al. 1990). Ac-cording to the findings of Knasmüller et al. (2004),HepG2 cells represent an excellent tool for thedetection of environmental and dietary genotoxicants.The comet assay results on HepG2 cells clearly revealthe genotoxic effects on the level of DNA damage byDNA breaks (which might potentially lead to carci-nogenicity) in water soil leachates for samples T2, T3,T5 and T6 (Fig. 1). These effects are not as clear forsamples T1 and T4. At the same time, the highestgenotoxic potential was proven for samples T3 andT5, which might correlate with the highest concen-trations of Pb, Zn and especially Cd for sample T3,with the highest concentrations of Hg for sample T5and with the highest concentration of organic carbonand nitrogen for both (Tables 2 and 3). It is importantto stress, that DNA damage is also suspected to bedue to increased chromium contents in all six soilsamples The possible interactions between the samplecompounds present must also be taken into account.HepG2 cells have been several times successfullyused to study genotoxic and cytotoxic effects andmechanisms of action of heavy metals. Tchounwouet al. (2004) clearly demonstrated on HepG2 cells thepotential of lead nitrate to undergo biotransformationin the liver, to cause the proliferation, protein damage,metabolic perturbation, and growth arrest and DNAstrand breaks. Fatur et al. (2002) used the comet assayto demonstrate the DNA damage caused by Cd inHepG2 cells and supported the hypothesis that directand indirect mechanisms are involved in Cd-inducedDNA damage.

The second chosen indicator cell line for DNAdamage was Caco-2. Even though this cell lineappears not to posses all the desirable xenobioticmetabolizing enzymes of phase I and phase II, itexhibits different degree of specialization and entero-cyte like functions. The intestinal epithelium perme-ability, which Caco-2 cell line resembles, is a criticalcharacteristic that determines the rate and extent ofhuman absorption and ultimately the bioavailability of


a xenobiotic compound (Duthie et al. 1997). Thedetected genotoxicity in all water soil leachates(Fig. 1, the left two graphs) could be partially, butnot specifically ascribed, to increased Cr contents inall six soil samples, to exceeded limit immissionvalues for Cd in sample T3 and T5 and to theexceeded limited immission value for As in sampleT6 (Table 3) like in HepG2 cells, although the twocell lines showed a bit different pattern of responses.

A lot of research work has been done regarding thegenotoxicity of heavy metals, especially of Cr and As.Hartmann and Speit (1994) and Basu et al. (2001)agree about the possible mechanism of the genotoxicaction of As, where arsenic indirectly inhibits theenzymes for DNA repair. Several studies report thegenotoxic, cytotoxic and carcinogenic effects of Cr(VI) and its reduced form (III), which is a reactiveintermediate and which in combination with oxidativestress in cells causes oxidative DNA damage (Singhet al. 1998; Cavas and Ergene-Gözükara 2005; Levinaand Lay 2005; Rudolf et al. 2005; Sargeant andGoswami 2006). However, Majer et al. (2002)reported that the genotoxic effects of soil are to alarge extent connected to the physico-chemical prop-erties of the soil and influence profoundly the effectsof heavy metal contamination. The interactionsamong the metals and among the metals and othersoil components should be taken into considerationwhen explaining the genotoxic potential ofcontaminated soils (White and Claxton 2004).

Water soil leachates T1, T2 and T4 show a trendtoward increasing numbers of micronuclei formationwith the Tradescantia MN test (Fig. 2), indicatingclastogenicity, which can be explained by elevatedlevels of Cr and relatively high Pb, Ni and Hgcontents in soil samples. Tradescantia MN assay,based on the formation of micronuclei resulting fromchromosomal breakage in the meiotic pollen mothercells of Tradescantia ssp. Inflorescences. has beenone of the most frequently applied genotoxicityassays for detecting clastogenicity of contaminatedsoils (White and Claxton 2004). The extensive studyof Majer et al. (2002) of soils contaminated withvarying amounts of heavy metals (As, Cd, Cr, Cu,Mn, Ni, Pb, Zn...) proved numerous significantpositive responses, even threefold above the meancontrol value by Tradescantia MN assay. Besidemicronuclei induction in plants, the induction ofchromosomal aberrations and sister chromatide ex-

change by heavy metals has been detected by differentplant bioassays (White and Claxton 2004).

In this study, a battery of bioassays detectingdifferent genetic end-points were used: point muta-tions due to base pair substitution or base insertion ordeletion, detected by the Ames test; DNA breaks/primary DNA damage detected by the comet assay andcytogenetic damages due to an alteration in the plantchromosomal integrity expressed as micronuclei anddetected by the Tradescantia micronucleus test.According to several scientific reports (Maron andAmes 1983; Knasmüller et al. 1998, 2004; Majer et al.2002; Crebelli et al. 2005; Mouchet et al. 2006)describing the involvement of genetic end-points inpathological processes, all these end-points should betaken into consideration while monitoring the environ-ment for genotoxic hazards. Crebelli et al. (2005)explain that chemical mutagens usually do not affectdifferent genetic end-points with the same degree ofefficiency. These conclusions are in agreement with theresults of our study, where we are mostly dealing withheavy metal soil contamination. Several differentbioassays used in our study complement each otherfor the genotoxic evaluation of soil samples. This studyis completed by the use of physico-chemical analyseswhich alone are insufficient for ecotoxicity monitoring,but are very useful in investigations of genotoxicitycausing factors and genotoxicity mechanisms.

Conclusions

Most of the mutagenic/genotoxic substances arepoorly water soluble, and the standard test systemsare not sensitive enough to detect these low concen-trations (Ehrlichmann et al. 2000). According to theresults of this study, it can be estimated that the cometassay and the micronucleus test are sensitive enoughfor the effective evaluation of genotoxicity in aqueoussoil leachates. In spite of the low extractability ofheavy metals and other xenobiotic compounds bywater, genotoxic effects were detected in two of theselected bioassays. To summarize, the so-calledpoliphase approach, combining physico-chemical,pedological and (geno)toxicological approaches inthe studies and monitoring of genotoxins in soilsamples, improves the evaluation of the ecotoxico-logical effects of environmental samples, due to manydifferent classes of xenobiotic compounds being


present in the environment. It might also be importantto consider the use of in vivo tests for genotoxicity,which would complement the in vitro results and givean even clearer picture of the genotoxic potential ofenvironmental samples.

Acknowledgements This study was supported by the SlovenianMinistry of Education, Science and Sports;Ministry of Agriculture,Forestry and Food and the Coal mining corporation RudnikVelenje. The authors are thankful to Mr Darryl Glover forreviewing the English language in the article.

References

Basu, A., Mahat, J., Gupta, S., & Giri, A. K. (2001). Genetictoxicology of a paradoxical human carcinogen arsenic: Areview. Mutation Research, 488, 17–194.

Békaert, C., Ferrier, V., Marty, J., Pfohl-Leszkowicz, A., Bispo,A., Jourdain, M. J., et al. (2002). Evaluation of toxic andgenotoxic potential of stabilized industrial waste andcontaminated soils. Waste Management, 22, 241–247.

Békaert, C., Rast, C., Ferrier, V., Bispo, A., Jourdain, M. J., &Vasseur, P. (1999). Use of in vitro (Ames and Mutatoxtests) assays to assess the genotoxicity of leachates fromcontaminated soil. Organic Geochemistry, 30, 953–962.

Bispo, A., Jourdain, M. J., & Jauzein, M. (1999). Toxicity andgenotoxicity of industrial soils polluted by polycyclicaromatic hydrocarbons (PAHs). Organic Geochemistry,30, 947–952.

Cavas, T., & Ergene-Gözükara, S. (2005). Induction of micro-nuclei and nuclear abnormalities in Oreochromis niloticusfollowing exposure to petroleum refinery and chromiumprocessing plant effluents. Aquatic Toxicology, 74, 264–271.

Chenon, P., Gauthier, L., Loubieres, P., Severac, A., &Delpoux, M. (2003). Evaluation of the genotoxic andteratogenic potential of a municipal sludge and sludge-amended soil using the amphibian Xenopus laevis and thetobacco: Nicotina tabacum L. var xanthi Dulieu. Scienceof the Total Environment, 301, 139–150.

Cottele, S., Masfaraud, J.-F., & Ferard, J.-F. (1999). Assessmentof the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleusassays. Mutation Research Fundamental and MolecularMechanisms of Mutagenesis, 426, 167–171.

Crebelli, R., Conti, L., Monarca, S., Feretti, D., Zerbini, I.,Zani, C., et al. (2005). Genotoxicity of the disinfectionby-products resulting from paracetic acid- or hypochlorite-disinfected sewage wastewater. Water Research, 39,1105–1113.

Duthie, S. J., Johnson, W., & Dobson, V. L. (1997). The effectof dietary flavonoids on DNA damage (strand breaks andoxidised pyrimidines) and growth in human cells. Muta-tion Research, 340, 141–151.

Ehrlichmann, H., Dott, W., & Eisentraeger, A. (2000).Assessment of the water-extractable genotoxic potential

of soil samples from contaminated sites. Ecotoxicologyand Environmental Safety, 46, 73–80.

Ejchart, A., & Sadlej-Sosnowska, N. (2003). Statistical evalu-ation and comparison of comet assay results. MutationResearch. Genetic Toxicology, 534, 85–92.

Duthie, S. J., & Collins, A. (1997). The influence of cell growthdetoxifying enzymes and DNA repair on hydrogen peroxide-mediated DNA damage (measured using the comet assay) inhuman cells. Free Radical Biology & Medicine, 4, 717–724.

Fatur, T., Tusek, M., Falnoga, I., Scancar, J., Lah, T. T., &Filipic, M. (2002). DNA damage and metallothioneinsynthesis in human hepatoma cells (HepG2) exposed tocadmium. Food & Chemical Toxicology, 40, 1069–1076.

Fent, K. (2004). Ecotoxicological effects at contaminated sites.Toxicology, 20, 22–240.

Gatehouse, D. G., Wilcox, P., Forster, R., Rowland, I. R., &Callander, R. D. (1990). In D. J. Kirkland (Ed.), Bacterialmutation assays UKEM recommended procedures guide-lines for mutagenicity testing (pp. 13–59). Massachusetts:Cambridge University Press.

Gauthier, L., Van der Gaag, M. A., Haridon, J. L., Ferrier, V., &Fernandez, M. (1993). In vivo detection of waste waterand industrial effluent genotoxicity: Use of the NewtMicronucleus test (Jaylet test). Science of the TotalEnvironment, 138, 249–269.

Gelman, A., Carlin, J. C., Stern, H., & Rubin, D. B. (2004).Bayesian data analysis (2nd ed., pp. 58488–388). NewYork: Chapman & Hall.

Giller, K. E., Witter, E., & McGrath, S. P. (1998). Toxicity ofheavy metals to microorganisms and microbial processesin agricultural soils: a review. Soil Biology & Biochemis-try, 30, 1389–1414.

Gichner, T., & Velemínský, J. (1999). Monitoring the genotox-icity of soil extracts from two heavily polluted sites inPrague using the Tradescantia stamen hair and micronu-cleus (MNC) assays. Mutation Research Fundamental andMolecular Mechanisms of Mutagenesis, 426, 163–166.

Glasenčnik, E., Ribarič Lasnik, C., Müller, M., Grill. D., &Batič, F. (2002). Impact of air pollution on geneticmaterial of the shallot (Allium cepa L var ascalanicum)exposed in the vicinity of major Slovene local emissionsources in 1999. Phyton, 42, 237–250.

Glatt, H., Gemperlein, I., Setiabudi, F., Platt, K. L., & Oesch, F.(1990). Expression of xenobiotic-metabolizing enzymes inpropagatable cell cultures and induction of micronuclei by13 compounds. Mutagenesis, 5, 241–249.

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 investigationsof the genotoxic effects of metals in the single cells gel(SCGE) assay and their sister chromatide (SCE) test.Environmental and Molecular Mutagenesis, 23, 299–305.

Ivask, A., François, M., Kahru, A., Dubourguier, H.-C., Virta,M., & Douay, F. (2004). Recombinant luminiscentbacterial sensors for the measurement of bioavailabilityof cadmium and lead in soils polluted by metal smelter.Chemosphere, 55, 147–156.


Kim, I.-Y., & Hyun, C.-K. (2006). Comparative evaluation ofthe alkaline comet assay with the micronucleus test forgenotoxicity monitoring using aquatic organisms. Ecotox-icology and Environmental Safety, 64, 288–297.

Knasmüller, S., Gottmann, E., Steinkellner, H., Fomin, A.,Pickl, C., Paschke, A., et al. (1998). Detection ofgenotoxic effects of heavy metal contaminated soils withplant bioassays. Mutation Research, 420, 37–48.

Knasmüller, 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 ofenvironmental and dietary genotoxicants: Current state ofknowledge. Toxicology, 198, 315–328.

Knasmüller, S., Uhl, M., Gottmann, E., Hölzl, C., & Majer,B. J. (2003). The Tradescantia micronucleus bioassay. InJ. Maluszynska & M. Plewa (Eds.), Bioassays in Plantcells for improvement of Ecosystem and Human Health(pp. 95–106). Katowice: Wydawnictwo UniwersytetuŚląskiego

Kugonič, N., Pokorny, B., & Šešerko, M. (2004). Pollution ofgarden soils and vegetables in the Šalek Valley. ActaBiologica Slovenica, 1, 21–25.

Kugonič N., & Stropnik M. (2004). Heavy metal contents inagricultural soil and plants in Šaleška 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 vitrocomet assay and Ames test. Food Technology andBiotechnology, 432, 139–146.

Lah, B., Zinko, B., Tisler, T., & Marinsek-Logar, R. (2005b).Genotoxicity detection in drinking water by Ames testZimmermann test and Comet assay. Acta ChimicaSlovenica, 523, 341–348.

Levina, A., & Lay, P. A. (2005). Mechanistic studies ofrelevance to the biological activities of chromium. Coor-dination Chemistry Reviews, 249, 281–298.

Liu, W., Zhou, Q.-X., Li, P.-J., Sun, T.-H., Yang, Y.-S., &Xiong, X.-Z. (2003). 124-trichlorobenzene induction ofchromosomal aberrations and cell division of root-tip cellsin Vicia faba seedlings. Bulletin of Environmental Con-tamination and Toxicology, 71, 689–697.

Loska, K., Weichula, D., & Korus, I. (2004). Metal contami-nation of farming soils affected by industry. EnvironmentInternational, 30, 59–165.

Lovell, D. P., Thomas, G., & Dubow, R. (1999). Issues relatedto the experimental design and subsequent statisticalanalysis of in vivo and in vitro comet studies. Teratogen-esis, Carcinogenesis, and Mutagenesis, 19, 109–119.

Majer, B. J., Tscherko, D., Paschke, A., Wennrich, R., Kundi, M.,Kandeler, E., et al. (2002). Effects of heavy metal contam-ination of soils on micronucleus induction in Tradescantiaand on microbial enzyme activities: A comparative inves-tigation. Mutation Research, 515, 111–124.

Ma, T. H., Cabrera, G. L., Cebulska-Wasilewska, A., Chen, R.,Loarca, F., Vandenberg, A. L., et al. (1994). Tradescantiastamen hair mutation bioassay. Mutation Research, 310,211–220.

Monarca, S., Donatella, F., Zerbini, I., Alberti, A., Zani, C.,Sergio, R., et al. (2002). Soil contamination detected using

bacterial and plant mutagenicity tests and chemicalanalyses. Environmental Research Agenda, 88, 64–69.

Maron, D. M., & Ames, B. N. (1983). Revised methods for thesalmonella mutagenicity test. Mutation Research, 113,173–215.

Martin, F. L., Piearce, T. G., Hewer, A., Phillips, D. H., &Semple, K. T. (2005). A biomarker model of sublethalgenotoxicity (DNA single-strand breaks and adducts)using the sentinel organism Aporrectodea longa in spikedsoil. Environmental Pollution, 138, 307–315.

Mouchet, F., Gauthier, L., Mailhes, C., Jourdain, M. J., Ferrier,V., Triffault, G., et al. (2006). Biomonitoring of thegenotoxic potential of aqueous extracts of soils and bottomash resulting from municipal solid waste incineration usingthe comet and micronucleus tests on amphibian (Xenopuslaevis) larave and bacterial assays (Mutatox® and Amestests). Science of the Total Environment, 355, 232–246.

Official Gazette (1996). Uredba o vnosu nevarnih snovi inrastlinskih 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 andrepair in tumor and normal cells measured using the»comet« assay. Radiation Research, 112, 86–94.

Patra, M., Bhowmik, N., Bandopadhyay, B., & Sharma, A.(2004). Comparison of mercury lead and arsenic withrespect to genotoxic effects on plant systems and the effectson plant systems and the development of genetic tolerance.Environmental and Experimental Botany, 52, 199–223.

R Development Core Team (2005). R: A language andenvironment for statistical computing. Vienna, Austria: RFoundation for Statistical Computing. ISBN 3-900051-07-0, URL http://www.R-project.org.

Radetski, C. M., Ferrari, B., Cotelle, S., Masfaraud, J.-F., &Ferard, J. F. (2004). Evaluation of the genotoxic muta-genic and oxidant stress potentials of municipal solidwaste incinerator bottom ash leachates. Science of theTotal Environment, 333, 209–216.

Rudolf, E., Cervinka, M., Cerman, J., & Schroterova, L. (2005).Hexavalent chromium disrupts the actin cytoskeleton andinduces mitochondria-dependent apoptosis in human fibro-blasts. Toxicology In Vitro, 19, 713–723.

Sauvant, M. P., Pepin, D., Bohatier, J., & Grolierere, C. A.(1995). Comparison of six bioassays for the assessing invitro acute toxicity and structure-activity relationships forvinyl chloride monomer its main metabolites and deriva-tives. Science of the Total Environment, 172, 79–92.

Sargeant, A., & Goswami, T. (2006). Hip implants-Paper VI-Ion concentrations. Materials & Design, 27, 287–307.

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, 1307–1318.

SIST ISO 11464 (1994). Soil quality pre-treatment of samplesfor physico-chemical analyses. Geneve, Switzerland:International Organization for Standardization.

SIST ISO 5666 (1996a). Water quality – Determination ofmercury. Geneve, Switzerland: International Organizationfor Standardization.


http://www.R-project.org

SIST ISO 10523 (1996). Water quality – Determination ofpH. Geneve, Switzerland: International Organization forStandardization.

SIST ISO 11083 (1996). Water quality – Determinationof chromium (VI)-spectrometric method using 15-diphenylcarbazide. Geneve, Switzerland: InternationalOrganization 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 ofdissolved anions by liquid chromatography of ions Part 2:Determination of bromide chloride nitrate nitrite ortho-phosphate and sulphate in waste water. Geneve, Switzer-land: International Organization for Standardization.

SIST DIN 38406-29 (2000). German standard methods for theexamination of water wastewater and sludge-Cations(group E) – Part 29: Determination of highly volatilehalogenated hydrocarbons – Gas-chromatographic meth-ods. Geneve, Switzerland: International Organization forStandardization.

SIST ISO 8245 (2000). Water quality Guidelines for thedetermination of total organic carbon TOC and dissolvedorganic carbon DOC. Geneve, Switzerland: Internationalorganization for standardization.

Spiegelhalter, D. J., Thomas, A., Best, N. G., Gilks, W. R., &Lunn, D. (2003). BUGS: Bayesian inference using Gibbssampling. Cambridge, England: MRC Biostatistics Unit.http://www.mrc-bsu.cam.ac.uk/bugs/.

Sturtz, S., Ligges, U., & Gelman, A. (2005). R2WinBUGS: Apackage for runningWinBUGS fromR. Journal of StatisticalSoftware, 12, 3, 1–16. 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 tran-scriptional activation of stress genes in human livercarcinoma Hep-G2) cells. Molecular and Cellular Bio-chemistry, 255, 161–170.

United States Environmental Protection Agency (1996). Healtheffects tests guidelines: OPPTS 8705265. The Salmonellatyphimurium reverse mutation assay. EPA712-C-96–219.Washington: United States Environmental ProtectionAgency.

Watanabe, T., & Hirayama, T. (2001). Genotoxicity of soil.Journal of Health Science, 47, 433–438.

White, P. (2002). The genotoxicity of priority polycyclicaromatic hydrocarbons in complex mixtures. MutationResearch, 515, 85–98.

White, P. A., & Claxton, L. D. (2004). Mutagens in contami-nated soil: A review. Mutation Research, 567, 227–345.


http://www.mrc-bsu.cam.ac.uk/bugs/

http://www.jstatsoft.org/v12/i03

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