Introduction

An unfortunate consequence of industrialization and industrial production is the generation and release of toxic waste products. Although these wastes can be treated, reused, and recycled, large quantities of toxic material is released into the atmosphere, hydrosphere, and lithosphere (Claxton et al. 1988, 1998; Claxton and George 2002). Although much of the waste is released directly into the atmosphere and hydrosphere (i.e., surface waters of lakes, rivers, and streams), land disposal activities, intentional or otherwise, contribute to direct contamination of surface soils and subterranean (groundwater) aquifers. Intentional land disposal activities such as landfills, lagoons, surface impoundments, ponds, septic systems, and land treatment are cost-effective disposal strategies that take advantage of the enormous capacity of soil to retain and degrade toxic pollutants (Chakraborty and Konar 2002; Chen and White 2004). However, inadequate information about waste toxicity and post-disposal behavior, poor planning, improper disposal, and poor management of disposal sites has resulted in serious contamination problems at industrial and hazardous waste disposal sites (White and Claxton 2004; Barra et al. 2005).

Pesticide effluents might be played an important role in the transmission of environmental carcinogens. These compounds are released into air eventually deposit on the ground and can be accumulated in surface soil; therefore, surface soil is thought to be a promising material for monitoring environmental pollution with mutagens and carcinogens. Indeed, many studies showed that organic extracts of soils from roadsides, parks (Watanabe et al. 2005), agricultural land (Edenharder et al. 2000), residential sites (Knize et al. 1987), and so forth exhibited mutagenicity and/or DNA damaging activity (Watanabe and Hirayama 2001; Courty et al. 2008; Watanabe et al. 2008). Genotoxic compounds in soil may have an effect on human health in an exposed population through pathways such as inhalation of dust which contains these compounds, ingestion of plants that uptake the compounds from soil, and leaching of the compounds from soil to groundwater and surface water used as drinking water (Ehrlichmann et al. 2000; Watanabe and Hirayama 2001; Covacia et al. 2005; Watanabe et al. 2008).

There are several reports on the genotoxicity of soil contaminated with chemicals originating from industrial sources. The contaminants of these soil samples varied widely, e.g., polychlorinated biphenyl (Donnelly et al. 1988; DeMarini et al. 1992; Cotelle et al. 1999), polycyclic aromatic hydrocarbons (Ehrlichmann et al. 2000), heavy metals (Cotelle et al. 1999; Ehrlichmann et al. 2000) solvents (Cotelle et al. 1999), munition wastes (Ehrlichmann et al. 2000), wood-preserving wastes (McDaniels et al. 1993; Randerath et al. 1994), etc. Some authors reported about the mutagenic activity of pesticides, applying many biological assays (Siroki et al. 2001; Celik et al. 2003; Asita and Makhalemele 2008; Steckert et al. 2009). These bioassays provide a means for assessing the genotoxicity of complex mixtures without the need of precise chemical characterization. The genotoxicity of unknown mixture 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; Lah et al. 2008). The Salmonella mutagenicity test for mutagens and environmental compounds is the most widely used short-term test (Claxton et al. 1992; Shukla et al. 2004). Soil samples need to be extracted before testing since they cannot be applied directly to aqueous test systems. Extractions are often performed using organic solvents (Courty et al. 2004, 2008).

Application of wastewater to agricultural lands for enhancing crop productivity is a common practice in India as well as in many other countries from several decades without knowing its ill effect. However, long-term irrigation with industrial effluents mixed with municipal wastewater has resulted in the excessive accumulation of toxicants in agricultural soils (such as heavy metals and polychlorinated substances from domestic and industrial wastes that enters the sewer system) and has brought a potential risk to human health due to the accumulation of toxicants through the food chain (Malik and Ahmad 1995; Aleem and Malik 2003; Ansari and Malik 2009).

The present study focuses on the mutagenicity of agricultural soil irrigated with wastewaters discharged from industries from about two decades and ground water irrigated soil using Ames Salmonella/mammalian microsome test with special reference to pesticide pollution.

Materials and methods

Sample collection

Composite soil samples were collected at a 15-cm depth from three different adjacent agricultural fields near a pesticide industry, Chinhat, Industrial area, Lucknow, UP (India) which had been irrigated with wastewater discarded from the factory located at around 200 m. For each field, 1 kg of soil from four different sites at around 50 m distance from each other was collected and 4 kg composite samples were prepared. Composite samples were transported and stored at 4°C.

Assessment of pesticides in contaminated soil

Extraction of pesticides from soil was done according to the method described by Gan et al. (1999). Soil (10 g) was shaken vigorously with 20 ml of solvent: water (4:1, v/v) for 1 h. The mixture was centrifuged at 10,000 rpm (8,940 ×g) for 15 min and the supernatant was decanted. This procedure was repeated twice and the volume of the extract was reduced to ~15 ml using a rotary evaporator at 40°C. The aqueous sample was then acidified to pH ~ 1 with HCl and partitioned three times with chloroform (30 ml; HPLC-grade) using a separatory funnel. The organic phases were again evaporated near to dryness and then redissolved in 1 ml of n-hexane (HPLC-grade) and finally analyzed by gas chromatography. Standard solutions were prepared according to the method of Singh et al. (1987) and stored at − 20°C. They were filtered through 0.45-μm membrane filters before use.

Gas chromatographic analysis of pesticides

Analyses of the extracts were performed by Perkin Elmer CLARUS 500 gas chromatograph equipped with an electron capture detector. Instrument parameters and operating conditions {Column, ELITE5; temperature: (injector 250°C, detector 325°C, oven:170 − 7°C/min ram to 220°C then increase to 250°C then 5°C/ram); hold time, 5.86 min, carrier gas flow rate of nitrogen, 1 ml/min then makeup 30 ml/min}.

The peaks were identified by comparing their retention times with the standards (Sigma-Aldrich Bangalore, India).

Extraction of soil samples with different solvents

Extraction of soil with different solvents (hexane, acetonitrile, methanol, chloroform, and acetone) was done according to the method of Knize et al. (1987). Ten grams of soil was extracted with 10 ml of the extraction solvent. The extracts were centrifuged at 7,000 rpm for 10 min. The extracts were evaporated to dryness and then re-dissolved in 1 ml of dimethyl sulfoxide (DMSO; SRL, India). These extracts were filtered sterilized through 0.45 μm filters and stored at − 20°C until testing was completed.

Bacterial strains

The Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104 employed for this study (a kind gift of Prof. B. N. Ames) were maintained in frozen stocks and grown as described by Maron and Ames (1983). Each strain was tested on the basis of associated genetic markers raising it from a single colony from the master plate (Maron and Ames 1983).

Salmonella mutagenicity testing

The preincubation test was performed as described by Maron and Ames (1983) with some modifications (Pagano and Zeiger 1992). Five doses of each soil extract, i.e., 1, 2, 4, 5, and 10 μl/plate (correspond to 10, 20, 40, 50, and 100 mg equivalent soil per plate) were plated in triplicate with 0.1 ml of the bacterial culture. After incubating the test sample and bacterial culture for 30 min at 37°C, 2.0 ml top agar containing traces of histidine and biotin was added and contents were poured on minimal glucose agar plates. Plates were incubated at 37°C for 48–72 h. Negative and positive controls were included in each assay. The negative control plates contained bacteria and solvent (DMSO) but no test sample. Methyl methane sulfonate and sodium azide served as positive controls. All the soil extracts were also tested in the presence of (+S9) microsomal fraction, to which 20 μl of S9 liver homogenate mix per plate was added. The criterion used to classify the results as positive was similar to those of Vargas et al. (1993, 1995): number of revertants double the spontaneous yields accompanied by a reproducible dose–response curve.

Mutagenic index

The number of his +  revertants in the sample was compared to the negative control by its mutagenic index value.

Mutagenic index = Number of his +  revertants induced in the samples / number of his +  revertants induced in the negative control

Mutagenic potential

The mutagenic potential of the test samples was calculated by the initial linear portion of the dose–response curve with various tester strains. The slope (m) was obtained by the least square regression of the initial linear portion of the curve of initial dose–response.

Induction factor (Mi)

The induction factor for various test strains for different soil extracts evaluated as follows.

$$ \mbox{Mi}=\ln \;n-c/c $$

Where n is the number of revertants in the samples and c is the number of revertants in solvent control. The induction factor was calculated to determine the difference between two samples if the sensitivity pattern based on the slope (m) was similar.

ANOVA

To determine the significance of the number of his +  revertants in the samples as compared to the control, one way ANOVA was done at P ≤ 0.05 using MINITAB Software, USA.

Results

Determination of pesticides in contaminated soil

Gas chromatography analysis of the soil samples revealed the presence of a high level of organochlorine pesticides. The concentration of lindane in the contaminated soil was 547 ng/g, while α-endosulfan and β-endosulfan were detected at the concentrations of 422 and 421 ng/g respectively. Some unidentified peaks in the gas chromatograms were also observed in the test samples indicating the presence of other contaminants apart from the pesticides.

Reversion of the Salmonella tester strains in the presence of soil extracts

The results of the mutagenicity assessment by Ames assay conducted in the presence and absence of S9 mix in different Salmonella tester strains at the dose of 10, 20, 40, 50, and 100 mg equivalent soil per plate for soil samples irrigated with wastewater and groundwater extracted with different solvents is summarized in Tables 1, 2, 3, 4 and 5. In general, maximum reversion of his −  tester strains were observed at the dose of 5 μl (50 mg equivalents soil per plate) in soil irrigated with wastewater and reversion of all Salmonella tester strains were declined beyond this dose. Reversion of his −  tester strains was found to be low at the same dose with extracts of soil irrigated with groundwater. A significant mutagenic activity was observed with the extracts of soil irrigated with wastewater as compared to groundwater irrigated soil (Tables 1, 2, 3, 4 and 5). TA98 was found to be most responsive strain in terms of mutagenic index, while other strains revealed weak response against the test samples. The order of responsiveness based on the mutagenic index in the presence and absence of S9 fraction for different soil extracts were as follows:

Table 1 Reversion of Salmonella tester strains in the presence of hexane extract of soil
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Table 2 Reversion of Salmonella tester strains in the presence of acetonitrile extract of soil
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Table 3 Reversion of Salmonella tester strains in the presence of methanol extract of soil
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Table 4 Reversion of Salmonella tester strains in the presence of chloroform extract of soil
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Table 5 Reversion of Salmonella tester strains in the presence of acetone extract of soil
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Hexane extract of soil irrigated with wastewater

  1. −S9:
    $$ \text{TA98 $>$ TA97a $>$ TA102 $\ge $ TA104 $>$ TA100} $$
  2. +S9:
    $$ \text{TA98 $>$ TA97a $>$ TA100 $>$ TA102 $>$ TA104} $$

Acetonitrile extract of soil irrigated with wastewater

  1. −S9 and +S9:
    $$ \text{TA98 $>$ TA97a $>$ TA100 $>$ TA102 $\ge $ TA104} $$

Methanol extract of soil irrigated with wastewater

  1. −S9:
    $$ \text{TA98 $>$ TA97a $>$ TA100 $>$ TA102 $>$ TA104} $$
  2. +S9:
    $$ \text{TA98 $>$ TA97a $\ge $ TA100 $>$ TA104 $>$ TA102} $$

Chloroform extract of soil irrigated with wastewater

  1. −S9:
    $$ \text{TA98 $>$ TA97a $>$ TA100 $\ge $ TA102 $>$ TA104} $$
  2. +S9:
    $$ \text{TA98 $>$ TA97a $>$ TA102 $>$ TA100 $>$ TA104} $$

Acetone extract of soil irrigated with wastewater

  1. −S9:
    $$ \text{TA98 $>$ 97a $>$ TA104 $>$ TA100 $\ge $ TA102} $$
  2. +S9:
    $$ \text{TA98 $>$ TA97a $>$ TA100 $\ge $ TA102 $>$ TA104} $$

TA98 was also the most responsive strain in terms of induction factor (Mi) for all extracts (soil irrigated with wastewater). In case of groundwater irrigated soil samples mutagenic index for all tester strains are nearly same for almost all the soil extracts, while induction factor (Mi) is very low as compared to the wastewater-irrigated soil. The mutagenicity of soil extracts of wastewater-irrigated soil was compared to ground water irrigated soil in terms of mutagenic potential. It was found that the slope (m) of initial dose–response curve have higher values in wastewater-irrigated soil as compared to ground water irrigated soil. The order of mutagenicity with soil extracts (soil irrigated with wastewater) in terms of mutagenic potential of initial linear dose–response for the most sensitive strain TA98 with and without S9 fraction is in the following order: hexane > acetonitrile > methanol > chloroform > acetone.

The significance of the reversion of tester strains with increasing dose was determined by one way ANOVA. The analysis showed that reversion of the tester strains increases significantly (P ≤ 0.05) in comparison to the negative control (Tables 1, 2, 3, 4 and 5).

Mutagenicity of soil extracts by different solvents in terms of net revertants is given in Table 6. Net revertants per gram of soil extracts (soil irrigated with wastewater) of hexane, acetonitrile, methanol, chloroform, and acetone for the most responsive TA98 strain in the presence of S9 fraction was 46,900, 45,700, 42,800, 26,500, and 25,200 revertants/g, respectively, and in the absence of S9 fraction was 42,000, 37,300, 32,500, 19,400, and 17,600 revertants/g, respectively (Table 6).

Table 6 Mutagenicity of soil extracts by different solvents in terms of net revertants per gram of soil for most responsive TA98 strain
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Discussion

Soil contamination is the result of human activity, including the entry of industrial wastes into soil through atmospheric deposition or application of agrochemicals and domestic waste to the land. These organic contaminants reduce the soil quality for agricultural production. Soils are very important in the fate and distribution of persistent toxic substances in the environment since they have a huge retention capacity and they may work as re-emission sources for the atmosphere (Sanghi and Sasi 2001; Barra et al. 2005). Organochlorines (OC) are the commonly used pesticides in agricultural field for crop protection in which lindane and endosulfan is most common insecticide. Lindane (hexachlorocyclohexane, HCH) contaminated soils can be found in most countries of the world (Abhilash and Singh 2010).

Hans et al. (1999) tested soil samples from dry bed of the River Ganges at Kanpur (India) and they reported pesticides HCH, dichlorodiphenyl trichloroethane (DDT) their isomers and metabolites in all the samples. Mean levels of 109.35, 136.76, and 145.93 μg HCH/kg and 6.64, 49.3, and 46.70 μg DDT/kg were obtained in the rural upstream, city, and downstream industrial areas, respectively. Prakash et al. (2004) evaluated 45 soil samples of surface and subsurface soils from agricultural sites of Delhi, Haryana, Haridwar, Uttar Pradesh and also around the pesticide industry, Chinhat industrial area, Lucknow, (India) for the presence of residues of HCH isomers. They found residues of β-HCH (2.5 μg/kg to 463 mg/kg of soil) in 39 of the 45 soil samples, γ-HCH (0.08 μg/kg to 43.00 mg/kg) in the remaining soil samples, whereas α-HCH (0.04 μg/kg to 98.00 mg/kg of soil) and δ-HCH (0.07 μg/kg to 458.00 mg/kg of soil) were detected less frequently. In the present study, the pesticides lindane, α-endosulfan, and β-endosulfan were detected at the concentration of 547, 422, and 421 ng/g in agricultural soils of industrial area.

Genotoxicity assessment of soil have employed many assays to detect DNA damaging ability and mutagenicity (Alexander et al. 2002), in which Ames test developed by Ames (1971), is one of the important assay for testing mutagenicity of environmental samples such as soil and water. In vitro Salmonella/microsome assay with rat liver microsomal fraction (+S9) is also one of the most frequently used test for assessing the mutagenic potential for both pure compounds and complex mixture (Aleem and Malik 2003; Karabay and Oguz 2005; Ansari and Malik 2009). Karabay and Oguz (2005) reported that TA98 and TA100 were sensitive strains at all doses of insecticides in the presence of metabolic activation S9. Tzoneva et al. (1985) monitored the genotoxic effect of pesticides (endodan and kilacar) by Ames test at the concentrations of 7.5 and 12.0 μg/plate together with S9 mix and observed that it induced base-pair substitutions in the TA100 strain of S. typhimurium at low level, while kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at the concentrations of 2.5 and 5.0 μg/plate together with S9 mix. Ruiz and Marzin (1996) tested the genotoxicity of six pesticides (atrazine, captafol, captan, chlorpyriphosmethyl, molinate, and tetrachlorvinphos) by two in vitro tests (Ames test and SOS chromotest) with and without S9 mix of rat liver homogenate (pre-treated with Aroclor 1254) and demonstrated that captan and captafol were genotoxic in both the Ames test and the SOS chromotest. Watanabe et al. (2008) collected surface soil from 12 sites in Kyoto City, Japan and their organic extracts were examined by the Ames/Salmonella assays. TA98 was the most responsive strain in the presence and absence of metabolic activation S9 for almost all the test samples. Šašek et al. (2003) used Ames test for the genotoxicity testing of contaminated soil; they also found frameshift-mutation type of mutagenic activity with S9 metabolic activation in S. typhimurium strain TA98. Knize et al. (1987) tested the genotoxicity of soil using different solvents employing Ames test and reported that TA98 was most responsive strain. They also reported net revertants/kg soil; acetonitrile extract produced 298,000 his +  revertants/kg of soil for the most sensitive strain TA98, while the strain TA100 was less sensitive producing 73,000 his +  revertants/kg of soil. Courty et al. (2004) also reported that TA98 strain exhibited maximum response with the test soil samples.

Much of the published Salmonella mutagenicity data revealed that TA98 strain was the most responsive in Ames testing for soil extracts, often obtained in the presence of S9 metabolic activation. It suggests that much of the mutagenicity in contaminated soils is S9 activated frameshift activity (Zemanek et al. 1997; DeMarini et al. 1992; Donnelly et al. 1991; Davol et al. 1989; Barbee et al. 1992; Aleem and Malik 2003; Ansari and Malik 2009). A schematic representation of proposed mechanism of action of genotoxicity of the soil extracts/pesticides in model system is shown in Fig. 1.

Fig. 1
figure 1

Schematic representation for a proposed mechanism of genotoxicity of the soil extracts/pesticides in model system

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Our study on mutagenicity assessment of contaminated soil in the vicinity of industrial area revealed that the soil was highly polluted with the pesticides lindane, α-endosulfan, and β-endosulfan and other mixture of genotoxins. In the present study, TA98 was the most responsive strain in terms of mutagenic index, mutagenic potential and induction factor. In S. typhimurium strains, frame shift tester strain TA98 and base-pair substitution strain TA100 have strings of GC (guanine–cytosine) base pairs at the critical site for reversion (growth without histidine). These strains are particularly sensitive to the mutagenicity of aromatic amines and polycyclic aromatic hydrocarbons. In contrast to the tester strains that detect mutagens with an affinity for damaging GC base pairs, TA102 has an AT (adenine–thymine) base pair at the critical site of reversion (Felton and Wu 2004). These findings clearly indicated that the test samples preferentially act on G-C base-pair mutants (frame shift) as compared to those having A-T base pairs at the site of mutation. In view of the common practice of application of untreated wastewater to agricultural land in the neighboring area should be strictly prohibited as the pollutants might enter into the food chain and cause health hazards to humans.