Physicochemical and heavy metal analysis of soil samples
Physiochemical analysis of agricultural soils are presented in Table 1. The pH of wastewater irrigated soil was found to slightly alkaline (pH 8.36), while it was 7.97 for soil irrigated with ground water. Carbonate and bicarbonate were found to be 147.8 and 129.13 mg/kg in wastewater irrigated soil, while 154 and 114.4 mg/kg were detected in ground water irrigated soil. The electrical conductivity (EC) of wastewater and ground water irrigated soils were recorded as 1.2 and 1.3 dS/m with loamy soil texture. Total organic carbon was 1.13% in contaminated soil, whereas, it was 0.15% in ground water irrigated soil. The amount of chloride (Cl− 1), phosphorous (P), and sulphur (S) was found to be 42.7, 20.55 and 12.64 mg/kg respectively while potassium (K) was 174.81 mg/kg in contaminated soil. On the other hand, the concentration of Cl− 1, P, S and K in ground water irrigated soil were found to be 24.7 mg/kg, 18.71 mg/kg, 10.8 mg/kg, and 96.07 mg/kg respectively (Table 1).
The soil samples contaminated with some heavy metals ions including Ni, Cd, Pb, Cu, Zn, Cr, Fe, and Mn as confirmed by AAS (Table 1). The concentration of Ni, Cd, Pb, Cu, Cr, Zn, Fe and Mn were 12.57, 3.33, 22.95, 17.6, 33.35, 11.86, 14.06, and 15.13 mg/kg in wastewater irrigated soil, whereas 1.41, 0.16, 1.29, 0.98, 2.78, 1.64, 9.12 and 4.24 mg/kg in ground water irrigated soil respectively (Table 1).
Quantitative determination of pesticides in soil samples
Gas chromatographic (GC) analyses of tested samples of soil revealed high-level of organochlorine and organophosphate pesticides. Soil irrigated with wastewater contained high-level of both organochlorine and organophosphate pesticide groups. The organochlorine pesticides like α-BHC, β-BHC, lindane, heptachlor, aldrin, α-endosulfan, 4-4” DDE, dieldrin, β-endosulfan, endrin aldehyde, endosulfan sulfate and endrin ketone in wastewater irrigated soil were 31.44, 6.21, 44.26, 16.75, 9.42, 30.7, 29.45, 114.18, 60.61, 12.22, 18.59 and 14.83 µg/g respectively (Table 2), whereas dichlorvos, disulfoton, parathion-methyl, chlorpyriphos, prothiofos, and azinphos-methyl (organophosphate pesticides) were found to be 4.45, 43.53, 14.39, 4.69, 41.31 and 14.93 µg/g detected respectively. The groundwater irrigated soil also contained both organochlorine and organophosphate pesticides and the concentration of α-BHC, β-BHC, lindane, heptachlor, γ-chlordane, α-endosulfan, dieldrin, β-endosulfan, 4-4” DDD and endosulfan sulfate were recorded to be 0.55, 0.34, 1.3, 0.22, 2.2, 0.18, 20.59, 0.35, 0.32 and 0.49 µg/g respectively while dichlorvos, disulfoton, prothiofos, and azinphos-methyl were 1.51, 1.24, 7.55 and 3.95 µg/g detected, respectively (Table 2).
Reversion of Ames Salmonella tester strains
Salmonella tester strains (TA97a, TA98, TA100, TA102, and TA104) were also used to evaluate the mutagenicity of soil samples. Hexane extract of contaminated soil sample was found to be high mutagenic compared to DCM extract of soil samples in the terms of mutagenic parameters (mutagenic index, induction factor, and mutagenic potential) (Table 3-6). The number of reversion colonies of Salmonella strains were increased with rising doses up to 20 μl/plate then decreased at 40 μl/plate. Both the solvent extracts (hexane and DCM) of contaminated soil showed the significant mutagenicity with tester strains (such as TA98, TA100 and TA102) in the absence as well as in presence of S9 fraction. In hexane extracts of wastewater irrigated soil, strain TA98 was most sensitive concerning of mutagenic index (13.41 and 13.46 without and with S9 fraction), induction factor (2.52 and 2.53 without and with S9 fraction) and mutagenic potential (6.37 and 7.72 without and with S9 fraction) (Table 3). The responsiveness order (on the basis of mutagenic index and induction factor) are as follow:
(TA98 > TA97a > TA100 > TA102 > TA104).
Whereas following trends was found in terms of mutagenic potential/slope:
TA98 > TA100 > TA102 > TA97a > TA104.
DCM extract of contaminated soil, strain TA98 was also found maximum response with mutagenic index (11.03 and 11.11 without and with S9 fraction), induction factor (2.30 and 2.31 without and with S9 fraction), whereas TA100 displayed highest mutagenic potential (5.74 without and 6.30 with S9 fraction) (Table 5). The responsiveness order (on the basis of mutagenic index along with induction factor) are as follow:
(TA98 > TA97a > TA100 > TA102 > TA104).
However, different trends was found in terms of mutagenic potential/slope:
TA100 > TA98 > TA102 > TA97a > TA104.
The response of all Salmonella strains were significantly greater when treated with wastewater irrigated soil extracts in comparison to uncontaminated soil extracts (Table 4 and 6). As expected, the uncontaminated soil extracts of hexane (ground water irrigated) displayed low mutagenic response with TA98 strains. The mutagenic index were 3.07 with S9 and 3.03 without S9 fraction and induction factor were found to be 0.73 and 0.71 (with and without S9 fraction) (Table 4). Whereas, the mutagenic potential was found to be 1.46 and 1.16 (with S9 and without S9) (Table 4).
Salmonella tester strains also showed low level of mutagenicity while treating with the DCM extract of the ground water irrigated soil (Table 6). Strain TA98 showed maximum responsive regarding mutagenic index (3.24 and 3.39 without as well as with S9 fraction) and induction factor (0.81 without and 0.87 with S9 fraction), while strain TA100 was most sensitive concerning mutagenic potential (1.83 and 2.03 without and with S9 fraction) (Table 6).
Survival of E. coli K-12 strains treated with test samples
E. coli K-12 DNA repair defective mutants (recA, lexA and polA) and isogenic wild types counterparts is shown in Fig. 1 when treated with soil extracts (at dose of 20 μl/ml of culture). All the strains displayed significant reduction in CFUs while treatment with hexane extract of contaminated soil sample and percent survival was 39% in polA, 47% in lexA and 55% in recAE. coli K-12 mutants after 6 h of treatment (Fig. 1a). DCM extract of wastewater irrigated soil exhibited survival of 25% in polA, 39% in lexA and 46% in recA mutants after 6 h of treatment (at dose of 20 μl/ml of culture) under similar experimental condition (Fig. 1c). Moreover, the mutant strains were also treated with uncontaminated soil (both DCM and hexane) and the survival was observed to be 56%, 64% and 78% in polA, lexA and recA mutants respectively with hexane extract (Fig. 1b), and 51% in polA, 59% in lexA and 77% in recA mutants with DCM extract under similar conditions (Fig. 1d).
Allium cepa chromosomal aberration test
The effect of aqueous soil extracts on the mitotic index (MI) of root meristematic cells of A. cepa was affected in dose dependent manner (5-100%). MI significantly decreased with increasing concentration of soil extracts. It was observed that decline of MI was more in contaminated soil (wastewater irrigated) than uncontaminated soil (ground water irrigated). MI was observed to be 9.1% at 100% concentration for wastewater irrigated soil, whereas it was 22.6% at 100% concentration for ground water irrigated soil. Mitotic index of ground water irrigated soil extract (31.9% at 5% soil extract) was comparable to that of MI of negative control (31.47%) (Table 7).
Statistically significant (P<0.05) frequencies of chromosomal abnormalities and percent aberrant cells were observed with soil extracts of contaminated soil compared to uncontaminated soil (Table 8). Chromosomal aberrations and percent aberrant cells were increased from 8.44% to 39.95% with increasing extract concentrations of soil irrigated with wastewater (5 to 100%). The soil samples caused different forms of aberrations in the root tips, such as breakage, anaphase spindle disturbance, disturbance at metaphase, C-mitosis, stickiness, multipolar anaphase with chromosomal break, anaphase bridge with vagrant chromosome and uneven proportions of chromosomes at anaphase stage (Fig. 2). Therefore, the wastewater irrigated soil sample displayed greater frequency of aberrations (39.95%) as compared to ground water irrigated soil (17.81%) at 100% concentration for wastewater and ground irrigated soil extracts (Table 8).
In vitro toxicity of soil extracts to Vigna radiata
Toxicity of contaminated soil (wastewater irrigated) and uncontaminated soil (ground water irrigated) were also assessed by V. radiata seed germination test. The germination rate of mung bean seeds and other plant parameters were found to be influenced and reduced when treated with different doses of wastewater irrigated soil extract (Fig. 3). At 100 % dose of wastewater irrigated soil extract, % seed germination, seedling vigour index (SVI), plumule length (PL), radicle length (RL), dry biomass of plumule (DBP) and dry biomass of radicle (DBR) were found to be 52.22%, 698 SVI, 7.8 cm, 5.53 cm, 0.19 g and 0.09 g, respectively (Fig. 4). The damage to root tip cells caused by pollutants in soil extracts were observed and easily visible under a fluorescent microscope using propidium iodide to produce red fluorescence. The fluorescence intensity increased as the concentration of wastewater irrigated soil extract increased (Fig. 5).
Plasmid nicking assay
DNA band pattern of plasmid nicking test with different doses of wastewater irrigated soil extract is shown in Fig. 6. Different concentration (5, 10, 15 and 20 µl) of wastewater irrigated soil extract were used to analyse the effect in partial transformation of pBR322 plasmid DNA from supercoiled state to the open circular (Fig. 6, lane b-e). The test samples also caused the conversion of supercoiled pBR322 DNA into linear (Fig. 6, lane b-e). Highest loss of pBR322 plasmid (supercoiled form) was observed in 20 µl of soil extract. The band intensity of pBR322 DNA (open circular form) was increased and supercoiled form was decreased in dose dependent manner. Positive control (MMS) caused in the compete loss of supercoiled pBR322 plasmid DNA (Fig. 6; lane f).