Genotoxicity Hazard Assessment of Industrial Effluent Discharge and Domestic Waste Discharge on Surface River Water

Research Article

Austin J Environ Toxicol. 2016; 2(2): 1014.

Genotoxicity Hazard Assessment of Industrial Effluent Discharge and Domestic Waste Discharge on Surface River Water

Mathur N¹*, Kaur K¹, Pareek S¹, Nepalia A¹ and Bhatnagar P²

¹Department of Zoology, University of Rajasthan, India

²Department of Life Sciences, IIS University, Jaipur, India

*Corresponding author: Mathur Nupur, Department of Zoology, University of Rajasthan, India

Received: June 07, 2016; Accepted: August 26, 2016; Published: August 31, 2016


Surface water is utilized by humans for the purpose of drinking, domestic usage, industrial usage, irrigation etc. In many parts of the world this surface water is receiving indiscriminate dumping of industrial effluents, domestic sewage and agricultural runoff, thereby, deteriorating the quality of water. Untreated or improperly treated wastewater effluent discharges often contain mutagens especially when the proportion of industrial wastewater in comparison to municipal wastewater is high. Some of the substances found in waste water are genotoxic and are suspected to be possible case of cancers observed in the last decades. This restricts the usage of surface water for potability and direct consumption by human population. Even then, the polluted water is being utilized continuously in many areas as they are the only natural sources of water. Also, toxicity and risk associated with the usage of such waters is ignored.

The present study focuses on genotoxicity assessment of water samples taken from upstream and downstream potability sites of Chambal River and also of effluent discharged into Chambal River from two big industries located in Kota (Rajasthan), India. The water samples taken from both upstream and downstream sites of Chambal River and also from the two effluent treatment plants were found to be highly genotoxic. The assays used for genotoxicity assessment were Salmonella typhimurium reverse mutation assay and E. coli. WP2 assay.

Genotoxicity tests were found to be an excellent means to study the toxicity and associated risk with these anthropogenic activities on natural water resources.

Keywords: Salmonella typhimurium; E. coli WP2 assay; Genotoxicity; Mutagenicity; Surface water; Water pollution


Water Pollution is one of the major consequences of urbanization. In the quest for higher standards for life, humans are deteriorating and depleting the natural resources. Anthropogenic urban-industrial effluents discharge, domestic waste discharge and agricultural waste discharge can add significant amounts of contaminants to surface water and sediments and, consequently, water pollution is becoming a serious problem for the aquatic biota and humans that interact with these aquatic ecosystems. It is a well known fact that the contamination of water resources by genotoxic compounds is a worldwide problem [1-5].

In India, there is a tendency of disposal of industrial effluents directly into municipal sewer system, which is further treated along with the domestic sewage in the municipal sewage treatment plant. However, many cities are still lacking municipal sewage treatment plants and are directly discharging raw sewage and industrial effluents in the surface waters of the rivers in their vicinity deteriorating its quality and adding to its pollution load and increasing its toxicity for the humans itself. Studies have shown that water quality is an important risk factor in cancer and relative risks have been estimated [6,7].

Many mutagenecity and genotoxicity tests have been used in combination with physical and chemical analysis in order to evaluate water quality [5,8-13]. The growing interest in these tests is due to the fact that despite the existence of different toxicity mechanisms for various organisms of different species, a substance that is toxic for an organism often demonstrates similar toxic effects on other organisms [14].

One of the most commonly used microbial bioassays is the Ames Salmonella mutagenicity assay. It has several advantages over the use of mammals for testing compounds. Also E. coli WP2 reverse mutation system is a valuable tool for mutagenesis research [15] by using a battery of different bioassay systems each with different mechanism of toxicity, the composite toxicological response to a waste water sample can be characterized.

Material and Methods

Sampling sites

Chambal River is an important water source that supplies drinking water for over 10 lakh habitants of the city of Kota in the Rajasthan state of India. Treated drinking water are received from Chambal itself and supplied to the houses of the city. Surface water samples were collected at three sampling sites, of the Chambal River.

Sampling was done during the months of June (summer) and December (winter) to account for seasonal variation.

The sample sites were as follows (Figure 1):

Site 1: Upstream site of the Chambal River, located in the vicinity of the city of Kota prior to Kota Barrage.

Site 2: Kota Super thermal power plant, situated on the left bank of Chambal river at upstream of Kota barrage. Thermal power station has set up its own treatment plant for treatment of effluents prior its discharge into Chambal River.

Site 3: Downstream site of river after Kota barrage receiving effluents from industrial areas of Kota.

Site 4: DCM Shriram Rayons, the Kota complex, consists of several manufacturing plants: power plant, calcium carbide plant, cement plant, chloralkali plant, fertilizer plant, PVC plant, PVC compounding plant and common supporting units.

Site 5: The Akelgarh pumping station, the only pumping station in Kota city supplying potable water to the inhabitants of the city the water sample was taken from the site from where the Chambal water is pumped into the pumping station.

Site 6: Potable water supplied by Akelgarh pumping station (Figure 1).

Sample collection

Wastewater samples from all the sites were collected in precleaned and sterilized glass bottles and refrigerated at 4°C until testing. No further fractionation or treatment of samples was done.

The samples were then tested for their genotoxic potential in the test doses 2 μl, 5 μl, 10 μl, 50 μl and 100 μl. These sample mixtures were treated as a single entity and were tested in their crude form.


Ames Salmonella/microsome reversion mutagenicity Assay: The Salmonella/microsome reversion assay was conducted using the plate incorporation procedure [16,17]. The tester strains of Salmonella typhimurium viz. TA98, TA100 and TA102 were obtained from Microbial Type Culture Collection & Gene Bank, Institute of Microbial Technology (IMTEC), Chandigarh (India). The samples were analyzed with and without the hepatic S9 fraction, which incorporates an important aspect of mammalian metabolism into the in vitro test. To prepare S9 mix, uninduced Swiss-Albino mice liver was used [18]. The S9 mix contains liver enzymes, from a rat. These enzymes can metabolize the agent being tested in order to predict the mutagenic properties within a living system. Five dose levels of individual samples were tested (2, 5, 10, 50 and 100 μl). The positive controls used in this assay were Sodium azide used for TA100 in absence of S9 mix, 2-Nitrofluorene, used for TA98 and TA 102 in absence of S9 mix and 2-Anthramine used as positive control for TA98, TA100 and TA102 in presence of S9 mix. All the plates were run in duplicate. Each set of experiment was repeated twice.

The S. typhimurium strains TA98, TA100 and TA102 were grown at 37°C, with shaking, for 10hrs to obtain final cell concentration of 109 bacterial cells. 0.1 ml of this fresh culture was mixed with 0.2 ml of histidine/biotin solution, 0.1 ml or less of test chemical, 0.5 ml of buffer or 0.5 ml of S9 mix and total volume was made up to 1.0 ml by autoclaved distilled water. This mixture was then shaken and poured on plates containing about 25 ml of minimal glucose agar medium. The test concentrations were selected from a set of standard test doses for liquids. The plates were immediately covered with paper to protect photosensitive chemicals present in the test compounds. Plates were then inverted and placed in a dark incubator for 48 h at 37°C. The revertant colonies were clearly visible in a uniform background lawn of auxotrophic bacteria. After 48 h the revertant colonies on the test and control plates were counted. All regents used were of analytical grade, supplied by Himedia Laboratories Limited (India) and Sigma- Aldrich (India).

E.coli. WP2 Bioassay: Escherichia coli strain WP2 and its repairdeficient derivatives are suitable strains for mutagen screening. In these strains, agents which cause base substitution mutations can be shown to increase the frequency of trp+ revertants. In addition, agents causing many types of DNA damage can be detected through increased killing of the repaired deficient derivatives. E.coli. tryptophan reversion system has been used extensively in microbial studies (including chemical screening, radiation studies and analysis of bacteria DNA-repair pathways and in numerous non-genetic applications). In contrast to the Salmonella strains that have different unique target DNA sequences in the Histidine operon, the four most commonly used WP2 strains carry the same tryptophan marker, trpE [19]. The assay is currently used by many laboratories in conjunction with the Ames Salmonella assay for screening chemicals for mutagenic activity [20]. The strain was obtained from Microbial Type Culture Collection and gene Bank (MTCC), Institute of Microbial Technology (IMTech), Chandigarh (India).

The cultures were grown overnight in 10 ml of growth medium and re-incubated with aeration for an additional 2.5 h at 37°C till the cell density reached 2x108 cells/ml. SA2 agar was used in this assay as it contains a trace of tryptophan. After solidification, 2, 5, 10, 50 and 100 μL of the test material was added and spread through spreader. The plates were incubated at 37°C for 48 h, permitting diffusion of the chemical into agar. All samples were tested in at least two independent experiments using five doses and three plates per dose. All reagents used were of analytical grade, supplied by Himedia Laboratories Limited (India) and Sigma-Aldrich (India).

Statistical analysis for mutagenicity assays

Non-statistical analysis: The most common method of evaluation of data from the mutagenecity assay is the ‘‘two fold rule’’ [20]. This rule specifies that if a test compound doubles or more than doubles mean spontaneous mutation frequency obtained on the day of testing, then the compound is considered significantly mutagenic. Using this procedure the following criteria were used to interpret results:

For all samples that showed dose dependent increase in the number of revertant colonies, mutagenicity ratios were calculated. Mutagenicity ratio is the ratio of average induced revertants on test plates (spontaneous revertants plus induced revertants) to average spontaneous revertants on negative control plates (spontaneous revertants) [4]. Mutagenicity ratio of 2.0 or more is regarded as a significant indication of mutagenicity.

Statistical analysis: The Quadratic regression model was used to observe the genotoxic effects of water samples from 7 sampling sites, during summer and winter seasons on 3 strains of S. typhimurium and 1 strain of E. coli WP2. The SPSS ver.2 program was used for the quadratic regression analysis [21]. Revertant colonies were taken as the dependent variable and dose as the independent variable; whereas the time and strains (TA98, TA100, TA 102 and E.coli WP2) were fixed for all the seven water samples. A comparison-wise P value of 0.05 was considered to be statistically significant and test was twotailed.


The results of Salmonella mutagenicity assay for four different sampling sites are summarized in (Table 1) as the mutagenicity ratio of average induced reversions to spontaneous reversions.