Mutagenicity of industrial wastewaters collected from two different stations in northern India Shams Tabrez and Masood Ahmad* ABSTRACT: Mutagenicity of wastewaters taken from two different cities was compared by means of Ames plate test and Ames fluctuation test. TA100 and TA98 strains of S. typhimurium exhibited the highest sensitivity against the Saharanpur sample (SWW) in terms of the slope (m) of the dose–response curve in the plate incorporation assay. However, the most sensitive strain against the test samples from Aligarh (AWW) was TA98. Interestingly, TA100 and TA98 strains also displayed the highest susceptibility towards the samples from Saharanpur in the fluctuation test. However, TA102 and TA100 responded maximally to AWW in this bioassay. Interestingly, S9 supplementation resulted in the decline in mutagenic potential of SWW contrary to significant increase with AWW by both the tests. Both samples were found to generate different types of ROS as predominant species. While SWW were shown to generate a high concentration of superoxide radicals and hydrogen peroxide, hydroxyl radicals were predominantly occurring in AWW. From our result, we conclude that both the test water samples were highly genotoxic. In view of the complementary nature of these two testing systems, we recommend both bioassays for the genotoxicity assessment of complex water samples. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: mutagenicity testing; Ames plate incorporation method; Ames fluctuation test; wastewater; ROS generation INTRODUCTION Human and industrial activities are the origin of the discharge of multiple chemical substances in the environment and are the main causes of environmental pollution (White and Rasmussen, 1998). The contamination of water resources by genotoxic compounds is a worldwide problem (Vargas et al., 1995; Claxton et al., 1998; Kong, 1998; Ohe et al., 2003; Buschini et al., 2004). With rapid strides in industrialization, there has been an alarming increase in the pollution of various water bodies in India during the past few decades (ISGE, 1990; Rehana et al., 1995, 1996; SOER, 2001; Aleem and Malik, 2003). Many toxic agents in the environment act through damaging DNA and hence causing mutations (Daniels et al., 1997; Zahm et al., 1997). Genotoxicity testing of surface waters or industrial effluents using a variety of bioassays demonstrates that these mixtures contain many unidentified and unregulated toxicants that may pose risks and carcinogenicity of unknown magnitude (Lerda and Prosperi, 1996; Magliola et al., 1997; Magdaleno et al., 2001; Ohe et al., 2004). Most of the toxicity testing systems rely on small mammals such as rats or mice and hence are time‐consuming, very expensive and attract considerable ethical criticism (Tsuda et al., 2001). For these reasons a number of in vitro tests have been developed which employ bacteria or plant cells (Ames, 1984; Wilcox and Denny, 1985; LeCurieux et al., 1995; Vargas et al., 1995; Liu et al., 1999). Among the tests that are routinely advocated for the genotoxicity evaluation of water, the Ames plate incorporation test and Ames fluctuation test occupy a prominent position (Claxton et al., 1998; White and Rasmussen, 1998; Siddiqui and Ahmad, 2003). These mutagenicity bioassays are the short‐term tests for the detection of environmental mutagens (Ames, 1984; Malik and Ahmad, 1995). Both the tests are based on the ability of chemicals to induce reverse mutations in certain histidine requiring strains of Salmonella typhimurium. The present study was carried out to evaluate and compare the genotoxicity of wastewater samples collected from Aligarh and Saharanpur cities of northern India, employing the Ames plate incorporation test and the Ames fluctuation test. MATERIALS AND METHODS Water Sampling Wastewater samples were collected from the industrial areas of Aligarh and Saharanpur cities of northern India, in sterile glass bottles strictly according to the method described in American Public Health Association (1998). Prior to the mutagenicity assay, the test samples were filter sterilized by passing through 0.45 μm filters. The S. typhimurium strains employed for this study were obtained from Professor Takehiko Nohmi, National Institute of Health Sciences, Japan. These strains were tested on the basis of associated genetic markers. Having satisfied the requirements with the tester strain, the culture was raised and streaked over minimal and nutrient agar slants. S9 fraction was prepared from liver of Sprague–Dawley male rats using Aroclor‐1254 as the inducer. The composition of S9 mix was as follows: rat liver S9 fraction (4%), magnesium chloride (1 M), glucose‐6‐phosphate (1 M) and NADP (0.1 M). The revertant colonies were screened using an electronic colony counter supplied by Mac India Ltd. *Correspondence to: M. Ahmad, Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh 202002, India. E-mail: masood_amua@yahoo.co.in Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh 202002, India J. Appl. Toxicol. 2011; 31: 783–789 Copyright © 2011 John Wiley & Sons, Ltd. Research Article Received: 20 September 2010, Revised: 3 November 2010, Accepted: 4 November 2010 Published online in Wiley Online Library: 25 January 2011 (wileyonlinelibrary.com) DOI 10.1002/jat.1635 783