## 4 Discussion  
PPCPs are found in majority of water bodies [34], so understanding the adverse effects of the environmentally relevant concentrations of these toxicants on aquatic organisms seems to be an integral component of environmental monitoring and management. Monitoring their effects on food fishes is very pertinent, as contaminated fish pose a direct threat to consumers, especially humans. The cyto-genotoxic effects of the selected concentrations of TCS were clearly visible from a concentration-dependent increase in the frequency of necrotic and apoptotic cells and the elevation of MN, NCA and comet parameters. Cytotoxicityicity of TCS seems mainly due to the formation of crosslinks by the chemical with DNA and the intercalating nature of its metabolites like 2, 8-dichlorodibenzo-P-dioxin [35]. Martin et al. [36] suggested that the main reason behind apoptosis caused by a xenobiotic isthe activation of endogenous nucleases and the formation of oligonucleotidesomal fragments of DNA. The fragmentation of DNA is directly associated with an increase in the frequency of apoptotic cells [37], as is clear from the concentration-dependent increase in nuclear fragmentation and cell lysis during the depruation period. TCS-induced stress is reported to elevate expression of caspase 3 and Bax in the neural stem cells of rats [38] and that of caspases (8, 9 and 3) and fas receptors in the neocortical neurons of Swiss mice [39]. Caspase 8 initiates exogenous pathway, while caspase 9 initiates endogenous pathway of apoptosis in stressed cells. TCS causes concentration-dependent membrane disruption, cell lysis and inhibition of protein synthesis, as suggested by Schweizer [14]. This directly supports our observations, and it could be the possible cause of cytotoxic effect of TCS. When defense system of a cell is unable to repair severe DNA damage, the damaged cell undergoes necroptosis or necrosis. In the present study, decreased cell viability and increased necrotic and apoptotic cell frequency in the hatchlings were supported by the results of Parenti et al. [17], who observed that 1 \(\upmu\)g/L TCS for 120 h significantly decreased cell viability and increased necrotic cell frequency in the embryos of _Danio rerio_. Our study is also corroborated by Binelli et al. [15], who observed that exposure of 1, 2 and 3 nM TCS for 96 h caused a dose-dependent increase in apoptotic cell frequency in the hemocytes of zebra mussel. Decreased cell viability and increased apoptotic cell frequency were also reported in hepatocyte cell lines of zebra fish after 24 h of exposure to 0.5, 1 and 2 mg/L TCS [40]. The clastogenic pollutants increase the mutation frequency in DNA, which leads to different types of nuclear and cellular abnormalities which can be easily observed with the help of MN assay. Micronuclei are the chromosomal fragments or the lagging chromosomes formed due to improper segregation of chromosomes during cell division or due to mitotic failure [41; 42]. It has been highlighted that TCS disturbs genetic balance by blocking cytokinesis of normal cells, which may lead to carcinogenesis [43]. We also observed a concentration-dependent increase in binucleated cells during the post exposure period. TCS induces formation of reactive oxygen species that cause deleterious effects on nucleic acids and macromolecules [44], and this could be the probable reason for the greater increase in the MN and NCA frequencies during the post exposure period in the present study. Our results are consistent with the results of Binelli et al. [15], Emery [45], Vijitha et al. [16] and Paul et al. [18; 19]. An increase in nuclear abnormalities has also been reported in blood cells of gold fish after exposure to 0.1399, 0.2798 and 0.5596 mg/L TCS for 14 days [46] and in _Clarias gariepinus_ after 14 and 28 days of exposure to 0.30 and 1.60 mg/L TCS [47]. Comet assay is a simple, rapid and sensitive tool to evaluate the extent of DNA damage.