There are various studies reported in literature that determines the acute toxicity (LC50) on fish in response to various pollutants. The acute toxicity of mercuric chloride for Channa punctatus was found to be 1.21mg/L and 0.81 mg/L (Gupta 2005; Maheshwari 2017). Similarly, acute toxicity of mercuric chloride for Percocypri spingi, Clarias batrachus and Oreochromis mossambicus was found to be 0.327 mg/L, 1.85ppm and 0.58ppm (Yuan et al. 2017; Manorama and Pundkar. 2018; Vasanthi et al. 2019). There occur variations among LC50 values because of difference in laboratory conditions, genetic variations of test organisms, environmental factors, mode of administration of toxicant and sensitivity of test species to broad range of environmental pollutant.
Fish serves as an early bioindicator to detect aquatic pollution as fish are the one of the most vital aquatic organisms in an aquatic ecosystem because they play multiple roles in the trophic web and responds to even low concentrations of toxicants and answers indirectly in terms of feeding on contaminated aquatic living organisms. Fish are the best indicator of heavy metal pollution in an ecosystem as their metabolic state changes in response to toxicants in surrounding medium (Tasneem and Yashmeen 2018).
Nucleic acid content marks the identity of an organism and is an index of the amount of protein synthesis. The exposure of genotoxic compounds leads to an increase in the activity of the DNase that digests the DNA of an organism and hence disturbs the central dogma of the cell of an organism (Tasneem et al. 2018). The physiology of fish in terms of growth and development depends on the ratio of DNA content and it serves as an important biochemical parameter. The growth of cell and protein synthesis are dependent on the DNA content of fish. There occur drastic changes in quality of DNA, when fish exposed to heavy metal loaded contaminated water (Tasneem and Yashmeen 2018). Genotoxicity leads to carcinogenesis (Malacarne et al. 2021)
There occur variations among the results of the comet assay from one study to another due to differences in the type of species, weight and the size of fish and also due to different environmental factors such as dissolved oxygen, salinity and temperature. Heavy metals are the positively charged compounds so they get directly bound to negatively charged DNA molecules and cause mutagenesis. The comet assay has an edge over other genotoxic techniques such as chromosome aberrations and sister chromatids exchange, as the cells used for comet assay need not to be mitotically active (Nagarani et al. 2012).
In the present study, there observed, concentration and duration dependent increase in DNA damage as tail length, %tail DNA and olive tail moment (OTM) in the fish, Channa punctatus in response to mercuric chloride. Similar observations were studied by previous workers too. The results on DNA damage in present study are in agreement with the findings of Matsumoto et al (2003) that showed the significant DNA damage in erythrocytes of Oreochromis niloticus when exposed to chromium. In another study, Channa punctatus when exposed to malathion showed significant DNA damage (Kumar et al. 2010). DNA damage observed in present study could be due to DNA single strand, DNA double strand breaks, DNA adducts formations, DNA-protein or DNA-DNA cross-links as proposed by (Kushwaha et al. 2012). The DNA damage was reported in Lates calcarifer in response to heavy metal, Cadmium and Mercury. There occurs significant (p < 0.01) increase in OTM in response to increase in duration to heavy metals. As per another theory, it was due to the formation of reactive oxygen species that leads to lipid peroxidation and increase in genotoxicity in the living system. The release of reactive oxygen species due to respiratory burst, by haemocytes are favourable to innate immune response. During the favourable environment, there is a balance between production and destruction of reactive species but in response to genotoxic compounds, this balance gets disrupted and causes DNA damage (Senthamilselvan et al. 2012).
The interaction of genotoxic compounds with DNA molecules leads to the formation of alkaline labile adducts that contributes to the DNA strand breakage through enzymatic removal of damaged nucleotides (Ahmed et al., 2017). It was reported that even low concentration of mercuric chlorideleads to DNA damage in human hepatic cell line and this damage was produced through non-apoptotic mechanism (Bucio et al. 1999; Ozer et al. 2000).
Nuclear abnormalities including fragmented nucleus, vacuolated nucleus, binucleated and nuclear bud, along with appearance of micronucleus are taken as authentic indicators of cytogenotoxicity. These cytogenetic abnormalities are caused due to the clastogenic effects of toxicants and it might cause gene amplification and chromosomal detachment. There occurs vacuole formation in blood cells, which is due to the unequal distribution of haemoglobin and swelling of cells were caused because of necrosis of cellular membrane (Sharma and Chadha 2020). A significant decrease was observed in DNA damage in both genotoxic assays after 60 days of recovery, that suggests that there is possibility of full turnover of fish cells. Similar findings were observed by Marques et al. (2011) where DNA damage returned to control after 24hours on cessation of exposure. Another study on Trout exposed to vineyard pesticide showed decline in DNA damage during recovery period (Bony et al. 2008).
The changes in the levels of various biochemical constituents in fish in response to environmental toxicants were reported by various workers. Jagadeshwarhu and Devi (2018) studied the effect of sub-lethal concentrations of copper (1/16th, 1/12th, 1/8th and 1/4th of 96h LC50 on the glucose, total lipids and glycogen in blood, liver and muscles of fish, Oreochromis mossambicus. There occurs concentration and time dependent increase in muscle, liver and blood glucose and decrease in lipid and glycogen levels. Mohiseni et al. (2016) studied the biochemical changes in the common carp in response to Lead and Cadmium exposure. It was observed that as compared to control, the level of plasma glucose were elevated throughout the exposure duration of lead, while in case of chromium exposure, plasma glucose levels increases in the beginning but after prolonged exposure, there occurs decline in its level till it gets depleted. The reason for this could be decline of energy reserves to handle the stress caused by heavy metal accumulation and this could be due to improper gluconeogenesis (Javed et al. 2017). A significant decrease was observed in glycogen content in liver in the freshwater fish, Channa punctatus in response to exposure to polluted water by waste of thermal power plant (Javed and Usmani. 2015). The increase in glucose content in response to heavy metals exposure is due to the enhanced glycogenolysis that results in the formation of more glucose to meet energy demand during stressful conditions (Javed et al. 2015). The alterations in glucose level might be due to renal injury, lack of nutrition and liver damage. During the environmental stress, glycogen level gets depleted and this leads to increase in glucose content. The glucose synthesis from non- carbohydrate sources such as amino acids and extra hepatic proteins, also leads to elevation in glucose levels (Thangamalathi et al. 2016). The decrease in protein content is due to the metabolic utilization of the ketoacids for the synthesis of glucose via gluconeogenesis pathway. The possible reason could be the formation of heat shock proteins that leads to heavy metal induced apoptosis (Sobha et al. 2007). The plasma protein contains albumins and this also causes decrease in albumin level. The necrosis of liver leads to liver damage and results in the increase in ALT, SGOT and SGPT levels in the blood and kidney and liver damage elevates cholesterol content in blood. The liver dysfunctioning shows hypothyroidism that results in increase in levels of total glycerides (Mohiseni et al. 2016). Lipids are the reserve source of energy. Mercuric chloride affects lipid metabolism of the fish and hence decrease in activity of NADPH and Glucose-6- phosphate dehydrogenase were observed and there occur high energy demands to survive in toxic environment (Bhilave et al. 2008).
The high concentrations of mercuric chloride showed increase in content of nitrogenous compounds and hence increases creatinine, bilirubin and uric acid in the blood due to that there occurs disruption to the functioning of kidney and gills (Mutlu et al. 2015).
MN and nuclear aberrations are formed during the process of cell division. There occurs change in their expression at different times after damage to DNA, and it depends on the mechanism of induction and cell cycle kinetics. MN assay mainly targets interphase cells regardless of its karyotype. So, this assay is widely used as biomarker in environmental biomonitoring studies and this assay is successfully applied to study genotoxicity in fish (Bolognesi and Hayashi 2011: Kaur and Dua 2016). Mercury is regarded as potent mutagen. Its toxicity on tubulin, the structural subunit of the cellular microtubules that plays a vital role in spindle fibres formation and cytoplasmic organization, interferes to tubulin polymerization that causes contraction of chromosomes at metaphase stage of cell division, and leads to delayed division of centromere and reduction in anaphasic movement (Rocha et al. 2009).