Effect of temperature changes on antioxidant enzymes, and oxidative stress in Nerita oryzarum collected along Atomic Power Plant sites

Effect of heated euent released from the Tarapur Atomic Power Station, India were investigated by analyzing the level of antioxidant enzyme activities on Nerita oryzarum. Seasonal variation of antioxidant enzyme (LPO, CAT, SOD, GPx and GST) were determined at six location. CAT increases in the pre-monsoon season (range of 0.12 to 2.1 mM of H 2 O 2 consume/min/mg of protein) and LPO activities also increasing trend (range of 0.4 to 2.3 nM of MDA/min/mg) during same pre-monsoon at NIII and SII respectively. Enzyme values encountered high during pre-monsoon, which indicating that increased in temperature is resulting in increased activities. In experimental condition also (30 0 C, 35 0 C, 40 0 C and 25 0 C as control), antioxidant enzyme activities were in increasing trend due to raises of water temperatures. Present study was prima facie work related to physiological response of N. oryzarum related to heated euent released from atomic power station and useful as baseline information for future research work.


Introduction
To generate the energy in living body, consumption of oxygen through breathing is needed. Most of the oxygen gets incorporated into harmless water molecule except up to 2 percent may be spun off in to our cell as free radical or ROS (Reactive Oxygen Species), which are greatly charged molecules. The production of ROS are dominant in aquatic ecosystem. The oxidative stress are very signi cant component as stress response, especially in marine environment were intertidal organisms get exposed to assortment of environmental as well as anthropogenic stressors, for example thermal related stress which is very predominant due industrialization, power plants and global climate change.
Oxidative stress can brie y de ne as, the production of ROS beyond the ability of organism to reduce and be able to cause damage to proteins, lipid and DNA (Halliwell and Gutteridge, 1999). The quenching of 1 O 2 on site of production and to decrease the uctuation of other ROS to ultimately inhibit the production of furthermost damaging ROS, i.e., HO. is the primary function of the antioxidant defense system (Cadenas, 1989;Fridovich, 1998). Organisms in marine ecosystem usually get exposed to multiple environmental stressors, such as increase in temperature, reduction in pH, uctuation in salinity and exposure to metals (Harley et al., 2006;Brierley and Kingsford, 2009;Sparks et al., 2019;Albayrak et al., 2019). Especially the organisms from rocky intertidal and shallow water areas have more negative impact (Millero, 2007) than pelagic zone organisms (Beuchel et al., 2006). Increase in temperature result in elevated activities of cytosolic and mitochondrial enzyme system that generates ROS in extra mitochondrial compartment which contribute to both increase rate of metabolism and ROS production (Sokolova et al., 2012). High temperature increases mitochondrial ROS production in mollusks (Heise et al., 2003;Abele et al., 2002). A moderate increase in temperature leads to stimulation of the antioxidant protection system and expression of antioxidant enzymes get induced (Ivanina et al., 2009;Gourgou et al., 2010). Since, gastropods are slow moving and inhabitant of intertidal regions, suggesting that they already get exposed to thermal stress due to exposure during tidal rhythm, any additional thermal stress, such as, heated e uent from nuclear power plant, may cross their optimum tolerance limit. In the present study, the gastropods of Nerita oryzarum was selected the study animal due to availability of all seasons around Tarapur Atomic Power Station, India and it could be the greatest demonstrative model for studies of in uence of stress on antioxidant defense mechanism.

Results
2.1 Measurement of lipid peroxidation level due to temperature changes The oxidative lipid degradation is known as lipid peroxidation. It is the development in which free radicals take electrons from the lipids in cell membranes, nally cells got damage. These process done by mechanism of free radical chain reaction. During present study the lipid peroxidation in whole body tissue of N. oryzarom was analysed spatially at six different locations as well as seasonally during six different seasons in close vicinity of nuclear power plant site, Tarapur (S Table-2). During the monsoon the minimum LPO activity was measured of 0.58 nM of MDA/min/mg at S I and maximum of 2.31nM of MDA/min/mg at SII location with (STDV) ± 0.41. Post-monsoon season the LPO activity measured in N. oryzarum got reduced in the range of 0.09 (at NIII) to 1.21 (at SII) nM of MDA/min/mg with (STDV) ± 0.29. During the pre-monsoon it again showed increasing trend and was in the range of 0.4 (at NIII) to 2.3 (at SII) nM of MDA/min/mg with (STDV) ± 0.5 (Fig. 1).

Catalase analysis
Catalase is present in all aerobic organisms. Within the cell, catalase in mainly located in peroxisomes and mitochondria as both soluble and membrane bound form. Catalase in alternative dismutase enzyme comprises heme moiety at its active position. It changes H 2 O 2 in H 2 O and molecular oxygen mostly in peroxisomes. It avoids conversion to hydroxyl radical and other toxic ROS. In the present study the CAT was measured in the range of 0.03 (at NI and NII) to 1.1 (at SI) mM of H 2 O 2 consume/min/mg of protein with (STDV) ± 0.35 at monsoon season. During the post-monsoon season CAT values does not showed any trend and was in almost similar range as of monsoon season i.e., 0.03 (at SI) to 1.1 (at SII) mM of H 2 O 2 consume/min/mg of protein with (STDV) ± 0.34 (S Table-2). The CAT increases in the pre-monsoon season and was in the range of 0.12 (at NIII) to 2.1 (at SII) mM of H 2 O 2 consume/min/mg of protein with (STDV) ± 0.42 (Fig. 2).

Effect of temperature changes on SOD level
Superoxide dismutase catalyzes the dismutation of superoxide (O 2 − ) radical into hydrogen peroxide (H 2 O 2 ) or ordinary molecular oxygen (O 2 ) providing cellular defense against reactive oxygen species. It was observed that the minimum activity of SOD was recorded at NII location during all the season with minimum of 1.8 U/mg of protein during post-monsoon followed by 2.3 U/mg of protein in monsoon and 3.5 U/mg of protein in pre-monsoon season. The maximum values of SOD activity was recorded at SII location throughout study period with similar trend i.e., minimum of 13.4 U/mg of protein in postmonsoon followed by 19.2 U/mg of protein in monsoon and maximum of 27.5 U/mg of protein during pre-monsoon season (Fig. 3). Glutathione peroxidase is selenium comprising antioxidant enzyme that e ciently reduces H 2 O 2 and lipid peroxides to water and lipid alcohol, one-to-one and in turn oxidizes glutathione to glutathione disul de. The Glutathione peroxidase helps to prevent lipid peroxidation and maintains intracellular homeostasis as well as redox balance. The GPx values were minimum during the post-monsoon season and it was in the range of 0.04 (at NII) to 1.1 (at SII) nmol glutathione oxidase /min/mg protein with (STDV) ± 0.24. During monsoon it was in the range of 0.4 (at NIII) to 2.154 (at SI) nmol glutathione oxidase /min/mg protein with (STDV) ± 0.37 and in pre-monsoon it varied in the range of 0.4 (at NIII and SIII) to 3.04 (at SII) nmol glutathione oxidase /min/mg protein with (STDV) ± 0.58 (Fig. 4).

Glutathione S-transferases level
Glutathione S-transferases (GST) are a group of enzymes that catalyze conjugation of reduced glutathione to a varied range of substrates, nally resulting in detoxi cation (Graham et al., 2013). They also function as transport proteins. It catalyses the conjugation of GSH via a sulfhydril group to electrophilic centers on wide variety of substrates to make the compound more water soluble. The GST values showed the increasing trend; minimum during post-monsoon followed by monsoon and premonsoon. It was minimum of 8.9 (at SIII) nmoles/min/mg Protein during post-monsoon season. During the monsoon it varied in the range of 10.2 (at NIII and SIII) to 17 (at NI) nmoles/min/mg Protein and it increased in pre-monsoon with the range of 10 (at SIII) to 20.1 (at SII) nmoles/min/mg Protein with (STDV) ± 2.09 (Fig. 5). Similarly, all the antioxidant enzyme activity was studied in experimental organisms and the result showed that, all the activity was in increasing trend and directly proportional to the water temperature. The result of experimental analysis is given S In the present study the in uence of heated e uent water released from the Atomic plant at Tarapur were investigated by examining the antioxidant enzymes as molecular biomarkers. The ve different assay's were analysed such as Lipid peroxidase, Catalase, Superoxide dismutase, Glutathione-S-transferase and Glutathione peroxidase in N. oryzarum collected from six different locations during three different seasons. The thermal impact was also studied in similar organism at laboratory condition for similar biomarkers as at environmental conditions by maintaining the organism at three different temperatures (30 0 C, 35 0 C, 40 0 C and 25 0 C as control). The correlation of these biomarkers with response to heated e uent was studied (Table-2).

Discussion
All aquatic animals, their diversity and community structure are in uence by temperature. Various studies have suggested that the temperature bring out considerably the physiological stress and higher metabolic activity which increases oxygen consumption and consequently produces ROS (O 2 − , H 2 O 2 and OH − ) (Lushchak and Bagnyukova, 2006;An and Choi, 2010). The temperature limits are different for different species within which they can successfully survive, for some species the range of this limit is very narrow. The metabolic scope and stress response mechanisms are primarily important factors that allow the survival of particular species in that range limit (Somero, 2002 andPortner, 2010). To maintain the physiological homeostasis within the temperature range various cellular mechanisms plays a vital role. Off this the important are, heat shock proteins, mechanism of general alteration of energetic metabolism and most primitive, antioxidative system (Arad et al., 2010, Pohlmann et al., 2011,Qian et al., 2012and Mizrahi et al., 2012. According to Handy et al. (2009), marine invertebrate's response to temperature by adjusting mitochondrial electron transport, which in turn produces the superoxide anions as a bi-product (Davidson and Schiestl, 2001). The antioxidant enzyme activity uctuates as per the production and scavenging of ROS produced. The organism is said to be in oxidative stress when the antioxidant defense mechanisms get oppressed by production of reactive oxygen species (Menghong Hu et al., 2015).
The use of biomarkers in environmental monitoring programs, both in freshwater and marine water, frequently uses molluscs as sentinel species (Marigómez et al., 2006;Laffon et al., 2006;Cajaraville et al., 2006;Lima et al., 2007). The antioxidant defense mechanism of marine mollusks has been the subject of many researchers. Many studies mostly assessed the response of antioxidant defense system of mollusks related to speci c pollutants, such as metals (Nikitha Divakaran, 2014), polycyclic aromatic hydrocarbons (Sarkar et al., 2008;Bhagat et al., 2016), oil spill (Moreira et al., 2004;Ana et al., 2009), organic-inorganic pollutants, Industrial and domestic waste disposal etc. In particular most of study focused on bivalve species (for review, Winston and Di Giulio, 1991). But little is known about antioxidant defense of marine gastropods, especially the data related to thermal stress due to heated e uent released from nuclear power plant sites is seldom. Present study investigates the physiological response of N. oryzarum in the form of response of antioxidant system towards heated e uent released from nuclear power plant site, Tarapur.
Antioxidant enzymes of SOD, CAT, GPx, GST and GR are signi cant in coping with oxidative stress produced by metabolism itself or/ and environmental factors also (Tripathi et al., 2006;Pinto et al., 2003).
The antioxidant system in mollusks involves some important enzymes which prevent oxidative damage mediated by ROS are Catalase, Superoxide dismutase, Glutathione-S-transferase and Glutathione peroxidase. In the present study most of the enzymatic activity and lipid peroxidation values encountered high during pre-monsoon and at SWIII location, which indicating that increased in temperature is resulting in increased activity. These results are in correlation with experimental condition where increased in temperature also leads to increased in the activity of antioxidant enzymes. Many authors studied the effect of temperature, pH and salinity on antioxidant systems of mollusks. Matozzo et al., 2013, studied the combined effect pH and temperature on bivalve and suggested that exposure of mollusks to the ROS generating stress full condition may either induce or inhibit the activity of antioxidant enzymes.
Lipid peroxidation can generally be described as the process in which the carbon-carbon double bond in lipid especially PUFA (Polyunsaturated fatty acids) get attacked by oxidants such as free radicals (Antonio et al., 2014). In the present study TBARs test was used to represent the level of LPO in Neritta oryzarum. The maximum LPO activity (1.88 ± 0.12 nM of MDA/min/mg of protein) was estimated during pre-monsoon 2014, when the maximum water temperature was recorded, indicates that, increased in temperature induces the LPO activity. Production of free radicals increased with increase temperature and it reacts with polyunsaturated fatty acids of the cell which induces the LPO activity (Jacky Bhagat et al., 2016). Under experimental treatments, the LPO activity was directly proportional to temperature (Table-1) and con rms the present nding. The similar result was also reported by Verlecar et al., 2007, in Perna viridis exposed to high temperature.
Superoxide dismutase accumulates in response to oxidative stress and is a main antioxidant defense pathways (Down et al., 2001). Increased SOD level shows that cells are answering to an oxidative stress (Fridovich, 1995). SOD being the primary scavenger of O 2 . − during heat stress indicates that it inhibiting accumulation of oxygen radicals. Verlecar et al., 2007 reported increased activity of SOD in digestive gland and gills of Perna viridis at higher temperature (32 0 C) than the control condition (26 0 C). Increased in temperature substantially increase the oxygen consumption in Nacella concinna (Peck, 1989) and also leads to increased SOD activity in digestive gland and gills (Abele et al., 1998). de Oliveira et al., 2015 have also reported the similar trend in N. concinna gastropods that increased in temperature leads to increased SOD activity. In the present study maximum SOD activity was detected at SW II location during pre-monsoon 2014 (22.57 ± 0.89 U/mg protein) when the temperature was recorded highest and was in the range of 28.2 0 C to 38.5 0 C (Avg. 31.20 0 C ± 3.78) followed by monsoon 2013 (16.71 ± 0.57 U/mg protein), where temperature was in the range of 28 0 C to 33.75 0 C (Avg. 30.71 0 C ± 1.99) indicating that increased in temperature leads to increased in SOD activity (although not linear in organism from natural habitat). The result was con rmed in experimental set up where organism was exposed to increased temperature for 8 hrs leads to an increased SOD activity (Table-1). This results is similar with Matozzo et al., 2013, encountered the upward trend of SOD activity with increased in temperature in clams and mussels. Parihar et al. (1997), have also described that, increased in SOD activity in gills of H. fossilis on short period temperature improvement. In present study the SOD values shows the seasonal as well as spatial trend. It was minimum during post-monsoon 2013 and maximum in pre-monsoon 2014. Spatially, intake locations i.e. NWI and SWI had less value for SOD activity than the discharge locations. SOD values shows positive correlation with pH (r = 0.439) and water temperature (r = 0.911) and also it is positively correlated with CAT (r = 0.798) and LPO (r = 0.784). M. Hu et al., 2015 also have similar results and stated that SOD activity was high in gills and digestive glands of M. coruscus kept at low pH and high temperature.
Increased in SOD activity carry out the procedure of O 2− dismutation to H 2 O 2 which should ultimately stimulate the activity of Catalase enzyme, which then bring out the reduction of H 2 O 2 and protects the tissue from ROS and hydroxyl radicals. Thus catalase act as very important scavenger in the cell. In present study the activity of catalase was recorded high at SWII location (1.52 ± 0.14 mM of H 2 O 2 consume/min/mg of protein) during pre-monsoon 2014 (Figure-3). Under the oxidative stress often activity of catalase also increases (Hermes- Lima, 2004), this is in consistent of current nding indicating increased in temperature also indices the activity of SOD and CAT. The result was also similar during the experimental condition where organisms were exposed to thermal stress also have shown similar trend of increased in catalase activity from 0.41 ± 0.2 mM of H 2 O 2 consume/min/mg of protein to 1.17 ± 0.3 mM of H 2 O 2 consume/min/mg of protein at 30 0 C and 40 0 C respectively (Table-1). The catalase activity was higher in experimental thermal set up (i.e. 30 0 C< 35 0 C< 40 0 C) than in control (25 0 C, CAT = 0.39 ± 0.1 mM of H 2 O 2 consume/min/mg of protein) con rmed that organism exposed to thermal stress induces the higher activity of catalase in order to cope up with oxidative stress. Abele et al., 1998, have demonstrated the thermal stress in Nacella concinna by exposing it to increased temperature for 24 hrs and concluded the increased in SOD and CAT activity in the gills. In the present nding CAT activity showed positive correlation with SOD activity (r = 0.798) and with water temperature (r = 0.911) and pH (r = 0.439) indicating both parameters are in uencing the enzymatic reactions in N. oryzarum (Table-2).
Along with catalase, glutathione peroxidase also acts as important scavenger in the cell. GPx are a large family of selenium dependent and selenium independent enzymes with peroxidase activities. Reduction of organic hydroperoxides to their corresponding alcohol and free H 2 O 2 to water are the main biological roles of GPx. In cellular matrix it is located in cytosol, mitochondrial matrix and membrane, thus it removes H 2 O 2 from various cellular compartments in combining with catalase (Halliwell and Gutteridge, 2007). During present study, the highest activity of GPx was measured at SWII location (1.98 ± 0.2 nmol glutathione oxidase/min/mg protein) during pre-monsoon 2014, followed by monsoon 2013 at NWII location (1.39 ± 0.1 nmol glutathione oxidase/min/mg protein). Both locations are the discharge point of TAPS 1&2 And TAPS 3&4, respectively and seasons when maximum values for water temperature was reported during study period, indicates that the increased in water temperature in uence the activity of GPx. Similarly, when the organism was exposed to thermal stress also shows directly proportional relationship between GPx and water temperature. In the present study GPx activity was positively correlated with water temperature (p = 0.808), SOD activity (p = 0.704) and salinity (p = 0.449) ( Table-2).
Glutathione S-transferases (GST), enzymes catalyses the conjugation process of condensed glutathione to extensive range of substrates and ultimately results in detoxi cation. It catalyses the reaction of glutathione compound with -SH group, thereby neutralizing their electrophilic sites and making the compound more water soluble. Temperature signi cantly affects the GST activity (Lynn et al., 2000), and it increases with increase in temperature. In present study the GST activity was maximum (16 ± 0.57 nmoles/min/mg protein) was measured during pre-monsoon 2014. At experimental condition the GST activity increased with increase in temperature from 30 0 C to 35 0 C and it reduces at 40 0 C, these could be reason that the organism has already cross the lethal temperature and further response of antioxidant system has stopped. Although, GST is frequently used as biomarker in many experimental studies, the systematic studies of its kinetics, ecologically relevant endpoints and affect of environmental factors are limited. In case of invertebrates, the Glutathione S-transferase activity does not give any predictable trends, because detailed study of its kinetic behavior under stress condition and characterization of several enzymes with in this family is seldom (Domingues et al., 2010).
Biomarkers, when used as diagnostic tool for environmental monitoring, it is important to link the response of biomarker at individual level, potential effect of pollutant on population, ecosystem and on communities of particular organism. However, often it is di cult as meaningful interpretation required deep knowledge of integration of various levels of biological organization and its mechanistic processes (Forbes et al., 2006). Moreover, one should also consider that spatial and temporal history related to particular contaminants which in uence the biochemical changes in organisms, and if these changes are in physiological response and ecologically relevant, then biomarkers are successfully acts as measure of effect (Ankley et al., 2007).

Sample collection
The Nerita oryzarum (around 20-25 organisms from each site) were collected during three different seasons (monsoon 2013 to pre-monsoon 2014) from six rocky intertidal locations (TAPS1&2, Light house, Varor, TAPS 3&4, Popharan and Nandgaon) around Tarapur nuclear power plant site (S Table-1 & S Fig. 1). The gastropod of similar size (23 to 32mm) and irrespective of their sex was collected for analysis. The organisms were freeze in liquid nitrogen at the site of collection and transported to laboratory. In laboratory, shell of gastropod was gently smashed and entire body tissues were cautiously dissected from other vicera. A soft tissue from 5 to 6 gastropods were pulled together to make 1gm of weight, which was used for preparation of tissue extract for biochemical assay.

Analaysis of protein and lipid peroxide (LPO):
The estimation of protein in N. oryzarum, The Lowry's method (Lowry et al., 1955). The standard procedure for estimation of level of lipid peroxide in organism tissue by their reaction with thiobarbituric acid developed by Ohkawa et al., 1979 was used. To estimate lipid peroxidation, 1 g of tissues was homogenized in 9 ml of ice cold 0.25 M sucrose. 0.2 ml of animal tissue homogenate was assorted with 0.2 ml of 8 % SDS followed by 2 ml of 20 % acetic acid and 2 ml of 0.8 % Thiobarbituric Acid (TBA).
Further, the mixture was make up to 4 ml by using distilled water and heated for 60 min in boiling water bath. After cooling the mixture was centrifuge at 3000 rpm for 10 min. The absorbance was measured at 532 nm and the results were expressed as nM of MDA min − 1 mg − 1 of protein. Lipid peroxide was considered as MDA equivalent.

Determination of Catalase (CAT):
The catalase assay was measured according to simple calorimetric method for rapid assay of catalase described by Sinha (1972). Tissue homogenate for catalase activity was prepared in 4 ml of phosphate buffer (0.1 M, pH 7.4) by using tissue homogenizer. The total contents were centrifuged at 4 0 C, 18,000 rpm for 30 min. The reaction mixture contents 0.5 ml of 0.2 M H 2 O 2 (Hydrogen Peroxide), one ml of 0.01 M sodium phosphate buffer (pH 7.0) and 0.4 ml of double distilled water. Further, two ml of dichromateacetic acid reagent, which contains glacial acetic acid and potassium dichromate and in 3:1 proportions, were added to stop the reaction. The reaction mixture heated for 10 min. in water bath and further kept for cool. After cooling, green color was developed and the absorbance was measured at 583 nm and blank also maintained. The CAT activity was expressed as mM of H 2 O 2 consumed/min/mg of protein.

Determination of Superoxide dismutase (SOD)
The SOD (Superoxide dismutase) activity was measured by the method of Misra and Fridovich (1972) with minor modi cation. The SOD activity in tissue homogenates were analysed by the level of autooxidation of epinephrine to adrenochrome. For SOD determination, 1 g of tissue was homogenized in 0.1M phosphate buffer (pH 7.4), 0.2 ml of chloroform (ice chilled), 0.15 ml of ethanol (ice chilled) followed by 1 ml of distilled water was added in chilled condition. The total contents were centrifuged for 10 min at 4 0 C and 3000 rpm in cooling centrifuge machine. The one ml of reaction mixture contained epinephrine (10 mM), sodium carbonate buffer (50 mM with pH 10.2) and EDTA (10 mM). The absorbance was measured for 3 min at 30 sec interval at 320 nm. The SOD activity results were reported as U mg − 1 (per mg of protein), where 1 U of SOD is distinct as the quantity of sample causing 50 % of inhibition of epinephrine auto-oxidation.

Determination of glutathione peroxidase (GPx)
The estimation of glutathione peroxidase in tissue homogenate was done by following the method of Paglia and Valentine (1967)  The activity of GSH (Glutathione-S-transferase) measured by the method describe in Habig et al., (1974). The reaction mixture content 0.1 ml of tissue homogenate mixed with 1 ml of phosphate buffer (0.1 M K 2 HPO 4, EDTA-Na 2, pH 6.5) followed by 0.1 ml of CDNB (1-Chloro-2,4-Dinitrobenzene). Further, make up the volume of reaction mixture to 2.5 ml by using double distilled water and incubated at 37 0 C for 5 min.
Then add 0.1 ml of 30 mM reduced glutathione (GSH). The change in absorbance was measured at 340 nm for 3 min at 15 sec interval. The reaction was based on the conjugation of CDNB solution and reduced glutathione reaction in reaction buffer. The GST activity was measured by using the extinction coe cient of 9.6 mM − 1 cm − 1 for CDNB. The results were stated as µmol/min/mg of protein.
4.7 Experimental set-up for analyzing antioxidant enzymes changes, and oxidative stress in Nerita oryzarum due temperature variation The gastropod species Nerita oryzarum were collected at rocky intertidal locations from Varor which was considered as reference location, as it is situated away from impact of heated e uent. The gastropods of similar size (18 to 20 mm) and irrespective of their sex was collected for our experiment study. The N. oryzarum was brought to library in a plastic bucket half lled with sand and seawater from their natural habitat. While collecting the animal, the temperature of sea water was measured (29.7 ± 1 0 C). In laboratory the organisms were transferred carefully to the glass tank of 20 L capacity (S Fig. 2). The sand from natural habitat of organism was spread at the bottom of a tank. The organisms were allowed to acclimatize in aerated seawater for 24 hours. Then the waterproof heater was inserted to each tank after setting it to desire temperature ranges. The 30 0 C was directly set in heater and for two other temperature ranges it was slowly increased with an interval of 2-3 0 C until the temperature of seawater in tank reach 35 0 C and 40 0 C. The water temperature, pH, dissolved oxygen and salinity were recorded from 0 hr. time to 8 hr at every 2 hr interval.
The acclimatized N. oryzarum samples were divided in three groups and each group had three replicates as tank A, B and C. In each tank, 8-10 animals were placed. Have three tanks labeled as A, B, C (maintained at 25 0 C temperature) details are mentioned in Table-1. The animals were sacri ced after 8 hrs of experimental duration. The temperature range and duration was xed based on led observations. Since, the N. oryzarum collected are inhabitant of intertidal region which are exposed to tidal rhythm of around 6 to 7 hrs of diurnal variation.

Statistical analysis:
The data received from all analytical methods were treated statistically using ORIGIN software. Two-way ANOVA with Duncun's test were run by using SPSS v.20. The graphs were plotted by using SIGMA PLOT software. The correlation analysis between environmental parameters and antioxidant values were analysed on STATISTIKA 7 software. Expressive data analysis were performed, including the calculation of mean, SD, maximum and minimum.

Conclusion
The results of present study is prima facie work related to physiological response of gastropod of N. oryzarum related to heated e uent released from atomic power station and useful as a baseline information for future research work. Further this study provide a meaningful understanding of how temperature changes affect the antioxidant defense system in marine intertidal organisms. Studies on mechanisms for individual function of enzymes in temperature induced stress nding also required in future. Declarations