Sublethal Toxicity of Quinalphos on Oxidative Stress and Antioxidant Responses in a Freshwater Fish Cyprinus carpio Devan Hemalatha, Antony Amala, Basuvannan Rangasamy, Bojan Nataraj, Mathan Ramesh Department of Zoology, Unit of Toxicology, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
Received 19 September 2014; revised 26 March 2015; accepted 30 March 2015 ABSTRACT: Extensive use of quinalphos, an organophosphorus pesticide, is likely to reach the aquatic environment and thereby posing a health concern for aquatic organisms. Oxidative stress and antioxidant responses may be good indicators of pesticide contamination in aquatic organisms. The data on quinalphos induced oxidative stress and antioxidant responses in carps are scanty. This study is aimed to assess the two sublethal concentrations of quinalphos (1.09 and 2.18 lL L21) on oxidative stress and antioxidant responses of Cyprinus carpio for a period of 20 days. In liver, the malondialdehyde level was found to be significantly increased in both the concentrations. The results of the antioxidant parameters obtained show a significant increase in superoxide dismutase, catalase, and glutathione-S-transferase activity in liver of fish. These results demonstrate that environmentally relevant levels of the insecticide quinalphos can cause oxidative damage and increase the antioxidant scavenging capacity in C. carpio. This may reflect the potential role of these parameters as useful biomarkers for the assessment of pestiC 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. cide contamination. V Keywords: quinalphos; sublethal; lipid peroxidation; antioxidants; Cyprinus carpio
INTRODUCTION Pesticides are widely used in Indian agriculture to enhance food production by eradicating unwanted insects and controlling disease vectors (Amin and Hashem, 2012). The use of pesticides was introduced in India during the mid-60s, which are now being used on a large scale and is a common feature of Indian agriculture (Bhanti and Taneja, 2007). These chemicals have made great contributions to plant protection but at the same time their unregulated and indiscriminate applications has resulted in serious health and environmental problems (Jeyasankar and Jesudasan, 2005). It has been estimated that only 0.1% of the applied pesticides reach the target pests and the remaining 99.9% find their Correspondence to: M. Ramesh; e-mail: [email protected] Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22145
way to different components of the environment. Aquatic ecosystems that run through agricultural or industrial areas have high probability of being contaminated by these hazardous chemicals (Todd and Leuwen, 2002). Pesticide residues reach the aquatic environment mainly via agricultural runoff, spray drift, and leaching, where it poses significant toxicological risks to the nontarget organisms and finally finding their way to the food chain threatening the ecological balance and the biodiversity of the nature (Cerejeira et al., 2003; Konstantinou et al., 2006; Asita and Makhalemele, 2008; Saravanan et al., 2011; Bacchetta et al., 2014). Among the pesticides, organophosphorus pesticides (OPs) are the most commonly used pesticides in the world owing to their high insecticidal property, low mammalian toxicity, less persistence, and rapid biodegradability in the environment (Singh et al., 2010; Ren et al., 2012). About 70% of the pesticides in current use are OP compounds that constitute a total consumption of around 90 million pounds
C 2015 Wiley Periodicals, Inc. V
1
2
HEMALATHA ET AL.
per year (Ojha et al., 2011). Unfortunately, OPs lack target specificity and can cause severe, long-lasting population effects on terrestrial and aquatic nontarget species, particularly fish (Schulz and Liess, 1999; Fulton and Key, 2001). The widely used OPs pesticides can exert their toxicity by inhibiting acetylcholinesterase (AChE) activity of organisms (Kavitha and Venkateswara Rao, 2007; Assis et al., 2012). In addition to neurotoxicity, OPs can induce a range of sublethal toxic effects to other body functions which may adversely affect the health and survival of exposed organisms (Canty et al., 2007; Yonar et al., 2014). Quinalphos, (QP: O,O diethyl O-2 quinoxalinoxalinyl phospharothionate) is a synthetic organophosphate, nonsystemic, broad spectrum insecticide, and acaricide extensively used in Indian agricultural field owing to its low bioaccumulation and high rate of biodegradation (Rahman et al., 2004; Mishra and Devi, 2014). But on degradation, quinalphos produce various metabolites through hydrolysis, oxidation dealkylation, and isomerization processes. These metabolities include quinalphos oxon, O-ethyl-Oquinoxalin-2-yl phosphoric acid, 2-hydroxy quinoxaline, and quinoxaline-2-thiol. Among these, 2-hydroxy quinoxaline and oxon were considered to be more toxic than the parent compound and persist in the environment for a longer period (Gupta et al., 2011). It is effective against wide range of pests of cotton, groundnuts, rice, tea, coffee, soybeans, and so forth. Quinalphos has been classified as moderately hazardous pesticide by WHO but has become a matter of concern because of its potentiality and hazardous effect to nontarget organisms. The primary target of quinalphos action is the inhibition of AChE activity, the enzyme that degrades the neurotransmitter acetylcholine in cholinergic synapses. Experimental evidence showed that quinalphos, besides its inhibitory effect on AChE, also induces oxidative stress. Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted owing to the depletion of antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, leading to damage (Scandalios, 2005). Pesticides are known to modulate antioxidant defense systems and to cause oxidative stress in aquatic organisms via ROS production (Bagchi et al., 1995; Livingstone, 2001; Sinhorin et al., 2014). ROS such as hydrogen peroxide (H2O2) and the free radicals superoxide (O2•2) and hydroxyl radical (HO•) can react with biological macromolecules and produce enzyme inactivation, lipid peroxidation (LPO), DNA damage, and protein oxidation, resulting in oxidative stress (Livingstone et al., 1993; Nordberg and Arner, 2001; Shi et al., 2005). To attenuate the adverse effects of ROS, fish possess an antioxidant defence system similar to other vertebrates that use enzymatic and nonenzymatic mechanisms. The most important antioxidant enzymes are superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.16), and glutathione-S-transferase (GST; EC.2.5.1.18, GST) (Storey, 1996; Droge, 2002).
Environmental Toxicology DOI 10.1002/tox
The SODs are a family of metalloenzymes responsible for catalyzing the transformation of the reactive superoxide anion (O22•) into hydrogen peroxide (H2O2) (McCord and Fridovich, 1969). CAT reduces hydrogen peroxide (H2O2) into oxygen and water (Mates, 2000; Atli and Canli, 2007). GSTs represent a group of multifunctional enzymes that catalyze the conjugation of electrophilic compounds (or phase I metabolites) with GSH which involved in the detoxification of xenobiotics (Monteiro et al., 2006). One of the most important targets of ROS is the membrane lipids which undergo peroxidation (LPO). Thus, the estimation of LPO has also been successfully employed to signify oxidative stress and most frequently used as biomarker in the evaluations of toxicological assays. Therefore, examining the change in the activity of SOD, CAT, GST, and LPO may indirectly reflect the presence of toxic contaminants and considered as an effective method of denoting oxidative stress in aquatic species. Fish are widely used to evaluate the health of aquatic systems, and physiological changes in fishes serve as biomarkers of environmental pollution, and thus can be used for the quality assessment of the aquatic system (Winger et al., 1990; Saiki et al., 1993; Kock et al., 1996; Saravanan and Ramesh, 2013). The freshwater fish, Cyprinus carpio, is of great commercial importance because it is the most common fish widely consumed worldwide. Therefore, it can be a good model to study the responses to various environmental contaminations. In addition, for pesticide contamination, there are very good correlations between fish species and sensitivity differences are small or irrelevant for most pesticide groups (Tremolada et al., 2004). To our knowledge, the impact of quinalphos toxicity on freshwater fish is very limited. Recently in India, the residues of quinalphos have been detected in many vegetables and fruits. This study is aimed to elucidate the sublethal toxicity of quinalphos on oxidative stress (LPO) and antioxidant defenses (SOD, CAT, and GST) in a freshwater fish C. carpio. This article may provide some consults for ecological risk assessment of quinalphos and its analogs.
MATERIALS AND METHODS Fish Species and Test Conditions The common carp fingerlings, C. carpio L. (mean weight, 5–6 g; mean length, 6–7 cm) were procured from Tamil Nadu Fisheries Development Corporation Limited, Aliyar Fish Farm, Tamil Nadu, India, and transferred to laboratory. The fish were acclimatized to laboratory conditions for 1 month in a large tank (capacity, 1000 L) with dechlorinated water before exposure. The tank was provided with continuous aeration and a 12:12 h light–dark cycle was maintained throughout the experiment period. During acclimation and toxicity tests, fish were fed ad libitum twice a day with commercial fish pellets and water renewal was performed once a
SUBLETHAL TOXICITY OF QUINALPHOS
day corresponding to 85% of the tank water to minimize contamination from metabolic wastes. Prior to exposure, the fish were examined carefully for any pathological symptoms. The physicochemical parameters of water were determined according to APHA (2005) and are as follows: temperature, 27.2 6 1.2 C, pH 7.1 6 0.08, dissolved oxygen 6.4 6 0.4 mg L21, total alkalinity 18.3 6 8.0 mg L21, and total hardness 18.2 6 1.5 mg L21.
Experimental Design After the initial acclimatization period, fish with an average length of 6–7 cm and weight of 5–6 g were selected to determine the 96-h LC50 value of quinalphos in static renewal system in laboratory as per standard methods (APHA, 2005). The range finding test was carried out prior to the definitive test to determine the concentration of the test solution. All experiments were conducted in 100-L rectangular glass aquaria (25 cm 3 60 cm 3 25 cm). Quinalphos (25% EC) was obtained from the local market of Coimbatore, Tamil Nadu, India, supplied by, The Tudiyalur Co-op. Agrl. Services, Tamil Nadu, India. Stock solution of quinalphos was prepared by dissolving 1 mL of quinalphos in appropriate amount of analytical grade acetone initially and then mixed well with tap water and the solution was made up to 1000 mL to obtain a stock solution. The volume of acetone was kept equal in all treatment groups and control groups.
3
in groups three and four were subjected to two sublethal concentrations of quinalphos that is 1.09 ll L21 (Treatment I) and 2.18 ll L21 (Treatment II) for a period of 20 days, respectively. Test medium was renewed every day to avoid dilution owing to active ingredient degradation. During the experiment period, the observation of toxic symptoms such as stress, movement, respiration, swimming, responses to the outer effects also recorded. Feeding was with held during experimentation; that is, fish were starved 24 h prior to dissection. After the stipulated time, fish were killed with a blow on the head and the liver tissue samples were collected and washed by 0.6% saline solution. The samples were immediately frozen and stored at 280 C until analysis. Frozen liver tissue samples were weighed and homogenized (1:10 w/v) in 50 mM ice cold potassium phosphate buffer, pH 7.0, containing 0.5 mM EDTA using glass homogenizer and then centrifuged for 30 min at 12,0003g at 4 C with a refrigerated centrifuge. Supernatants were immediately used to determine the activities of antioxidant enzymes and total protein content by using a spectrophotometric assay.
Assay for LPO LPO was measured by the method of Devasagayam and Tarachand (1987). The malondialdehyde (MDA) of the samples was expressed as nanomoles of MDA formed/mg protein.
Measurement of Enzymatic Antioxidants Toxicity Assessment and Determination of 96-h LC50 Value To determine the LC50 value for 96-h, a set of 10 fish specimen were randomly exposed to each of the quinalphos concentrations (10.6, 10.7, 10.8, 10.9, and 11 lL L21). Another set of 10 fish were also simultaneously maintained in tap water (0.00 lL L21) as the control. The experiment was set in triplicate and fish were starved 24 h prior to experiment to avoid prandial effects and to maintain the quality of water. During the exposures, mortality was monitored at 1-h interval for the initial 12-h, and then at each 12-h interval to the end of the test. The criteria for death were no gill movement and no reaction to gentle prodding. Dead fish were removed after each observation. The median lethal concentration (LC50 value) for C. carpio was calculated by log probit analysis with the help of SPSS 20 statistical software for Windows with a confident limit of 95% value. Of the calculated LC50 value, one-tenth (1.09 lL L21) and one-fifth (2.18 lL L21) of quinalphos concentrations were selected for sublethal toxicity studies.
The SOD (EC 1.15.1.1, SOD) activity was determined by the method of Marklund and Marklund (1974). This assay depends on the autoxidation of pyrogallol. The enzymatic activity was expressed as the amount of enzyme per milligram of protein. The activity of CAT (EC1.11.1.6, CAT) was determined according to the procedure of Aebi (1974) by following the absorbance of hydrogen peroxide at 590 nm and expressed as micromoles of H2O2 utilized/min/ mg protein. GST (EC.2.5.1.18, GST) activity was measured in liver according to Habig et al. (1974) using 1-chloro-2,4dinitrobenzene (CDNB) as a substrate. The activity of GST was expressed as micromole GSH-CDNB conjugate formed/ min/mg/protein using an extinction coefficient of 9.6 mM21 cm21.
Measurement of Protein Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Statistical Analysis Sublethal Toxicity Studies To evaluate the sublethal effect, fish were divided into four main groups each with 50 individuals. Group one was kept in pesticide-free fresh water and treated as the control. Group two was maintained as solvent (acetone) control. Fish
Statistical analysis was carried out using a statistical package (SPSS 20.0 for Windows). The results obtained from each experimental group were analyzed by one-way analysis of variance (ANOVA). The significant means were compared by Duncan’s Multiple Range Test (DMRT). A p-value of
Environmental Toxicology DOI 10.1002/tox
4
HEMALATHA ET AL.
TABLE I. LC50 value and confidence limits (95%) of quinalphos for the fish C. carpio 95% Confidence Limits Pesticide
96-h LC50 (lL L21)
Upper Limit
Lower Limit
Quinalphos
10.9 lL L21
10.842
11.011
Received 19 September 2014; revised 26 March 2015; accepted 30 March 2015 ABSTRACT: Extensive use of quinalphos, an organophosphorus pesticide, is likely to reach the aquatic environment and thereby posing a health concern for aquatic organisms. Oxidative stress and antioxidant responses may be good indicators of pesticide contamination in aquatic organisms. The data on quinalphos induced oxidative stress and antioxidant responses in carps are scanty. This study is aimed to assess the two sublethal concentrations of quinalphos (1.09 and 2.18 lL L21) on oxidative stress and antioxidant responses of Cyprinus carpio for a period of 20 days. In liver, the malondialdehyde level was found to be significantly increased in both the concentrations. The results of the antioxidant parameters obtained show a significant increase in superoxide dismutase, catalase, and glutathione-S-transferase activity in liver of fish. These results demonstrate that environmentally relevant levels of the insecticide quinalphos can cause oxidative damage and increase the antioxidant scavenging capacity in C. carpio. This may reflect the potential role of these parameters as useful biomarkers for the assessment of pestiC 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. cide contamination. V Keywords: quinalphos; sublethal; lipid peroxidation; antioxidants; Cyprinus carpio
INTRODUCTION Pesticides are widely used in Indian agriculture to enhance food production by eradicating unwanted insects and controlling disease vectors (Amin and Hashem, 2012). The use of pesticides was introduced in India during the mid-60s, which are now being used on a large scale and is a common feature of Indian agriculture (Bhanti and Taneja, 2007). These chemicals have made great contributions to plant protection but at the same time their unregulated and indiscriminate applications has resulted in serious health and environmental problems (Jeyasankar and Jesudasan, 2005). It has been estimated that only 0.1% of the applied pesticides reach the target pests and the remaining 99.9% find their Correspondence to: M. Ramesh; e-mail: [email protected] Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22145
way to different components of the environment. Aquatic ecosystems that run through agricultural or industrial areas have high probability of being contaminated by these hazardous chemicals (Todd and Leuwen, 2002). Pesticide residues reach the aquatic environment mainly via agricultural runoff, spray drift, and leaching, where it poses significant toxicological risks to the nontarget organisms and finally finding their way to the food chain threatening the ecological balance and the biodiversity of the nature (Cerejeira et al., 2003; Konstantinou et al., 2006; Asita and Makhalemele, 2008; Saravanan et al., 2011; Bacchetta et al., 2014). Among the pesticides, organophosphorus pesticides (OPs) are the most commonly used pesticides in the world owing to their high insecticidal property, low mammalian toxicity, less persistence, and rapid biodegradability in the environment (Singh et al., 2010; Ren et al., 2012). About 70% of the pesticides in current use are OP compounds that constitute a total consumption of around 90 million pounds
C 2015 Wiley Periodicals, Inc. V
1
2
HEMALATHA ET AL.
per year (Ojha et al., 2011). Unfortunately, OPs lack target specificity and can cause severe, long-lasting population effects on terrestrial and aquatic nontarget species, particularly fish (Schulz and Liess, 1999; Fulton and Key, 2001). The widely used OPs pesticides can exert their toxicity by inhibiting acetylcholinesterase (AChE) activity of organisms (Kavitha and Venkateswara Rao, 2007; Assis et al., 2012). In addition to neurotoxicity, OPs can induce a range of sublethal toxic effects to other body functions which may adversely affect the health and survival of exposed organisms (Canty et al., 2007; Yonar et al., 2014). Quinalphos, (QP: O,O diethyl O-2 quinoxalinoxalinyl phospharothionate) is a synthetic organophosphate, nonsystemic, broad spectrum insecticide, and acaricide extensively used in Indian agricultural field owing to its low bioaccumulation and high rate of biodegradation (Rahman et al., 2004; Mishra and Devi, 2014). But on degradation, quinalphos produce various metabolites through hydrolysis, oxidation dealkylation, and isomerization processes. These metabolities include quinalphos oxon, O-ethyl-Oquinoxalin-2-yl phosphoric acid, 2-hydroxy quinoxaline, and quinoxaline-2-thiol. Among these, 2-hydroxy quinoxaline and oxon were considered to be more toxic than the parent compound and persist in the environment for a longer period (Gupta et al., 2011). It is effective against wide range of pests of cotton, groundnuts, rice, tea, coffee, soybeans, and so forth. Quinalphos has been classified as moderately hazardous pesticide by WHO but has become a matter of concern because of its potentiality and hazardous effect to nontarget organisms. The primary target of quinalphos action is the inhibition of AChE activity, the enzyme that degrades the neurotransmitter acetylcholine in cholinergic synapses. Experimental evidence showed that quinalphos, besides its inhibitory effect on AChE, also induces oxidative stress. Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted owing to the depletion of antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, leading to damage (Scandalios, 2005). Pesticides are known to modulate antioxidant defense systems and to cause oxidative stress in aquatic organisms via ROS production (Bagchi et al., 1995; Livingstone, 2001; Sinhorin et al., 2014). ROS such as hydrogen peroxide (H2O2) and the free radicals superoxide (O2•2) and hydroxyl radical (HO•) can react with biological macromolecules and produce enzyme inactivation, lipid peroxidation (LPO), DNA damage, and protein oxidation, resulting in oxidative stress (Livingstone et al., 1993; Nordberg and Arner, 2001; Shi et al., 2005). To attenuate the adverse effects of ROS, fish possess an antioxidant defence system similar to other vertebrates that use enzymatic and nonenzymatic mechanisms. The most important antioxidant enzymes are superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.16), and glutathione-S-transferase (GST; EC.2.5.1.18, GST) (Storey, 1996; Droge, 2002).
Environmental Toxicology DOI 10.1002/tox
The SODs are a family of metalloenzymes responsible for catalyzing the transformation of the reactive superoxide anion (O22•) into hydrogen peroxide (H2O2) (McCord and Fridovich, 1969). CAT reduces hydrogen peroxide (H2O2) into oxygen and water (Mates, 2000; Atli and Canli, 2007). GSTs represent a group of multifunctional enzymes that catalyze the conjugation of electrophilic compounds (or phase I metabolites) with GSH which involved in the detoxification of xenobiotics (Monteiro et al., 2006). One of the most important targets of ROS is the membrane lipids which undergo peroxidation (LPO). Thus, the estimation of LPO has also been successfully employed to signify oxidative stress and most frequently used as biomarker in the evaluations of toxicological assays. Therefore, examining the change in the activity of SOD, CAT, GST, and LPO may indirectly reflect the presence of toxic contaminants and considered as an effective method of denoting oxidative stress in aquatic species. Fish are widely used to evaluate the health of aquatic systems, and physiological changes in fishes serve as biomarkers of environmental pollution, and thus can be used for the quality assessment of the aquatic system (Winger et al., 1990; Saiki et al., 1993; Kock et al., 1996; Saravanan and Ramesh, 2013). The freshwater fish, Cyprinus carpio, is of great commercial importance because it is the most common fish widely consumed worldwide. Therefore, it can be a good model to study the responses to various environmental contaminations. In addition, for pesticide contamination, there are very good correlations between fish species and sensitivity differences are small or irrelevant for most pesticide groups (Tremolada et al., 2004). To our knowledge, the impact of quinalphos toxicity on freshwater fish is very limited. Recently in India, the residues of quinalphos have been detected in many vegetables and fruits. This study is aimed to elucidate the sublethal toxicity of quinalphos on oxidative stress (LPO) and antioxidant defenses (SOD, CAT, and GST) in a freshwater fish C. carpio. This article may provide some consults for ecological risk assessment of quinalphos and its analogs.
MATERIALS AND METHODS Fish Species and Test Conditions The common carp fingerlings, C. carpio L. (mean weight, 5–6 g; mean length, 6–7 cm) were procured from Tamil Nadu Fisheries Development Corporation Limited, Aliyar Fish Farm, Tamil Nadu, India, and transferred to laboratory. The fish were acclimatized to laboratory conditions for 1 month in a large tank (capacity, 1000 L) with dechlorinated water before exposure. The tank was provided with continuous aeration and a 12:12 h light–dark cycle was maintained throughout the experiment period. During acclimation and toxicity tests, fish were fed ad libitum twice a day with commercial fish pellets and water renewal was performed once a
SUBLETHAL TOXICITY OF QUINALPHOS
day corresponding to 85% of the tank water to minimize contamination from metabolic wastes. Prior to exposure, the fish were examined carefully for any pathological symptoms. The physicochemical parameters of water were determined according to APHA (2005) and are as follows: temperature, 27.2 6 1.2 C, pH 7.1 6 0.08, dissolved oxygen 6.4 6 0.4 mg L21, total alkalinity 18.3 6 8.0 mg L21, and total hardness 18.2 6 1.5 mg L21.
Experimental Design After the initial acclimatization period, fish with an average length of 6–7 cm and weight of 5–6 g were selected to determine the 96-h LC50 value of quinalphos in static renewal system in laboratory as per standard methods (APHA, 2005). The range finding test was carried out prior to the definitive test to determine the concentration of the test solution. All experiments were conducted in 100-L rectangular glass aquaria (25 cm 3 60 cm 3 25 cm). Quinalphos (25% EC) was obtained from the local market of Coimbatore, Tamil Nadu, India, supplied by, The Tudiyalur Co-op. Agrl. Services, Tamil Nadu, India. Stock solution of quinalphos was prepared by dissolving 1 mL of quinalphos in appropriate amount of analytical grade acetone initially and then mixed well with tap water and the solution was made up to 1000 mL to obtain a stock solution. The volume of acetone was kept equal in all treatment groups and control groups.
3
in groups three and four were subjected to two sublethal concentrations of quinalphos that is 1.09 ll L21 (Treatment I) and 2.18 ll L21 (Treatment II) for a period of 20 days, respectively. Test medium was renewed every day to avoid dilution owing to active ingredient degradation. During the experiment period, the observation of toxic symptoms such as stress, movement, respiration, swimming, responses to the outer effects also recorded. Feeding was with held during experimentation; that is, fish were starved 24 h prior to dissection. After the stipulated time, fish were killed with a blow on the head and the liver tissue samples were collected and washed by 0.6% saline solution. The samples were immediately frozen and stored at 280 C until analysis. Frozen liver tissue samples were weighed and homogenized (1:10 w/v) in 50 mM ice cold potassium phosphate buffer, pH 7.0, containing 0.5 mM EDTA using glass homogenizer and then centrifuged for 30 min at 12,0003g at 4 C with a refrigerated centrifuge. Supernatants were immediately used to determine the activities of antioxidant enzymes and total protein content by using a spectrophotometric assay.
Assay for LPO LPO was measured by the method of Devasagayam and Tarachand (1987). The malondialdehyde (MDA) of the samples was expressed as nanomoles of MDA formed/mg protein.
Measurement of Enzymatic Antioxidants Toxicity Assessment and Determination of 96-h LC50 Value To determine the LC50 value for 96-h, a set of 10 fish specimen were randomly exposed to each of the quinalphos concentrations (10.6, 10.7, 10.8, 10.9, and 11 lL L21). Another set of 10 fish were also simultaneously maintained in tap water (0.00 lL L21) as the control. The experiment was set in triplicate and fish were starved 24 h prior to experiment to avoid prandial effects and to maintain the quality of water. During the exposures, mortality was monitored at 1-h interval for the initial 12-h, and then at each 12-h interval to the end of the test. The criteria for death were no gill movement and no reaction to gentle prodding. Dead fish were removed after each observation. The median lethal concentration (LC50 value) for C. carpio was calculated by log probit analysis with the help of SPSS 20 statistical software for Windows with a confident limit of 95% value. Of the calculated LC50 value, one-tenth (1.09 lL L21) and one-fifth (2.18 lL L21) of quinalphos concentrations were selected for sublethal toxicity studies.
The SOD (EC 1.15.1.1, SOD) activity was determined by the method of Marklund and Marklund (1974). This assay depends on the autoxidation of pyrogallol. The enzymatic activity was expressed as the amount of enzyme per milligram of protein. The activity of CAT (EC1.11.1.6, CAT) was determined according to the procedure of Aebi (1974) by following the absorbance of hydrogen peroxide at 590 nm and expressed as micromoles of H2O2 utilized/min/ mg protein. GST (EC.2.5.1.18, GST) activity was measured in liver according to Habig et al. (1974) using 1-chloro-2,4dinitrobenzene (CDNB) as a substrate. The activity of GST was expressed as micromole GSH-CDNB conjugate formed/ min/mg/protein using an extinction coefficient of 9.6 mM21 cm21.
Measurement of Protein Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Statistical Analysis Sublethal Toxicity Studies To evaluate the sublethal effect, fish were divided into four main groups each with 50 individuals. Group one was kept in pesticide-free fresh water and treated as the control. Group two was maintained as solvent (acetone) control. Fish
Statistical analysis was carried out using a statistical package (SPSS 20.0 for Windows). The results obtained from each experimental group were analyzed by one-way analysis of variance (ANOVA). The significant means were compared by Duncan’s Multiple Range Test (DMRT). A p-value of
Environmental Toxicology DOI 10.1002/tox
4
HEMALATHA ET AL.
TABLE I. LC50 value and confidence limits (95%) of quinalphos for the fish C. carpio 95% Confidence Limits Pesticide
96-h LC50 (lL L21)
Upper Limit
Lower Limit
Quinalphos
10.9 lL L21
10.842
11.011
