Effects of Quinalphos 25 EC and Dimethoate 30 EC on Activities of AchE, Catalase, GOT and GPT in the Freshwater Prawn Macrobrachium rosenbergii

The commercially important freshwater prawn, Macrobrachium rosenbergii post larvae (PL), 1.5±0.2 cm and 0.1±0.03 g were subjected to static renewal type acute toxicity bioassays against two orgnophophate insecticides, quinalphos (Ekalux EC 25) and dimethoate 30% EC (TAFGOR). The 96 hr LC50 values were determined to be 0.774 μgl-1for quinalphos and 0.856 mgl-1for dimethoate. The PL were exposed to lethal (the 96 hr LC50) and sub-lethal (1/2 nd and 1/4th of the 96 hr LC50) concentrations of these insecticides (quinalphos: 0.774, 0.384 and 0.193 μgl -1; dimethoate: 0.856, 0.428, 0.214 mgl-1) for a duration of 4, 8 and 12 days to study their acute and chronic impacts on whole body activities of enzymes, the neurotransmitter, acetyl cholinesterase (AChE), the antioxidant, catalase and metabolic enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). The activity levels of AChE and catalase were found to be significantly (P<0.05) decreased in test prawns when compared with control, whereas, there were significant elevations in GOT and GPT levels (P<0.05). Among GOT and GPT, the impact was more on GOT than GPT. The dosage and time dependent manner of inhibition or elevation in activities of these enzymes were recorded. Among these two insecticides, quinalphos showed more impacts than dimethoate on this non-target organism. Introduction Pesticides have tremendous benefits to man by increasing crop protection and thereby increasing food production, and controlling the vectors of man and animal diseases. They are transported over long distances by global circulation, and through run-off, find their way into aquatic systems. At the same time the pollution of freshwater ecosystem by chemical pesticides has become one of the most critical environmental problems (Northoff and William, 2004). This causes extensive damage to the activities of the living resources of food-web due to their toxicity, persistency with half-lives of decades and tendency to accumulate in the organisms (Joseph and Raj, 2010; Joseph et al., 2010; Joseph and Raj, 2011). Quinalphos (C12H15N2O3PS), O,O-diethyl O-quinoxalin-2-yl phosphorothioate, an ester of OP is used as insecticide and acaricide having a quick knock down effect through contact and stomach poisoning (David and Kumaraswami, 1988; Hassal, 1990). It is frequently used in many countries and represents a source of toxicity to humans and vertebrate animals (Kegley et al., 2010). Dimethoate (C5H12NO3PS2), O,O-dimethyl S-[2-(methylamino)-2-oxoethyl] dithiophosphate is a broad-spectrum OP insecticide and acaricide exhibit both contact and systemic activity (David and Kumaraswami, 1988; Hassal, 1990). Its degradation by esterases and amidases are very low in insects as compared with those of mammals (Rose and Hodgson, 2004). The effects of pesticides, such as endosulfan, carbaryl, dichlorvos, lindane, chlorpyrifos, monocrotophos, carbofuran and methomyl have been studied on acute toxicity (Bhavan et al., 1997a, b, 2008; Key and Fulton, 2006; Satapornvanit et al., 2009) and biochemistry (Bhavan and Geraldine, 1997, 2000a, b; 2001, 2002, 2004, 2007, 2009; Geraldine et al., 1999; Bhavan et al., 2011) of freshwater prawns. However, no data is available pertaining to quinalphos and dimethoate toxicity induced changes on the activities of the neurotransmitter, acetyl cholinesterase (AChE), the enzymatic antioxidant, catalase and metabolic enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in Macrobrachium. Therefore, it was necessitated to generate information on these pesticides in Macrobrachium rosenbergii to establish the potential of predictive biomarkers for use in water pollution monitoring. Materials and Methods The post larvae of freshwater prawn, M. rosenbergii were purchased from Happy Bay Aqua Nova Hatchery, Mugaiyur, Marakanam Taluk, and Kancheepuram District, Tamilnadu, India. They were safely brought to the laboratory in polythene bags filled with hatchery water and well-oxygenated. They were stocked in large cement tank (6’ x 4’ x 3’) and acclimatized for 2 weeks in ground water. During which they were fed with boiled egg albumin, Artemia nauplii and commercially available scampi crumble feed alternatively thrice a day. The excreta, unfed feed and exuvia if any were removed daily, three fourth of the water was renewed daily and adequately aerated. Quinalphos (Ekalux EC 25) and dimethoate 30% EC (TAFGOR) were purchased from local agro service centre. Ten concentrations of each quinalphos (0.250-1.375 μgl-1) and dimethoate (0.060-1.350 mgl-1) were prepared by mixing in distilled water afresh on every day. Post larvae (PL) of M. rosenbergii (1.5 ±0.2 cm and 0.1 ± 0.03g) were transferred to plastic aquaria of 10 l capacity (each with 10 PL) with ground water for 2 days. Out of eleven groups one PL group was served as control and others were exposed to ten different known concentrations (quinalphos: 0.250-1.375 μgl-1; dimethoate: 0.060-1.350 mgl-1) of each insecticide for 96 hours to assess their LC50 value as per the guidelines prescribed by ASTM (1980). The experiment was conducted


Introduction
Pesticides have tremendous benefits to man by increasing crop protection and thereby increasing food production, and controlling the vectors of man and animal diseases. They are transported over long distances by global circulation, and through run-off, find their way into aquatic systems. At the same time the pollution of freshwater ecosystem by chemical pesticides has become one of the most critical environmental problems (Northoff and William, 2004). This causes extensive damage to the activities of the living resources of food-web due to their toxicity, persistency with half-lives of decades and tendency to accumulate in the organisms (Joseph and Raj, 2010;Joseph et al., 2010;Joseph and Raj, 2011).
Quinalphos (C 12 H 15 N 2 O 3 PS), O,O-diethyl O-quinoxalin-2-yl phosphorothioate, an ester of OP is used as insecticide and acaricide having a quick knock down effect through contact and stomach poisoning (David and Kumaraswami, 1988;Hassal, 1990). It is frequently used in many countries and represents a source of toxicity to humans and vertebrate animals (Kegley et al., 2010). Dimethoate (C 5 H 12 NO 3 PS 2 ), O,O-dimethyl S-[2-(methylamino)-2-oxoethyl] dithiophosphate is a broad-spectrum OP insecticide and acaricide exhibit both contact and systemic activity (David and Kumaraswami, 1988;Hassal, 1990). Its degradation by esterases and amidases are very low in insects as compared with those of mammals (Rose and Hodgson, 2004).
The effects of pesticides, such as endosulfan, carbaryl, dichlorvos, lindane, chlorpyrifos, monocrotophos, carbofuran and methomyl have been studied on acute toxicity (Bhavan et al., 1997a(Bhavan et al., , b, 2008Key and Fulton, 2006;Satapornvanit et al., 2009) and biochemistry (Bhavan and Geraldine, 1997, 2000a2001, 2002, 2007Geraldine et al., 1999; of freshwater prawns. However, no data is available pertaining to quinalphos and dimethoate toxic-ity induced changes on the activities of the neurotransmitter, acetyl cholinesterase (AChE), the enzymatic antioxidant, catalase and metabolic enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in Macrobrachium. Therefore, it was necessitated to generate information on these pesticides in Macrobrachium rosenbergii to establish the potential of predictive biomarkers for use in water pollution monitoring.

Materials and Methods
The post larvae of freshwater prawn, M. rosenbergii were purchased from Happy Bay Aqua Nova Hatchery, Mugaiyur, Marakanam Taluk, and Kancheepuram District, Tamilnadu, India. They were safely brought to the laboratory in polythene bags filled with hatchery water and well-oxygenated. They were stocked in large cement tank (6' x 4' x 3') and acclimatized for 2 weeks in ground water. During which they were fed with boiled egg albumin, Artemia nauplii and commercially available scampi crumble feed alternatively thrice a day. The excreta, unfed feed and exuvia if any were removed daily, three fourth of the water was renewed daily and adequately aerated.
Quinalphos (Ekalux EC 25) and dimethoate 30% EC (TAF-GOR) were purchased from local agro service centre. Ten concentrations of each quinalphos (0.250-1.375 µgl -1 ) and dimethoate (0.060-1.350 mgl -1 ) were prepared by mixing in distilled water afresh on every day. Post larvae (PL) of M. rosenbergii (1.5 ±0.2 cm and 0.1 ± 0.03g) were transferred to plastic aquaria of 10 l capacity (each with 10 PL) with ground water for 2 days. Out of eleven groups one PL group was served as control and others were exposed to ten different known concentrations (quinalphos: 0.250-1.375 µgl -1 ; dimethoate: 0.060-1.350 mgl -1 ) of each insecticide for 96 hours to assess their LC 50 value as per the guidelines prescribed by ASTM (1980). The experiment was conducted ReseaRch PaPeR in triplicates. The toxic water medium was renewed daily by siphoning method, causing minimum disturbance to the prawns and freshly prepared concentrations of quinalphos and dimethoate were added separately to maintain the toxic level in a steady state. During the experiment the prawns were neither fed nor aerated. The concentrations and their respective mortality percentage were subjected to computation for calculation of the median lethal concentration. The 96 h LC 50 value with 95% confidence limits was assessed using computerized program of Finney (1971) method of probit analysis.
Based on 96 hr LC 50 values of quinalphos and dimethoate, three concentrations each for quinalphos (0.774 µgl -1 , 0.384 µgl -1 and 0.193 µgl -1 ) and dimethoate (0.856 mgl -1 , 0.428 mgl -1 and 0.214 mgl -1 ) were selected for treatment during 12 days. A common control was also maintained. Each group comprised 5 aquaria (15 l capacity) and each aquarium housed 20 PL. Sampling was done on day 4, 8 and 12. The entire quantity of medium in each aquarium was gently siphoned out daily and replaced by medium containing freshly prepared concentrations of quinalphos and dimethoate with minimal disturbance to the prawns. During the period of the experiment, the toxic medium was not aerated and the animals were fed with commercial scampi feed. The dead post larvae prawns were removed during the experiment. The activities of enzymes in post larvae prawns, such as AChE (Ellman et al., 1961), catalase (Sinha et al, 1972), and GOT and GPT (Reitman and Frankel, 1957) were assayed on 4 th , 8 th and 12 th day of exposure by sacrificing the test PL in each group. Control prawns were similarly assayed at the same time as the test prawns. The differences between control and pesticide exposed groups were analyzed by adopting student-'t' test using SPSS software (version 16.0). All measurements were performed in triplicates and the results are expressed as mean ± SD of three individual observations. P<0.05 was fixed to assess the statistical significance.

Results and Discussion
The 96 hr LC 50 of quinalphos and dimethoate for M. rosenbergii PL was assessed to be 0.774 μgl -1 and 0.856 mgl -1 respectively (Tables 1 and 2). During bio-assay tests, the mortality of PL was found to be increased in response to higher concentrations of quinalphos and dimethoate (Tables 1 and  2). A comparison of the 96 hr LC 50 values assessed in the present study revealed that quinalphos was >1000 fold more toxic than that of dimethoate to M. rosenbergii PL (Tables  1 and 2). Therefore, it is clear that M. rosenbergii was more sensitive to quinalphos toxicity than that of dimethoate. The toxicity of quinalphos and dimethoate caused severe metabolic distress, which was evident from the escaping tendency of test PL from the aquaria and such behavior was based on dosage of these pesticides, which eventually leads to death of test PL.
Available literature revealed that the 96 hr LC 50 values reported for quinalphos in a another species of freshwater prawn, Macrobrachium lamarrai (0.461 mgl -1 ) was many more times higher toxic (Omkar and Shukla, 1985) when compared with the result observed in the present study, 0.774 μgl -1 (Table 1). In the case of dimethoate, the reported value of 96 hr LC 50 (540 µgl -1 (0.540 mgl -1 ) in a marine crustacean species, the opossum shrimp, Neomysis integer (Roast et al., 1999) was closer to the result recorded in the present study, 0.856 mgl -1 (Table 2). However, in a report on the freshwater shrimp, Paratya australiensis, the reported 96 hr LC 50 value of dimethoate (8.00 µgl -1 (0.008 mgl -1 ) by Kumar et al., (2010) was 100 times lower than that of the value observed in the present study (Table 2). It is important to mention here that toxicity of a xenobiotic is governed by many factors, like water temperature, purity of the toxin, life stage of an organism, size of the individual etc.
Similar inhibition in AChE activity has been reported in the grass shrimp, Palaemonetes pugio embryos exposed to OP pesticides, chlorpyrifos and malathion (Lund et al., 2000), in Macrobrachium malcolmsonii exposed to dichlorvos, endosulfan, and carbaryl (Geraldine et al., 1999;Geraldine, 2001, 2002), in the freshwater shrimp Paratyaaus traliensis exposed to dimethoate (Kumar et al., 2010), in the clam, Ruditapes decussatus exposed to malathion (Nadji et al., 2010), in the Riceland prawn, Macrobrachium lanchesteri on exposure to chlorpyrifos (Tongbai and Damrongphol, 2011) and in freshwater fairy shrimp, Streptocephalus dichotomus exposed to malathion and glyphosate (Kumar and Ali, 2013). The inhibition of AChE recorded in M. rosenbergii PL indicates impairment in hydrolysis of ACh, which suggests disruption of synaptic transmission in the cholinergic system.
Catalase is a sensitive antioxidant biomarker against oxygen free radicals, the reactive oxygen species generated due to oxidative stress (Regoli et al., 2004;Atli and Canli, 2007). In the present study, due to toxicity of quinalphos and dimethoate excessive hydrogen peroxide or superoxide radical may have produced, which in turn inactivated the catalase activity (Table 3). Therefore, the protective mechanism was hampered in test PL even at lower sub lethal level of quinalphos and dimethoate (0.193µgl -1 and 0.214 mgl -1 respectively). OP inhibited catalase activity due to oxidative damage has also been reported in the brackish water prawn Penaeus monodon exposed to fenvalerate (Vijayavel and Balasubramanian, 2009), in the clam, Ruditapes decussates (Nadji et al., 2010) and in freshwater fish, Labeo rohita (Thenmozhi et al., 2011) exposed to malathion.
Increases or decreases in GOT and GPT activity levels are suggested as reflection of tissue damage or organ disfunction (Oluah, 1999;Rao, 2006). In the present study, the increase recorded in GOT activity suggests that an important reaction of the molecular rearrangement involving amino acids linked to the citric acid cycle at two points (oxaloacetic and ketoglutaric acids) that GOT catalyzes was affected. Similarly, the increase in GPT indicates the fact that the test PL terribly required intensive glycogenesis to coop-up the severe energy crisis occurred due to quinalphos and dimethoate toxic stress.
In conclusion, quinalphos and dimethoate exhibited dose and time dependent responses to coop-up with toxic stress, and thus, adversely modulate the activities of AChE, catalase, GOT and GPT in M. rosenbergii. Therefore, these enzymes can be taken as biomarkers for monitoring water pollution by these pesticides in natural environment.

Acknowledgement
The Bharathiar University, Coimbatore, Tamilnadu, India is gratefully acknowledged for providing infrastructure facility. The University Grants Commission, Government of India, New Delhi is also acknowledged for utilizing the facility acquired through Major Research Project sanctioned to the first author. The authors are gratefully acknowledged Mr. N. Manickam and Dr. S. Radhakrishnan for their help in conducting the experiment.  Each value is mean ± SD of 3 individual observations. Values in parentheses are % increase (↑)/ decrease (↓).
Values are significant at P< 0.05. NS , Not significant statistically.