Abstract Tilapia were exposed to sub??lethal concentrations of 0， 0.2， 2， 20 or 200 ??g/L for 30 d， and then transferred to methomyl??free water for 18 d. CAT and SOD in tilapia serum were examined at 0， 6， 12， 18， 24 and 30 d after methomyl exposure and at 18 d after transferring to methomyl??free water. There were no significant changes in antioxidants activities in tilapia exposed to 0.2 ??g/L. Significant increases in SOD， CAT were observed following methomyl exposure to 2， 20 or 200 ??g/L， suggesting the presence of oxidative stress. Thus， the 0.2 ??g/L methomyl might be considered the no observed adverse effect level. Recovery data showed that the effects produced by lower concentration of 20 ??g/L were reversible but not at the higher concentration of 200 ??g/L.
　　Key words Methomyl； Serum； Tilapia； Antioxidants； Recovery
　　Pesticides are used worldwide in agricultural activity， mostly to promote the harvest of products. However， due to their physico??chemical properties， such as water solubility， vapor pressure or partition coefficients between organic matter （in soil or sediment） and water， these compounds can disperse in various environmental media provoking serious health problems after they are released into the environment[1]. Carbamates are systemic and contact pesticides used as substitutes for organochlorine insecticides because of their high efficiency and relative low persistence in the environment[2]. Methomyl （C5H10N2O2S）， S??methyl??1??N??[（methylcarbamoyl）??oxy]??thioacetimidate， is an insecticide belonging to the family of carbamate pesticides， and it is one of the environmental estrogens having endocrine disrupting effects. Because of its broad biological activity， relatively rapid disappearance and high efficiency against insects， methomyl is widely used in many agricultural countries for crop protection and soil or plant treatment[3]. Methomyl has high water solubility （57.9 g/L at 25 ??） and a weak??to??moderate adsorption to soils， and therefore poses a contamination risk to surface and groundwater， especially the methomyl applied in the agricultural area is expected to infiltrate into the groundwater and threatens the safety of drinking water resources[4].
　　In aerobic cells， reactive oxygen species （ROS）， such as superoxide anion， hydroxyl radical etc.， are generated during normal metabolism， particularly as a result of oxidative metabolism at mitochondrial membranes， and these intermediates might be detrimental to the cell， leading to a state called oxidative stress[5]. Most components of cellular structure and function are likely to be the potential targets of oxidative damage， and the most susceptible substrates for autoxidation are polyunsaturated fatty acids of the cell membrane， which undergo peroxidation rapidly. This may lead to muscle degradation， impairment of the nervous system， haemolysis， general deterioration of the cellular metabolism and eventual cell death[5]. However， Aerobic organisms have evolved defense system to protect themselves from the toxic effects of the increased ROS production， activating the antioxidant system， such as antioxidant scavengers， e.g. glutathione， and specific antioxidant enzymes， e.g. SOD， CAT， GPx， GR， GST[6-9]. Defense systems protect against attacks from either endogenous （physiological production） or exogenous （xenobiotic??related） sources of ROS. Thus， there is mediation between ROS production and antioxidant scavengers in aerobic cells. Under normal physiological situation， the production of ROS and other oxygen reactive species are thought to be removed by antioxidant defense systems； however， a severe oxidative stress suppresses the activities of these enzymes and lead to oxidative damage[10]. Hence， the use of biochemical measurements in organisms as pollution indicators gives valuable information about deleterious responses of organisms[11]. Biochemical effects of pollutants occur more quickly， thus they provide earlier warning signal before other toxicological endpoints become evident[12]. Monitoring of the biomarkers in living organisms including fish is a validated approach and serves as early warning of adverse changes and damage resulting from chemical exposure[13]. 　　There are some reports about the acute toxicity of methomyl on aquatic organisms[14-17]. However， the chronic toxic effects of methomyl on aquatic organisms， especially on fish， has been scarcely investigated. Thus， the aim of the present study was to investigate the chronic toxic effects of the pesticide methomyl on tilapia （Oreochromis niloticus）， by analyzing the responses of the fish serum antioxidant enzymes CAT and SOD.
　　Materials and methods
　　Fish and chemicals
　　Male Nile tilapia was chosen for this study because methomyl is one of the environmental estrogens having endocrine disrupting effects and tilapia is commonly available in most fish farms worldwide. Specimens of tilapia with an average weight of （150.7 ?? 9.7） g and length of （19.0 ?? 1.4） cm were supplied by the fish farm of freshwater fisheries research center， Chinese Academy of Fishery Science （Wuxi， China）. Before the experiments， fish were acclimated under laboratory conditions for 4 weeks at a population density of 30 specimens in 200 L glass aquaria supplied with dechlorinated tap water. The physicochemical characteristics of the water used in the aquaria were analyzed according to methods in "Standard Method for the Examination of Water and Wastewater"[18]. The water had a pH of （7.3 ?? 0.3） and a temperature of （25.0 ?? 0.5） ??. Water hardness was 107 mg/L （as CaCO3）， and dissolved oxygen concentration was 6.5-7.0 mg/L. The stock fish and experimental fish were all fed by 2% body mass daily， with the commercial fish feed （Ningbo Tech??bank Co.， Ltd， China） and submitted to a 12??h light and 12??h dark photoperiod. Fish were used when no mortality was observed in the acclimation population. Methomyl （97% w/w） was produced by Shanghai Focus Biological Technology Co.， Ltd， China. All other chemicals used were of analytical grade and obtained from Sigma??Aldrich （St. Louis， MO， USA） and Sangon （Shanghai， China）.
　　Experimental design
　　Male tilapia were randomly distributed into the 200 L glass aquaria containing different concentrations of methomyl （0， 0.2， 2， 20 and 200 ??g/L）， and the range of exposure concentrations was based on the information from the previous study on 96 h LC50 （430 ??g/L） for tilapia [with an average weight of （3.9 ?? 0.4） g and length of （6.3 ?? 0.6） cm]， the residue level （0-55.3 ??g/L） of methomyl in environmental water[19] and the U.S. EPA on drinking water quality established a maximum permissible concentration for methomyl of 200 ??g/L[20]. The actual methomyl concentrations in the test water were measured by the method of Chen et al.[21]. The actual methomyl concentrations of 0， 0.2， 2， 20， 200 ??g/L groups were 0， 0.24， 2.06， 21.55， 205.57 ??g/L respectively at 0 h of exposure （the initial concentrations）， and were 0， 0.20， 1.95， 19.55， 198.57 ??g/L respectively after 24 h of exposure. And the results were discussed in relation to the nominal concentrations. 　　Thirty fish were introduced into each concentration in a semi??static system and water was renewed daily. The experiment lasted for 48 d， and after 30 d of exposure， the remaining fish were transferred to methomyl??free water for 18 d and then the same parameters were carried out for studying the recovery response. Sampling of exposed and control fish （n = 6 group-1） was done at 10 min （day 0）， 6， 12， 18， 24， and 30 d after starting the experiment and at 18 d （R18） after transferring to methomyl??free water for recovery. Feed was withheld 24 h prior to sampling. Blood was collected from the caudal vein of fish. The blood left to clot was then centrifuged at 1 040 g for 15 min. The separated serum samples were stored at -80 ?? for biochemical analysis.
　　Samples were stored at -80 ?? with an Ultra??low temperature freezer （Forma??86C， America）. Centrifugations were done with a refrigerated centrifuge （Sigma 2??16K， Germany）. Spectrophotometric readings were carried out with a UV¨Cvis spectrophotometer （UV??759S， China）.
　　Biochemical analysis
　　Catalase （CAT） activity was assayed by ultraviolet spectrophotometry[22]， and one unit of enzyme activity was defined as the amount of enzyme which decreased the concentration of H2O2 by 50% in 100 s at 25 ??. Superoxide dismutase （SOD） was assayed by the method of Marklund et al.[23]， and one unit of SOD activity was defined as the amount of the enzyme which gave 50% inhibition of the oxidation rate of 0.1 mM pyrogallol in one ml of solution at 25 ??.
　　Statistical analysis
　　CAT and SOD activities of treated tilapia were compared with control group in each sampling day， including recovery group （R18）， and expressed as % of the control. All data were expressed as means ?? standard deviation （n = 6）. A Two??Sample Student??s t??test was used to test significant differences between groups. Differences were statistically significant when P
　　Results and Analysis
　　Neither mortality nor visible disease signals were observed in the tilapia exposed to sublethal concentrations of methomyl during the performance of the experiment. Changes in the activities of antioxidant enzymes SOD and CAT in experimental fish were shown in Fig.1 & 2.
　　Compared with the control， no significant changes in SOD and CAT activities were observed in 0.2 ??g/L group. However， SOD and CAT activities were significantly increased in 2 ??g/L group after 24 and 18 d of methomyl exposure respectively. As the dose was increased to 20??g/L， significant elevation was noted for SOD and CAT at 12 d. In 200 ??g/L group， SOD and CAT activities significantly increased after 6 d of methomyl exposure， however， with the exposure time delaying， SOD activity was significantly （P 　　When fish were transferred into methomyl??free water， there was a recovery response in SOD and CAT activities in all groups compared with the 30??day exposure group. And compared with the control， no significant difference in SOD and CAT activities in 0.2， 2 and 20 ??g/L recovery groups were observed. However， the SOD and CAT activities in 200 ??g/L recovery group were significant difference from the control.
　　Discussion
　　Fish exposed to environmental pollutants exhibit a variety of physiological responses， including oxidative metabolism imbalances[24]. Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants or excessive accumulation of the ROS， or both， leading to damage[25]. Many xenobiotics， such as pesticides， may cause oxidative stress leading to the generation of ROS and alteration in antioxidants or free oxygen radicals scavenging enzyme systems in aquatic organisms[10]. The evaluation of antioxidant biomarkers is critical to the investigation of oxidative stress in organisms[26]. Antioxidant systems regulate ROS in the cells to maintain a steady??state balance of ROS production and elimination. Disturbance of this dynamic equilibrium can lead to enhanced lipid peroxidation processes and damage to cell constituents linked to changes in the antioxidant systems[26-27]. Organisms are equipped with interdependent cascades of enzymes to alleviate oxidative stress and repair macromolecules damaged during normal metabolism or by exposure to xenobiotics[28]. An important feature of antioxidants is their altered activity and content under conditions of oxidative stress[29]. The changes in activity and content of antioxidants demonstrate a pollutant??induced adaptive response in fish， and an attempt to neutralize the generated ROS[30]. As reported， the levels or activities of antioxidants are potential biomarkers revealing a contaminant??mediated biological effect on the organism[31-32]. Therefore， the change of CAT and SOD activities in the present study reflected the oxidative stress by balancing ROS in tilapia.
　　The first defense line against oxidative stress consists of the antioxidant enzymes SOD， and CAT[33]. SOD is the first enzyme to deal with oxyradicals[5]， and it serves to protect cells against the oxidative damage of free radicals by catalysing the conversion of superoxide anion to molecular oxygen （O2） and hydrogen peroxide （H2O2）， which is then catalyzed by CAT. Decrease in the activity of these enzymes changes the redox status of the cells[34]. Thus， it is possible that an increase in the activity of these enzymes contributes to the elimination from the cell of ROS induced by pesticide exposure[35]. Induction of SOD could occur during high production of superoxide anion radical[36]. In the present study， the significant induction （P 　　CAT is the enzyme that can catalyze hydrogen peroxide to oxygen and water， therefore reduce the oxidative stress of hydrogen peroxide to organisms. Pesticide??induced inhibition and induction of CAT activity has been reported in studies of various fish species. Jin et al.[35] reported increased CAT activity in zebrafish after 14 d atrazine exposure. Ballesteros et al.[41] stated that CAT activity was significantly decreased in Jenynsia multidentata exposed to endosulfan. However， Moraes et al.[42] reported a decrease in CAT activity in teleost fish and silver catfish after exposure to herbicides. Keramati et al.[43] reported that Diazinon caused fluctuated levels in fish Rutilus rutilus. It has also been reported that lower concentrations of endosulfan increase the CAT activity in Oncorhynchus mykiss while the higher concentrations reduce its activity[44] The increase in CAT activities in tilapia serum as observed in the present study might be in response to H2O2 produced by SOD activity since CAT is responsible for the detoxification of H2O2 to oxygen and water. The results of this study are consistent with the previous reports by Zhang et al.[5] and Yi et al.[45] that CAT activities could be elevated in fish after chronic exposure to 2，4??dichlorophenol and alachlor.
　　When tilapia exposed to 0.2 ??g/L methomyl， there were no significant changes in SOD and CAT activities （Fig.1） compared with the control， so 0.2 ??g/L methomyl could be suggested as the threshold dose for no effect to tilapia. When tilapia exposed to 0.2， 2 and 20 ??g/L methomyl were transferred to methomyl??free water for 18 d， SOD and CAT activities （Fig.1） in fish serum returned to the control values， which showed that these parameters exhibited a recovery pattern. However， when tilapia exposed to 200 ??g/L methomyl were transferred to methomyl??free water for 18 d， SOD and CAT activities （Fig.1） in fish had a recovery trend to some extent， but couldn??t return to the control values. Therefore， 200 ??g/L could be the threshold dose for methomyl??induced unreversible oxidative damage in serum of tilapia.
　　Conclusion
　　Antioxidants could be affected by a slight oxidative stress due to compensatory response. The present results show there is a very rapid response in SOD and CAT activities after tilapia contacting with methomyl， SOD and CAT activities in the serum of tilapia exposed to 2， 20 and 200 ??g/L are affected significantly during 30 d exposure compared with the control. These changes indicate the presence of oxidative stress. However， there are no significant changes in SOD and CAT activities in the serum of tilapia exposed to 0.2 ??g/L methomyl compared with the control， so 0.2 ??g/L methomyl might be the threshold dose for no effect to tilapia. And the results of the recovery data show that the toxicity produced by lower concentration of methomyl （no more than 20 ??g/L） is reversible， while the toxicity produced by higher concentration of methomyl （no less than 200 ??g/L） is unreversible within 18 d after stimulus withdraw. Therefore， 200 ??g/L could be the threshold dose for methomyl??induced unreversible oxidative damage in serum of tilapia. In conclusion， biochemical change has been related to methomyl exposure duration and concentration， warning for the potentially negative impact of this pesticide for wild fish， especially in case of persistence contact with methomyl contaminated water. 　　References
　　[1] GRAMATICA P， GUARDO AD. Screening of pesticides for environmental partitioning tendency[J]. Chemosphere， 2002， 47（9）： 947-956.
　　[2] SAINT??DENIS M， NARBONNE JF， ARNAUD C， et al. Saint??Denis M， Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil， effects of carbaryl[J]. Soil Biology & Biochemistry， 2001， 33： 1123-1130.
　　[3] World Health Organization. Methomyl Environmental Health Criteria 178[S]. Geneva： WHO， 1996： 74.
　　[4] STRATHMANN TJ， STONE AT. Reduction of the carbamate pesticides oxamyl and methomyl by dissolved FeII and CuI[J]. Environmental Science & Technology， 2001， 35： 2461-2469.
　　[5] ZHANG J， SHEN H， WANG X， et al. Effects of chronic exposure of 2， 4??dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus[J]. Chemosphere 2004， 55（2）： 167-174.
　　[6] FREEMAN BA， CRAPO JD. Biology of disease： free radicals and tissue injury[J]. Laboratory Investigation， 1982， 47（5）： 412-426.
　　[7] WINSTON GW， GIULIO RTD. Prooxidant and antioxidant mechanisms in aquatic organisms[J]. Aquatic Toxicollogy， 1991， 19 （2）： 137-161.
　　[8] FREI B. Molecular and biological mechanisms of antioxidant action[J]. Faseb Journal Official Publication of the fede， 1999， 13： 963-964.
　　[9] HOGG N， KALYANARAMAN B. Nitric oxide and lipid peroxidation[J]. Biochim Biophys Acta， 1999， 1411： 378-384.
　　[10] LIVINGSTONE DR. Contaminant??stimulated reactive oxygen species production and oxidative damage in aquatic organisms[J]. Marine Pollutution Bulletin， 2001， 42（8）： 656-666.
　　[11] TORTELLI V， COLARES EP， ROBALDO RB， et al. Importance of cholinesterase kinetic parameters in environmental monitoring using estuarine fish[J]. Chemosphere， 2006， 65（4）： 560-566.
　　[12] LIVINGSTONE DR. The fate of organic xenobi¨?ticos inaquatic ecosystems： quantitative and qualitative differences in biotransformation by invertebrates and fish[J]. Comparative Biochemistry and Physiology， 1998， 120： 43-49.
　　[13] VERMEULEN NP. Fish bioaccumulation and biomarkers in environmental risk assessment： a review[J]. Environmental Toxicology Pharmacology， 2003， 13： 57-149.
　　[14] HASHIMOTO Y， NISHIUCHI Y. Establishment of bioassay methods for the evaluation of acute toxicity of pesticides to aquatic organisms[J]. Journal of Pesticide Science， 1981， 6（2）： 257-264.
　　[15] FARRE M， FERNANDEZ J， GRANADA L， et al. Analysis and toxicity of methomyl and ametryn after biodegradation[J]. Analytical & Bioanalytical Chemistry， 2002， 373（8）： 704-709. 　　[16] PEREIRA JL， GONCALVES F. Effects of food availability on the acute and chronic toxicity of the insecticide methomyl to Daphnia spp[J]. Science and the Total Environment， 2007， 386： 9-20.
　　[17] LI H， JIANG H， GAO X， et al. Acute toxicity of the pesticide methomyl on the topmouth gudgeon （Pseudorasbora parva）： mortality and effects on four biomarkers[J]. Fish Physiology & Biochemistry， 2008， 34： 209-216.
　　[18] State Environmental Protection Agency of China. Standard method for the examination of water and wastewater[M]. Beijing： China Environmental Science Press， 2002： 725-729.
　　[19] VAN SCOY AR， YUE M， DENG X， et al. Environmental fate and toxicology of methomyl[J]. Reviews of Environmental Contamination & Toxicology ， 2013， 222： 93-109.
　　[20] U.S. EPA. 2012 Edition of the drinking water standards and health advisories. EPA 822??S??12??001. Office of Water U.S. Environmental Protection Agency， Washington， DC， 2012.
　　[21] CHEN YJ， LU YH， LIU J， et al. The liquid??solid extraction of methomyl in environmental water and gas chromatographic analysis[J]. Chinese Journal of Environmental Science， 1996， 17（2） ： 68-70.
　　[22] XU JB， YUAN XF， LANG PZ， et al. Determination of catalase activity and catalase inhibition by ultraviolet spectrophotometry[J]. Environmental Chemistry， 1997， 16： 73-76.
　　[23] MARKLUND S， MARKLUND G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase[J]. European Journal of Biochemistry， 1974， 47： 469-474.
　　[24] VELISEK J， STARA A， KOLAROVA J， et al. Biochemical， physiological and morfological responses in common carp （Cyprinus carpio L.） after long??term exposure to terbutryn in real environmental concentration[J]. Pesticide Biochemistry & Physiology， 2011， 100（3）： 305-313.
　　[25] SCANDALIOS JG. Oxidative stress： molecular perception and transduction of signals triggering antioxidant gene defenses[J]. Brazilian Journal of Medical and Biological Research， 2005， 38（7）： 995-1014.
　　[26] LUSHCHAK VI. Environmentally induced oxidative stress in aquatic animals[J]. Aquatic Toxicology， 2011， 101（1）： 13-30.
　　[27] TAMAGNO E， ARAGNO M， BOCCUZZI G， GALLO M， et al. Oxygen free radical scavenger properties of dehydroepiandrosterone[J]. Cell Biochemistry & Function， 1999， 16（1）： 57-63.
　　[28] SK UH， BHATTACHARYA S. Prevention of cadmium induced lipid peroxidation， depletion of some antioxidative enzymes and glutathione by a series of novel organoselenocyanates[J]. Environmental Toxicology & Pharmacology， 2006， 22（3）： 298-308. 　　[29] STARA A， KRISTAN J， ZUSKOVA E， et al. Effect of chronic exposure to prometryne on oxidative stress and antioxidant response in common carp （Cyprinus carpio L.）[J]. Pesticide Biochemistry and Physiology， 2012， 105， 18-23.
　　[30] KAVITHA P， RAO JV. Oxidative stress and locomotor behavior response as biomarkers for assessing recovery status of mosquito fish， Gambusia affinis after lethal effect of an organophosphate pesticide， monocrotophos[J]. Pesticide Biochemistry and Physiology， 2007， 87（2）： 182-188.
　　[31] CHEUNG CC， ZHENG GJ， LI AM， et al. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels， Perna viridis[J]. Aquatic Toxicology， 2001， 52： 189-203.
　　[32] CAMUS L， PAMPANIN DM， VOLPATO E， et al. Total oxyradical scavenging capacity responses in Mytilus galloprovincialis transplanted into the Venice lagoon （Italy） to measure the biological impact of anthropogenic activities[J]. Marine Pollution Bulletin， 2004， 49： 801-808.
　　[33] STARA A， MACHOVA J， VELISEK J. Effect of chronic exposure to simazine on oxidative stress and antioxidant response in common carp （Cyprinus carpio L.）[J]. Environmental Toxicology & Pharmacology， 2012， 33（2）： 334-343.
　　[34] OJHA A， YADUVANSHI SK， SRIVASTAVA N. Effect of combined exposure of commonly used organophosphate pesticides on lipid peroxidation and antioxidant enzymes in rat tissues[J]. Pesticide Biochemistry and Physiology， 2011， 99（2）： 148-156.
　　[35] JIN Y， ZHANG X， SHU L， et al. Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish （Danio rerio）[J]. Chemosphere， 2010， 78（7）： 846-852.
　　[36] ORUC EO， UNER N. Combined effects of 2， 4??D and azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus[J]. Comparative Biochemistry and Physiology C， 2000， 127（3）： 291-296.
　　[37] MISRA S， NIYOGI S. Selenite causes cytotoxicity in rainbow trout （Oncorhynchus mykiss） hepatocytes by inducing oxidative stress[J]. Toxicology In Vitro， 2009， 23（7）： 1249-1258.
　　[38] ORUC EO， USTA D. Evaluation of oxidative stress responses and neurotoxicity potential of diazinon in different tissues of Cyprinus carpio[J]. Environmental Toxicology & Pharmacology， 2007， 23（1）： 48-55.
　　[39] DIMITROVA MS， TISHINOVA V， VELCHEVA V. Combined effect of zinc and lead on the hepatic superoxide dismutase??catalase system in carp （Cyprinus carpio）[J]. Comparative Biochemistry and Physiology C， 1994， 108（1）： 43-46. 　　[40] PUERTO M， PICHARDO S， JOS A， et al. Differential oxidative stress responses to pure Microcystin??LR and Microcystin??containing and non??containing cyanobacterial crude extracts on Caco??2 cells[J]. Toxicon Official Journal of the International Society on Toxicology， 2010， 55（2）： 514-522.
　　[41] BLLESTEROS ML， WUNDERLIN DA， BISTONI MA Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan[J]. Ecotoxicology & Environmental Safety， 2009， 72（1）： 199-205.
　　[42] MORAES BS， LORO VL， PRETTO A， et al. Toxicological and metabolic parameters of the teleost fish （Leporinus obtusidens） in response to commercial herbicides containing clomazone and propanil[J]. Pesticide Biochemistry and Physiology， 2009， 95（2）： 57-62.
　　[43] KERAMATI V， JAMILI S， RAMIN M. Effect of diazinon on catalase antioxidant enzyme activity in liver tissue of Rutilus rutilus[J]. Journal of Fisheries & Aquatic Science， 2010， 5（5）： 368-376.
　　[44] SHARBIDRE AA， METKARI V， PATODE P. Effect of methyl parathion and chlorpyrifos on certain biomarkers in various tissues of guppy fish， Poecilia reticulate[J]. Pesticide Biochemistry and Physiology， 2011， 101（2）： 132-141.
　　[45] YI X， DING H， LU Y， et al. Effects of long??term alachlor exposure on hepatic antioxidant defense and detoxifying enzyme activities in crucian carp （Carassius auratus）[J]. Chemosphere， 2007， 68（8）： 1576-1581.