Effects of diazinon on biochemical parameters of blood in rainbow trout (Oncorhynchus mykiss)

Existence of diazinon, an organophosphorous pesticide, in river waters of Iran near rice paddy fields has been reported by some authors. The present research aimed to determine the acute toxicity and evaluate the effect of sub-lethal concentrations of diazinon on some biochemical parameters of rainbow trout, Oncorhynchus mykiss after 7, 14 and 28 days. No significant differences were observed in the plasma levels of creatinine among the treatment groups at different sampling intervals. Acetylcholinesterase activity and the levels of total protein, albumin as well as globulin in plasma were significantly reduced at both concentrations tested (p < 0.05). Lactate dehydrogenase activity was only decreased on 7th day in 0.1 mg/L diazinon treatment (p < 0.05). Creatine kinase activity was significantly lower in 0.1 mg/L diazinon group at 14th and 28th sampling periods, whereas its activity significantly increased in fishes exposed to 0.2 mf/L diazinon only on 7th day (p < 0.05). Aspartate aminotransferase, alanine aminotransferase activities and glucose levels in diazinon treated groups were significantly higher than the controlled group at experimental periods (p < 0.05). In conclusion, long-term exposure to diazinon at sub-lethal concentrations induced biochemical alterations in rainbow trout, and offers a simply tool to evaluate toxicity-derived alterations.     

Mutagenic, genotoxic and enzyme inhibitory effects of carbosulfan in rainbow trout Oncorhynchus mykiss

Rainbow trout (Oncorhynchus mykiss; 116.88 ± 21.69 g) were exposed to sublethal concentrations (25 μg/L) of carbosulfan for 60 days to test if the long term exposure of fish to carbosulfan affects red blood cells acetylcholinesterase (AChE), δ-aminolevulinic acid dehydratase (ALA-D) and paraoxonase (PON) enzyme activity and induces genotoxic and/or mutagenic effects. The exposure resulted in inhibition of AChE and ALA-D activity of rainbow trout when compared to control fish. The activity of PON was not affected by carbosulfan. Interestingly, carbosulfan was found to induce DNA damage in red blood cells (comet assay) and proved to be mutagenic as revealed by the Ames test. Results indicate that blood AChE and ALA-D of rainbow trout may be a sensitive biomarker for assessment of carbosulfan contaminated water bodies. Furthermore, because the Ames test and comet assay were proven successful to detect the genotoxicity of carbosulfan, we proposed that nonlethal techniques such as blood collection from caudal vein of fish should be used to determine potential toxic effects of other pesticides to surrounding environment.     

Impacts of hypersaline water on the biotransformation and toxicity of fenthion on rainbow trout (Oncorhynchus mykiss), striped bass (Morone saxatilis X Morone chrysops) and tilapia (Oreochromis mossambicus)

Hypersaline water derived from agricultural practices primarily in arid landscapes can impact fisheries in streams receiving run-off from fields. Previous studies have indicated significant elevation in the toxicity of thio-ether pesticides by hypersaline water in certain species of euryhaline fish due to enhanced formation of sulfoxide metabolites which may be more toxic than the parent compounds. The objectives of this study were to evaluate the effects of salinity on the toxicity of the thio-ether organophosphate pesticide, fenthion in three species of euryhaline fish: rainbow trout (Oncorhynchus mykiss), striped bass (Morone saxatilis X Morone chrysops), tilapia (Oreochromis mossambicus) and determine whether a relationship was observed between toxicity and enantioselective sulfoxidation. Stereochemical formation and total sulfoxide formation did not mirror acute toxicity in the three species exposed in fresh or hypersaline conditions. Mortality of striped bass and rainbow trout due to fenthion exposure was enhanced by hypersaline treatments and a trend towards increased toxicity was observed in tilapia. In liver microsomes of rainbow trout, inhibition of cytochrome P450 (CYP) caused a respective 7- and 3-fold increase in sulfoxide formation in liver microsomes fresh and saltwater fish, and a significant increase in the formation of the (+)R-sulfoxide. CYP inhibition also caused a significant elevation of the (+)R-sulfoxide in freshwater striped bass, but not in hypersaline-treated bass. The results indicated contribution by CYP in the sulfoxidation of fenthion as well as the formation of other metabolites in all three species. In summary, hypersaline conditions impacted fenthion toxicity under conditions that appeared to be independent of fenthion sulfoxide formation indicating a much more complex mechanism of action for compounds with phosphorothioate and thio-ether structural features potentially involving multiple oxidative pathways.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Changes in selected immunological parameters and antioxidant status of rainbow trout exposed to malachite green (Oncorhynchus mykiss, Walbaum, 1792)

In this study, the effects of malachite green on selected immunological parameters, oxidative stress and antioxidant status biomarkers in blood, liver, kidney, spleen and gill of rainbow trout, Oncorhynchus mykiss, was examined. During 5 days the malachite green was applied at concentrations of 1/15,000 and 1/150,000 for 30 s and 60 min, respectively. Immunological parameters (nitroblue tetrazolium (NBT) activity, total plasma protein (TP), total immunoglobulin (TI)) and biochemical parameters (lipid peroxidation (MDA), catalase (CAT) activity, reduced glutathione (GSH) levels) were evaluated after exposed to malachite green. It has been observed that NBT activity (p < 0.05, p < 0.001), total protein (p < 0.01, p < 0.001) and total immunoglobulin (p < 0.05, p < 0.001) levels were decreased compared with control group. In the rainbow tout exposed to malachite green duration 5 days significantly increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione and catalase activity in the whole tissues of O. mykiss (p < 0.05, p < 0.01, p < 0.001 for each cases).     

Effects of N-acetylcysteine on oxidative responses in the liver of fenthion exposed Cyprinus carpio

The aim of this study was to evaluate the effects of different N-acetylcysteine doses on the tolerance to fenthion-induced oxidative stress, alterations in glutathione metabolism and cholinesterase specific activities in the liver by using freshwater fish Cyprinus carpio (Cyprinidae) as a model organism. An acute toxicity study was carried out to determine 96-h median lethal concentration of fenthion for this species (2.16 mg/L) and 80% of this concentration was applied in toxicity studies. Four groups, each containing eight fish were constituted as follows: Control group, fenthion treated group, 0.5 or 400 mg/kg NAC-injected + fenthion-treated groups. Biochemical analyses were carried out spectrophotometrically. Fenthion treatment significantly decreased total glutathione and glutathione levels, glutathione/glutathione disulfide ratio together with glutathione reductase and γ-glutamylcysteine synthetase specific enzyme activities. The higher dose of N-acetylcysteine increased the toxic effects of fenthion and γ-glutamyl transpeptidase specific activity while decreasing glutathione S-transferase specific activity. However, injection of the lower dose provided a limited protection against fenthion toxicity. In all exposure groups, lipid peroxidation increased and total protein levels decreased, while protein depletion was prevented by low dose of N-acetylcysteine application. Acetylcholinesterase and butyrylcholinesterase activities were at similar levels in the liver of C. carpio. A dose-dependent inhibition was observed in butyrylcholinesterase activity by N-acetylcysteine application. The results showed that fenthion had a significant oxidative stress inducing potential through the reduction of glutathione redox capacity. The critical point for overcoming oxidative stress by N-acetylcysteine in fenthion toxicity was the selection of the dose; N-acetylcysteine exerted its toxic effects by means of oxidative stress in fish liver at the higher dose.     

In vitro and in vivo effects of some pesticides on glucose-6-phosphate dehydrogenase enzyme activity from rainbow trout (Oncorhynchus mykiss) erythrocytes

We investigated the in vitro and in vivo effects of some pesticides on rainbow trout erythrocyte glucose-6-phosphate dehydrogenase enzyme. The enzyme was purified 1691-fold with a specific activity of 16.235 U/mg protein and a yield of 63%. Cypermethrin, and propoxur inhibited glucose-6-phosphate dehydrogenase enzyme in vitro and deltamethrin inhibited both in vivo and in vitro. The obtained IC₅₀ values for deltamethrin, cypermethrin, and propoxur were 0.63, 1.02, and 12 mM, respectively. The activity of the control was determined as 5.17 ± 0.62 U/g Hb in in vivo studies. The enzyme activities of the groups treated with 0.25 g/L deltamethrin were measured at 6, 12, 24, 48, and 72 h, and found to be 4.32 ± 0.47, 3.57 ± 0.39, 3.47 ± 0.45, 2.86 ± 0.37, and 2.31 ± 0.32 U/g Hb. In vivo experiments showed that deltamethrin significantly inhibited the G6PD enzyme activity after the 48th h (p < 0.05).     

虫螨腈胁迫对甜菜夜蛾保护酶系和解毒酶系的诱导效应

为探明虫螨腈对甜菜夜蛾幼虫体内生化代谢的影响及酶抑制剂对虫螨腈的增效潜力,采用点滴法测定了虫螨腈对甜菜夜蛾3、4、5龄幼虫的毒力和顺丁烯二酸二乙酯(DEM)、增效醚(PBO)、磷酸三苯酯(TPP)对虫螨腈的增效作用,并采用生化分析法测定了LD₂₅和LD₅₀剂量虫螨腈对甜菜夜蛾体内保护酶系和解毒酶系的影响。结果表明,虫螨腈对甜菜夜蛾不同龄期幼虫的毒力差异不大,处理后24h和48h的LD₅₀基本相同。PBO对虫螨腈有拮抗作用,增效比为0.21,而DEM和TPP对虫螨腈无增效作用。虫螨腈对甜菜夜蛾幼虫体内超氧化物歧化酶具有激活作用,对过氧化氢酶、过氧化物歧化酶及羧酸酯酶具有先抑制后诱导的作用;虫螨腈处理后6~24h,谷胱甘肽-S-转移酶活性显著提高,最高为对照的1.85倍;处理6h后,甜菜夜蛾体内细胞色素P450含量明显下降,而12~24h后,又明显上升,最高时为对照的1.86倍。     

Effect of celangulin V on detoxification enzymes in Mythimna separata and Agrotis ypsilon

Celangulin V (CA-V), a β-dihydroagrofuran sesquiterpene polyol ester, is extracted from the root bark of Chinese bittersweet, Celastrus augulatus Maxim. It exhibited selective toxicity against different insects. By CO-difference spectral and biochemical method, the effects of CA-V on two kinds of detoxification enzymes, cytochrome P450 (P450 and NADPH-cytochrome P450 reductase) and glutathione S-transferase, were investigated in oriental armyworm, Mythimna separata and black cutworm, Agrotis ypsilon. CA-V showed higher induction against P450 of M. separata than that of A. ypsilon. Treated by CA-V, the maximum absorption of M. separata increased 1.2 and 0.8 nm than the control, respectively. Meanwhile, compared with the control, the P450 content and NADPH-P450 reductase activity in treated M. separata larvae increased 1.46-, 2.26- and 1.26-, 2.56-fold, respectively. But in treated A. ypsilon larvae, they all increased a little more than those of control. So far as M. separata and A. ypsilon, whether there is exposure of CA-V or not, the P450 content and GST activity in A. ypsilon were obviously higher than those in M. separata. It suggested that the content or activity difference of these two kinds of detoxification enzymes may have important roles in the selective toxicity of CA-V in M. separata and A. ypsilon.     

The effect of pretreatment with S,S,S-tributyl phosphorotrithioate on deltamethrin resistance and carboxylesterase activity in Aphis gossypii (Glover) (Homoptera: Aphididae)

The synergism of S,S,S-tributyl phosphorotrithioate (DEF) and its effect on carboxylesterase activity were investigated in deltamethrin-selected resistant (DRR) and susceptible (DSS) strains of cotton aphids, Aphis gossypii (Glover). Compared to the DSS strain, the DRR strain showed 23,900-fold resistance to deltamethrin, and 7560- and 99-fold cross-resistance to bifenthrin and ethofenprox, respectively. The synergist, DEF, increased the toxicity of both deltamethrin and bifenthrin, but not of ethofenprox when DEF was pretreated of 15 h. DEF exhibited significant inhibition on the carboxylesterase activity in the DRR strain, but no significant effect on that of the DSS strain in vitro. After the cotton aphids exposing to DEF, the carboxylesterase activity decreased gradually until 15 h and then gradually recovered until 24 h in the DRR strain, which fluctuated according to the effect of DEF on the deltamethrin toxicity detected using DEF pretreatment in the DRR strain. Therefore, our studies suggested that the effect of DEF on carboxylesterase was associated with deltamethrin resistance in the DRR strain.     

Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos

Recovery study was performed at regular intervals to establish the time course of 50% and 100% recovery in neurotransmitter enzyme (acetylcholinesterase, AChE, EC 3.1.1.7) and locomotor behaviour response of mosquito fish, Gambusia affinis exposed to lethal concentration (20.49 mg L⁻¹) of an organophosphorous pesticide, monocrotophos (MCP) for 96 h. In vitro AChE activity studies indicated that MCP could cause 50% inhibition (I₅₀) at 10.2 × 10⁻⁵ M. A positive correlation was observed between brain AChE activity and swimming speed during the recovery study. Also, the recovery response of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) as well as lipid peroxidation (LPO) as biomarkers of oxidative stress were assessed in viscera of G. affinis. The results showed that the MCP besides its inhibitory effect on target enzyme AChE activity and induction in antioxidant enzyme activities as a characteristic of oxidative stress, which can be used as biomarkers in the pesticide contaminated aquatic streams.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.     

Comparison of biochemical and toxicological characterizations of glutathione S-transferases and superoxide dismutase between Liposcelis bostrychophila Badonnel and L. entomophila (Enderlein) (Psocoptera: Liposcelididae)

The toxicological and biochemical characteristics of glutathione S-transferases (GSTs) and superoxide dismutase (SOD) in Liposcelis bostrychophila and L. entomophila were comparatively investigated. Compared to their counterparts in L. entomophila, the activities of both GSTs and SOD from L. bostrychophila were significantly higher (P < 0.05), indicating that there was a stronger ability to eliminate the toxicants in the latter. The inhibition kinetics of insecticides revealed that dichlorvos and paraoxon possessed excellent inhibitory effects on GSTs in vitro, while there were some activated effects of chlorpyrifos on GSTs, more significant in L. entomophila. As for SOD in vivo, dichlorvos and chlorpyrifos had some inhibitory effects under the condition of sublethal concentration. However, the situation of carbosulfan was a little complex. Within the first 3 h treatment period there were facilitated effects on SOD in L. bostrychophila, and some expressed inhibitory effects. While in L. entomophila carbosulfan always possessed inhibitory effects on SOD.     

Molecular and biochemical mechanisms of organophosphate resistance in laboratory-selected lines of the oriental fruit fly (Bactrocera dorsalis)

Genetic and biochemical factors leading to resistance to various organophosphate (OP) based insecticides were studied in lines selected for OP-resistance in the oriental fruit fly Bactrocera dorsalis. Lines were separately selected for resistance to naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion. Overall, these lines showed increased resistance ratios ranging from 13.7- to 814-fold relative to a susceptible (S) line. Also, in these newly selected lines the same three point mutations in the ace gene, previously identified in resistance studies and designated as I214V, G488S and Q643R, were found. As expected, the enzyme from the resistant lines showed lower overall activity and reduced sensitivity to inhibition by fenitrothion, methyl-paraoxon and paraoxon compared to the wild type acetylcholinesterase (AChE) enzyme. The apparent V_max values for esterase from the resistant lines were 1.2-3.69 times higher than that of the S line. Although only the naled-, trichlorfon- and fenthion-r lines showed lower esterase affinities (based on apparent K_m values) compared with the S line, all of the V_max/K_m ratios were higher in the resistant lines compared to that of the S line. The OP-resistant lines also displayed an overall similar pattern of isozyme expression, except for one additional band found only in the naled-r line and one band that was absent in the trichlorfon-, malathion-, and fenthion-r lines. Our results also show that overall, multiple examples of high OP resistance in selected lines of B. dorsalis exhibiting the same genetic alterations in the ace gene seen previously resulted in different effects on esterase enzyme activity in relation to various OP compounds.     

The effects of metribuzin on early life stages of common carp (Cyprinus carpio)

Metribuzin belongs to the triazine group of herbicides, which are frequently used in agriculture. The aim of this study was to assess the impact of metribuzin on growth and development of early life stages of fish. Subchronic toxic effects of metribuzin at concentrations of 0.9, 4, 14, and 32 mg/L on embryos and larvae of common carp (Cyprinus carpio) were investigated during a 30 day toxicity test under experimental conditions. Exposure to metribuzin at 32 mg/L was associated with increased mortality. Negative effects on total body length, weight, and inhibition of specific growth rate were induced by all experimental concentrations. Length and weight parameters were the most sensitive. The negative impact of metribuzin was observed in the highest tested concentration beginning on day 6 of exposure. Retardation of early ontogeny was associated with concentrations ?4 mg/L. Histological examination revealed changes in liver and caudal kidney after 30 days exposure to 32 mg/L. Based on growth parameters, development, and histological examination, the Lowest Observed Effect Concentration (LOEC) value was 0.9 mg/L.     

亚致死浓度氯虫苯甲酰胺对小菜蛾药剂敏感度和解毒酶活性的影响

采用叶片药膜法,使用亚致死浓度(LC10、LC25)的氯虫苯甲酰胺对小菜蛾Plutella xylostella(L.)3龄幼虫连续处理5代后,试虫对氯虫苯甲酰胺的敏感度分别比敏感品系下降了57.3% 和67.7%,同时对多杀菌素的敏感度也分别下降了60.2% 和51.5%,但对毒死蜱和高效氯氰菊酯的敏感度变化不明显。采用该浓度的氯虫苯甲酰胺分别处理小菜蛾3龄幼虫24、48和72 h,可诱导其羧酸酯酶(CarE)比活力上升,但对细胞色素P450 O-脱乙基酶(ECOD)、谷胱甘肽S-转移酶(GSTs)和芳基酰胺酶(AA)有明显的抑制作用;连续处理5代后,小菜蛾CarE和ECOD的比活力显著高于对照组,分别为对照组的1.16、1.40倍和1.65、1.56倍,但GSTs和AA的比活力则分别比对照下降了11.0%、27.5%和43.6%、52.5%。结果表明,小菜蛾对氯虫苯甲酰胺产生抗性的风险较高;羧酸酯酶和多功能氧化酶可能与小菜蛾对氯虫苯甲酰胺的敏感度下降有关。     

虱螨脲亚致死浓度对小菜蛾保护酶系和解毒酶系活力的影响

为阐明虱螨脲对小菜蛾亚致死效应的生化机制,采用室内生物测定法测定了LC_(10)和LC_(25)亚致死浓度处理小菜蛾3龄幼虫1、6、12、24、48和72 h后其体内保护酶系和解毒酶系的活力变化。结果表明:虱螨脲LC_(10)处理对小菜蛾幼虫体内超氧化物歧化酶(SOD)活力表现为早期诱导、后期抑制的作用,而对过氧化物酶(POD)则表现为先抑制后诱导的作用,对过氧化氢酶(CAT)仅在72h时具有诱导作用;LC_(25)处理对SOD表现为诱导-抑制-诱导-抑制的作用,对POD具有抑制作用,而对CAT具有诱导作用。经虱螨脲亚致死浓度处理后,小菜蛾幼虫体内酯酶和多功能氧化酶活力被明显抑制,且基本表现为浓度越高抑制作用越强;而谷胱甘肽S-转移酶(GST)活力仅在处理后1 h被诱导,之后被显著抑制。表明虱螨脲进入小菜蛾幼虫体内后,初期可诱导SOD、CAT和GST活力升高,以适应外界毒害的影响;但随着时间的延长,3种解毒酶活力被抑制,使其难以代谢,从而增强了对小菜蛾的毒性。     

Influence of three diets on susceptibility of selected insecticides and activities of detoxification esterases of Helicoverpa assulta (Lepidoptera: Noctuidae)

The oriental tobacco worm, Helicoverpa assulta Guenée, is one of the most destructive pests of tobacco and peppers in China. We determined the susceptibility of H. assulta reared on an artificial diet, chili pepper and tobacco to four insecticides (fenvalerate, phoxim, methomyl, indoxacarb) under laboratory conditions associated with the activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) in its larvae. H. assulta larvae that were fed with chili pepper were more susceptible to fenvalerate, indoxacarb, and phoxim than those that were fed with tobacco and the artificial diet, but not to methomyl. The larvae that were fed with chili pepper were 3.65-, 2.49-, 1.92- and 2.44-fold more susceptible to fenvalerate, phoxim, methomyl, and indoxacarb than those fed with tobacco, respectively. The AChE activities of H. assulta larvae that were fed with chili pepper and tobacco were 2.12 and 1.07 μmol mg⁻¹ 15 min⁻¹, respectively, almost 2-fold difference. The CarE activity of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 4.12, 7.40 and 7.12 μmol mg⁻¹ 30 min⁻¹, respectively. Similarly, the GST activities of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 52.02, 79.37 and 80.02 μmol mg⁻¹ min⁻¹, respectively. H. assulta larvae that were fed with chili pepper were more resistance to the tested insecticides. The low activities of AChE and the high activities of CarE and GST lead to H. assulta become more susceptible to the tested insecticides.     

Effects of the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and liver biomarkers in the common carp (Cyprinus carpio)

The aim of the study was to evaluate the effect of subchronic exposure to the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and important liver biomarkers in the common carp (Cyprinus carpio). One year old fish were exposed for 28 days to LASSO MTX added to the tank water at concentrations of 240 and 2400 μg L⁻¹. The exposure did not affect fish biometric parameters. Glutathione-S-tranferase (GST) activity in liver (hepatopancreas) remained unchanged in exposed fish when compared to controls. However, significant induction of total cytochrome P 450 (CYP 450), ethoxyresorufin-O-deethylase (EROD) activity and elevated glutathione (GSH) in liver of exposed fish were detected.