Insecticides induced stress response and recuperation in fish: Biomarkers in blood and tissues related to oxidative damage.

The present research investigated the growth, blood, antioxidant response (liver), AChE (brain and muscle) and Na+/K + ATPase in gills of Clarias batrachus exposed to 0 (control), two insecticides, 1.65 mg L-1 chlorpyrifos (CPF) and 2.14 mg L-1 monocrotophos (MCP) for a fixed interval time of 3, 6, 9, 12 and 15 days and follow up depuration process in fresh water for 30 days (at an interval of 7, 15 and 30 days). The toxicants exposed fish indicated significantly (P < 0.05) lower weight gain and HSI. The RBC, Hb, Hct, plasma total protein, glucose, albumin, globulin and respiratory burst activity was reduced. However, WBC, plasma glucose, serum creatinine, and triglycerides were enhanced. The weight gain, HSI and all haematological parameters were reversed following depuration of CPF and MCP exposed fish. Hepatic superoxide dismutase, catalase, lipid peroxidation, reduced glutathione, and glutathione S-transferase activities were significantly activated whereas glutathione peroxidase was inhibited in both tested groups. All the antioxidant enzymes were reversed on day 15 in MCP concentration, whereas CPF on day 30 of depuration process. The inhibition of acetylcholinesterase (brain, muscle) and gill Na+/K + ATPase activities were more in CPF exposure and early recovery in MCP. The results indicated that depuration process might help in detoxification of fish and improve growth, haematological conditions, oxidative stress and AChE, Na+/K + ATPase activity. However, further studies are needed in different fish species with different toxicants to support this strategy of depuration process in order to detoxify polluted fish.

The role of vitamin C as antioxidant in protection of biochemical and haematological stress induced by chlorpyrifos in freshwater fish Clarias batrachus.

The study was conducted to explore the modulatory effects of chlorpyrifos and protective role of vitamin C in tissues of Clarias batrachus. Treatments include E1 group (basal diet plus 1.65mgL(-1) CPF) and E2 group (basal diet+200mgkg body weight vitamin C and 1.65mgL(-1) CPF) along with a control group of fishes (fed on basal diet only). After 1, 7, 15, and 30d of treatment, fish tissues (brain, blood and liver) were used for the estimation of growth, biochemical and haematological parameters. The results of E1 group indicated significantly lower weight gain and survival rate. Brain AChE activity was inhibited. The RBC, Hb, respiratory burst activity, total protein and HSI were also reduced whereas WBC count, plasma glucose and haematocrit were elevated. In contrast, liver glycogen content, lactate dehydrogenase, alkaline and acid phosphatase activities were inhibited and malate dehydrogenase, aspartate, alanine amino transferase were enhanced. The E2 group of fish exhibited significant improvement in growth, survival, haematological indices, brain AChE, liver glycogen and oxidative enzyme activity. The findings support that dietary vitamin C supplementation might be helpful in abrogation of chlorpyrifos toxicity and improves growth, survival, biochemical and haematological conditions in fishes.

Evaluation of the biochemical stress response to chlorpyrifos in tissues of the edible crab Barytelphusa guerini: withdrawal of exposure improves the nutritional value.

The sublethal stress of the organophosphate insecticide chlorpyrifos was investigated in different tissues of the freshwater crab (Barytelphusa guerini). Crabs were exposed to 1/3 of LC50 concentrations for 7, 14, 21, and 28 days. After 28 days, they were released into fresh water and kept for 18 days for recovery. The study was conducted by estimating total proteins, amino acids, ammonia, urea, and glutamine levels, and protease, transaminases, and phosphatases activities. Total proteins level was decreased whereas amino acids and ammonia were increased. The urea content was decreased in all tissues and glutamine exhibited a mixed response. Protease activities and those of alanine and aspartate aminotransferase, respectively, were elevated. Acid phosphatase activity was reduced in hepatopancreas and brain and induced in gills and muscle. Alkaline phosphatase activity was enhanced in gills and hepatopancreas and reduced in muscle and brain. The crabs recovered from the biochemical stress caused by chlorpyrifos after their release into fresh water.

Development of an inhibitive assay using commercial Electrophorus electricus acetylcholinesterase for heavy metal detection.

Near-real-ime assay is anassay method that the whole process from sampling until results could be obtained in approximately Iess than one hour. The ElIman assay for acetyl cholinesterase (AChE) has near real-time potential due to its simplicity and fast assay time. The commercial acetylcholinesterase from Electrophorus electricus is well known for its uses in insecticides detection. A lesser known fact is AChE is also sensitive to heavy metals. A near real-time inhibitive assay for heavy metals using AChE from this source showed promising results. Several heavy metals such as copper, silver and mercury could be etected with IC50 values of1.212, 0.1185 and 0.097 mg I-1, respectively. The Limits of Detection (LOD) for copper, silver and mercury were 0.01, 0.015 and 0.01 mg I-1, respectively. TheLimits of quantitation (LOQ) or copper, silver and mercury were 0.196, 0.112 and 0.025 mg I-1, respectively. The LOQvalues for copper, silver and mercury were well below the maximum permissible limit for these metal ions as outlined by Malaysian Department of Environment. A polluted location demonstrated near real-time applicability of the assay with variation oftemporal levels of heavy metals detected. The results show that AChE from Electrophorus electricus has the potential to be used as a near real-time biomonitoring tool for heavy

Toxic impact of two organophosphate insecticides on biochemical parameters of a food fish and assessment of recovery response.

Sublethal effects of chlorpyrifos (CPF) and monocrotophos (MCP) on fish biochemical constituents were investigated along with the assessment of recovery response after cessation of intoxication. The fish, Clarias batrachus were exposed to 1.656 mg(-l) and 2.114 mg(-l) of CPF and MCP for 28 days. After 28 days, they were released in freshwater and allowed to recover for 21 days. The CPF exposure resulted in the decrease of carbohydrate and glycogen content, whereas MCP intoxication caused mixed response. Pyruvate and lactate contents were altered under the stress of CPF and MCP. Recovery of these alterations was observed after the cessation of toxicity. Exposure of C. batrachus to CPF and MCP resulted in decreased activity of lactate dehydrogenase in the kidney, liver and muscle but its activity increased in the gills. The CPF caused inhibition of succinate dehydrogenase enzyme in all tissues. Induction in the activity of malate dehydrogenase was caused by both insecticides. Glycogen phosphorylase a was induced in all tissues, whereas glycogen phosphorylase ab showed both induction and inhibition. Of the two insecticides, CPF was more toxic and the recovery response was less. These results are important in the assessment of the risk caused by organophosphate insecticides on nontarget organisms, especially the food fish.

In vivo impact of monocrotophos on biochemical parameters of a freshwater fish during subacute toxicity and following cessation of exposure to the insecticide.

In vivo toxicity of monocrotophos on key metabolites and enzymes of the protein metabolism was investigated in important tissues of the freshwater fish Clarias batrachus. Fish were exposed to 1/10 and 1/20 of LC50 concentration for 28 days. After 28 days of exposure, some fish were transferred to monocrotophos-free water and kept in the same for 21 days (recovery period) in order to study the recovery response. Total protein, amino acid, and ammonia contents were decreased in gill, kidney, liver, and muscle tissues, and recovery was slight at the end of 21 days of transfer of fish into freshwater. Urea and glutamine levels were elevated, except in kidneys, and recovered at the end of the recovery period. The activities of protease, transaminase, and phosphatase enzymes were elevated in all tissues during 28 days of exposure and at both concentrations. Recovery of the activity of enzymes was more significant at the lower concentration as compared to the higher concentration.

Organ-specific ATPase and phosphorylase enzyme activities in a food fish exposed to a carbamate insecticide and recovery response.

The acute effect of carbofuran, a carbamate insecticide, was studied on adenosine triphosphatase enzymes in gill, kidney, liver and muscle tissues of a food fish, Clarias batrachus. Glycogen and glycogen phosphorylase were investigated in gill and kidney only. Thirty-six fish were exposed to sublethal concentration (7.6 mg/L) for 6 days. After 6 days, 18 fish were released into freshwater in order to study the recovery response. Eighteen fish were kept in clean water as control. Tissues were isolated from control, exposed and recovery fish at the end of 1, 3 and 6 days and used for the assay of enzymes. Total ATPase was inhibited in kidney and muscle tissues throughout the exposure period, whereas branchial and hepatic tissues showed initial induction followed by inhibition. Na(+)-K(+) ATPase activity was induced in gill till day 3, whereas in other tissues inhibition was throughout the exposure period. Mg(+2) ATPase activity was inhibited in all tissues except liver. When the fish were released into freshwater, liver recovered almost to control values and other tissues showed organ-specific response. Glycogen content of gill increased initially followed by decrease, and in kidney initial decrease was noted. The recovery response was more in kidney than in gill. Induction in the activity of glycogen phosphorylases was observed in kidney, whereas gill tissue showed mixed response. Recovery was not observed in phosphorylases. Thus, the results of the present study demonstrated the acute effect of carbofuran on a food fish and organ-specific recovery response to insecticidal treatment.

Enzymes as biomarkers of cypermethrin toxicity: response of Clarias batrachus tissues ATPase and glycogen phosphorylase as a function of exposure and recovery at sublethal level.

The present study was conducted to evaluate the sublethal effects of a synthetic pyrethroid, cypermethrin on ATPase, and glycogen phosphorylase in gill, kidney, liver, and muscle tissue of freshwater fish, Clarias batrachus (Linn) during exposure and cessation of exposure. Thirty-six fish were exposed to 1/3 of LC 50 concentration i, e. 0.07 mg/L cypermethrin for 10 days. After 10 days, 18 fish were transferred to freshwater and 10 days recovery response was observed. Thirty-six fish were kept as a control group. The effect of cypermethrin intoxication was studied on total, Mg(+2), Na(+)-K(+) ATPase, glycogen, and glycogen phosphorylase (a) and (ab) in various physiological tissues at the end of 1, 5, and 10 days of exposure and recovery period. Cypermethrin intoxication resulted in a significant inhibition in the activities of total, Mg(+2), and Na(+)-K(+) ATPase enzyme and glycogen content, whereas it caused a significant induction in the levels of glycogen phosphorylase (a). The activity level of glycogen phosphorylase (ab) showed mixed response. When exposed fish were released into freshwater, they were able to normalize the activities of ATPase enzyme in liver tissue whereas gill, kidney, and muscle tissues showed slight recovery. At the end of 10 days of recovery period, glycogen and glycogen phosphorylase activities in kidneys recover better than muscle. Therefore, the present study clearly suggests that ATPase and glycogen phosphorylase enzymes can be used as biomarkers of exposure to aquatic organisms under cypermethrin intoxication.

Assessment of biochemical markers of carbofuran toxicity and recovery response in tissues of the freshwater teleost, Clarias batrachus (Linn).

The effects of a sublethal concentration (7.66 mg/L) of carbofuran, were assessed on Clarias batrachus. The fish were exposed to 7.66 mg of carbofuran/L for 6 days. After 6 days, fish were released into carbofuran-free water in order to study the recovery pattern. Proteins were decreased in gill and kidney and recovery was greater in gill than in kidney. Total amino acids were increased in both tissues. Ammonia level declined in gill and enhanced in kidney throughout the study period. The activities of all enzymes measured were induced in both tissues, except for aspartate aminotransaminase, which was inhibited in gill tissues.

Cypermethrin-induced biochemical perturbations in freshwater fish Clarias batrachus at sublethal exposure and after released into freshwater.

Cypermethrin-induced alterations in the fish, Clarias batrachus were studied. The fish were exposed to cypermethrin at a concentration of 0.07 mg/L for 10 days. After 10 days, fish were released into freshwater to observe the recovery response. At the end of 1, 5, and 10 days of exposure and after transfer into freshwater, different parameters were estimated in muscle and kidneys. Reduction in proteins was observed in both tissues, and recovery response was seen in muscle and kidneys. Free amino acid and ammonia content was enhanced in muscle and kidney for the 10 days. Transaminases (ALAT and AAT) and glutamate dehydrogenase activities were increased in both tissues for 10 days exposure span. Enzyme activities did not show much of the recovery.

Sublethal effects of profenofos on locomotor behavior and gill architecture of the mosquito fish, Gambusia affinis.

Subacute studies of profenofos on mosquito fish, Gambusia affinis, were carried out for 20 days to assess the locomotor behavior and structural integrity of gill in relation to bioaccumulation and targeted enzyme acetylcholinesterase (AChE; EC 3.1.1.7). The sublethal concentration of 0.13 mg/L (1/5 of LC50) altered locomotor behavior such as distance traveled and swimming speed in exposed fish. This could be due to inhibition in the activity of acetylcholinesterase and deformities in the primary and secondary lamella of gill. The bioaccumulation values indicated that the accumulation of profenofos was highest in viscera followed by head and body. The average bioconcentration factor values are 254.83, 6.18, and 2.52 microg/g for viscera, head, and body. The findings revealed that profenofos is highly toxic even at sublethal concentrations to the mosquito fish, Gambusia affinis.

Sublethal effects of monocrotophos on locomotor behavior and gill architecture of the mosquito fish, Gambusia affinis.

Subacute studies of monocrotophos [Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate] on mosquito fish, Gambusia affinis, were carried out in vivo for 24 days to assess the locomotor behavior, structural integrity of gill, and targeted enzyme acetylcholinesterase (AChE, EC: 3.1.1.7) interactions. Monocrotophos (MCP) can be rated as moderately toxic to G. affinis, with a median lethal concentration (LC(50)) of 20.49 +/- 2.45 mgL(-1). The fish exposed to sublethal concentration of LC(10) (7.74 mgL(-1)) were under stress and altered their locomotor behavior, such as distance traveled per unit time (m min(-1)) and swimming speed (cm sec(-1)) with respect to the length of exposure. Inhibition in the activity of brain AChE and deformities in the primary and secondary lamellae of gill may have resulted in failure of exchange of gases. The maximum inhibition of 95% of AChE activity was observed on days 20 and 24. Morphological aberrations in the gills were also studied during exposure to the sublethal concentration of monocrotophos for a period ranging from 8 to 24 days. The extent of damage in gill was dependent on the duration of exposure. The findings revealed that inhibition in brain AChE activity and structural alteration in gill were responsible for altering the locomotor behavior of exposed fish.

Changes in behavior and brain acetylcholinesterase activity in mosquito fish, Gambusia affinis in response to the sub-lethal exposure to chlorpyrifos.

Sub-lethal studies of chlorpyrifos, O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate on mosquito fish, Gambusia affinis were carried out in vivo, for 20 days to assess the locomotor behavior in relation to bioaccumulation and interaction with a targeted enzyme, acetylcholinesterase (AChE, EC: 3.1.1.7). Fish exposed to sub-lethal concentration of 60 microg/L (1/5 of LC 50) were under stress, and reduced their locomotor behavior like distance travelled per unit time (m/min) and swimming speed (cm/sec) with respect to the length of exposure. The alteration in locomotor behavior of fish may be due to an accumulation of acetylcholine (ACh), a neurotransmitter at synaptic junctions, due to the inhibition of AChE enzyme activity (40 to 55%) in brain and also bioaccumulation of the toxicant in different parts of fish. The bioaccumulation values indicated that the accumulation of chlorpyrifos was maximum in viscera followed by head and body. The average bio-concentration values are 0.109, 0.009 and 0.004 microg/g for viscera, head and body with depuration rates of 2.24, 1.69 and 0.39 ng/h respectively. It is evident from the results that the sub-lethal concentration [1/5 of LC 50; equivalent to Lowest Observed Effect Concentration (LOEC)] of chlorpyrifos can able to alter the locomotor behavior of G. affinis in relation to the length of exposure. The findings revealed that the locomotor activity of test organism could be considered as a suitable marker to evaluate the affect of toxicant even at LOEC levels.

Carbofuran insecticide induced biochemical alterations in liver and muscle tissues of the fish Clarias batrachus (linn) and recovery response.

The effect of carbofuran insecticide on certain metabolites and enzymes of protein and carbohydrate metabolism was evaluated in liver and muscle tissues of the freshwater fish, Clarias batrachus during exposure and following cessation of exposure. Thirty-six fish were exposed to 7.66mg of carbofuran/l for 6 days. After 6 days, 18 fish were transferred into carbofuran-free water for 6 days (recovery period) in order to study the recovery response. Eighteen fish were kept in clean water as controls. Liver and muscle tissues were isolated from exposed, recovery and control fish at the end of 1, 3 and 6 days in each condition, and used for the estimation of protein, amino acids, ammonia, glycogen, aminotransaminases, glycogen phosphorylase (a and ab) and glutamate dehydrogenase. Total protein showed a delayed decrease in liver and muscle tissues but recovered by the end of the recovery period. Free amino acid content was affected little in liver, but was elevated in muscle, and ammonia levels were elevated in both tissues throughout the exposure period, and ammonia levels in liver remained elevated during recovery. Glycogen content of liver declined substantially, and rebounded after transfer of fish into clean water. The activity levels of alanine aminotransaminase, aspartate aminotransaminase, glutamate dehydrogenase and glycogen phosphorylase a were found to increase in both tissues during the exposure period. The glycogen phosphorylase ab activity in liver was suppressed on exposure to carbofuran. The enzymes exhibited different recovery pattern in liver and muscle tissues of C. batrachus. The patterns of effect of carbofuran on intermediary metabolism suggest avenues to determine the mechanisms by which such effects occur.