Toxic Effect of Insecticides Mixtures on Antioxidant Enzymes in Different Organs of Fish, Labeo rohita

In present era, rapid development of industries and green revolution had led to serious environmental problems, water contamination being the most important (Samantha et al., 2005). The potentially harmful agrochemical chemicals such as pesticides are released in to the freshwater environment and had significantly unfavorable effects on non-target species like aquatic animals (John, 2007; Naz and Javed, 2012).

Endosulfan belong to organochlorine pesticides, is known be highly toxic to fish (Wan et al., 2005). Chlorpyrifos is widely used organophosphate pesticide, is a non-systemic insecticide designed to be effective by direct contact, ingestion and inhalation (Tomlin, 2006). This pesticide inhibits the AChE activity (Barata et al., 2004).

Fish are the keystone species which reflect the toxic effect of chemicals in water bodies (Slaninova et al., 2009). Pesticides may induce the production of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals (Kumar et al., 2011). The production of ROS in aquatic organisms exposed to pesticides is linked with the existence of toxicants and causes oxidative stress a possible mechanism of toxicity (Oropesa et al., 2008). The defensive mechanism of fish against free radicals consists of superoxide superoxide dismutase, catalase, peroxidase and glutathione S-transferase (Guven et al., 2008). Superoxide dismutase is primary enzyme, responsible for transformation of the O-2 into H2O2. Hydrogen peroxide further transformed into oxygen and water by catalase. Peroxidase also converts the group of peroxides, including hydrogen peroxide (Hermes, 2004; Maran et al., 2009). Glutathione-S-transferase is involved in the biotransformation process of toxic substances into less toxic so fish can excrete easily from the body. In present research work, toxic effect of endosulfan and chlorpyrifos on antioxidant enzymes of fish Labeo rohita was observed.

For semi-static acute toxicity bioassay, fingerlings of fish, L. rohita (90-day old; Average weight, 8.24±0.32) were obtained from local Fish seed Hatchery and transferred to the Fisheries Research Farm, University of Agriculture, Faisalabad, Pakistan. Fish were acclimatized to laboratory condition for 14 days. During acclimatization period, L. rohita were fed with commercial feed (3% of wet body weight). After acclimatization, fish were shifted to glass aquaria containing 70-liter water.

Technical grade pesticides viz. chlorpyrifos (CPF) and endosulfan (END) were used as test chemicals. The methanol was used to prepare stock-1 solution while solutions of CPF+END mixture of required concentrations (10ppm and 1:1 ratio) were prepared by its further dilutions in deionized water.

The acute toxicity tests (96-h LC50 and lethal concentration) of pesticides mixture for L. rohita were performed with three replicates. Sixteen concentrations (0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40, 1.60, 1.80, 2.00, 2.20, 2.40, 2.60, 2.80 and 3.00 μg/L) of CPF+END mixture were used. All aquaria were supplied with exceeding aeration for 2 h in order to get a uniform concentration of the insecticides, and then 10 fish were shifted into each aquarium. Observation on fish mortality was assessed at 24, 48, 72 and 96 h after the start of experiment and dead fish were removed instantaneously. Probit Analysis method was applied to calculate the LC50 and lethal concentration of CPF+END mixture for L. rohita.

After determination of 96 h LC50 concentration, a group (n=20) of fish were separately, exposed to this concentration for 4-days. For enzymatic study fish (n=5) sampling was done after 24, 48, 72 and 96-h. To calculate the antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione S-transferase (GST) were obtained from gills, liver, kidney, brain, heart and muscle of fish. All the organs were, separately, homogenized in cold phosphate buffer (pH 6.5, 0.2 M) by the ratio of 1:4 (w/v) using a blender. After that homogenates were centrifuged at 10,000 rpm and 4ºC for 15 min. Clear supernatants were obtained which were used for enzyme assay. The activity of SOD was checked according to the method of Giannopolitis and Ries (1977). CAT activity was measured by its ability to decrease the H2O2 concentration at 240 nm (Chance and Mehaly, 1977). The activity of POD was measured by following the procedure of Civello et al. (1995). The activity of GST was determined by following the method of Mannervik (1985).

Probit analysis method was applied to calculate the tolerance limits (LC50 and lethal concentration) of L. rohita for CPF+END mixture (Finney, 1971). ANOVA was performed on obtained data to find out the statistical differences among studied variables.

Table I.- Superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione S-transferase (GST) (UmL-1±SD) in gills of Labeo rohita during acute exposure of chlorpyrifos (CPF)+endosulfan (END) mixture.

Means sharing similar letter in a row or in column are statistically non-significant (P>0.05).

The LC50 concentration for 96 h was calculated as 1.95±0.02 μgL-1 with the 95% upper and lower confidence limits as 1.685 and 2.144 μgL-1, respectively. The 96 h lethal concentration was estimated as 3.23±0.05 μgL-1 with the 95% upper and lower confidence limits as 2.906 and 3.858 μgL-1, respectively. It was observed that the fish mortality increased with increasing the exposure concentration. Water contamination by toxic chemicals caused mass mortality of aquatic fauna like fish (Kumari 2005; Gupta et al., 2012). Mortality of fish due to insecticides contact primarily depends upon its tolerance to the chemical, its dose and time of exposure (Al-Rudainy and Kadhim, 2012). Several authors have been conducted experiments on pesticides toxicity (organochlorines, pyrethroids organophospates and carbamides) for many fish species (Ambreen and Javed 2015; Ghazala et al., 2014; Shrivastava et al., 2002; Shailkh and Yeragi, 2004; Visvanthan et al., 2009).

Means sharing similar letter in a row or in column are statistically non-significant (P>0.05).

For abbreviations, see Table I.

Means sharing similar letter in a row or in column are statistically non-significant (P>0.05).

For abbreviations, see Table I.

Tables I-VI show effect of insecticide mixture on SOD, CAT, POD and GST in gills, liver, kidney, brain, heart and muscles, respectively of Labeo rohita. Results of this study showed that SOD activity significantly (P<0.05) increased in liver, gills, kidney, brain, heart and muscle of CPF+END exposed L. rohita compared to control (Tables I-VI). The SOD activity increased in selected organs of fish as following order: liver>brain>kidney>gills>heart>muscle. The increased SOD activity under oxidative stress induced by CPF may be due to conversion of superoxide radicals into hydrogen peroxide which is further transformed by CAT into oxygen and water (Sanchez et al., 2005). SOD is also a first crucial antioxidant enzyme which plays an important role to cope with oxiredicals and comprises a basic defense against the lethal effects of reactive oxygen species (ROS) (Kohen and Nyska, 2002). Oruc (2010) also reported the significant increase in the SOD activity in fish exposed to chlorpyrifos. According to Kumar et al. (2011) SOD activity increased in bronchi and hepatic tissue of Oreochromis mossambicus after acute exposure to endosulfan.

Means sharing similar letter in a row or in column are statistically non-significant (P>0.05).

For abbreviations, see Table I.

Tables I, II and III show that CAT activity significantly (P<0.05) increased in liver, gills and kidney of exposed L. rohita as compared to control while it was decreased in brain (Table IV), heart (Table V) and muscle (Table VI). The decreased in CAT activity changes the redox condition of the cells. Thus, increased level of CAT may be due to the removal of ROS from the cell generated by insecticides exposure (Stara et al., 2012). CAT activity in liver of common carp significantly increased in the presence of endosulfan (Salvo et al., 2012). According to Oruc and Usta (2007), CAT activity in muscle of C. carpio was inhibited after exposed to diazinon. Exposure of chloprpyrifos resulted in a significant reduction in CAT activity in the skeletal muscle and brain of Heteropneustes fossilis (Tripathi and Shasmal, 2010). Chlorpyrifos exposure caused reduction in gills, kidney and liver CAT activity of Ctenopharyngodon idellus (Kaur and Jindal, 2017).

Means sharing similar letter in a row or in column are statistically non-significant (P>0.05).

For abbreviations, see Table I.

Exposure of CPF+END mixture significantly (P<0.05) increased the POD activity in all selected organs of L. rohita as compared to control (Tables I-VI). The mean POD activity was more pronounced in liver (Table II) of L. rohita followed by that of brain, gills, kidney, heart and muscle. The higher POD activity in liver may be due to its role in detoxification of toxicants. The production of ROS during the biotransformation of toxicants can harm the cell through oxidation (Van der Oost et al., 2003). The increased POD activity might be expected to scavenge the ROS by converting hydrogen peroxide (Hinton et al., 2008). Alteration in peroxidase activity in liver of tilapia exposed to, 4-D+azinphosmethyl mixture was reported by Oruc and Uner (2000). A duration-specific increase in peroxidase activity in gills, brain, liver and muscle of L. rohita exposed to acute concentration of endosulfan was observed by Ullah et al. (2016).

Results showed a significant increase in liver, gills, kidney, heart, brain and muscle GST activity of insecticides exposed L. rohita compared to control (Tables I-VI). The GST activity increase in the following order: brain>muscle>liver>gills>kidney>heart. These results are also confirmed by Kumar et al. (2011) who reported the significantly increased GST activity in gills and liver of tilapia after acute exposure to endosulfan. The activity of GST increased under certain agrochemicals exposure (Dorval et al., 2003; Monteiro et al., 2006). Exposure of chlorpyrifos increased the GST activity in common carp and zebra fish (Nunes et al., 2018). The increase in GST activity was due to the increase metabolism of lipoperoxides produced through Fenton reaction or due to the biotransformation of toxicants, representing the adaptive mechanism of fish (Modesto and Martinez, 2010). According to the Gonçalves et al. (2018) GST activity can be changed in contaminated area containing the organic chemical.

Means sharing similar letter in a row or in column are statistically non-significant (P>0.05).

For abbreviations, see Table I.

This study showed that the exposure of insecticides in mixture form can lead to important alterations in antioxidant enzymes of fish. These enzymes can be used as valuable biomarker for identification of insecticides pollution in aquatic bodies. Results also indicated that the existence of pesticides in water bodies may be injurious to the health of aquatic animals especially for the fish.

Authors have declared no conflict of interest.