INTRODUCTION

Growth of unwanted plants in agricultural fields is controlled by herbicides. Use of these synthetic chemicals has been increased enormously in last few decades. Globally herbicide consumption accounts for approximately 47.5% of total pesticides consumption (De et al., 2014). Mostly herbicides are used in paddy fields from where they enter nearby water systems along with the surface runoff. Excessive and improper use of herbicides resulted in their detection in surface and ground water (Gilliom, 2007). After entering water bodies, herbicides of different chemical nature may act as toxic substance and impose harmful effects on inhabitants of those systems including fish. Herbicides adversely affect the overall physiology of fishes which are the critical component of food chains in any aquatic ecosystems.

Oxygen free radicals are continuously produced in living cells as a result of different metabolic processes and play major role in control of overall physiology of the cells (Halliwell and Gutteridge, 1999). Oxygen free radicals are reactive oxygen species (ROS) and are produced in living cells in controlled manner. Thus during normal conditions a ‘redox homeostasis’ is maintained in every physiologically active cells. The maintenance of this redox homeostasis is necessary for physiological health of the organisms (Ames et al., 1993). When organisms are exposed to certain toxicants, this homeostasis is disturbed and more ROS is produced which leads to cellular dysfunction and damage of cell or cellular components (Ahmed and Ahmad, 2006). But cells have different defense pathways which regulate the number of ROS and protect cells from oxidative damage. One of the major defense pathways constitutes production of antioxidant enzymes. The produced antioxidant enzymes act as free radical scavengers and thus control the amount of ROS in cells (Vander et al., 2003; Akhgari et al., 2003; Song et al., 2006).

Lipid peroxidation is one the most extensively used method to determine the level of oxidative damage caused by a toxicant in ecotoxicological studies with fish. Enzymes like catalase (CAT), xanthine oxidase (XOD), superoxide dismutase (SOD), glutathione redox cycle enzymes, glucose 6-phosphate dehydrogenase, glutathione peroxidase and glutathione reductase (GR) are important component of antioxidant enzymatic system.

Herbicides of different chemical nature have been reported to induce oxidative stress in fishes from time to time by several workers. The increased level of lipid peroxidation was observed by Dar et al. (2015) in endosulfan exposed gold fish (Carassius auratus) and by Husak et al. (2016) in sencor exposed gold fish (Carassius auratus). Increased activity of antioxidant enzymes: SOD, CAT and GST were observed in the liver of fish Cyprinus carpio exposed to quinalphos (Devan et al., 2016). Jonsson et al. (2017) observed a significant increase in GST activity in the liver of fish tilapia exposed to herbicide mixture of hexazinone, ametryn, diuron and tebuthiuron after 14 days of exposure. Ruiz de Arcaute et al. (2019) observed increased activities of CAT and GST in dicamba (DIC) and 2,4-dichlorophenoxyacetic acid (2,4-D) exposed fish Cnesterodon decemmaculatus. GSH level and the activities of enzyme CAT and GPx were found decreased in the fish Gammarus pulex exposed to herbicide glyphosate (Pala, 2019). A significant decrease of GST activity, increased CAT and GPx activity was observed in zebra fish exposed to 2,4-D (Gaaied et al., 2019). Decrease in the superoxide dismutase (SOD) and catalase (CAT) activity and increased malondialdehyde (MDA) content was noted in pendimethalin exposed Oreochromis niloticus (Neamat- Allah et al., 2020).

Pretilachlor is a systemic herbicide of chloroacetamide group and is being used worldwide in paddy fields for the control of annual weeds. Half life of pretilachlor in paddy water is about 3.0 to 3.6 days (Fajardo et al., 2000) and in soil is about 20 to 50 days (Worthing and Hance, 1991). Chloroacetamide herbicides including pretilachlor have been detected considerably in surface water (Hladik et al., 2008), soil (Chao et al., 2007) and sediments (Xue et al., 2005). They are found toxic to fishes. Butachlor is found as endocrine disruptor (Chang et al., 2013), and immuno-toxic to zebra fish (Tu et al., 2013). It induces genotoxicity in C. batrachus (Ateeq et al., 2006) and behavioural toxicity in R. rutilus caspicus and S. lucioperca (Mohammad and Hedayati, 2017). Pretilachlor is less studied and is reported to induce 100% mortality in Gambusia at a concentration of 25 mg/L (Sadeghi and Imanpoor, 2013). It is also responsible for reproductive toxicity in Chinese rare minnow (Zhu et al., 2014) and C. batrachus (Soni and Verma, 2020). However little is known regarding ability of this herbicide to induce oxidative stress in fishes which is urgently needed for environmental risk assessment.

In the present study it has been proposed to evaluate the antioxidant status of fish C. batrachus exposed to herbicide pretilachlor through lipid peroxidation level and assay of three different antioxidant enzymes i.e. SOD, CAT and GR. SOD transform superoxide radical to hydrogen peroxide. CAT decomposes hydrogen peroxide to water and oxygen. Oxidized glutathione (GSSG) is converted to two molecules of reduced gluthatione in the presence of enzyme GR.

Fresh water fish C. batrachus has been selected in the present study to evaluate the effect of pretilachlor on antioxidative status. This animal model has been selected because it is widely distributed, available in all seasons and easily acclimatize to laboratory conditions.

Toxicity of herbicides may be evaluated through both nominal and measured concentrations of the toxicant. Most of the test guidelines (OECD, 1992; USEPA, 1996; ASTM, 2007) recommend using environmentally relevant concentrations of the toxicant for its toxicity evaluation. But large toxicity data is required to establish an eco-toxicological model (Raimondo et al., 2009). Thus to establish C. batrachus as eco-toxicological model for pretilachlor induced oxidative stress also to fill the existing gap of knowledge in available literature the present study has been carried out with nominal concentration of herbicide.

MATERIALS AND METHODS

Experimental Design

Healthy individuals of C. batrachus were collected from fish farms. After collection, specimens were washed with 0.05% potassium permanganate and were acclimatized to laboratory conditions for 20 days. Glassy aquariums of size 60 × 30 × 30 cm were used for the same. Physicochemical characteristics of aquarium water were determined using standard protocols (APHA, 2012). Commercial formulation of pretilachlor named ‘Rifit’ with 50% EC was used to evaluate its toxicity. Based on the obtained 96 h LC50 value of pretilachlor to C. batrachus (5.84 mg/L) in our previous study (Soni and Verma, 2018), three sub lethal test concentrations were chosen to evaluate antioxidant status of C. batrachus exposed to herbicide. These concentrations were SL-I (1/20th 96 h LC50), SL-II (1/15th 96 h LC50) and SL-III (1/10th 96 h LC50). Fishes were exposed to these concentrations for 30, 45 and 60 days. For herbicide exposure, a total of 54 fish were divided in to 9 groups with each group containing 6 individuals. First three groups were exposed to SL-I, next three groups were exposed to SL-II and last three groups were exposed to SL-III. The durations of exposure for three groups of each test concentration (SL-I, SL-II and SL-III) were 30, 45 and 60 days. During their exposure to herbicides, fishes were kept in glassy aquariums of size 60 × 30 × 30. For each duration of exposure, 06 individuals were maintained in an aquarium without exposure of herbicide and treated as control (C).

Oxidative stress parameters were evaluated in liver tissues of fish after their exposure to pretilachlor. Fish were sacrificed at predefined duration of herbicide exposure from both treated and control groups and the whole liver was dissected out carefully. Some part of liver tissue was used to prepare tissue homogenate. For preparation of tissue homogenate the tissue was thoroughly washed in ice-cold potassium chloride (1.15%) solution. After which it was blotted and weighed. Finally the liver tissue was homogenized in homogenizing buffer consisted of 1.15 KCL and 50 mM Tris-HCl maintained at pH 7.4.

Evaluation of Antioxidative Status

Estimation of Lipid Peroxidation

Estimation of lipid peroxidation was done by the method developed by Sharma and Krishnamurthy (1968). The prepared tissue homogenate was incubated at 37°C for 30 minutes. After which 1 mL of 10% trichloroacetic acid (TCA) was added to it. Now the homogenate was centrifuged at 2000 g for 15 min. After centrifugation 1 mL of supernatant was taken to which 1 ml of thiobarbituric acid reacting substances (TBA) solution was added. Now the tubes were kept in boiling water bath for around 10 min. After incubation of 10 minutes the test tubes were cooled and the optical density was measured at 535 nm. TBARS produced was calculated using a molar extinction coefficient of 1.56 × 105 M–1 cm–1 and the lipid peroxidation was expressed as nmoles of TBARS formed per mg of protein.

Estimation of Antioxidant Enzymes Activities

Superoxide Dismutase (SOD) Activity

The activity of enzyme superoxide dismutase (SOD) in liver tissue was evaluated through the protocol described by Misra and Fridovich (1972). The prepared liver tissue homogenate was centrifuged at 2000 g for 15 min. After centrifugation the supernatant was collected. 2.96 µL sodium carbonate- bicarbonate buffer, 0.02 µL EDTA, 0.01 µL epinephrine are mixed. To this mixture 0.01 µL enzyme sample was added and the reaction mixture was incubated for 60 seconds at room temperature. After incubation period the absorbance was read at 450 nm. The activity of SOD was expressed as superoxide dismutase units per gram of protein.

Catalase (CAT) Activity

Catalase (CAT) activity was evaluated through the protocol of Clairborne (1995). The liver tissue homogenate was centrifuged at 2000 g for 15 min. 10 µL of hydrogen peroxide was taken in a cuvette to which 2.4 mL of phosphate buffer was added. To this mixture 50 μL of supernatant collected after centrifugation was added. Reaction started and the absorbance was noted at 240 nm. The CAT activity was expressed as μmol of hydrogen peroxide consumed/min/mg protein.

Glutathione Reductase (GR) Activity

The Glutathione reductase (GR) activity was evaluated through the protocol as described by Massey and Williams (1965). The prepared liver tissue homogenate in potassium chloride was centrifuged at 2000 g for 15 min. 2.7 mL of phosphate buffer (50 mM) was taken in a cuvette to which 0.1 mL of NADPH and 0.1 mL of GSSG were added at an interval at one minute. After 4–5 minutes 0.1 mL of diluted sample was added to this mixture. Decrease in absorbance was noted at 340 nm against blank. The enzyme activity was expressed as nmol NADPH oxidized/min/mg protein.

Statistical Analysis

Two ways ANOVA was used to compare the mean differences in obtained results (SPSS, Chicago, IL, USA, Version 16). Intergroup comparisons were done by post hoc testing using the least significant difference test. ‘p’ value less than 0.05 were considered statistically significant.

RESULTS

Lipid Peroxidation

TBARS formation was measured in the liver tissue of C. batrachus after their exposure to predefined test concentrations of pretilachlor. The obtained values of TBARS are represented in the Fig. 1. TBARS formation in different group of herbicide exposed fish was found completely dependent on the concentration of the herbicide and duration of its exposure. Maximum level of LPO was observed in fish exposed to maximum concentration of herbicide at all duration of exposure. On the other for a particular sub lethal test concentration the maximum TBARS formation was seen in fish at 45 days of exposure which was followed by decrease at 60 days of exposure.

Fig. 1.
figure 1

Graph elucidating changes in TBARS formation/mg protein in C. batrachus exposed to herbicide pretilachlor. Value are expressed as mean + SE (n = 6). The alphabets (A, B, C, D) represent statistically significant difference (p < 0.05) between concentrations for a particular duration of exposure. The numerals (1, 2, 3, 4) represent statistically significant difference (p < 0.05) between exposure durations for a particular concentration.

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Antioxidant Enzymes

Alterations in the activity of CAT and SOD were seen in herbicide exposed fish but no alterations in GR activity were observed. In herbicide exposed fish, CAT and SOD exhibited the same pattern of alteration in their activities in relation with concentration of the herbicide and duration of its exposure. CAT and SOD activity was found significantly increased in herbicide exposed fish. This increase was dependent on the concentration of herbicide and duration of its exposure. Maximum increase was observed in fish exposed to maximum concentration of herbicide while for a particular concentration maximum increase was observed at 45 days of exposure. After 45 days, decrease in the activity of both enzymes was observed at 60 days of exposure. SOD activity in liver tissue of herbicide exposed fish is shown in Fig. 2 whereas CAT activity in herbicide exposed fish is represented in Fig. 3. No significant changes in GR activity was observed in pretilachlor exposed fish (Fig. 4).

Fig. 2.
figure 2

Graph elucidating changes in superoxide dismutase (SOD) activity in liver tissues of C. batrachus exposed to herbicide pretilachlor. Value are expressed as mean + SE (n = 6). The alphabets (A, B, C, D) represent statistically significant difference (p < 0.05) between concentrations for a particular duration of exposure. The numerals (1, 2, 3, 4) represent statistically significant difference (p < 0.05) between exposure durations for a particular concentration.

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Fig. 3.
figure 3

Graph elucidating changes in catalase (CAT) activity in liver tissues of C. batrachus exposed to herbicide pretilachlor. Value are expressed as mean + SE (n = 6). The alphabets (A, B, C, D) represent statistically significant difference (p < 0.05) between concentrations for a particular duration of exposure. The numerals (1, 2, 3, 4) represent statistically significant difference (p < 0.05) between exposure durations for a particular concentration.

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Fig. 4.
figure 4

Graph elucidating changes in glutathione reductase (GR) activity in liver tissues of C. batrachus exposed to herbicide pretilachlor. Value are expressed as mean + SE (n = 6).

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DISCUSSION

Whenever there is an imbalance between rate of production of reactive oxygen species (ROS) and its neutralization in organisms, it leads to oxidative stress in them. It has been reported that interference of various toxic substances including herbicides with the antioxidant defense system is one of the major cause of oxidative stress (Kaya and Yigit, 2012; Muthulakshmi et al., 2018). Oxidative stress is harmful as it leads to disturbances in normal physiological process and also responsible for reduction in cell viability (Poletta et al., 2016). Like many other herbicides, in the present study pretilachlor is found to induce oxidative stress which is evidenced by increase in LPO and changed activity of antioxidant enzymes.

Herbicide concentration and its duration of exposure dependent LPO were noted in liver tissue of pretilachlor exposed C. batrachus. Similar concentration and duration of exposure dependent results on LPO were also reported by Peeuba (2008) in fishes exposed to alachlor and Farombi et al. (2008) in fishes exposed to butachlor. Alachlor and butachlor are also chloroacetamide herbicide like that of pretilachlor. The increased level of LPO is mainly because of increased production of ROS. Detoxification of xenobiotics occurs in the liver and the metabolism of pretilachlor in liver is responsible for increased production of ROS. This increased level of ROS can damage nuclear membrane and so responsible for DNA damage (Mutlu-Turkoglu et al., 2003).

The ROS produced are controlled by cellular defense pathways and antioxidant enzyme system of a cell is one of the important components of these cellular defense pathways (Vander et al., 2003; Akhgari et al., 2003). Young and Woodside (2001) are of opinion that the level of induced oxidative stress in an organism depends on the prevailing internal or external environment and the activities of antioxidant enzymes increases or decreases as per the change in the environment whether internal or external. Impact of herbicide pretilachlor on the activities of three important antioxidant enzymes i.e. catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) in C. batrachus were evaluated in the present study. Activities of these enzymes are important as SOD transforms harmful superoxide radical to hydrogen peroxide. The produced hydrogen peroxide is converted to water and oxygen by CAT. Oxidized glutathione (GSSG) is converted in to two molecules of reduced gluthatione by GR. The antioxidant enzyme activities are found to be dependent on the sub lethal test concentration of the pretilachlor and duration of its exposure. Activities of SOD and CAT increases with increasing duration of exposure initially and then decreases. The obtained results are in accordance to the results obtained by Stara et al. (2012) in Cyprinus carpio exposed to simazine, by Samanta et al. (2014) in fish Heteropneustes fossilis exposed to glyphosate, by Persch et al. (2017) in fish Rhamdia quelen exposed to glyphosate, by Wang et al. (2018) in fish Danio rerio exposed to diquat and by Gupta and Verma (2020) in fish C. batrachus exposed to pendimethalin. On the basis of results obtained it can be said that increased activity of CAT during initial period of exposure was observed because of increased SOD activity. Increased activity of SOD was because of increased rate of formation of superoxide radical due to metabolism of herbicide. Thus both CAT and SOD act together to control the pretilachlor induced oxidative damage. We agree with the reviewer that evaluation of correlative relationship between activity of CAT and SOD enzymes will further help to analyze the results. As per suggestion correlation co-efficient was calculated and a strong correlation (r = 0.789) was obtained between the activity of these two enzymes. At highest period of exposure i.e. at 60 days, decreased activities of these two enzymes were noted which was because of increased production of H2O2 which resulted in decreased rate of reaction and so decreased activities of enzymes concerned (Nwani et al., 2010). However Ahmad and Ahmad (2016) reported no altered activities of CAT and SOD in pendimethalin exposed C. punctatus. The enzyme GR transforms oxidized glutathione (GSSG) to two molecules of reduced gluthatione (GSH) and maintains a constant ratio of GSH: GSSG. The lack of reaction of the GSH system was observed in the present study as glutathione peroxidase (GP) enzyme which is responsible for conversion of GSH to GSSG is having more affinity for hydrogen peroxides in comparison to CAT. Increased CAT activity was observed in the present study which indicates that exposure to pretilachlor produced significant amount of hydrogen peroxides and GP is mainly involved in the conversion of hydrogen peroxide to water and not available for GSH to GSSG conversion. Thus no increased activity of GR was observed which is responsible for GSSG to GSH conversion.

The obtained results on the oxidative stress parameters indicated that herbicide pretilachlor induce oxidative stress in fish C. batrachus which is because of increased production of ROS. Increased production of ROS resulted in increased level of LPO which further alters the antioxidant enzyme activity in the liver of herbicide exposed fish. Therefore it can be concluded that herbicide pretilachlor induced oxidative stress in fishes which may disturb the entire physiology of fish which further reduces the chances of fish to survive, reproduce and maintain its population. Therefore excessive use of this herbicide in agricultural field must be avoided.