Acetylcholine esterase and antioxidant responses in freshwater teleost, Channa punctata exposed to chlorpyrifos and urea

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Highlights

  • Chlorpyrifos and urea showed concentration and duration-dependent toxicities.

  • ~38–49% (max.) inhibition in AChE activity for U/CPF doses was noted.

  • SOD, CAT and GPx activities increased concomitantly with increase in MDA contents.

  • Binary mixtures (1CPF + 1U; 2CPF + 1U, 1CPF + 2U) showed complex interactions.

  • AchE and key antioxidant enzymes showed synergism at more U in the mixture.

Abstract

We aimed to understand the toxic effects of two crop protecting agents, organophosphate pesticide, chlorpyrifos (CPF) and fertilizer, urea (U), and their binary mixtures at sublethal concentrations for 28-d in a freshwater fish Channa punctata with a battery of biochemical biomarkers in gill and liver. The study has practical value as such mixtures, so often present together in water in the agro-intensive areas, might be predicted to cause cocktail effects. Both CPF and U inhibited AChE, augmented SOD, CAT, GPx activities, and caused lipid peroxidation and depletion in tissue macromolecules in a concentration and duration-dependent manner. While U alone had less severe effects compared to CPF treatments, complex interactions were observed for three combination doses (1CPF + 1U, 2CPF + 1U, 1CPF + 2U). In their mutual effects, antagonism prevailed over other interactions when CPF and U were in equal proportion in the mixture, while synergism was observed for AchE and key antioxidant enzymes when more U was in the mixture. The present study concluded that urea in water bodies might impart adverse effects in combination with pesticides in non-target aquatic organisms such as fish, and there should be a restriction in its excessive usage.

Introduction

In the recent past, widespread usage of organophosphate (OP) pesticides in agriculture have lead to a significant build-up of residues in water, as runoff, which is the primary sources of these contaminants to the aquatic environment, where they subsequently deteriorate water quality and fish health (Bonifacio et al., 2017). Often persistent in water, Chlorpyrifos (CPF) (o-o-diethyl-o-{3,5,6 trichloro-2-pyridyl}-phos- phorothioate), an OP pesticide, has been widely used in India to control a variety of insect pest (Duraisamy et al., 2018). While the individual pesticide residue in water should not exceed 0.1 μg L−1, in several agriculturally active regions of India, CPF residues in the range of 0.11–0.25 μg L−1 in groundwaters and 0.26–0.44 μg L−1 in surface waters were detected (Lari et al., 2014). CPF residues in soil and water near agricultural fields in Haryana, India, in the range of 0.61–1.12 μg L−1 was reported (Kumari et al., 2008). In a fragile Ramsar site in an Indian coastal lagoon, CPF in the range of 0.019–2.73 μg L−1 was also reported recently (Nag et al., 2020). It is predicted that less than 0.1% of applied pesticide reaches the target pests, leaving the bulk to impact the non-target organisms such as fish (Li et al., 2008), frog (Rutkoski et al., 2020), and earthworm (Zhu et al., 2020).

Concomitant with the rise in pesticide usage, there has been a rise in the application of nitrogen fertilizers in agriculture fields (Bouwman et al., 2017). In freshwater ecosystems, high levels of human-generated nitrogen sources such as ammonia, nitrite, and nitrate may produce harmful effects on aquatic organisms (Wang et al., 2017; Yu et al., 2015). Urea is an inexpensive nitrogen fertilizer used in Indian agriculture with an estimated 33 million tonnes of urea consumption for the year 2017–18, making the country the second most fertilizer consuming nation after China (Chakraborty and Singh, 2017). Although biodegradable, urea often has a slower rate of degradation and can potentially affect survival, food utilization, and oxygen consumption of fish (Palanivelu et al., 2005). Works on fertilizers and their impact on fish physiology are rare. In a study on silver carp Hypophthalmichthys molitrix, bighead carp Aristichthys nobilis, and gibel carp Carassius auratus gibelio, the 96-h LC50 values of un-ionized ammonia were 0.35, 0.33, and 0.73 mg L−1, respectively, which indicated that fertilizers were toxic to fish at low concentrations (Wang et al., 2017).

CPF is known to cause oxidative stress by generating reactive oxygen species (ROS) in fish, which induce lipid peroxidation (LPO) as one of the molecular mechanisms involved in its toxicity (Zhang et al., 2019a). Antioxidant defenses such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) help to counteract the toxicity of ROS that cause oxidative damage at the cellular and molecular levels (Nunes et al., 2018). CPF is also known to inhibit acetylcholinesterase (AchE) in fish, which plays a vital role in neurotransmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine to choline and acetate. This leads to the impairment of nerve impulses and the general health of fish (Bernal-Rey et al., 2020). Tissue macromolecules are other essential health indices in fish, alterations in the contents of which often imply changes in their overall turnover. In a study, CPF was found to decrease the macromolecular contents in the brain, liver, gill, and skeletal muscle of Heteropneustes fossilis as a function of an increase in CPF concentrations from 2 to 6 mg L−1 (Tripathi and Shasmal, 2011). Tilak et al. (2005) also found a decrease in glycogen and protein levels in tissues of Catla catla, Labeo rohita, and Cirrhinus mrigala exposed to CPF.

Fish are sensitive to xenobiotic stress, studies on the impacts in fish can provide quantitative information on the ecological integrity and hence act as a suitable bioindicator of polluted environments (Stalin et al., 2019). The Indian freshwater teleost, Channa punctata (Bloch) (Anabantiformes), is considered a suitable model for xenobiotics-associated risk evaluation due to ease of handling and availability throughout the year. The species is most likely to come in contact with agrochemicals due to its widespread distribution in shallow waters, near crop fields (Bhattacharjee et al., 2020). In such mixed environments, assessing the mixture toxicity with various biomarkers offers an important tool to understand how an organism responds to such situations. Multiple biomarker approach helps to obtain an integrated vision of the toxic substance effects on an organism and has high predictive importance in the evaluation of the risk associated with such mixtures (Bonifacio and Hued, 2019). In fact, most mixtures do not respond in an additive or independent manner, implying that prediction of mixture toxicity of agrochemicals offers a potential challenge, as synergism or antagonism in such combinations might occur (Bonansea et al., 2016). Gill and liver of fish are considered appropriate for biomonitoring of toxic stress at the cellular level owing to their respective role as active absorption site and metabolism of xenobiotics (Stalin et al., 2019). However, little is known about the impact of the nitrogen fertilizer, urea, or the binary mixtures of pesticide and urea in fish, which is so often present together in the water. It is, thus, predicted that such combinations might cause cocktail effects. Hence, the present study aimed at understanding the impact of CPF and urea and their binary mixtures in fish with a battery of biochemical biomarkers in fish tissues.

Section snippets

Fish maintenance

C. punctata (25.2 ± 0.8 g and 14.25 ± 0.5 cm) were bought from a local fishery and transferred to a 100 L glass aquarium and acclimatized for three weeks. Aquarium was supplied with oxygen by aeration. Dechlorinated tap water having pH 6.5 ± 0.5, dissolved oxygen 7.0 ± 0.85 mg L−1, temperature 22.5 ± 0.5 °C, electric conductivity 82 ± 1.5 μS cm−1, total hardness 250 ± 8 mg L−1, and nitrite 390 ± 4 mg L−1 was used. Fish were nourished with commercial fish food twice a day (‘Tokyu’ fish pellet

Oxidative stress in fish

Significant (p < 0.05) time and dose-dependent augmentation in the MDA contents over the control was observed in fish exposed to CPF and U after 7-d and 28-d of exposures, which indicated oxidative stress in fish (Fig. 1). In the fish gill, 0.225 μL L−1 CPF (1/2 CPF) induced a 4.5–5.6 fold increase, while 0.045 μL L−1 CPF (1/10 CPF) induced a 3.8–4.5 fold increase in the MDA levels. For 9.87 g L−1 U (1/2 U), 4.5–5.4 fold augmentation, while, at 1.97 g L−1 U (1/10 U) exposure, there was a

Conclusion

In the present study, two crop protecting agents, a pesticide (CPF) and a fertilizer (urea), were found to be extremely toxic to freshwater fish, C. punctata, at sublethal concentrations for 28-d. The toxicities of CPF is well established; however, the effects of urea in fish has not been addressed before, taking into consideration a battery of biochemical biomarkers, such as AChE, SOD, CAT, GPx activities along with MDA and tissue macromolecules. Practically nothing has been known about the

Declaration of competing interest

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors also declare that there is no competing interest in this article.

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