The use of herbicides to control weeds has been recognized as a part of agricultural practices throughout the world. Unfortunately, the indiscriminate use of these herbicides to improve agricultural production and yield may have impacts on non-target organisms, especially aquatic life forms and their environment. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is one of the most commonly used herbicides found in the rural environments. It is extensively used on corn, sorghum, sugarcane, pineapples, and to some extent on landscape vegetation. Rated as moderately toxic to aquatic species, atrazine is mobile in the environment and is among the most detected pesticides in streams, rivers, ponds, reservoirs and ground waters [1–3]. It has a hydrolysis half-life of 30 days and relatively high water solubility (32 mg·L⁻¹), which aids in its infiltration into ground water [4]. Atrazine concentrations of 20 μg·L⁻¹ have been commonly detected in surface water runoff, while concentrations as high as 700 μg·L⁻¹ have been reported [5–7]. Due to the low persistence of atrazine herbicide, repeated applications are practiced for the control of weeds in agricultural fields, and as a result, large quantities of the herbicide find their ways into water bodies. The mode of action of atrazine is blocking electron transport in photosystem 11 leading to chlorophyll destruction and blocking photosynthesis. When atrazine was first released for agricultural use, it was thought that since photosynthesis is limited to plants, animals would be immune to any effects of atrazine. It was soon suspected that atrazine might have non-target action in animals including genotoxic [8], clastogenic [9] and biochemical effects [10]. The indiscriminate use of this herbicide, careless handling, accidental spillage or discharge of untreated effluents into natural water ways have harmful effects on the fish populations and other aquatic organisms and may contribute to long term effects in the environment. Sublethal effects with biochemical and histopathological alteration of fish tissues may occur with long term exposure to levels of less than 2 mg·L⁻¹ of atrazine [11].

Fish can serve as bio-indicators of environmental pollution and can play significant roles in assessing potential risk associated with contamination in aquatic environment since they are directly exposed to chemicals resulting from agricultural production via surface run-off or indirectly through food chain of ecosystem [12]. Fish are endowed with defensive mechanisms to counteract the impact of reactive oxygen species (ROS) resulting from metabolism of various chemicals or xenobiotics. Oxidative stress develops when there is an imbalance between pro-oxidants and antioxidants ratio, leading to the generation of ROS. Environmental contaminants such as herbicides, heavy metals and insecticides are known to modulate antioxidant defensive systems and to cause oxidative damage in aquatic organisms by ROS production [13–16]. ROS such as hydrogen peroxide (H₂O₂), superoxide anion O₂⁻ and hydroxyl radical (OH^·) at supranormal levels can react with biological macromolecules potentially leading to enzyme inactivation, lipid peroxidation (LPO), DNA damage and even cell death [17,18] but at low concentrations their effects are less pronounced.

Several ecotoxicological characteristics of C. punctatus such as wide distribution in the freshwater environment, availability throughout the year, easy acclimatization to laboratory conditions and commercial importance make this species an excellent test species for toxicity and biochemical studies [19]. There have been studies on toxicities of atrazine-based herbicides; however meager information is available about their toxicity and effects on fish and aquatic organisms especially on sub-chronic exposure. Determination of the toxicity is essential for determining sensitivity of the animals to the toxicants and also useful for evaluating the degree of damage to the target organs and the consequent physiological, biochemical and behavioral disorders. Thus, to supplement risk assessment studies of these herbicides, it is important to obtain information on their toxicity and effects on some local species like C. punctatus.

The aim of the present study was to assess the toxicity of a commercial formulation of the herbicide atrazine (Rasayanzine) and the impact on some biochemical indices in C. punctatus. The information obtained may be useful for management and monitoring of atrazine (Rasayanzine) contamination in the environment.

Fish are often used as sentinel organisms for ecotoxicological studies because they play number of roles in the trophic web, accumulate toxic substances and respond to low concentration of mutagens [34] therefore, the use of fish biomarkers as indices of the effects of pollution are of increasing importance and can permit early detection of aquatic environmental problems [35,36].

Acute toxicity data has been used to derive water quality guidelines for regulatory measures [37]. The result of the LC₅₀ (median lethal concentration) for atrazine in the present study at 96 h was 42.381 mg·L⁻¹. The results show that the toxicity of atrazine for C. puctatus is both time and concentration dependent, thus, accounting for differences in LC_10–90 values obtained at different concentrations and time of exposure. However, some other researchers have shown that exposure time is not significant in LC₅₀ determinations for fish [38]. The LC₅₀ value obtained for C. puctatus in this study is higher than that reported by Bathe et al. [39]; Neškovic et al. [11] and Hussein et al. [40], who reported LC₅₀ vales of 16.0, 18.8 and 9.37 mgl⁻¹ for Lepomis macrochirus (Bluegill sunfish), Cyprinus carpio and Oreochromis niloticus, respectively, exposed to atrazine. Toxicity of chemicals to aquatic organisms has been shown to be affected by age, size and health of the species [41]. Physiological parameters like quality, temperature, pH, dissolved oxygen and turbidity of water, amount and kind of aquatic vegetation, concentration and formulation of chemical and its exposure also greatly influence such studies [42,43]. Sprague [44] observed variability in acute toxicity even in single species and single toxicant depending on size, age and condition of the test species along with experimental factors. The estimated safe level as calculated by multiplying the LC₅₀ with application factor (AF) varied from 4.24 × 10⁻⁴ to 8.657. However, the large variation in safe levels determined by different methods has resulted in controversy over its acceptability [19,45]. Kenega [46] emphasized that the major weakness in calculation of accumulation factor (AF) is its dependence on LC₅₀. Warren [47] and Pandey et al. [48] had reported that the introduction of toxicant into an aquatic system might decrease the dissolved oxygen concentration which will impair respiration leading to asphyxiation. Fish exposed to atrazine were stressed progressively with time before death. The respiratory impairment due to the toxic effect of atrazine on the gills of C. punctatus is similar to the reports of Abdul-Farah et al. [41]; De Mel and Pathiratne [49]; Tilak et al. [50] and Ayoola [51] that pesticides impair respiratory organs. Death could have, therefore, occurred either by direct poisoning or indirectly by making the medium unconducive for the fish or even by both. The abnormal behavior observed during the exposure period like incessant jumping, restlessness, surface to bottom movement, vigorous jerks were similar to the observations of Hussein et al. [40]; Pandey et al. [19] and Chandra [52].

The elevated level of lipid peroxidation in the liver of C. punctatus in response to the exposure to atrazine as observed in the present investigation suggests that there is increased production of ROS. Increased ROS production may, thus, be associated with the metabolism of atrazine herbicide leading to the peroxidation of membrane lipids of the liver. The liver is noted as site of multiple oxidative reactions and maximal free radical generation [53–55]. Previous investigations have reported the induction of LPO by pesticides such as deltamethrin [56,57], alachlor [58], malathion [52] and butachlor [59] in fish. Lushchak et al. [60], however, did not record elevation of lipid peroxidation in the brain and liver of goldfish Crassius auratus exposed to sublethal concentration of Roundup^®. The different responses probably are functions of species, the time of exposure, type and concentration of stressors. The observed LPO resulting from ROS generated by the atrazine may lead to cell apoptosis. ROS and oxidative stress have been demonstrated to be triggers of apoptosis [61]. Oxidative stress may also be due to the depletion of cellular GSH content below the critical level which prevents the conjugation of xenobiotics like atrazine to GSH and thus enables them to freely combine covalently with cell proteins [62]. However, organisms are equipped with interdependent cascades of enzymes to alleviate oxidative stress and repair damaged macromolecules, produced during normal metabolism or due to exposure to xenobiotics. In this cascade, SOD and CAT are the major enzymes in eliminating ROS formed during bioactivation of xenobiotics in the hepatic tissues [63] and the induction of SOD/CAT system provides a first line of defense against ROS. SOD help to dismutase superoxide radical O₂⁻ to hydrogen peroxide (H₂O₂). The increase in CAT activities in the liver as observed in the present study may be in response to H₂O₂ produced by SOD activity since CAT is responsible for the detoxification of H₂O₂ to water. Enhancement of CAT activity was observed in Cichlid fishes from polluted waters [64], in mullets [65], Lepomis macrochirus [66] and Prochilobus lineatus [67] exposed to herbicides. CAT activity, however, decreased after day 7 of exposure to atrazine, although the values obtained were significantly (p < 0.05) higher than control. Decrease in CAT activity after the 7th day of exposure could be due to decrease in the rate of reaction as a result of the excess production of H₂O₂. The inhibition of CAT by the superoxide radical has been reported [68,69]. In all, the increase in LPO, CAT and SOD activities in the liver tissue as reported in the present investigation supports the hypothesis that sublethal concentrations of atrazine induce oxidative stress in C. punctatus and could be an adaptive response to protect the fish from the atrazine-induced free radical toxicity. GR activity was unchanged in the liver of C. punctatus following exposure to different concentrations of atrazine, except on day 10 and 15. Similar to the present observation, the hepatic GR activity in the liver of bluegill sunfish (Lepomis macrochirus) was not affected following exposure to atrazine for 96 h [66]. It, thus, appears that dose and exposure time affected the activity of GR in C. punctatus exposed to atrazine. The elevated GR observed on day 10 and 15 could be part of the protective response in the tissue which suggest that GR pathways could efficiently be used to regulate the ROS formation during the contaminated period. This compensatory response accompanied with the induction of other antioxidants (SOD and CAT) evidently helps to prevent accumulation of free radicals and their products in stressed organisms. The result of these studies evidence that the biochemical responses are dependent on stressor type, species and exposure time. Furthermore, the herbicide may lead to the occurrence of transformation products in water with a potential or actual similar or higher toxicity than their parent [70]. Metabolites/breakdown products of atrazine potentially could have affected the biochemical measurements in exposed C. punctatus. Thus further studies are required to determine these biochemical changes after the exposure of fish to atrazine and its transformation products.

The present investigation indicated that the herbicide atrazine is toxic to fish and further substantiate earlier findings that antioxidant enzymes such as SOD, CAT and GR and LPO in fish could be effectively used as biomarkers of herbicide toxicity.