In recent years, the use of pesticides has been on the increase in different parts of the world [1]. Its application on the field has made a very positive impact in the overall increase in agricultural food production especially in the tropical region [2]. Conversely, pesticides are included in a wide range of organic micro-contaminants that have terrific ecological impacts especially in the aquatic environment [3]. Various groups of pesticides have diverse effects on living organisms; therefore, generalization of their effect is improper [4]. Among the myriads of biotas existing in the aquatic environment, teleost fishes are the one that are comparatively more responsive to modification in their surrounding environment. That is the reason the level of pesticide concentration in the aquatic organisms seems to be several times greater than that in the ecosystem [5]. This is consequent of bioaccumulation process, in which noxious materials are taken up from the environment by the organism and get accumulated in different organs and tissues of the fish [6]. 

However, some organophosphorus insecticides are thought to be toxic only after metabolism by enzyme systems and they could trigger physiological alterations in the exposed fish [7]. Atrazine is one of the most intensely used herbicides in the world. It is part of the 5-triazine chemical group. This herbicide inhibits photosynthesis by blockage of electron transport in the plant system [8]. It is included in the European Union (EU) priority substance list used for water chemical status definition due to its high mobility and persistence in the environment, causing adverse effects at low concentrations [9]. While Metolachlor is a chloroacetanilide that promotes the inhibition of cell division in seedling shoots and roots [10]. 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 [11]. 

Moreover, contaminants in the aquatic medium   have a very high capability to stimulate oxidative stress in aquatic organisms through production of free radicals and reactive oxygen species (ROS) and provoke an imbalance between intracellular ROS levels and antioxidant protection, which ultimately results in oxidative stress in the systems of the organisms [12, 13]. Oxidative stress can be described as a disruption of the prooxidant-antioxidant balance in favor of the former, leading to potential damage [14]. It is a result of an increase in Reactive Oxygen Species (ROS) and impairment of antioxidant defense systems or incapacity to repair oxidative damage [15]. The activities and expression levels of antioxidant enzymes were used as biomarkers to evaluate the influence of pollution on the biochemical pathway and enzymatic function in fish [16]. It can also be used for monitoring unacceptable levels of environmental contamination [17]. Reactive Oxygen Species generated can cause damage to proteins, lipids, carbohydrates and nucleic acids [18]. The resulting damage may alter cell functions, eventually leading to cell death [19] The antioxidants in fish could be used as biomarkers of exposure to aquatic pollutants. Keeping in view, present work has been undertaken to evaluate the oxidative stress parameters in the plasma of T.guineensis exposed to the different sub-lethal concentrations of atrazine/ metalochlor. 

Experimental Location and Fish 

The study was carried out in African Regional Aquaculture Center, an outstation of Nigerian Institute for Oceanography and Marine Research, Buguma, Rivers State, Nigeria. A total of 180 T.guineemsis comprised of 90 each of juvenile and adult size were sourced from ponds during the low tide. The fishes were transported in six open 50l open plastic containers to the laboratory and acclimated for a period of seven days.

Procurement and preparation test solution 

A commonly used pesticide and herbicide with brand name “Delta Force” (Atrazine and Metalochlor) was purchased off shelf, from “Analytical” chemical shop, Garrison, Port Harcourt, Rivers State. The solution of the chemical in water was prepared by serial dilution using the dilution formula of Puerto et al [20].

Experimental Design and Procedure 

The experimental design was a completely randomized design (CRD) with five treatments levels and a control with each level having three replicates. Five T.guineensis  juveniles were introduced individually 
 into 18, aquaria tanks of 1.5 m × 1 m × 0.5 m dimension, containing 0.00 (control), 0.01, 0.02, 0.03, 0.04 and 0.05 mg/L of Delta Force (metalochlor/atrazine). The same thing was repeated for the adult sizes. Each treatment and control had three replicates and lasted for 15 days. The solution for each concentration was renewed daily, with freshly prepared solution of Delta Force (metalochloclor/atrazine). The tank was covered with netted materials and supported with heavy objects to prevent the fish from escaping.

Analytical procedure

At the end of each experimental period, 2ml of fresh blood sample was collected by making a caudal puncture with the help of fine needle and poured in heparinized sample bottles. Blood samples were centrifuged immediately for 15 minutes at 5000 rpm. Plasma specimens were separated, pipetted into eppendorf tubes and stored in a refrigerator at -20°C until assayed [21]. The results were read using a universal microplate reader on a Jenwayvisible spectrophotometer (Model 6405). The activity of antioxidants in centrifuged plasma was determined spectrophotometrically using the method of Beechey et al. [22] (1975). Water quality parameters were also determined using the methods APHA [23].

Statistical Analysis

All the data were expressed as mean and standard deviation of mean. The statistical package, SPSS Version 22 was used for the data analysis. The means were separated using two ways ANOVA and the two means were considered significant at 5 %(p<0.05). 

The water quality parameters (Table 1) were within the same range except in DO, where lesser values were obtained at higher concentration of the chemical. The effects of atrazine/metalochlor on the antioxidants in the plasma of T.guineensis juveniles are presented in Table 2. It was observed that the values of SOD and GSH decreased with increasing concentrations of the herbicide. While CAT and LPO increased significantly when compared to the control values. The same trend was observed in the antioxidants of adult fish exposed to the chemical in Table 3.

The comparative values of CAT in the plasma of T.guineensis exposed to atrazine/metalochlor are presented in Figure 1. From the results the adult fish consistently had higher values of CAT in all concentrations. Also,  in  GSH  Figure 2,  the  values of GSH decreased as the concentrations of the chemical increased, the reduction was however more pronounced in juveniles’ fish, when compared to adult. The same trend was equally observed in the SOD of the fish exposed to the chemical. Also, in the comparative values of LPO Figure 4, the lowest values in both sizes were observed in the control and the highest in the 0,05mg/L concentration of the chemical.

Table1: Physico-Chemical Parameters of Water in Experimental Tanks of T. guineensis Exposed to Atrazine/Metalochlor 

Means within the same column with different super scripts are significantly different (p<0.05)

Table 2: Antioxidants Levels in T.guineensis Juveniles Exposed to Atrazine/Metalochlor 

Means within the same column with different super scripts are significantly different (p<0.05)

Table 3: Antioxidants Levels in Adults of T.guineensis Exposed to Atrazine/Metalochlor 

Means within the same row with different super scripts are significantly different (p<0.05)

Figure 1: Comparative Values of CAT in the Plasma of T.guineensis Exposed to Atrazine/Metalochlor 

Figure 2: Comparative Values of GSH in the Plasma of T.guineensis Exposed to Atrazine/Metalochlor

Figure 3: Comparative Values of SOD in the Plasma of T.guineensis Exposed to Atrazine/Metalochlor

Figure 4: Comparative Values of LPO in the Plasma of T.guineensis Exposed to Atrazine/Metalochlor

Recently in toxicological research herbicides induced oxidative stress has extensively been used to express and validate the system and mechanism of chemical toxicity in aquatic organisms especially fish [24,25]. Conversely, Livingstone [26], reported that the oxidative stress caused by herbicides in aquatic organisms may lead to ROS production and modification in antioxidants enzymes. The production of ROS may damage nearby molecules resulting in damage of the molecular structure and function or dysfunction of many organs and systems [27]. Therefore, the unnecessary production of ROS and their harmful consequence can be reduced by the cellular antioxidant systems [28].  In general, organisms under stress conditions utilize the antioxidant enzymes to adjust to environmental stress and altered activities depend on the dose, species and route of exposure [29]. The enzymes such as SOD and CAT plays a key role in eliminating the ROS produced during bioactivation of xenobiotics and the induction of SOD/CAT system may be the first defence mechanism against ROS [30]. In this study, the observed increase in CAT levels in the plasma of the exposed fish indicates a detoxifying mechanism against the toxicity. A similar observation was also noted by Romeo et al.[31] in the  tissues of Dicentrachus labrax exposed to some heavy metals . The increase in CAT activity in Mullus barbatus after exposure to some toxicants indicates an adaptive response to increased generation of ROS [32]. However, Salah et al. [33] have suggested that the alterations in the activity of antioxidant enzymes in Ctenopharyngodon idella exposed to sublethal concentrations of mercury and zinc indicate the adaptive response of the fish to ROS. The generated ROS on MP exposure might have caused damage to gills and liver resulting in leakage of these enzymes into plasma as noted in the present study [34]. 

In this study, the values of SOD decreased considerably in the plasma of the exposed fish. Correia et al. [35] observed similar results in gilt head seabream (Sparus auratus) and suggested that the O2 radicals or their transformation to H2O2 may cause oxidation of cysteine in the enzyme which results in decrease of SOD activity. Excess production of ROS according to Monteiro et al. [36], may also inhibit the SOD activity. The fluctuations in the values of SOD activities in the plasma of T.guineensis exposed to mixture of Atrazine and metalochlor may indicate a cellular oxidative stress consequent of chemical exposure [37] The inhibition of SOD activity  as observed in this study, may also be due to binding of toxicants –SH groups of enzymes, increased H2O2 and/or superoxide radical [38]. The significant decrease in SOD activity might have equally resulted from its inactivation by the superoxide radical triggered by exposure [38]. Results from the present investigation imply that the fish utilizes enzymatic mechanisms to tolerate the effects caused by generated ROS due to atrazine accumulation.

The Glutathione (GSH) enzymes are usually present in various tissues and injuries to the cells of these tissues may result in the release of GSH enzymes into plasma. Moreover, the response of these enzyme activities to stressors may occur through direct enzyme inhibition or induction and changes in metabolic pathways or fluxes [39]. In the present study, the significant reduction in the values of GSH activity in the plasma of T.guineensis during the sublethal exposure might have resulted from the organal damage due to atrazine/metalochlor accumulation[40]. Lipid peroxidation (LPO) has been reported as a major contributor to the loss of cell function under oxidative stress [41]. Moreover, some pesticides may increase lipid peroxidation by direct interaction with the cell membrane [42]. Conversely, Avellini et al. [43] regarded LPO as the first step signifying the cellular damage caused by toxicants. In the present study the level of LPO in the plasma of Atrazine treated fish increased remarkably when compared to the control value. A similar increase in plasma LPO level was noted in Elongatulus eucirrus exposed to cypermethrin[44]. The elevation of LPO content in the plasma  indicates an increase in free radicals due to MP toxicity or metabolism [45]. 

The results of the present investigation indicate that sublethal exposure of T.guineensis to atrazine induces significant changes in the antioxidant activities of the fish. However, the presence of this chemical in the aquatic  environment is hazardous to the ecosystem and will definitely affect the survival of fish. Hence, caution should be taken to prevent this chemical from gaining entry in to aquatic environment. 

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