Pesticides have been accepted as part of agricultural practices all over the world. Herbicides, one of the most hazardous and widely used groups of them, could reach water bodies from the soil and this can cause some ecological problems that threaten the existence of living creatures in aquatic environments [1]. Herbicides at high concentration are known to reduce the survival, growth and reproduction of fish and can produce many visible effects on them [2]. Herbicides are chemicals used to manipulate or control undesirable vegetation. Its application occurs most frequently in row-crop farming, where they are applied before or during planting to maximize crop productivity by minimizing other vegetation [3]. Atrazine is a widely-used herbicide in many countries for the control of broadleaved and grassy weeds in agricultural crops. It is the most commonly patronized herbicide in Nigeria [4]. The prolonged use of Atrazine and its persistence involves the risk of its retention in crops and soils. Moreover, these compounds may also pass from surface to ground waters. The chemicals through surface run off may reach unrestricted areas like ponds and rivers and alter the physio-chemical properties of water and consequently affect aquatic organisms [5]. Water contamination by herbicides whether directly or indirectly can cause fish death, low fish productivity and increased levels of unwanted compounds in wholesome fish tissue which can adversely affect the health of humans via consumption [6].

Clarias gariepinus is the most cultured fish in Nigeria and indeed Africa [7]. Clarias gariepinus is a genus of clariid (order Siluriformes) of the family Clariidae, the air breathing catfish. It is widely distributed and accepted by many farmers in Africa because of its fast growth, large size, low bone content, tolerance to poor water quality parameters, omnivorous in its feeding habit, adaptability to overcrowding, high market value and has been successfully propagated artificially thereby making its fry and fingerlings easily available [8]. For sustainable fish production in Nigeria, the ecotoxicology monitoring programmes need to incorporate proper management programmes for herbicide use and disposal in aquatic habitat. The use of haematological technique in fish culture for toxicological research, environmental monitoring and fish health conditions has grown rapidly in recent times. Haematological indices are of different sensitivity to various environment factors and chemical[9].

Atrazine is a widely used herbicide in agricultural practices, and its persistent presence in aquatic systems has been linked to toxicological effects on non-target organisms, including fish. Clarias gariepinus, a commonly consumed freshwater fish, may be exposed to atrazine through contaminated water sources [10]. Atrazine is known to be haematotoxic, causing alterations in the blood parameters of fish, which can affect their overall health and survival. However, limited information is available on the haematotoxic effects of atrazine on Clarias gariepinus. The continuous use of pesticides at high concentrations has resulted in the reduction in survival, growth and reproduction of fish [11]. Among all forms of chemicals atrazine is considered to be the most hazardous with respect to environmental pollution once they are very persistent, non-biodegradable and capable of bio-magnification as they move up in the food chain. Exposure of fish to these compounds can result in mortality as well as sub-lethal impacts [12].

Blood is the most essential and abundant body fluid and is a vehicle for quickly mobilizing defence against trauma and ill health [13]. In assessing the toxic effects of chemicals in aquatic organisms, the use of haematological techniques has become more relevant in recent times, because of the relevance of blood in maintaining homeostasis and life functions of fishes [14]. Studies have revealed that when the aquatic quality is affected by contaminants, any physiological variations will be revealed in values of one or more haematological parameters of aquatic animals [14]. Moreover, Samprath et al. [15] observed that the relevance of haematological studies in fish, lies in the possibility that the blood will reveal anomalies within the body of the fish long before there is any outward manifestation of symptoms of disease or effects of unfavourable environmental factors [16]. To this end, many laboratory studies have also elucidated effects of toxicants on the haematology of Clarias gariepinus including exposure to chlorpyrifos and DDforce [17], cypermethrin [18,19], as well as dichlorvos [20], thereby supporting earlier assertions.

Moreover, Ololade and Oginni [22] opined that the most common haematological variables measured during stress included Red and White blood cells count, haemoglobin content, and haematocrit value and red blood cells indices [23]. In their independent research works, Abdulkareem and Owolabi [23] and Nwani et al. [24] have reported significant changes in the white blood cell (WBC), red blood cell (RBC) and other haematological parameters of Clarias gariepinus exposed to various pesticides [25]. This study aims to investigate the haematotoxic effects of atrazine in Clarias gariepinus to assess its potential risk to the fish and its consumers. The results of this study will contribute to the understanding of the toxicological effects of atrazine on fish health and provide useful information for regulatory agencies in setting guidelines for safe use of atrazine in agricultural practices. Furthermore, the study will provide insights into the potential impact of atrazine on the fish population, which can inform conservation efforts. This study is generally aimed to determine the haemototoxicity of atrazine to Clarias gariepinus juveniles.

Experimental Location

The experiment was carried out at the Wet Laboratory in the Department of Fisheries and Aquaculture Management, Faculty of Agriculture, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.

Source of Experimental Fish

One Hundred and Fifty (150) Clarias gariepinus of equal size (mean length 11.74±2.64cm and mean weight 256.68±1.81g) were sourced from House Tully Fish Farms, Okpuno, Awka, Anambra State, Nigeria. They were transferred in two 50 litre plastic tanks to the laboratory for acclimation process.

Acclimation and Feeding of Fish

The experimental fish were acclimated in four 150L capacity circular plastic tanks containing 150L de-chlorinated water, for 7 days to experimental conditions at room temperature Netted materials with central slits was tied to the tops of the tanks to prevent escape of fish.  Water renewal was done every two days. The fish were fed with a commercial feed at 5% body weight throughout this period.

Experimental Design

The experimental design was a completely randomized design (CRD) with four treatments levels and a control with each level having three replicates.

Procurement of Test Solution

A commonly used selective herbicide Vestrazine (Atrazine 100.0%) was purchased off shelf, from “Analytical” chemical shop, Eke-Akwa Market, Akwa, Anambra State, Nigeria.

Preparation of Test Solution

The solution of the chemical in water was prepared by serial dilution protocols using the dilution formula of Reynolds [26]

N1 V1 = N2 V2

Where N1 = is the manufacture concentration of sodium bromide

           V1 = Volume of original solution added

           N2 = Concentration of the test solution desired

           V2 = Volume of test solution

Exposure of Fish to Atrazine

Ten C. gariepinus each were introduced individually into 15 aquaria tanks of 1.5m x 1m x 0.5m dimension, containing 0.00 (control), 0.05, 0.10, 0.15, and 0.20 of Atrazine.  Each treatment(s) and control were replicated three times and the experimental duration lasted for a period of 96 Hours. The tank was covered with netted materials and supported with heavy objects to prevent the fish from escaping.

Evaluation of Physico-Chemical Parameters of Water

During the experiment, the following water quality parameters namely: Temperature, pH, Dissolved Oxygen, Nitrate and Ammonia levels of control and other treatment exposures were determined and the readings taken at 0, 24, 48, 72 and 96hr intervals in three replicates. Temperature was determined using the mercury-in-glass thermometer, which was inserted in water and the temperature (°C) reading was taken after four minutes. pH was determined using a Jenway® type pH meter (Model 3015). The probe was first inserted in the buffer for 5 minutes to standardize the meter to pH 7, thereafter, it was dipped into the water and the static pH was read 60 seconds later. Dissolved Oxygen was measured by Winklers method APHA, [27]. Ammonia and nitrate were determined by automation using a multi-parameter photometer (Hanna instrument H183200).

Collection of blood samples

Blood samples were collected at 0hrs, 24 hours, 48 hours, 72 hours and 96 hours of the experimental period. Each blood collection was completed within 5 minutes of fish removal from the experimental tank. 5ml samples were drawn once and poured into Eppendorf tubes containing EDTA used as an anticoagulant. The blood samples were put in ice chest box and transported within 6 hours of collection to biochemistry laboratory for analysis.

Analysis of Haematological Parameters

The following blood parameters: Packed Cell Volume (PCV), Haemoglobin (Hb), Red Blood Cell (RBC), White Blood Cell (WBC), Platelets, Red Blood cell indices, were determined based on the methods of Blaxhall and Daisely ]28] and Wedemeyer et al. [29]. Red blood indices Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were derived from the RBC, PCV and Hb as described by Jain [30]. MCV was calculated in femtoliters = PCV/RBC x 10, MCH was calculated in picograms = Hb/RBC x 10 and MCHC = (Hb in 100mg blood / Hct) x 100.

Statistical Analysis

Date obtained from the experiments were collated and   subjected to ANOVA using Statistical Package for the social Sciences, (SPSS) version 22, differences among means were separated by Turkeys Comparative Test at 0.05%.

Table 1 shows the   results   for   the   physiochemical   parameters   of   water   in   tanks of C. Gariepinus exposed to acute concentrations of Atrazine (0.00, 0.05, 0.10, 0.15, and 0.20mg/l) respectively for 96hrs. The result showed no significant differences (P>0.05) in temperature in all the concentrations after an exposure time of 96hrs with temperatures ranging from 28.33?-28.45?in all concentrations. There were significant increases (P<0.05) in pH from 6.53 for the control to 6.80 for 1.0mg/l concentration of Atrazine. Dissolved oxygen (DO) decreased significantly as the concentration of Atrazine increased from 6.67mg/l in the control to 4.03mg/l in the 1.0mg/l concentration at p=0.05. Nitrite significantly increased from 0.00 in the control to 0.076mg/l in the 1.0mg/l concentration. There were significant increases in the mean content of ammonia with increase in Atrazine concentrations with the highest increase seen in 1.0mg/l of Atrazine (0.36mg/l).

The changes in the various hematological parameters (PVC, Hb, RBC, WBC and PLAT) in the fish juveniles exposed to acute concentrations of Atrazine are presented in Table 2 – Table 6. The RBC, PCV, Hb and platelets values in the exposed fish significantly decreased (P<0.05) compared to the control group. However, parameters such as WBC increased progressively as the toxicant concentrations increased when compared to the control. The variations observed were proportional to the herbicide concentration. PCV reduced from 37.00mg/l in the control to 13.88mg/l in the 0.20mg/l atrazine concentration. HB also reduced from 12.73mg/l in the control to 4.23mg/l in the 0.20mg/l herbicide exposure. Platelets reduced significantly and progressively from 276.66mg/l in the control to 187.97mg/l in the 0.20mg/l atrazine concentration. These reductions are statistically significant at P<0.05.

The comparative values of PCV in C. Gariepinus exposed to atrazine for a period of 96 hours is shown in Figure 1. The results revealed that the values of PCV declined across the experimental period, with the highest value of 37.33% at the control and the lowest (13.88%) in 20.00mg/l concentration of the chemical at 96 hours. Comparatively, the values of Hb in C. Gariepinus exposed to atrazine for a period of 96 hours is shown in Figure 2. The results indicated that the values of Hb decreased across the experimental period, with the highest value of 12.99g/dl at the control and the lowest (4.22g/dl) in 20.00mg/l concentration of the chemical at 96 hours. In addition,

the values of RBC in C. Gariepinus exposed to atrazine for a period of 96 hours are presented in Figure 3. The results indicated that the values of RBC decreased across the experimental period, with the highest value of 5.43 Cells x 1012 at the control and the lowest (2.43 Cells x 1012) in 20.00mg/l concentration of the chemical at 96 hours.

Moreover, the values of WBC increased across the experimental period, with the lowest value of 8.27 Cells x 109 at the control and the highest (26.67 Cells x 109) in 20.00mg/l concentration of the chemical at 96 hours (Figure 4). Furthermore, the values of Platelets in C. Gariepinus exposed to atrazine for a period of 96 hours is shown in Figure 5. The results revealed that the values of Platelets declined across the experimental period, with the highest value of 285.00 Cells x 1012 at the control and the lowest (187.97 Cells x 1012) in 20.00mg/l concentration of the chemical at 96 hours. The values of MCV varied among the treatments across the experimental period, while MCV values in the control were within the same range (Figure 6). Moreover, the values of MCH and MCHC varied among all the treatments with no definite pattern (Figures 7 and 8).

Table 1: Physicochemical Parameters of Water in Tanks of C. gariepinus exposed to acute concentrations of Atrazine for 96 Hours (Mean ±SD).

Means within the same column with different superscripts are significantly different (P<0.05).

Table 2: Haematological Parameters of C. gariepinus exposed to acute concentrations of Atrazine for 0 Hours (Mean ±SD).

Means within the same column with different superscripts are significantly different (P<0.05)

Key: PCV – Packed Cell Volume, HB – Haemoglobin, RBC – Red Blood Cell, WBC – White Blood Cell, MCV – Mean Corpuscular Haemoglobin, MCH – Mean Corpuscular Haemoglobin, MCHC – Mean Corpuscular Haemoglobin Concentrations.

Table 3: Haematological Parameters of C. gariepinus exposed to acute concentrations of Atrazine for 24 Hours (Mean ±SD).

Means within the same column with different superscripts are significantly different (P<0.05)

Key: PCV – Packed Cell Volume, HB – Haemoglobin, RBC – Red Blood Cell, WBC – White Blood Cell, MCV – Mean Corpuscular Haemoglobin, MCH – Mean Corpuscular Haemoglobin, MCHC – Mean Corpuscular Haemoglobin Concentrations.

Table 4: Haematological Parameters of C. gariepinus exposed to acute concentrations of Atrazine for 48 Hours (Mean ±SD).

Means within the same column with different superscripts are significantly different (P<0.05)

Key: PCV – Packed Cell Volume, HB – Haemoglobin, RBC – Red Blood Cell, WBC – White Blood Cell, MCV – Mean Corpuscular Haemoglobin, MCH – Mean Corpuscular Haemoglobin, MCHC – Mean Corpuscular Haemoglobin.

Table 5: Haematological Parameters of C. gariepinus exposed to acute concentrations of Atrazine for 72 Hours (Mean ±SD).

Means within the same column with different superscripts are significantly different (P<0.05)

Key: PCV – Packed Cell Volume, HB – Haemoglobin, RBC – Red Blood Cell, WBC – White Blood Cell, MCV – Mean Corpuscular Haemoglobin, MCH – Mean Corpuscular Haemoglobin, MCHC – Mean Corpuscular Haemoglobin.

Table 6: Haematological Parameters of C. gariepinus exposed to acute concentrations of Atrazine for 96 Hours (Mean ±SD).

Means within the same column with different superscripts are significantly different (P<0.05)

Key: PCV – Packed Cell Volume, HB – Haemoglobin, RBC – Red Blood Cell, WBC – White Blood Cell, MCV – Mean Corpuscular Haemoglobin, MCH – Mean Corpuscular Haemoglobin, MCHC – Mean Corpuscular Haemoglobin.

Figure 1: Changes in Packed Cell Volume (PCV) in C.gariepinus exposed to Atrazine for 96 hours.

Figure 2: Changes in Haemoglobin (Hb) in C.gariepinus exposed to Atrazine for 96 Hours.

Figure 3: Changes in the values of Red Blood Cells in C.gariepinus exposed to Atrazine for 96 hours.

Figure 4: Changes in the values of White Blood Cells in C.gariepinus exposed o Atrazine for 96 hours.

Figure 5: Changes in the values of Platelets in C.gariepinus exposed to Atrazine for 96 hours.

Figure 6: Changes in Mean Corpuscular Volume (MCV) in C.gariepinus exposed to Atrazine for 96 Hours.

Figure 7: Changes in the values of Mean Corpuscular Haemoglobin (MCH) in C.gariepinus exposed to Atrazine for 96 hours.

Figure 8: Changes in the values of Mean Corpuscular Haemoglobin Concentration (MCHC) in C.gariepinus exposed to Atrazine.

At exposure of the toxicant for 96hours, the recorded increase in ammonia and nitrite and decrease in dissolved oxygen and the fluctuations that occur between the various concentration and time could be due to the fact that these parameters are highly unstable. Although herbicides cause changes in the quality of water in and around sprayed areas and decrease the dissolved oxygen in the water, along with an increase in temperature which may pose a threat to the survival of fish species, the result of the present study indicates that atrazine application does not result in significant changes in the physicochemical parameter to a point that is capable of causing visually observable negative impacts in fish [30[. The water quality parameters under study are within the standard meant for aquaculture purposes. This study is in line with the work of Owhonda et al. [31], who reported that pH of 6.5 – 9.0 supports fish life. Ahmed et al. [32] also reported that catfish and other air breathing fish can tolerate low Dissolved oxygen concentration of 4 mg/l. Hence the dissolve oxygen content is within the values necessary for fish life. Similar findings were also reported by Gabriel et al. [33]. Basically, in aquatic ecosystem temperature, pH and other physiochemical properties of water are very essential for the survival of fish through metabolism. As such inability of fish to adapt to the environment could cause a change in their physiological responses which could lead to mortality. The temperature, pH, Dissolved oxygen and other parameters are within the ambient values of the area and values of surface water resources in Nigeria [34]. Hence the fishes may not have died due to temperature or other of the physiochemical parameters recorded in this study. The values recorded in this study have been reported to be within the tolerance ranges of warm water fish species.

Haematology is used as an index of fish health status in a number of fish species to detect physiological changes following different stress conditions like exposure to pollutants, diseases, heavy metals, hypoxia, etc. [35]. Studies have shown that when the water quality is affected by toxicants, any physiological changes will be reflected in the values of one or more of the haematological parameters [36]. Thus, water quality is one of the major factors responsible for individual variations in fish haematology. In this respect the present study was designed to investigate the effects of sub-chronic doses of atrazine on haematology of Clarias gariepinus over a period of 96 hours. The significant reductions observed in some of the hematological parameters (PCV, HB, RBC, Platelets, Neutrophils and Lymphocytes) of C. gariepinus exposed to atrazine demonstrates the fact that pesticides cause a significant reduction in the haematological parameters of fish [37]. This result agrees with the findings of Akinrotimi et al. [38] in Tilapia guineensis treated with industrial effluents.  These reductions could also be the product of impaired erythropoiesis and rapid haemolysis of the RBC. Furthermore, the reduction in MCV in the present study reveals erythrocytic shrinkage leading to microcytic anaemia. Also, the reduction in the Hb concentration could also suggest that atrazine could have inhibited the Hb biosynthetic pathway by interfering or inhibiting the utilization of delta-aminolevulinic acid. This may have an adverse impact on the oxygen carrying capacity of the blood This reduction in MCV in the atrazine exposed fish could also arise due to osmoregulatory imbalances [39]. Moreover, Alalibo   et al. [40] reported that sub lethal doses of formalin reduced the erythrocyte count, Hb and PCV in Clarias gariepinus. Reduction in the values of these parameters was also reported by Pereira et al. [41] in Prochilodus lineatus exposed to clomazone.  and in Labeo rohita exposed to fenvalerate as reported by Prusty et al., [42]. The results from this study are also consistent with the trend in Heteropneustes fossilis, exposed to adrin and fenvalerate [43] and in Salmo gardneri and Mystus vittatus exposed to pesticides [44]. 

 Generally, WBC modulate immunological functions in animals, including fish [45]. The observed increase in WBC in the present study indicated abnormal immune protective response to atrazine intoxication. It also suggested that atrazine stimulated the immune system with a concomitant release of WBC from the lymphomyeloid tissue as a defense response. It resulted in the leucocytosis, which altered body physiology [46]. Leucocytosis was also reported by Jee et al. [47] in Korean rock fish, Sebastes schegeli exposed to cypermethrin and that of Jasmin et al. [48] in Labeo rohita exposed to deltamethrin in the laboratory. White blood cells are the smaller number compared with red blood cells, and they have the defensive role in the body of organisms. Changes in the levels of white blood cells following exposure to paraquat may be due to disturbances in the process of hematopoiesis and subsequent reduction or non-specific immune weakening in fish [49]. In this study, the values of Platelets count reduced with increasing concentrations of the chemical.  This was attributed to the presence of the toxicants. According to Lai et al. [50], blood clotting is often accelerated under stress conditions as a result of thrombocytes rise in the blood. Thrombocytosis was induced to arrest the bleeding that occurred in fishes after laceration of tissues or organs leading to blood clotting. At the same time a report by Rao et al. [51], states that the stress condition is not always accompanied by significant increase in thrombocytes count; cortisol may affect fish thrombocytes in similar way as leukocytes reducing their number.

Based on the data and evidence recorded in this study, atrazine is found to be toxic and posed stress to Clarias gariepinus and the effect increased with increase concentration of atrazine. Studies have revealed that exposure to atrazine leads to significant alterations in red and white blood cell counts, hemoglobin levels, hematocrit values, and other hematological parameters in Clarias gariepinus. These changes indicate disturbances in the normal functioning of the fish's hematopoietic system, which is responsible for the production and regulation of blood cells. The haematotoxic effects of atrazine on Clarias gariepinus highlight the potential ecological risks associated with the use of this herbicide in aquatic environments. Based on the findings in this study it is therefore recommended that discharge of this herbicide in the aquatic environment should be restricted in order to reduce its potential risk to fishes as well as humans. There should be constant monitoring of the aquatic environment so as to prevent the overloading of the river systems with chemicals and effluents.

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