Global agriculture has embraced the use of herbicides for the sole purpose of increasing food production. The introduction of herbicides for agricultural purposes has been on the increase, thereby making them the most used chemical worldwide [1]. Herbicides simply refer to chemical substances used for the control of unwanted organisms in agricultural processes in order to increase crop yields. However, excessive use of these chemicals can exert negative effects on non-target organisms, both those living within and outside the target ecosystems. Major disadvantages of herbicides include not being biodegradable, being toxic to both target and non-target organisms even at low concentrations, accumulation in various food chains, and consequently death [1]. The pollution of aquatic ecosystems by herbicides of all kinds has been of global concern due to the excessive use of these products in the control of aquatic plants, leachate, and runoff from agricultural areas [1].

Atrazine is a well-known herbicide invented in 1958 in the Geigy laboratories as the second of a series of 1,3,5-triazines with a molecular weight of 215.69 and chemical formula 2–chloro-4ethylamino-6-isopropylamino-S-triazine [2]. It is a systemic herbicide used in the control of grass and broad-leafed crops.  It functions by binding to the plastoquinone-binding protein in photosystem II, and through starvation and oxidative damage quickened by high light intensity as a result of breakdown in the electron transport processes, plant death occurs. [3]. Atrazine is one of the most widely used herbicides in the world, thereby making it the most common herbicide contaminant of groundwater and surface water [4]. It has a persistent effect on the environment and covalently binds to a large number of mammalian proteins [5]. Atrazine can be transported over 1000km from the point of application through runoff, and as a result, it pollutes other habitats and organisms where it is not being used. In Nigeria, atrazine exists under the trade names Atraforce^®, Cotrazine 80, Actazin, Delzin®, and Atraz. [6]. Aquatoxicological studies usually make use of various fish species to assess environmental quality due to their ability to detect the presence of toxic substances at both acute and chronic levels. Toxicology studies in fish are a paramount issue due to their adverse effect on human health upon consumption; thus, toxicology studies are vital for the determination of animal sensitivity to various toxicants and the evaluation of the degree of damage to target organs based on physiological, hematological, histological, biochemical, and behavioral disorders [7].

In Nigeria, the aquaculture sector is dominated by the culture of Clarid fishes, mainly Clarias gariepinus and Heterobranchus longifilis, due to their rapid growth, high market value, acceptability, resistance to disease, and harsh environmental conditions, especially for the Clarias species. In search of maximum profit, most aquaculturists have embraced the production of hybrids, which is a breeding technique involving the mating of genetically differentiated organisms in order to produce offspring with specific desirable traits or a general improvement in performance. Several crosses in clarid fish species include crossing between male Clarias gariepinus and female Heterobranchus longifilis, popularly known as "Heteroclarias,” and crossing between male Heterobranchus longifilis and female Clarias gariepinus, popularly called “Clarobranchus [8]. Food security in Nigeria has increased the quest for optimal crop yield in order to meet the demand of the ever-growing population of 220 million inhabitants. Thus, most crop farmers have embraced the maximum use of herbicides such as atrazine for increased crop production. The resultant effects of these agricultural practices, together with industrialization and urbanization, are environmental threats to freshwater ecosystems [9,10]. Despite the existence of several toxicology studies with herbicides to determine their effects on target organisms, there is a need to study the chronic effects of these various herbicides on aquatic organisms, which are consequently non-target organisms.

Study Area

This study was carried out in the Department of Fisheries and Aquaculture Laboratory, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. It is a federal university located in the tropical rainforest zone of south-eastern Nigeria, within latitude 6°14'38.4" North and longitude 7°07'18.7" East. The major climatic seasons in this study area are controlled by south-west and north-east trade winds from the Atlantic Ocean and the Sahara Desert, respectively.

Procurement of Experimental Organisms

One hundred and twenty (120) juveniles each of Clarias gariepinus and its hybrid (male Clarias gariepinus X female Heterobranchus longifilis) were sourced from Nnamdi Azikiwe University Fish Farm, Awka, Anambra State, Nigeria, and transported to the Department of Fisheries and Aquaculture's wet laboratory, where they were subjected to a prophylactic treatment, acclimatized for two weeks (14 days) under laboratory conditions, and fed commercial fish pellets of 4% body weight daily in two rations at 9.00 a.m. and 5.00 p.m. The fish pellets used are from a brand named Skretting Fish Feeds, manufactured by Skretting Nigeria LTD, Niger West Building, Ibadan, Nigeria.

Preparation of the Test Solution

For this study, the atrazine-based herbicide used is Cotrazine (Atrazine 80% W.P.), manufactured by Nantong Foreign Trade Mehco, Shanghai, China. This was obtained as a commercially available herbicide from an agrochemical outlet in Awka, Anambra State, Nigeria.

The preparation of the test solution was according to the dilution method of Reish and Oshida [11].

Based on the results obtained from the acute toxicity, the sub-lethal concentration of one-fifth (1/5), one-tenth (1/10), and one-twentieth (1/20) of LC₅₀ was determined as recommended by Oladimeji and Ologunmeta [12] and Mohammed [13] for the static experiment. Sub-lethal concentrations were renewed every 3 days to maintain a fresh solution for chronic toxicity testing.

Experimental Design

The experimental design used is a completely randomized design made up of four (4) treatments (including a control) in three (3) replications each for Clarias gariepinus and hybrid juveniles.  A total of 24 plastic aquaria, each of size 30.5 x 30.5 x 92.5 cm, contain 20 liters of dechlorinated water. Ten (10) juveniles each of Clarias gariepinus and hybrid juveniles (of mean weight 7.60 ± 0.10g and mean standard length 3.51 ± 0.13cm, regardless of sex) were used for the sub-lethal experiment, which lasted for a period of 84 days (twelve weeks).

All the plastic aquaria were covered with a 1 mm mesh-size net to prevent the fish from jumping out of the tanks. Fish were fed with pelleted commercial feed at 5% body weight daily in two rations at 9.00 a.m. and 5.00 p.m. Water quality parameters such as temperature, pH, and dissolved oxygen were monitored on a weekly basis throughout the period of the experiment using standard methods [14]. The effect on hematological and biochemical parameters and atrazine accumulation in the muscle were determined after exposure for 84 days to sub-lethal doses of atrazine.

Haematological Analysis

At the end of the chronic toxicity bioassay of 84 days, five (5) each were taken from the control and exposed groups of both fish species. Blood samples of both the control and exposed groups were collected from the caudal peduncle using a 2 ml heparinized syringe and needle and preserved in disodium salt of ethylenediamine tetraacetate (EDTA) sample bottles for analysis [15]. All samples were properly labeled. Indices analyzed for hematology include packed cell volume (PCV), hemoglobin concentration (Hb), red blood cell (RBC) count, white blood cell count (WBC), neutrophils, and lymphocytes. All hematological analyses were carried out in the laboratory according to standard methods [16].

Determination of Serum Biochemical Indices

At the expiration of sublethal exposure for 84 days, blood samples from both the control and exposed groups were collected using a 2 ml sterile syringe and needle from the caudal peduncle. The blood samples collected were transferred into plain bottles in order to obtain serum for biochemical parameter analysis. The clotted blood meant for serum biochemistry was separated from the clear serum by centrifugation at 2000 rpm for 15 minutes and properly labeled. Biochemical indices analyzed include Alanine Amino Transferase (ALT), Aspartate Amino Transferase (AST), total protein, high-density lipoproteins, low-density lipoproteins, and blood glucose [17].

The assay kits used for the estimation of biochemical parameters were commercial diagnostic kits from Randox^® United Kingdom, following the manufacturer’s instructions using a spectrophotometer. The blood glucose of the experimental specimens was measured using glucometer test strips.

Determination of Atrazine Accumulation in Fish Muscle

This accumulation of atrazine in the muscles of both the control and exposed groups of both species was determined using gas chromatography techniques [18]. Ten grams (10g) of both control and exposed samples were collected at the end of the 84-day sublethal testing, and atrazine accumulation in the body was determined through gas chromatography. The instrument used for this was a Buck 530 gas chromatograph, CA, USA.

Statistical Analysis

The data obtained were statistically analyzed with the statistical package (SPSS version 20). The data was subjected to ANOVA, and Duncan’s multiple range test was used to determine the significant difference between the means at the 5% probability level.

Results from this research showed that atrazine herbicide was toxic to fish, and there were significant differences among exposed groups and control groups of both fish species. Effects of sub-lethal toxicity manifested in the hematological, biochemical, and atrazine accumulation in the muscle.

The results of the hematological parameters of C. gariepinus and its hybrid exposed to sub-lethal concentrations of atrazine are shown in Table 1. The result showed no significant difference (P>0.05) between the two species of fish, but significant differences (P<0.05) exist among various concentrations of atrazine in both species when compared to their control. Packed cell volume (PCV) across the various sublethal concentrations for both species showed a significant difference (P<0.05) when compared to control. PCV decreased in a dose-dependent manner, with the highest means (35.00±1.00) recorded in the control group of both organisms and the lowest value (27.00±3.00) recorded in the hybrid juvenile with the highest atrazine concentration (2.5ml).Haemoglobin also decreased with an increase in atrazine concentration in both fish species, having the highest values (11.80±0.10) in the control hybrid and the lowest value (10.03±0.56) in the hybrid with the highest concentration.

The results of the red blood cell count (RBCC) recorded the highest and lowest values in the control group of hybrids and hybrids with the highest concentration of atrazine, respectively.

The values of total white blood cell count (TWBC) showed a significant difference (P<0.05) in test organisms based on sub-lethal doses of atrazine exposure. TWBC had the lowest values in the control of both species (1.80±1.13) and the highest values (2.65±0.50) in the Clarias gariepinus group exposed to a 0.6 mL atrazine concentration. The highest value of neutrophils (29.67±0.57) was recorded in the control group of hybrids, while the least value (17.67±0.58) was recorded in the hybrid with the highest concentration of atrazine exposure.

Lymphocyte values showed a dose-dependent increase with an increase in concentration, whereby the highest mean value (82.33±0.58) of lymphocytes was found in the hybrid with the highest dose of atrazine and the lowest mean value (69.67±0.57) was found in the hybrid (0.0 mg/l). The hematological studies showed no detection of monocytes, basophils, or eosinophils.

The comparative mean values of packed cell volume (PCV), hemoglobin (HB), red blood cell count (RBC), total white blood cell count (TWBC), neutrophils, and lymphocytes in the two experimental fish exposed to 0.00 mg/l, 0.60 mg/l, 1.30 mg/l, and 2.50 mg/l of atrazine are presented in Figures 1, 2, 3, and 4, respectively. The results indicated that all hematological parameters were within the same range; however, higher values were observed in the lymphocytes of hybrid fish exposed to 2.50 mg/L of atrazine.

Biochemical Parameters

The result of the biochemical properties in this study showed significant differences (P<0.05) between the control and exposed groups for both species, but no significant differences (P>0.05) across different species, as shown in Table 2. Aspartate amino transferase (AST) and Alanine amino transferase (ALT) increased from 10.33±0.58 to 27.33±0.58 and 8.00±0.00 to 21.00±1.00, respectively. Serum protein decreased from 4.14±0.02 to 2.00±0.01. Cholesterol increased from 92.90±0.10 to 135.4±0.25. Low-density lipoproteins (LDL) showed a significant increase from 14.03±0.15 to 71.43±0.06, while high-density lipoproteins (HDL) recorded a significant decrease from 99.97±0.05 to 64.36±0.12. Serum glucose was found to be the lowest (28.33±0.58) in the control group and the highest (45.67±0.58) in the exposed group.

The comparative mean values of aspartate aminotransferase (AST), Alanine aminotransferase (ALT), protein, cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and glucose in the two experimental fish exposed to 0.00 mg/l, 0.60 mg/l, 1.30 mg/l, and 2.50 mg/l of atrazine are presented in Figures 1, 2, 3, and 4, respectively. The results indicated that all the biochemical variables were within the same range; however, higher values were observed in cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) of hybrid fish exposed to atrazine in all concentrations.

Atrazine Accumulation in the Muscle

Atrazine accumulation in the muscles of exposed fish species was observed to be dose-dependent, with juveniles of hybrid species having higher atrazine accumulation than the exposed groups of C. gariepinus, as shown in Table 3. Atrazine accumulation was found to be highest (5.76±0.04) in hybrid species exposed to 2.5 ml of atrazine concentration, followed by hybrid (0.6 ml), and then hybrid (1.3 ml) before the juveniles of Clarias gariepinus. The lowest values were recorded in the control of both species. Figure 9 represents the mean concentration of atrazine (in parts per million, ppm) detected in the muscle tissues of Clarias gariepinus juveniles and its hybrid with Heterobranchus longifilis exposed to varying concentrations of atrazine.

Table 1: Effect of Atrazine on the Haematological Parameters of C.Gariepinus and Hybrids Exposed to Varying Concentrations pf Atrazine.

*Means with the same superscript along the columns are not significantly different (P>0.05).

*PCV = Packed Cell Volume, Hg = Haemoglobin, WBC = White blood count, RBC= Red   Blood Cell count.

Figure 1: Comparative Values of Haematology in The Control Group of C. Gariepinus Juveniles and Its Hybrid with H. Longifilis Exposed To 0mg/L of Atrazine.

Figure 2: Comparative Values of Haematology in C. Gariepinus Juveniles and Its Hybrid with H. Longifilis Exposed To 0.6mg/L Of Atrazine.

Figure 3: Haematology of Clarias gariepinus juveniles and its hybrid with Heterobranchus longifilis exposed to 1.3mg/l of Atrazine.

Figure 4: Haematology of Clarias gariepinus juveniles and its hybrid with Heterobranchus longifilis exposed to 2.5mg/l of Atrazine.

Table 2: Effect of Atrazine on the Biochemical Parameters of C. gariepinus and Hybrid Exposed to varying Concentrations of Atrazine.

*Means with the same superscript along the columns are not significantly different (P>0.05).

Figure 5: Serum Biochemistry Analysis of The Control Group of C. Gariepinus Juveniles and Its Hybrid with H. Longifilis Exposed To 0mg/L Of Atrazine.

Figure 6: Serum Biochemistry Analysis of C. Gariepinus Juveniles and Its Hybrid with H. Longifilis Exposed To 0.6mg/L Of Atrazine.

Figure 7: Serum Biochemistry Analysis of C. Gariepinus Juveniles and Its Hybrid with Heterobranchus Longifilis Exposed To 1.3mg/L Of Atrazine.

Figure 8: Serum biochemistry analysis of Clarias gariepinus juveniles and its hybrid with H. longifilis exposed to 2.5mg/l of Atrazine.

Table 3: Accumulation of Atrazine in muscles of C. gariepinus and Hybrid exposed to varying Concentrations of Atrazine.

*Means with the same superscript along the columns are not significantly different (P>0.05).

Figure 9: Atrazine Accumulation in The Muscles of Clarias Gariepinus Juveniles and Its Hybrid with Heterobranchus Longifilis Exposed to Varying Concentrations (0mg/L, 0.6mg/L, 1.3mg/L, And 2.5mg/L) Of Atrazine.

The result of both acute and sub-lethal concentration studies on the behavioral patterns of test organisms in the present study suggests that atrazine affects the behavioral patterns of C. gariepinus and hybrid juveniles. Several toxicology studies have shown that the introduction of chemicals in culture mediums exerts significant changes in the hematological parameters of aquatic organisms. Haematology studies indicate the immunological status and provide a definitive diagnosis of fish during acute and chronic toxicity testing [19,20]. Haematological indices possibly reveal the diseased conditions of cultured fish species long before outward manifestations [21].

In this study, the results of hematological parameters in C. gariepinus and hybrid juveniles exposed to atrazine herbicide for 84 days showed a consistent reduction relative to the control in the values of packed cell volume (PCV), hemoglobin (Hb), red blood cells (RBC), and neutrophils, while the values of WBC and lymphocytes increased. The decrease in PCV, RBC, and Hb could be attributed to gill damage, impaired osmoregulation causing anemia and hemoglobin, or it could be a result of the lysing of erythrocytes [22]. The variations observed in the hematological characteristics of the exposed fish species, as well as their corresponding reactions, could be predominantly influenced by the concentration of atrazine. A decrease in the PCV of exposed organisms could be attributed to deteriorating toxic conditions and the development of anemia. This conforms to findings by Omoniyi [7], who reported that a reduction in the concentration of PCV, Hb, and RBC in the blood of test organisms could be attributed to the presence of toxic factors such as haemagglutin, which directly affects blood formation, or could be due to the response to stress imposed on them by atrazine. Inhibition of erythropoiesis and an increase in the rate of erythrocyte destruction in hematopoietic organs could be the major causes of the decrease in RBC count [23].

Hemoglobin (Hb) concentration signifies the oxygen-carrying capacity of the blood. Every organism strives to maintain its hemoglobin concentration in the event of external stressors. The dose-dependent reduction in the hemoglobin concentration of the exposed groups of both species indicates the toxic effect of atrazine at various concentrations. Similar results have been reported for several freshwater fish by Aderolu et al. [24] and Adekunle [25]. Changes in the erythrocyte profile could be due to the compensation of an oxygen deficit in the body caused by gill damage; thus, the nature of these changes triggers the release of erythrocytes from the blood depots [26]. However, this result deviates from the findings of Ajani [26] and Gabriel et al. [27], who reported an increase in PCV, RBC, and hemoglobin levels in the blood of C. gariepinus exposed to different doses of atrazine concentrations, stating that the increase in these blood parameters was dose-dependent.

White blood cells and lymphocytes are known to fight antibodies in the body systems of various organisms. Total white blood cells (TWBC) together with lymphocytes exert immunological system function, and their concentration is directly proportional to the increase in exposure period to toxicants. A significant increase in the concentration of TWBC in this study could be due to an increase in the production of antibodies needed for the survival of exposed fish species. An increase in TWBC count might be a result of an increase in lymphoresis or the enhanced release of lymphocytes from lymphoid tissues [28]. This result is in agreement with Adi [29], who reported an increase in WBC on an acute hematological study of cichlid fish Sarotherodon melanotheron exposed to toxicants, stating the rise in TWBC shows an immune response to the toxicants. However, this finding is in disagreement with Luiz et al. [30] and Naji et al. [31], who recorded a significant decrease in the level of TWBC and lymphocytes in silver catfish juveniles exposed to atrazine when compared to the control.

Serum biochemistry analysis of an organism is vital for monitoring the health condition of every organism. Biochemical changes depend on the fish species, age, sexual maturity, and health condition. Analysis of serum biochemical parameters has shown useful information in the detection and diagnosis of metabolic disturbances and diseases in fish [32]. Biochemical indicators such as enzymes serve as biomarkers to identify possible environmental contamination and prevent deleterious effects on the health of aquatic organisms [33].

Alanine amino transferase (ALT) and aspartate amino transferase (AST) are plasma enzymes found in the liver, but upon destruction of cells or in diseased conditions, these enzymes are released into the blood, and their high concentration in the blood indicates abnormality. A change in the activities of transaminase indicates amplified transamination processes, and an increase in transamination occurs with amino acid input into the TCA cycle to cope with the energy crisis during pesticide stress [34]. In the present study, an increase in ALT and AST was observed to increase with an increase in the concentration of atrazine in the exposed group, which indicates a diseased condition of the liver. This is in agreement with the findings of Wegwu and Omeodu [35], who reported an increase in transaminase activities in Clarias gariepinus exposed to an aqueous extract of Nigerian crude oil, and Odo et al. [36], who observed an increase in serum biochemical properties (ALT and AST) of Clarias gariepinus exposed to Vestaline® (Pendimethalin) herbicide.

The diagnostic significance of protein synthesis in an organism is crucial due to its role in the production of antibodies, enzymes, and hormones. The total protein in the study decreased with an increase in the concentration of atrazine. This reduction in protein indicates an increase in proteolytic activities and possible utilization of their products for metabolic purposes to overcome stress. This conforms to Adeyemo [37] but deviates from Osman et al. [38], who reported an increase in the total protein concentration in the blood of Nile tilapia and African catfish exposed to heavy metals. The difference in the concentration of total protein might be a result of variations in the type of toxicant used for various studies.

Cholesterol is the most important sterol occurring in animal fats. Changes in the blood cholesterol and triglyceride concentrations are sensitive indicators of liver dysfunction, as homeostasis of lipids is one of the principle liver functions [39]. The cholesterol level in the present study significantly increased in a dose-dependent manner across the various exposed groups. This may be due to the necrosis of the liver by the toxicant, which leads to an impairment in the metabolism of cholesterol. However, this result disagrees with Anand et al. [39], who observed a decrease in the level of cholesterol in Channa punctatus exposed to phorate. Total cholesterol is made up of high-density lipoproteins (HDL) and low-density lipoproteins (LDL), which were observed to be significantly different based on various concentrations of atrazine. HDL is usually expected to be higher in normal organisms and lower in the case of unfavorable conditions, while LDL is expected to be lower under normal circumstances and higher under unfavorable or diseased conditions; thus, the higher the better (for HDL) and the lower the better (for LDL). The study showed that HDL and LDL in exposed groups were affected by atrazine concentration, as the control group had higher HDL than the exposed group and lower LDL than the exposed group [40].

Serum glucose levels are one of the most important signs of stress in fish. An increase in glucose indicates conditions of stress, and affected organisms strive to combat such stress with energy reserves such as glycogen in muscles and liver [41]. Blood glucose has been shown to be a sensitive indicator of environmental stress for any chemical pollutant, including herbicides. ⁵³ The glucose level in the present study was observed to increase as concentrations of atrazine increased. This finding is in agreement with Omoniyi [7], who observed a significant increase in glucose levels in C. gariepinus juveniles exposed to different atrazine concentrations as a result of a conventional indicator of liver injury in fish. Several authors also reported increased levels of glucose in the blood of fish exposed to ultraviolet radiation [42], heavy metals [43], and other pollutants [44], which can be attributed to the alteration in the activity of glucose-6-phosphate dehydrogenase and lactate dehydrogenase. An increase in glucose levels could be a result of the production of energy by exposed organisms to fight the effects of atrazine in the body. Blood glucose level has been used as an indicator of environmental stress to reflect changes in carbohydrate metabolism under stress conditions [45]. Aquatic animals in polluted waters tend to accumulate many chemicals in high concentrations, which is a potentially hazardous situation for the entire food chain [46,47]. From the result of atrazine accumulation in the present study, it can be concluded that juveniles of Clarias gariepinus were more resistant to atrazine accumulation than the juveniles of Hybrid.

Excessive use of herbicides for maximum crop production has posed a threat to non-target organisms both in terrestrial and aquatic habitats. Most widely used herbicides, such as atrazine, enter the aquatic environment through runoff and leachates and exert deleterious effects on aquatic organisms. In the aquatic ecosystem, fish species are one of the most abundant organisms, and their increased susceptibility to environmental pollution makes them significant organisms for toxicity testing. Atrazine has been found to be toxic to Clarias gariepinus and its hybrid with Heterobranchus longifilis, even at sublethal doses. More toxicology research should be carried out on hybrids in order to gain more knowledge of their adaptations under stressed conditions.

Conflict of interest

There was no conflict of interest among the authors in this publication

Authors Contributions

Okpala-Ezennia, K.P – Conceptualization, Methodology, Project Administration, Resources, Writing – Original draft.

Nlewadim, A.A – Supervision, Visualisation, Writing – review and editing.

Ikeogu, C.F – Data curation, formal analysis and validation.

Acknowledgement

We sincerely appreciate the staff and management of Department of Fisheries and Aquaculture, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria, for their genuine contribution to the successful completion of this research.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors that could have influenced its outcome.

Ethical Approval

We confirm that all aspect of the work covered in this manuscript involving animal subjects was carried out with the ethical approval of the Veterinary Services Department, Ministry of Agriculture and Rural Department, Awka, Anambra State, Nigeria with Reference number: MOA/ANV/441/Vol.1/43.

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