Haematological Changes in African Catfish (Clarias Gariepinus) Exposed to Mixture of Atrazine and Metolachlor in the Laboratory

Seventy Two (72) male and female African catfish (Clarias gariepinus) juveniles of mean length (10.74 ± 1.81 g) were exposed to different concentrations of atrazine and metolachlor (0.00 mg/1-control, 0.01, 0.02, 0.03, 0.04 and 0.05mg/1) for 14 days to determine its effect on haematological parameters of the fish. The results obtained indicated significant (P<0.05) reductions with increased concentrations of the chemical in haemoglobin (Hb), Red blood Cell (RBC), packed cell volume (PCV), lymphocytes, platelets and mean corpuscular volume (MCV). The white blood cell (WBC), neutrophils, monocytes, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentrations (MCHC) in fish exposed to the pesticides were significantly (P<0.05) higher than that of the control. The data obtained from this work will contribute to the base line haematological parameters for use in monitoring health status of Clarias gariepinus in the wild and culture medium


Introduction
Herbicides are widely used all over the world to control the harmful effects of pests and weeds on agricultural productions and fish farm. However, despite the good results of using herbicides in agriculture, their use in the environment is usually accompanied by deleterious environmental and public health effects (Nwani et al., 2010). The herbicide after being used, ultimately find their way into different aquatic ecosystems and have been found to be highly toxic to non-target organisms, especially aquatic life form and their environment (Nwani et al., 2010).Herbicides pollution severely affects aquatic organism strophic levels including human beings. The effects of herbicides on fishes are of great concern (Bagheri and Nezami, 2000;Nwani et al., 2010).
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 (Roses et al., 2013). 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 (European Commission, 2008). While Metolachlor is a chloroacetanilide that promotes the inhibition of cell division in seedling shoots and roots (Selck et al., 2012).
The African catfish Clarias gariepinus is an ecologically and commercially important fish for the Nigerian aquaculture industry (Ita, 1980). These mud fish are frequently and widely cultured in ponds and they also occur freely in Nigerian's natural freshwater. Musa and Omoregie, (1999) reported that fish are intimately associated with the aqueous environment, such that physical and chemical changes in the environment are rapidly reflected as measurable physiological changes in fish.
The use of haematological technique in fish culture for toxicological research, environmental monitoring and fish health conditions have grown rapidly in recent times Akinrotimi, 2008;Akinrotimi et al., 2011a). Many works have been conducted on haematological changes of in the fish exposed to pesticides include but not limited to that of Das and Mukherjee (2000); Adebayo et al. (2005); Patnaik and Patra (2006); Gabriel et al. (2007b); Gabriel et al. (2012); Akinrotimi et al. (2011b); Akinrotimi and Amachree (2016). Haematology is an indicator of immunological status and can provide definitive diagnosis of fish during toxicant exposure Campbell and Ellis, 2007;. Haematological indices are of different sensitivity to various environment factors and chemical (Akinrotimi et al., 2013). Studies have shown that when the water quality is affected by toxicants, any physiological changes will be reflected in values of one or more haematological parameters of aquatic organisms Gabriel et al., 2007c). However, Sampath et al. (1993) and noted that there is a possibility that studies on fish blood might reveal conditions within the body of the fish long before there is any outward manifestation of disease.
Among all forms of chemicals, atrazine and metolachlor are considered to be the most hazardous with respect to environmental

Statistical Analysis
Data obtained from the experiments were collated and subjected to ANOVA using Statistical Package for the social Sciences, (SPSS) version 10, differences among means were separated by Tukeys Comparative test at 0.05%.

Results
The results of water quality variables in the experimental tanks during the exposure period are shown in Table 1. The result indicated an increase in the values of Alkalinity from 89.10 in the control to 156.34 mg/l at 0.05 mg/l of the chemical. Conductivity also increased from 121.50 to 271.21 µS/cm. However, reductions were recorded in the values of dissolved oxygen and total dissolved solids, while the values for Temperature and pH were within the same range in all concentrations of exposure.
The result of hematological parameters in C. gariepinus juvenile males exposed to Atrazine/ metalochlor, for 14 days is presented in Table 2. The result indicated a consistent reduction relative to the control in the values of haemoglobin (Hb), red blood cell (RBC), packed cell volume (PCV), lymphocytes, platelets and mean corpuscular volume (MCV). While the values of WBC increased from 21.00 to 36.71, Neutrophils 40.00-59.12, monocytes 4. MCH 16.47 to 22.71 and MCHC 31.11 to 32.61.These was a decrease in the values of Hb 11.20 to 8.20, RBC 6.30 to 6.10 PCV 36.00 to 10.00%, Lymphocyte 59.66 to 36.12%, platelets 501.21 to 299.33% and MCV 52.94 TO 27.70 fl, with increasing concentration from control to 0.5 mg/L of the chemical. The result of hematological parameters, in female C. gariepinus juveniles exposed to Atrazine/metalochlor for 14 days is shown in Table 3.The results indicated that there was increase in the values of WBC from 24.00 to 40.81, Neutrophilis 40.21 to 59.33%, and MCHC 34.00 to 74.00%.There was decrease in: Hb from 13.61 to 7.20, RBC 7.40 to 4.51, PCV 39.11 to 10.14, Monocytes 44.21-9.02%, Lymphocytes 59.21 to 35.62%, platelets 614 to 279.33% MCV 52.85 to 22.48 fl andMCH 18.39 to 15.96 pg. The summary of haematological parameters in C. gariepinus exposed to Atrazine and matelolachlor for 14 days is shown in Table 4. The values of Hb, RBC, PCV, Lymphocytes Platelets, and MCV reduced significantly (P<0.05) with increasing concentrations of experimental tanks on weekly basis. Ammonia, Alkalinity, Conductivity, Dissolved oxygen (DO), was determined using the methods described by APHA (1998). The water pH was measured using pH meter, and while Temperature was evaluated by using mercury in glass thermometer.

Blood sampling and analysis
Blood sampling was conducted at the expiration of 14 days. Blood samples were collected from a total of 25 fish (five fish/ treatment) with heparinized plastic syringe, fitted with 21 gauge hypodermic needle and preserved in disodium salt of ethylenediamine tetraacetic acid (EDTA) bottles for analysis. The Blaxhall and Daisley (1973), and Wedemeyer et al. (1983) haematological methods were adopted for this study. The cyano-haemoglobin method was used to determine haemoglobin (Hb) using diagnostic kits from Sigma diagnostics USA, and packed cell volume (PCV) was determined by the microhaematocrit method. Red blood cell (RBC), leucocrit (LCT) and thrombocyte count were determined with the improved Neubauer haemocytometer according to (Dacie and Lewis, 1991). White blood cells (WBC) was determined with the improved Neubauer counter, while differential counts such as neutrophils, lymphocytes and monocytes were determined on blood film stained with May-Grunwald-Giemsa stain (Mirale, 1982). The values of haematological indices were calculated Brown (1980).

Mean cell volume (MCV)
Values of MCV were calculated using the formula:

Mean cell haemoglobin concentration (MCHC)
Values of MCHC were calculated using the formula:

Discussion
The temperature values recorded during the experiment maybe considered to be good enough for the normal growth of Clarias. gariepinus as it falls within the range reported by Hogendoorn (1983) at 27.5-32°C for optimal growth of fish. The chemical also did not significantly change the pH of the various treatments, such that control and treatment were all slightly alkaline. The values of dissolved oxygen recorded for all treatment were below recommended values of 5-6 ppm for the rearing of Clarias    gariepinus (Janseen, 1985). This may be due to the fact that water was contaminated by the toxicants and therefore the amount of dissolved oxygen was relatively low. There was an increase in the values of alkalinity and conductivity. The rise in alkalinity and conductivity falls in line with the study at Eleiyele reserviour at Ibadan, Nigeria (Mombeshora et al., 1981), where all the physical and chemical parameters were within acceptable limits for fishes, but were increased above tolerant limits as the parent river was used as a depository for wastes. The changes in the physical and chemical composition of natural aquatic environment from internal and external influences and their effects on fauna and flora are well documented (Binay et al., 1994).
The result obtained in the exposure of C.gariepinus to different concentrations of atrazine and metalochlor indicated that there was increase in WBC, neutrophils, monocytes, MCH, MCHC in both male and female C. gariepinus. This falls in line with the works of Akinrotimi et al. (2011b) on acute haematological study of cichlid fish. Sarotherodon melanotheron exposed to toxicants. The rise in WBC, neutrophils and monocytes shows an immune response to the toxicants. The result is in agreement with work of Akinrotimi and Gabriel (2012a) on submission of remarkable richness of toxicants on the fish blood, where they found that more of these white blood cells and its components are recruited to combat the stressor in the blood stream of the fish. The result shows the values of RBC, Lymphocytes, platelets, haemoglobin and packed cell volume of C. gariepinus were higher in the control group than the experimented group exposed to Atrazine/Metolachlor for 14 days, which was in agreement with the studies of Akinrotimi et al. (2011c) and Oriakpono et al. (2012). The derived variables of the haemoglobin indices such as MCV, and MCH excluding MCHC were observed to be lower than expected and the reason could be as result of the size, and age of fish used and duration of exposure .
Literature shows that changes in haematological indices of fish caused by toxicants and their responses varied depending on the species, chemical and duration of exposure. They are also influenced by the type, quality and concentration of the chemicals in the water. These factors can cause reversible and irreversible alterations in the haematological parameters of fish (Sovio et al., 2004;Cameron, 2010;. It is well known that Atrazine/metolachlor promotes the reticence of cells, causes premature mortality of mature red blood cells and inhibition of haemoglobin formation. This lead to anaemia at high exposures and erythropoiesis at lower exposure of the chemical (Hodson et al., 1984;Akinrotimi et al., 2010a). In the light of the present study, the mean value of PCV decreased progressively in the experimented group compared to the control. The result agreed with the work of Akinrotimi et al. (2009) in haematological indices of Tilapia guineensis subjected to handling stress. The decrease in the PCV indicates the worsening of the condition of the organism and developing of anaemia.
Haemoglobin concentration reveals the status of an organism oxygen level and the organism itself tries to maintain them as much as possible in the face of any stressor. This study shows that the mean haemoglobin reduced considerably when compared to the control values. Reduction in the values of haemoglobin in the blood of exposed fish, is usually caused by the effect of chemicals on the blood, as well as decrease in its oxygen carrying capacity, which also imply anaemia or validate the toxic effect of Atrazine/ metolachlor on Clarias gariepinus (Akinrotimi et al., 2010b;. A dose dependent reduction in RBC level was observed in C. gariepinus treated with atrazine/metolachlor. Haematological indices like RBC count, concentration of haemogloblim and PCV have been reported to indicate secondary responses of an organism to pollutants (Rogers et al., 2003;Akinrotimi et al., 2012c). The MCV, MCH, and MCHC increased considerably in all treatment compared to the control. The increase in MCV, MCH and MCHC is in agreement with the work of Ariweriokuma et al. (2011) following a shoot term exposure of C. gariepinus to cypermethrin in the laboratory. These alterations were attributed to direct or feedback response of structural impairment to RBC membranes resulting in haemolysis and impairment in haemoglobin producyion, stress-related release of RBCS from the spleen and hypoxia stimulated by exposure to atrazine and metolachlor (Shah, 2006;Akinrotimi and Gabriel, 2012b).

Conclusion
The result obtained in this study revealed that atrazine/ metalochlor is highly toxic to the C. gariepinus. Toxicity of atrazine /metalochlor on C. gariepinus increased with increasing concentration of the pesticides. In the aquatic environment, this can negatively affect the ecosystem. Although, the aquatic environment is not the target of such pesticide but the widespread use of them has led to some serious problem in aquatic biota. Hence discharge of this pesticide in aquatic environment should be restricted.