Pesticides are known to cause severe environmental problems, especially in the aquatic environment. This phenomenon is more pronounced during the dry season, because during this period, the dilution capacity of the water systems is low, thus increasing the risk of high concentrations of toxic chemicals. Moreover, the dry season is often the critical period for many animals, especially fish and birds [1-2]. Water pollution by pesticides enters aquatic environment mostly through run offs from agriculture activities. These can lead to fish mortality, reduced fish productivity or elevated concentrations of undesirable chemicals in edible fish tissue, which are detrimental to the health of humans consuming these fishes [3-4].

Analysis of blood is important in the field of fisheries research, especially in the area of toxicology, health management and environmental monitoring [5]. Haematological parameters are very critical indices used in assessment of physiological changes in fish when compared to the control values. These alterations depend on species, age, sex and exposure of fish to diseases [6]. In warm-blooded animals, changes in the blood parameters of fish, which occur because of injuries or infections of some tissues or organs, can be used to determine and confirm the dysfunction or injuries of the latter (organs or tissues). [7]. However, in fish, these parameters are more related to the response of the defensive mechanism in fish. Although the defensive mechanisms of fish to xenobiotics have been investigated, it is obvious that species differences of these mechanisms exist. Thus, evaluation of leucocytes in response to contaminants is crucial in toxicological studies in fish.

One of the important leucocytes commonly assessed in fish is lymphocytes.   A lymphocyte is one of the subtypes of white blood cell or differential counts in a vertebrate's immune system [8]. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). They are the main type of cell found in lymph, which prompted the name "lymphocyte”. Lymphocytes are the key effectors cells of adaptive immunity [9]. For activation, lymphocytes require exposure to self and non-self antigens in the context of major histocompatibility complex (MHC) molecules. Antigen–MHC complexes are presented to lymphocytes on the surface of antigen presenting cells [10].

Many authors have reported the effect of diverse pesticides on the behaviors and haematological responses in different species of fish, [11-13], and have found varying responses after exposing the fish to varying sublethal concentrations of toxicants. The purpose of this study is to assess and contributes to knowledge on the changes in lymphocytes composition in different sizes of C. gariepinus exposed to different chemicals in the laboratory.

Experimental Fish

Seventy-five each of juveniles (mean length 13.54±1.78 cm; mean weight 100.65±2.89g), sub adults (mean length 17.99±1.64 cm; mean weight 400.98±2.07g) and adults (mean length 27.08±2. 02cm; mean weight 1000.04±2.88g) of C. gariepinus were purchased from a commercial farm in Port Harcourt, Nigeria. The fishes were transported in six open 50l plastic containers to the African Regional Aquaculture Center Aluu, Port Harcourt and acclimated for a period of seven days.

Preparation of Test Solutions and Exposure of Fish

Three pesticides paraquat dichloride (PARAQ), 2,2-dichlorovinyl phosphate (DDVP) and dimethoate (DMC) used in this experiment were purchased from a commercial outlet in Port Harcourt, Nigeria. Three sizes of C. gariepinus were exposed to each of the chemical   at the concentrations of 0.00 control, 1.00, 2.00, 3.00 and 4.00 mg/L in triplicates. Five fishes were randomly distributed into each test tank. The experiment lasted for a period of 21 days. The water in the tanks was renewed daily. The fish were fed twice daily at 3% body weight with  a commercial feed.

Haematological Studies

 Haematological studies were carried out on the fishes before and after the experiment. The fishes were taken out individually using a small hand net and placed belly upward on a table. Blood samples of about 5 mL was collected from the caudal peduncle  with the aid of a 2 mL plastic syringe, 2 mL of the blood was dispensed into Ethylene Diamine Tetra-acetic Acid (EDTA) anticoagulant for haematological studies .Leukocyte count (WBC) were determined using the improved Neubauer haemocytometer after appropriately diluted. Differential leukocyte counts were determined by scanning Giemsa’s stained slides in the classic manner [14].

Statistical Analysis

All the results were subjected to analysis of variance (ANOVA) using 17.0 version. Duncan multiple range test (Duncan, 1955) was further used to evaluate the mean differences at 0.05 significant levels.

The effects of paraquat dichloride (PARAQ), 2,2-dichlorovinyl   phosphate   (DDVP) and dimethoate (DMC) on the lymphocytes of   juveniles, sub adults and adult sizes of C.gariepinu  are shown in Figures 1,2 and 3 respectively.  The result indicated that the lymphocytes levels in the treatment groups were significantly higher (P < 0.05) than the control in all sizes. These increments in all the sizes of fish exposed to the chemical, progressively increased with increasing concentrations of the chemicals. In this study, the highest values of lymphocytes was observed in the fish exposed to paraquat, while the lowest was in dimethoate (DMC) in all sizes of C.gariepinus.

Figure 1: Changes in Lymphocytes in Juveniles of C.gariepinus Exposed to three Chemicals

Figure 2: Changes in Lymphocytes in Sub -Adults of C.gariepinus Exposed to three Chmicals

Figure 3: Changes in Lymphocytes in Adults of C. gariepinus Exposed to three Chemicals

Fish exposed to environmental contaminants usually show evidence of physiological distortions such as disturbances in blood stability, ion regulator and oxygen uptake. This becomes noticeable, since blood is an indicator of the physiological condition of animals. Hematological analysis and assessment blood plasma are useful in monitoring the physiological status of fish and as indicators of the health of the aquatic environment. Blood parameters are frequently considered when clinical analysis of fish physiology is applied to determine the sublethal concentration effects of toxicants both in the wild and laboratory. In this study, effects of pesticides on lymphocytes were observed, due to the fact that differential counts are sensitive to application of pesticides [14]. One of the most elementary ways to assess the immune system is to explore changes in the white blood cells count and its types such as lymphocytes [15].

The lymphocytes are reported to be responsible for immune response [16]. The nucleus occupies virtually the whole of the cell, leaving only a narrow rim of basophilic cytoplasm in which there are a few mitochondria and isolated ribosomes. The number of lymphocytes in the blood is noticeably greater in fishes than in mammals [17]. Significant increase in lymphocytes concentration in fish exposed to different concentrations of pesticides was observed in this study.  High levels of lymphocytes counts indicate damage due to infection of body tissues and severe physical stress. Similar findings were observed in fish exposed to toxicants in the laboratory [18]. Also, Akinrotimi et al. [19], documented significantly higher values of lymphocytes  in fish exposed to higher industrial  effluents concentrations. In this assay, exposure of different sizes of C. gariepinus to different pesticides concentrations caused leukocytosis, characterized by higher number of lymphocytes. Leukocytosis has been attributed to an increase in leukocyte to protect the organism against infections in pesticides -damaged tissue [20].

Toxicological and environmental issues resulting from the widespread use of pesticides in agriculture have raised concerns, particularly with respect to the potential toxic effects in humans and animals. The exposure of C.gariepinus   to paraquat dichloride (PARAQ), 2,2-dichlorovinyl phosphate (DDVP) and dimethoate (DMC) were associated with alterations in lymphocytes  levels, resulting in stress to the organism. These pesticides are therefore classified as belonging to substances toxic for fish. Long-term exposure to these chemicals can affect  immunity profiles of C.gariepinus.

1. Bradbury S.P and Coats J.R. "Comparative toxicology of the pyrethroid insecticides." Rev Environ Contam Toxicol, 1989, pp. 133–177.

2. Bunn K.E, Thompson H.M and Tarrant K.A. "Effect of agrochemical on the immune system." J Bull Environ Contam Toxicol, vol. 57, no. 4, 1996, pp. 632–639.

3. Chauhan R.R.S, Saxena K.K and Kumar S. "Rogor induced haematological alterations in Cyprinus carpio." Adv Bios, 1994, pp. 57–62.

4. Chen X et al. "Immunotoxicity of penta chlorophenol on macrophage immunity and IgM secretion of the crucian carp (Carassius auratus)." J Bull Environ Contam Toxicol, 2004, pp. 153–160.

5. Adhikari S et al. "Effect of cypermethrin and carbofuran on certain hematological parameters and prediction of recovery in a freshwater teleost, Labeo rohita (Hamilton)." Ecotoxicol Environ Saf, 2004, pp. 220–222.

6. Agarwal K and Chaturvedi L.D. "Anomalies in blood corpuscles of Heteropneustes fossilis induced by alachlor and rogor." Adv Bios, 1995, pp. 73–80.

7. Adeyemo O.K et al. "Acute toxicity and blood profile of adult Clarias gariepinus exposed to lead nitrate." Internet J Hematol, 2008.

8. Anderson D.P. "Immunostimulants, adjuvants and vaccine carriers in fish: applications to aquaculture." Annu Rev Fish Dis, 1992, pp. 281–307.

9. Arkoosh M.R and Kaattari S.L. "Development of immunological memory in rainbow trout (Oncorhynchus mykiss). I. An immunochemical and cellular analysis of the B cell response." Dev Comp Immunol, 1991, pp. 279–293.

10. Arma N.R et al. "Characterization of expressed genes in kidney cells of Japanese flounder Paralichthys olivaceus treatment with ConA/PMA and LPS." Fish Pathol, 2004, pp. 189–196.

11. Armstrong P.B and Quigley J.P. "α-2-Macroglobulin: an evolutionarily conserved arm of the innate immune system." Dev Comp Immunol, 1999, pp. 375–390.

12. Alexander J.B and Ingram G.A. "Noncellular nonspecific defense mechanisms of fish." Annu Rev Fish Dis, 1992, pp. 249–279.

13. Bayne C.J and Gerwick L. "The acute phase response and innate immunity of fish." Dev Comp Immunol, 2001, pp. 725–743.

14. Elcombe B.M et al. "Evolution of antibody structure and effector functions: comparative hemolytic activities of monomeric and tetrameric IgM from rainbow trout, Salmo gairdnerii." Comp Biochem Physiol, 1985, pp. 697–706.

15. Schroder M.B et al. "Ontogeny of lymphoid organs and immunoglobulin producing cells in Atlantic cod (Gadus morhua L.)." Dev Comp Immunol, 1998, pp. 507–517.

16. Secombes C.J. "Will advances in fish immunology change vaccination strategies?" Fish Shellfish Immunol, 2008, pp. 409–416.

17. Akinrotimi O.A et al. "Haematotoxicity of cypermethrin to African catfish Clarias gariepinus under laboratory conditions." J Environ Eng Technol, 2012, pp. 20–25.

18. Akinrotimi O.A and Amachree D. "Changes in haematological parameters of Tilapia guineensis exposed to different concentrations of detergent under laboratory conditions." J Aquat Sci, 2016, pp. 95–103.

19. Akinrotimi O.A et al. "Haematological responses of Tilapia guineensis treated with industrial effluents." Appl Ecol Environ Sci, 2013, pp. 10–13.

20. Gabriel U.U et al. "Pollutant induced altered behaviours in fish: a review of selected literature." J Technol Educ Niger (JOTEN), 2011, pp. 9–23.