Alterations in Biochemical and Haematological Indices in Bufo

regularis (Amphibian) and Clarias gariepinus (Fish) Exposed to Endosulfan.

Presenter:Ogbomida Emmanuel

1Tongo Isioma, 2Ogeleka Doris, 3Ikpesu Thomas, 4Ogbomida Emmanuel, 5Enuneku Alex, 6Ezemonye Lawrence.1,3 Department of Animal and Environmental Biology (AEB) University of Benin, Benin City2 Department of Chemistry, Western Delta University, PMB 10, Oghara, Delta State5Department of Environmental Science, Western Delta University, PMB 10, Oghara, Delta State4,6 National Centre for Energy and Environment (NCEE), Energy Commission of Nigeria, University of Benin

CSTS/SETAC Africa 2011 Meeting31 May–3 June 2011, University of Buea, Cameroon

INTRODUCTION

Pesticides have the potential to affect many aquatic taxa, they destroys the delicate balance between species that characterizes a functioning ecosystem (Khan and Francis, 2005).

Presence of pesticides in streams and lakes is largely due to the runoff from agricultural fields and outfall from pesticide manufacturing factories (Anandkumar, 1988).

Pesticides are not highly selective, up to 90% of the pesticides applied never reach the intended targets; as a result, many other organisms sharing the same environment as pests are accidentally poisoned. These non-target organisms include amphibians (Fulton and Chambers, 1985; Berrill et al., 1994; Sparling et al., 2001) and fish (Ayoola, 2008; Franklin et al., 2010).

Among pesticides, the chlorinated hydrocarbon insecticides are generally much more persistent and have long residual properties that have placed them in excessive usage in agriculture and forestry through out the world (Anandkumar, 1988).

Endosulfan is a broad spectrum organochlorine insecticide of the cyclodiene group. The chemical is marketed by many different companies and under a variety of names including: Agrosulfan, Aginarosulfan, Endorifan, Hildan, Redsun, Seosulfan, and Thiodan.

The pesticide kills indiscriminately, not only the pests, but also a wide range of other non-target organisms. (Fulton and Chambers, 1985). Endosulfan not only acts as a hormone disruptor, but it also affects biochemical and haematological biomarkers of many species such as fish and amphibians (Ikpesu, 2010; Tongo 2010).

Assessing arrays of biochemical and haematological biomarkers is therefore essential for designing appropriate mitigating measure for the ecological risk assessment of endosulfan pesticide.

This study incorporated the use of biochemical and haematological biomarkers as alternative endpoints in the Ecological Risk Assessment of Endosulfan pesticide.

RATIONALE FOR THE STUDY

In Nigeria, particularly in the Niger Delta, existing data and report are lacking in toxicity and risk assessment of pesticides to aquatic fauna especially fish and toad.

Endosulfan is persistence in the environment and has been reported to disrupt endocrine system, causing reproductive and developmental damage in both animal and human (PMRA, 2003).

The pesticide is commonly used in agricultural farms in Nigeria for the control of insects on food crops (Badejo and Sosan, 2005).

RATIONALE FOR THE TEST ORGANISMS

Availability all year round

High prolific nature

Ease of collection ,transportation, handling and maintenance under laboratory conditions

consumed by humans.

AIMS AND OBJECTIVES OF THE STUDY

The aim of this study therefore, was to determine the effect of Endosulfan at sub-lethal doses on a battery of biochemical and haematological biomarkers in the amphibian species, Bufo regularis and the fish species, Clarias gariepinus.

MATERIALS AND METHODS

Collection, Transportation and Acclimatization of The Test Organisms

•Adult toads, B. regularis of both sexes were collected from their spawning ponds in unpolluted areas in Oghara, Delta State (5°30′N 6°00′E),while juveniles of C.gariepinus of uniform length were collected from Patiby Agro Industrial Enterprise, Erawa Owhe, Delta State, Nigeria.

•Toad and fish samples were collected with hand net from their natural environment and transported in covered baskets and plastic tank to the Laboratory.

•The samples were acclimatized to laboratory condition in holding glass tanks containing dechlorinated tap water for two weeks prior to the experiment. The holding tanks were aerated with the help of air pump, cleaned and water renewed daily. Organisms were fed twice daily and unconsumed food and faecal wastes were removed.

SUBLETHAL BIOASSAY TEST

•The Sublethal toxicity test was carried out for approximately 28 days.

•Sub lethal concentrations of 0.01, 0.02, 0.03, 0.04 g/l (toad) and 0.00, 0.0025, 0.005, 0.0075, 0.01 g/ l(fish)were prepared by serial dilution of stock solution

• Alterations in Biochemical and Heamatological parameters in Serum, were used as indices of toxicosis.

• Parameters were measured in triplicates and averaged for statistical analysis.

HAEMATOLOGICAL ANALYSIS

•For heamatological analysis, fresh blood samples of the amphibians were collected using 2ml hypodermic syringe into 2ml lithium heparin’s tubes. Heparin was used as an anticoagulant.

•Whole blood was used for the estimation of the parameters. The red blood cell and white blood cell profile were determined using various methods described by Svobodova et. al., 1991.BIOCHEMICAL ANALYSIS

•Determination of GST activity was adopted from the procedure described by Habig et al (1974) .•Determination of AChE activity was adopted from colorimetric procedure of Ellman et al(1961).•Glucose level was estimated following the method of Folin-Wu (1926). . •Cortisol levels was estimated by the method as described by Barseghian et al.(1982) . •Total protein was estimated according to the methods of Lowry et al. (1951).

RESULTS Biochemical Indices Glutathione S-Transferase (GST)

• GST activities in the serum of the fish and toad species showed an increase in GST activities.

• The changes in GST activity in the serum increased compared to the control (Figure 1). However, increase in GST activities in the fish was more than that of the toad. 

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GST Levels

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Figure 1: Glutathione-S-transferase activity in the serum of C.gariepinus and B. regularis exposed to different concentrations of endosulfan.

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AChE Level

0 0.0025 0.005 0.0075 0.01

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0 0.01 0.02 0.03 0.04

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Figure 2: Acetyl cholinesterase (AChE) activity in the serum of C.gariepinus and B. regularis exposed to different concentrations of endosulfan.

Acetyl cholinesterase (AChE)No significant changes in activity were observed (P >0.05) for both species (Figure 2).

Glucose levels•Exposure to Endosulfan pesticide showed dose dependent elevations in glucose levels for both species. The changes in glucose activity in the serum increased compared to the control and the increase was significant compared to the control in all the test concentrations.

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Figure 3: Percentage Increase in Glucose Levels in serum of C.gariepinus and B. regularis exposed to Endosulfan for 28 days.

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Cortis

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0 0.0025 0.005 0.0075 0.01

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0 0.01 0.02 0.03 0.04

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Figure 4: Reduction in Cortisol Levels in serum of C.gariepinus and B. regularis exposed to Endosulfan for 28 days.

Cortisol Levels•The exposure of C.gariepinus and B. regularis to Endosulfan pesticide showed a

decrease compared to the control. •However, reduction of Cortisol levels in the fish was more than that of the toad (Figure 4).

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0 0.0025 0.005 0.0075 0.01

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0 0.01 0.02 0.03 0.04

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Figure 5: Reduction in Protein Levels in serum of C.gariepinus and B. regularis exposed to Endosulfan for 28 days.

Total Protein Levels•The results showed significant changes in the protein levels in both species

examined compared to the control (P<0.05). •Reduction of Protein levels in the fish was also more than that of the

toad (Figure 5).

Haematological Indices

Red blood cell indices•Red blood cell indices of C.gariepinus and B. regularis exposed to different

sublethal concentrations of Endosulfan showed a decrease in Erythrocyte Sedimentation Rate (ESR), Erythrocyte count (RBC), Haemoglobin (Hb), Pack Cell Volume (PCV), Mean Corpuscular Volume (MCV) and Mean Corpuscular Haemoglobin (MCH) with increasing concentrations. (Figure 6)

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Figure 6: Alteration in Red Blood Cell Indices in C.gariepinus and B. regularis Exposed to Endosulfan for a period of 28 days.

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Figure 7: Alteration in White Blood Cell Indices in C.gariepinus and B. regularis Exposed to Endosulfan for a period of 28 days.

White blood cell indices•Sublethal exposure to Endosulfan pesticide after 28 days resulted in lower White

Blood cell (WBC), Lymphocytes, Basophils and Monocytes counts (Figure 7). •The reduction in White Blood Cell Indices were statistically significant compared

with control (P<0.05) for both species. •In contrast, there was an increase in Neutrophile granulocytes and Eosinophils

when compared with the control. Increase in Neutrophile granulocytes and Eosinophils were also statistically significant compared with control (P<0.05).

•Conclusion

The sublethal effects on biochemical and haematological indices of C.gariepinus and B. regularis exposed to endosulfan pesticide revealed different degrees of alterations.

Alteration in GLUCOSE levels and WHITE BLOOD CELL COUNTS in both species proved to be the most effective biochemical and haematological biomarkers respectively for Endosulfan pesticide toxicity assessment

Biomonitoring of chemical pollutants especially pesticides on natural populations of organisms is therefore imperative.The biomarker array described in this study can therefore be used as alternative endpoints in the Ecological Risk Assessment of Endosulfan pesticide when monitoring aquatic ecosystems in Nigeria.

ACKNOWLEDGEMENT

This study was generously supported by the National Centre for Energy and Environment (NCEE) Energy Commission of Nigeria (ECN), University of Benin, Benin City, Research Grant.

The technical assistance of members of the thematic group at the centre is highly appreciated.

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