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Alterations in Some Haematological Parameters of The African Snakehead: Parachanna africans Exposed to Cadmium

Ovie KORI-SIAKPERE*, Urowoli IKOMI*

Delta State University, Department of Animal and Environmental Biology, Abraka, Nigeria; [email protected] (*corresponding author)

Abstract

The haematological alterations produced on exposure of the African snakehead, Parachanna africans to the sublethal concentration (0.0, 0.1, 1.0 and 10.0 mg/L) of cadmium (Cd2+) for 21 days have been studied. Red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (Hct), the mean corpuscular haemoglobin (MCH) and the mean corpuscular volume (MCV) levels were decreased with an increase in exposure concentration, but the level of the mean corpuscular haemoglobin concentration (MCHC) was increased. These alterations could be attributed to haemolysis and impairment of haemoglobin synthesis, resulting in a hypochromic microcytic anaemia, induced by exposure to cadmium. Cadmium exposure also caused significant decreases in white blood cell (WBC) count. The primary consequence of the observed changes in total and differential leucocyte counts in stressed fish was attributed to suppression of the immune system and increased susceptibility to disease. Plasma glucose and total plasma protein concentrations were significantly decreased; showing that cadmium affects the fish energy metabolism. The present study thus confirmed that haematological parameters are very sensitive indicators of fish organism response to chemicals in this case cadmium.

Keywords: cadmium, glucose, haematological parameters, Parachanna africans, protein, sublethal effect

Introduction

Heavy metals are inorganic elements essential for growth in trace or very minute quantities, toxic and poi- sonous in relatively higher concentrations; biologically undegradable but easily assimilated and bioaccumulated in the protoplasm of aquatic organisms (Egborge, 1994).

They are introduced into the aquatic environments by in- dustrial, domestic and mining activities (Pourang, 1996).

Heavy metals are attributed to adverse effects on the health of biota and indigenous populations due to their toxicity and bioaccumulative tendencies within the environment (Vare et al., 2006). Cadmium is one of the listed extremely hazardous substances. It belongs to the group IIb in the pe- riodic table, occurring as a soft bluish white metal, solid in its standard state. It enters surface water with the discharge of industrial wastes or by leaching of soil to which sewage sludge is deposited. It accumulates in the tissues of mam- mals and aquatic organism causing acute haemolytic crisis resulting in severe anaemia, hyperglycaemia among other effects in mammals (Freiberg et al., 1974). The toxicity and toxic effects of cadmium generally result from the binding of the metal with reactive and/or complexity group lead- ing to inhibition of enzymatic process, possibly disturbing general growth, development and reproduction (WHO, 1992). It is biologically very reactive and therefore gives rise to both acute and chronic poisoning. Nariagu and Sprague (1987) emphasized elaborately on effects of cad- mium on aquatic organisms. Many reports are available on the effect of Cd on fish blood. Blood is a good bio indi-

cator or a diagnostic tool to study the problem in organ function. The measurement of haematological changes of fish under exposure to any toxicant may be used to predict effects upon chronic exposure. The objective of present work was to determine the effects of sublethal concentra- tions of cadmium to haematological parameters in the Af- rican snakehead, Parachanna africans.

Materials and methods

Healthy live specimens of Parachanna africans were obtained were obtained from local fish market at Ughelli, Delta State, Nigeria; transported to the laboratory and kept in glass aquaria supplied with clean water. The fish were checked for injury and disease, and then washed in 0.1% potassium permanganate (KMnO4) solution for 5 minutes. All fish were maintained in the laboratory for a minimum period of two weeks during which they were fed with commercial fish food.

Stock solution of cadmium was prepared from 2.37 g of cadmium acetate (CH3COO)2.Cd 2H2O equivalent to 1 g cadmium metal was dissolved in 1 litre of distilled water to form the stock solution. From this stock solution, various concentrations used in the investigations were pre- pared.

The containers used, consisted of large plastic contain- ers of 150 litres capacity. The upper part of each containers were covered with a lid made of fine polyethylene gauze screen of 1 mm mesh size. Each experimental set up and

Received 15 July 2011; accepted 12 November 2011

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Kori-Siakpere O. and Ikomi U. / Not Sci Biol, 2011, 3(4):29-34

30

mean corpuscular volume (MCV) were calculated from the equations given by Anderson and Klontz (1965).

t(%) 100 Haematocrin(g%) Haemoglobi

MCHC(%) u

(per/L) 10 count e Erythrocyt

(g%) n Haemoglobi g)

MCH(U u

(per/L) 10 count e Erythrocyt

(%) t Haematocri )

MCH(μ3 u

The plasma glucose and total plasma protein were de- termined with aid of Randox commercial diagnostic kits.

All results obtained from the sublethal tests were sub- jected to statistical analysis using one-way analysis of vari- ance (ANOVA) to test differences between the various levels of sublethal concentrations of cadmium. Multiple comparisons of the means were analyzed by the Duncan tests. All analyses were performed using the software pro- gramme (GraphPads Prism® Software version 5.0, San Di- ego, CA). Results were considered significant at the 95%

confidence level (P< 0.05).

Results

The water quality parameters of the diluting water used in the tests and determined by standard methods are pre- sented in Tab. 1.

Haematological alterations resulting from the expo- sure of Parachanna africans to the various sublethal con- centrations of cadmium in the water are presented in Fig.

1. Red blood cell count (RBCC), haemoglobin (Hb) con- centration, haematocrit (Hct) and white blood cell count (WBCC) were decreased with an increase in exposure concentration; being statistically significant (P< 0.05) at the higher concentrations of 1.0 and 10 mg/L cadmium.

Alteration in the haematological indices of MCHC, MCH and MCV; and plasma glucose and total plasma protein in Parachanna africans following exposure to the various concentrations of cadmium in the water are presented in Fig. 2. The mean corpuscular haemoglo- bin (MCH) and the mean corpuscular volume (MCV) mean levels were decreased with an increase in exposure concentration, but the level of the mean corpuscular hae- moglobin concentration (MCHC) was increased. Plasma glucose and total plasma protein concentrations were also significantly (P< 0.05) decreased.

control (each aquarium) were stocked with 10 fish speci- men in each of the concentrations tested.

After acclimatization, the experimental fish were di- vided into four (4) groups to assess the sublethal effect of cadmium on haematological parameters. The first group was kept in fresh untreated water and used as control while the other three groups were exposed to the sublethal con- centrations (0.1, 1.0 and 10.0 mg/L) of cadmium in the water. Fish specimens were starved for 24 hours, weighed and then introduced into the test solution/water (10 spec- imens in each aquarium).

Water quality parameters were determined at three days interval using standard laboratory methods as described by APHA (1998) and the exposure media replaced. The parameters measured were temperature, pH, dissolved oxygen, free carbon dioxide and total alkalinity.

At the end of the exposure period of twenty-one (21) days the fish were taken from the control and test tanks, sacrificed and subjected to the analysis described below.

Five fish were caught individually in a small hand net from the containers. After the preliminary investigation of the length and weight, the fish were then placed belly upwards and blood samples obtained from the caudal circulation with the aid of a heparinised 2 cm3 dispos- able plastic syringe and a 21 gauge disposable hypodermic needle. The use of plastic syringe is a necessary precau- tion with fish blood because contact with glass results in decreased coagulation time (Smith et al., 1952). The site chosen for puncture (about 3-4 cm from the genital open- ing) was wiped dry with tissue paper to avoid contamina- tion with mucus. The needle was inserted at right angle to the vertebral column of the fish and gently aspirated during penetration. It was then pushed gently down un- til blood started to enter as the needle punctured a caudal blood vessel. Blood was taken under gentle aspiration until about 1 cm3 has been obtained; then the needle was with- drawn and the blood gently transferred into heparinized plastic containers. The samples were then mixed gently but thoroughly. Some blood samples were used for the mea- surement of haematocrit, haemoglobin concentration, red blood cell count and total white blood cell count. Plasma was obtained from blood samples by centrifugation and then drawn into a 1 cm3 plastic syringe transferred into a universal bottle and stored in a refrigerator and later used for the determination of plasma glucose and total plasma protein. All determinations were carried out in duplicates for each sample.

The haemoglobin concentration of the blood samples was determined in duplicate by the cyanmethaemoglo- bin method (Larsen and Snieszko, 1961). The haemat- ocrit was determined by the microhaematocrit method of Snieszko (1960). The red blood cells were enumerated in an improved Neubaeur haemocytometer, using Hendricks diluting fluid. The total white blood cell counts were similarly enumerated in an improved Neubaeur haemo- cytometer using Shaw’s diluting fluid. The haematologi- cal indices: mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH) and

Tab. 1. Water quality parameters of dilution water used in the sublethal tests

Parameter Values

pH 6.56 + 0.52

Temperature 28.8 + 1.2oC

Dissolved oxygen 6.96 + 0.72mg/L

Free carbon dioxide 5.45 + 0.09 mg/L

Alkalinity 34.6 + 1.34 mg/L

Hardness 126.84 + 9.08 mg/L

Turbidity 0.34 + 0.07 mg/L

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Discussion

The results obtained in the present study revealed a significant response in the haematological variables in the cadmium (Cd2+) exposed fish both in respect to con- centration and exposure time. The statistically significant (P < 0.05) decrease in many values of the haematologi- cal parameters studied is not uncommon in fish exposed to sublethal concentrations of toxicants and therapeutic agents. Similar reduction in haematological indices has been reported by Musa and Omoregie (1999) following exposure of C. gariepinus to sublethal concentrations of the therapeutant: malachite green. Omoregie et al. (1994) also made similar observations when Oreochromis niloticus was exposed to sublethal concentrations of formalin. The general reduction of the blood parameters is an indication of anaemia caused by exposure of Parachanna africans to cadmium in this study.

The decreases in haemoglobin concentration signifies that the fish’s ability to provide sufficient oxygen to the tis- sues is restricted considerably and will result in decrease of physical activity (Nussey et al., 1995a; Wepener et al., 1992). The significant decrease in the haemoglobin con- centrations may also be due to either an increase in the rate at which the haemoglobin is destroyed or to a decrease in the rate of haemoglobin synthesis (Reddy and Bashani- hideen, 1989). Buckley et al. (1976) reported that pro- longed reduction in haemoglobin content is deleterious to oxygen transport and any blood dyscrasia and degenera- tion of the erythrocytes could be ascribed as pathological conditions in fishes exposed to toxicants.

Significant decreases in the haematocrit values record- ed after exposure to cadmium are indicative of anaemia and haemodilution possibly due to gill damage or/and im- paired osmoregulation (Larsson et al., 1985).

Fig. 1. Alteration in haemoglobin (a), haematocrit (b), total erythrocyte count (c) and total leucocyte count (d) in Parachanna afri- cans following 21days exposure to the various concentrations of cadmium in the water. Each column represents the mean value and vertical bars indicate the standard error of the mean. Asterisks represent statistical significance at 0.05 level

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Kori-Siakpere O. and Ikomi U. / Not Sci Biol, 2011, 3(4):29-34 32

in erythrocytes reported in the present study indicated that the experimental fish, became anaemic, which Wede- meyer et al. (1984) attributed to haemodilution resulting from impaired osmoregulation across the gill epithelium.

Changes in erythrocyte counts have been reported by Wedemeyer and Yasutake (1977) and Clarke et al. (1979) to be strong indicators of stress due to presence of toxi- cants or pollutants in the aquatic environment. Reduction

Fig. 2. Alteration in MCHC (a), MCH (b), MCV (c) plasma glucose (d) and total plasma protein (e) in Parachanna africans fol- lowing 21days exposure to the various concentrations of cadmium in the water. Each column represents the mean value and vertical bars indicate the standard error of the mean. Asterisks represent statistical significance at 0.05 level

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Conclusions

Haematological parameters related to oxygen transport (RBCC, Hct and Hb), defense mechanisms (WBCC) and the calculated indices showed overall differences between control and experimental groups. Therefore, haematologi- cal parameters are involved in the response of the African snakehead to cadmium under the experimental condi- tions. The present study thus confirmed that haematologi- cal parameters are very sensitive indicators of fish organ- ism response to chemicals in this case cadmium.

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