Eduved ISSN 2348-6775 (Online)

Edu ed v ISSN 2348-6775 (Online)

International Journal of Interdisciplinary Research 2349-5480 (Print)

SOME HAEMATOLOGICAL STUDIES IN CLARIAS BATRACHUS

AFTER INTOXICATION WITH CADMIUM CHLORIDE AND

MERCURIC CHLORIDE IN COMBINATION WITH ANTIOXIDANT

ABSTRACT

Heavy metals and their salts cons�tute a very important group of environmental pollutants since they are potent

metabolic inhibitors. The inherent toxicity of metal depends upon its capacity to disturb the dynamic life processes in

biological system by combining with cell organelles, macromolecular and metabolites. Toxic effects of heavy metals can

be modulated through an�oxidant therapy. The present study highlights the use of an�oxidant ascorbic acid to

modulate the toxicity induced by cadmium chloride and mercuric chloride with respect to haematology.

INTRODUCTION

Heavy metal such as lead, mercury, chromium, cadmium, zinc etc. are neither created nor be destroyed in significant

amount in the environment so they persist forever. In small quan��es certain heavy metals are nutri�onally essen�al

for a healthy life, but they have poten�al to become major pollutant in the environment. These heavy metal become

toxic when they are not metabolized by the body and accumulated in the so� �ssue. Most of metal in environmental

media is of anthropogenic sources and its exposure mainly occurs through primarily air, drinking water, pes�cides,

ba�eries, piping, paints, Ni-Cd ba�eries, motor oil, rubber goods etc.

Cadmium and mercury has a�racted the a�rac�on of many workers to evaluate the toxicity level and their biochemical

and histopathological impact on various organs of fish. Some researchers have given emphasis to inves�ga�ons with

data for treatment, which is not only cura�ve but also prophylac�c such as the case of use of vitamins on the

supplementa�on of commercial diets. Among these inves�ga�ons, the use of vitamins as A, D, C and E is paramount,

because they are closely related to the performance of fish immune system. The need for the vitamins differ according

to the species, age and raising period, and the research in this field is s�ll much limited.

Ascorbic acid has a wide range of benefical proper�es like it detoxifies the liver of the fishes, heals wounds, effec�ve in

comba�ng lateral line disease when used in the proper amount prevent large degree of bacterial infec�on and build up

and strengthens the immune system of the fishes by allowing the evacua�on of heavy metals. Keeping this in view the

present study has been undertaken and it embodies the results of the experiment conducted and facts observed on

Clarias batrachus, as a representa�ve live fish, subjected to various limits, acute, sub-lethal and chronic doses of the

aforesaid heavy metals and their detoxifica�on with an�oxidant. Blood is a good bioindicator or a diagnos�c tool to

study the problem in organ func�on. The measurement of haematological changes in blood of fish under exposure to

any toxicant may be used to predict effects upon acute, sub-lethal and chronic exposure.

MATERIALS AND METHODS

Clarias batrachus were collected from the local fish market. They were carefully examined for any injury and then kept

in one percent solu�on of potassium permagnate for few minutes to get rid off any dermal infec�on. Finally they were

stored in large glass aquaria measuring 75 cm. X 37.5 X 37.5 cm. and fed on boiled egg yolk and fish food. Clarias

batrachus, is a species of freshwater airbreathing ca�ish found primarily in Southeast Asia. Although known in English

as "walking ca�ish", its "walk" is o�en just a sort of wriggling mo�on with snakelike movements, using its pectoral fins

to keep it upright. However, it can use its tail so vigorously that it hops like a one-legged frog. Walking ca�ish are around

30 cm (a foot or so) in length and have an elongated body shape. This ca�ish has long-based dorsal and anal fins as well

Sushi�ma Singh Department of Zoology, R.B.S. College, Agra

Department of Zoology, R.B.S. College, AgraK.K. Gaur

Vol. 3 issue 04 April 201622

as several pairs of sensory barbels. The skin is scaleless but covered with mucus, which protects the fish when it is out of

water.

Experimental metallic compounds and an�oxidant:

Cadmium chloride : Cd-Cl₂

Mercuric chloride : Cl₂-Hg or Hg-Cl₂

L-ascorbic acid : C₆-H₈-O₆

Experimental Protocol: To assess the effect of experimental compounds on Clarias batrachus, the following 7 sets were

established with each set consis�ng 5 fishes-

1. Control set (without any compound)

2. Cadmium chloride treated set

3. L-ascorbic acid+cadmium chloride treated set

4. L-ascorbic acid pellet+cadmium chloride treated set

5. Mercuric chloride treated set

6. L-ascorbic acid+mercuric chloride treated set

7. L-ascorbic acid pellet+mercuric chloride treated set

Double oxalate vials were used for storing the blood samples for various haematological studies. 800 mg potassium

oxalate and 1200 mg ammonium oxalate was dissolved in 100 ml dis�lled water. One drop of this solu�on was added to

each empty sterilized vial, shaken and dried in the oven at 800C. Five fishes from each set were sacrificed for the

morphological studies of blood a�er 4 days, 20 days and 60 days. The blood was collected a�er severing the caudal

peduncle of the living fish by a scissor. In each vial two to three drops of an an�coagulant EDTA were added prior to

collec�on of blood.

Haematological studies: The haemoglobin concentra�on of blood was es�mated by the Standard Sahli's method out

lined by Wintrobe (1968). Total leucocytes were counted by using improved Standard Neubauer Haemocytometer

(Dacie and Lewis, 1968) and Wintrobe's method. The packed cell volume or haematocrit value expresses the

percentage sample occupied by the erythrocytes. It was determined by Wintrobe (1968).

RESULTS AND DISCUSSION

In Clarias batrachus, the toxicity of metal leads to enormous extrusion of mucous which prac�cally covers the fact that

mucous hinders the exchange of gasses resul�ng in the deficiency of oxygen in the blood which in turn effects the

general physiology of the body and results in death, almost similar observa�ons have been made by Sastry and Siddique

(1982) in Channa punctatus in response to the toxicity of carbamate pes�cide Sevin. Fish behaviour in different test

concentra�ons of insec�cides were observed and it was found that each group of fish exhibit certain physical reac�ons,

fish swims very rapidly with pronounced excita�on followed by jerky movement of the body and made jumped

frequently. Present findings gain support with the finding of Dalela et al. (1978) and Verma et al. (1976). In the present

study metallic toxicity stress are altered by the use of an�oxidant as reported by Agarwal et al. (1978).

The haemoglobin concentra�on showed a decreasing trend in 4, 20 and 60 days on treatment of cadmium chloride and

mercuric chloride as compared to control. The same result were observed by Rai and Qayyam (1984) in Catla catla due

to intoxica�on of lead; Thakur and Sahai (1987) in Channa punctatus exposed to BHC; Garg and Tyagi (1989) in

Heteropneustes fossilis due to manganese poisoning, Goswami and Du�a (1991) in Heteropneustes fossilis due to vit. A

deficient diet; while Singh and Shrivastava (1991) in Heteropneustes fossilis due to propoxur toxicity; Nath and Banerjee

(1995) in Heteropneustes fossilis treated with devithion; Singh (1995) in Channa punctatus due to copper sulphate and

potassium dichromate poisoning; Raizada and Rana (1998) in Clarias batrachus, Gupta and Gupta (2000) in

Heteropneustes fossilis, Ananadkumar et al. (2001) in Heteropneustes fossilis, Saxena and Seth (2002) in Channa

punctatus a�er cypermethrin treatment, Das et al. (2004) a�er nitrate toxicity in Labeo rohita. Masud et al. (2005) in

Cyprinus carpio following mercuric chloride intoxica�on, Kumar et al. (2006) in Clarias batrachus and Singh and Singh




(2007) in Heteropneustes fossilis.

The reduc�on in haemoglobin content may be due to the forma�on of a mucous envelope on the en�re body surface

and par�cularly on gills. Bha�acharya (1995) also assigned the similar reason for the decrease in haemoglobin content

due to cadmium treatment in fishes.

Toxicity of mercury and cadmium reduces when treated with ascorbic acid which is be�er than mercury or cadmium

alone and less than control in 4, 20 and 60days. This reduc�on in toxicity with ascorbic acid supplementa�on was also

reported by Shalaby (2001) in Oreochromis nilo�cus against mercury intoxica�on. The animals fed with the diets only

containing iron showed a slightly lower value of haemoglobin in ca�ish. Further, ascorbic acid without iron can increase

a mycrocit anemia rate. Thus, the decrease in levels of iron can be occur by deficiency in food iron and supplement in

ascorbic acid, because this vitamin have an important role in iron libera�on of ferra�n to liver and transport of this to

plasma as reported by Lim et al. (2000). This support the preven�ve ac�on of ascorbic acid used in the present study.

The data has also revealed that the toxicity of mercuric chloride is more as compared to cadmium chloride and more

recovery with ascorbic acid pellet may be due to its high dose as compared to ascorbic acid dissolved in water directly.

The total leucocyte count showed decreasing trend in 4, 20 and 60days on the treatment of cadmium chloride and

mercuric chloride exposure as compared to control. The same result were observed by Garg and Tyagi (1989) in

Heteropneustes fossilis due to manganese poisoning, while Goswami and Du�a (1991) in Heteropneustes fossilis due

to aldrin and fenvalerate intoxica�on; Saxena and Chauhan (1994) in Heteropneustes fossilis due to copper sulphate

intoxica�on; Nath and Banerjee (1995) in Heteropneustes fossilis treated with devithion; Singh (1995) in Channa

punctatus due to copper sulphate and potassium dichromate induced toxicity, Raizada and Rana (1998) in Clarias

batrachus, Gupta and Gupta (2000) in Heteropneustes fossilis, Ananadkumar et al. (2001) in Heteropneustes fossilis,

Kumar et al. (2006) in Clarias batrachus, Sanjib and Ashok (2006) in Heteropneustes fossilis a�er chromium sulphate

treatment and Singh and Singh (2007) in Heteropneustes fossilis. Similar results were obtained by Hudercova and

Ginter (1992) who recorded increase in the biochemical stress by heavy metal due to deteriora�on of metabolic

cascade.

An�oxidant ac�vity of ascorbic acid makes it a hunter of free radicals, thus preven�ng the autointoxica�on of

immunological cells such as macrophages which are the first precursors of the informa�on about the alien bodies and

maximizing the defense of fish as stated by Brake (1997) in fish. Qinghui et al. (2004) also assigned similar reason for

decreased TLC and its normaliza�on by ascorbic acid supplementa�on which improves immune system to counteract

the stress of heavy metals in Japanese seabass, Lateolabrax japonicus.

Toxicity of mercury and cadmium reduces when treated with ascorbic acid show be�er result than mercury or cadmium

alone and more than control in 4, 20 and 60days treatment.

The packed cell volume showed decreasing trend on the treatment of both metals cadmium chloride and mercuric

chloride in 4, 20 and 60days exposure as compared to control. The present results find support from the observa�ons of

Mishra and Shrivastava (1979) in Colisa fasciatus due to zinc sulphate; Garg and Tyagi (1989) in Heteropneustes fossilis

due to manganese; Singh and Shrivastava (1992) in Heteropneustes fossilis due to propoxur intoxica�on; Nath and

Banerjee (1995) in Heteropneustes fossilis treated with devithion; while Singh (1995) in Channa punctatus due to

copper sulphate and potassium dichromate poisoning respec�vely; Thakur and Bais (2000) in Heteropneustes fossilis

due to aldrin and fenvalerate intoxica�on, Dhanapakin and Ramaswamy (2001) in Cyprinus carpio, Gupta et al. (2001) in

Heteropneustes fossilis exposed to washing effluents, Nussy et al. (2002) in Oreochromis mossambicus exposed to

copper and zinc mixture, Tripathi and Verma (2004) a�er endosulphan intoxica�on in Clarias batrachus, Acharya et al.

(2005) in Labeo rohita and Johal et al. (2007) in Cyprinus carpio respec�vely.

There may be three possible causes for the decrease of the hematocrit (PCV) during the stress: increase on the volume

of the plasma, loss of water in the erythrocyte and hemolysis of the erythrocytes in the blood stream. The response to

stress is characterized by hormone change (catecholamine and cor�costeroid) that induce altera�on on the

hematological parameter, plasma ion and so on. Dugan and Moon (1998) and Ruane et al. (1999) have also confirmed

the similar reason for decreasing packed cell volume in fish. The obtained results revealed that toxicated fish show an

increasing trend in 4, 20 and 60days treatment in the PCV when treated with ascorbic acid and reduce the toxicity of




metal which show be�er result than mercury and cadmium alone treatment as compared to control. Supplemen�ng

ascorbic acid to the diet of fishes has shown its higher efficiency on the produc�on of an�bodies in the phagocyte

ac�vity, lysozine level to fight bacteria in addi�on to the posi�ve role it plays on the stress reduc�on (Montero et al.,

2001).

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TABLE-1 HAEMOGLOBIN CONCENTRATION (mg/dl)

TABLE-2 TOTAL LEUCOCYTE COUNT (109/L)




TABLE-3 PACKED CELL VOLUME (%)

* Non-significant (P>0.05)

** Significant (P<0.05)

*** Highly significant (P<0.01)

**** Very highly significant (P<0.001)







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Eduved ISSN 2348-6775 (Online)