Introduction

The effect of heavy metals on aquatic organism is currently attracting wide spread attention particularly in studies related to industrial pollution. High toxicity of industrial pollutions have been known since long time, but their hazardous nature as pollution of aquatic environment has been matter of concern only after a large number of deaths of fishes occurring in different areas due to different metals.

Fish live in very intimate contact with their environment, and are therefore very susceptible to physical and chemical changes which may be reflect in their blood component (Wilson and Taylor,1993).In fish , exposure to chemical pollutants can induce either increase or decrease in haemotological levels. Early diagnosis is also possible when evaluating haematological data,

particularly blood parameters (Folmar,1993;Golovina,1996;Luskova,1997).Furthermore, should be noted that haematological indices are of different sensitivity to various environmental factors and chemicals (Lebedeva et al.,1998;Vosyliene,1999a and 1999a)

A survey of literature on heavy metals toxicity clearly shows that heavy metals cause several haematological and biochemical disorders both in laboratory animals as well as on aquatic organisms. The toxicity of lead, copper and other metals has been studied since mid 1920, (Qasim 1923, Dilling et al., 1926, Carpenter 1927, Cardift 1937, Ellis 1937 Laurie et al., 1938 Newton 1944, Jones 1947, Davidson 1949, Fry 1957, Cairns And Scheir 1958, Imanishi et al 1959, Hynes 1960, Lloyd 1961, Zavon 1964 ,and Ray et al., 1964) Sprangue (1970) Van Vuren (1986), Lebedeva et al;(1998), Vosy liene (1999), Olifia et al.,(2002) Andhrabi et al; (2006), Shah,(2006), Adeyemo,(2008). The present paper reports the sub-lethal effect of lead nitrate on haematological profile of Clarias batrachus (Linn.).

Materials and Methods

Alive, healthy and disease free fishes (Clarias batraclus weight 80-100gm and length 18-22cm) were collected from local fish market, Bhimavaram and bought to the laboratory. The fishes were kept in the glass aquarium to observe any visible pathological symptoms. Before introducing into the aquarium fishes were treated with 0.1% KMno₄ solution to obviate any dermal infection. Then the fishes were acclimatized in laboratory condition for a period of one weak. No mortality was recorded during this period. The fishes were fed chopped meat daily during acclimatization.

Lead nitrate ( Pb (No₃ )₂ ) were used for the preparation of various concentration (stock solution) by adopting the dilution techniques. Adequate quantity of distilled water was used to get the required concentration. Sub-lethal level of the above metal was determined on the above said species by the prohibit analysis method (Finney, 1971). Sixteen fishes were exposed to sub- lethal concentration for 8, 16 and 24 hours under acute studies. While chronic studies were also conducted for 45 days. Haematological studies were made after 15, 30, 45 days of exposure. Blood was drawn from posterior caudal vein according to Schmitt et al., (1999). In acute and chronic studies feeding was stopped one day before the experiment and under chronic studies refeeding was done after one day of exposure.

Large size glass aquaria were chosen to avoid space problem to fish, Various water quality parameters such as water temperature, hydrogen ion – concentration (PH), dissolved oxygen, carbon dioxide, total alkalinity, calcium hardness and total hardness were analyzed before sacrifice of fish. Water quality parameters were analyzed by following the procedure of APHA (2000).

Fish behavior was observed and recorded accordingly. Control groups were maintained for all the above experiments, after exposure the fish was sacrificed for haematological examination. Each experiment was done in triplicates.

Statistical Analysis

Results

Acute studies

Serum protein profile

Chronic studies

Serum protein profile

Physico-chemical parameters

Discussion

Aquatic environment is constantly polluted from a variety of sources and presently it has assumed a dangerous proportion for aquatic life and fish species are no exception. In reality, heavy metals intoxication cause deleterious effect such as enzyme inactivation, reduction in RBC’s life span and haemoglobin surface area, mitochondrial dysfunction, breakage of genetic material, interference with immunology, alterations in haematological and biochemical organization of different fish species.

Among the heavy metals, lead is one of the metal known to man since medieval times. It is a non - essential element being released into the media either terrestrial or aquatic and is causing several toxicological problems to aquatic animals and man. The natural water is continuously being contaminated by lead due to increase anthropogenic activity and industrial exploitation of metal (Chandravathi and Reddy, 1996). The harmful effects caused by lead include haematological, biochemical and physiological alterations in several aquatic species (Chandravathi and Reddy, 1996).

The uptake and accumulation of lead by aquatic organisms from water and sediment are influenced by various environmental factors such as temperature, salinity, pH, dissolved oxygen, alkalinity, hardness etc. in sediment, only a minor fraction is dissolved in water. Lead is accumulated mostly in gill, liver, kidney and bone, fish accumulates lead from water as well as sediments, aquatic uptake is influenced by presence of cation and oxygen content of water (IPCS, 1989). Heavy metals probably exact their toxic effect on fish by reacting with the mucous on the surface of the gills causing precipitation, coagulation and thus interfere with the normal exchange of gases (Carpenter 1927, 1930). The toxic effects of heavy metals on fish are multidirectional and manifested by numerous changes in the physiological and chemical process of their body system (Dimitrova et al., 1944). Sub-lethal toxicity of lead to fish produces haematological and neurological effects (Hodson et al .,1984). Literature shows the changes in haematological indices of fishes caused by heavy metals and their mixture are different. They are pre determined both by the concentration of heavy metals in the water and time of exposure, and both this factors can cause reversible and irreversible changes in the homeostatic system of fish. Haemoglobin concentrations reflect the supply of an organism with oxygen and the organism itself tries to maintain them as much stable as possible.

In present study it has been observed that, in the exposed fishes the number of RBCs and haemoglobin percentage decreased significantly from normal values. However, the differential leucocyte counts were deviating significantly from normal values. The increase was observed in the number of lymphocytes and eosinophils while decrease was noticed in the number of monocytes and neutrophils. the total serum protein was also decreased as compared to normal value. This is in the agreement with the work of Shan (2006) and Olanike K. Adeyemo et al (2008).

Figure 1

Figure 1: showing RBC count and Haemoglobin percentage of lead nitrate Exposed in (Linn) with (10 mg/l) for 8, 16, 24 Hours

Figure 2

Figure 2: showing RBC count and Haemoglobin percentage of lead nitrate Exposed in (Linn) with (50 mg/l) for 8, 16, 24 Hour

Figure 3

Figure 3: showing RBC count and Haemoglobin percentage of lead nitrate Exposed in (Linn) with (100 mg/l) for 8, 16, 24 Hours

Figure 4

Figure 4: showing Leucocyte differential count of lead nitrate exposed in (Linn) with (10 mg/l)for 8, 16, 24 Hours

Figure 5

Figure showing Leucocyte differential count of lead nitrate Exposed in (Linn) with (50 mg/l) for 8, 16, 24 Hour

Figure 6

Figure 5 showing Leucocyte differential count of lead nitrate Exposed in (Linn) with (100mg/l) for 8, 16, 24 Hours

Figure 7

Figure 6 showing Albumin, Globulin and Total serum protein of lead nitrate Exposed in (Linn) with (10 mg/l) for 8, 16, 24 Hours

Figure 8

Figure 7 showing Albumin, Globulin and Total serum protein of lead nitrate Exposed in (Linn) with (50 mg/l) for 8, 16, 24 Hours

Figure 9

Figure 8 showing Albumin, Globulin and Total serum protein of lead nitrate Exposed in (Linn) with (100 mg/l) for 8, 16, 24 Hours

Figure 10

Figure 9 showing RBC count and Haemoglobin percentage of lead nitrate exposed in (Linn) with (10 mg/l)for 15,30,45 days .

Figure 11

Figure 10 showing Leucocyte differential count of lead nitrate exposed in (Linn) with (10 mg/l)for 15,30,45 days .

Figure 12

Figure 11 showing Albumin , Globulin and Total serum protein of lead nitrate exposed in (Linn) with (10 mg/l)for 15,30,45 days .

Figure 13