1401
ABOU HADEED, A. H et al.
EXPERIMENTAL STUDIES ON NICKEL TOXICITY IN NILE TILAPIA HEALTH
ABOU HADEED, A. H.1, K. M. IBRAHIM1, N. I. EL-SHARKAWY1,
F. M. SALEH SAKR2 AND S. A. ABD EL-HAMED.2
1- Faculty of Vet. Med. Forensic Medicine and Toxicology dept.
2- Central Lab. for Aquaculture Researches (Abbassa)
Abstract
Ths work was planned to investigate the toxic effect of nickel chloride on health status of widely cultured fresh water fish tilapia species. one hundred and twenty nile tilapia fish divided into 3 groups; the first group exposed to 1/5 lc50 (7.2 mg/l) of nickel chloride for one week, the second group exposed to 1/10 lc50 (3.6 mg/l) of nickel chloride for 8 weeks. the third group left as control. the results showed respiratory disorder, abnormal swimming behavior, rapid opercular movements and skin lesion besides white discoloration of skin. blood parameters of tilapia species exposed to 1/5 lc50 of nickel chloride for one week revealed increase in rbcs count, decreased hb, mch and wbcs count. tilapia fish which exposed to 1/10 lc50 for 8 weeks showed increased rbcs count and hb content and decreased mch and wbcs count. the liver and kidney functions of tilapia fish exposed to 1/5 lc50 of nickel chloride for one week and 1/10 lc50 of the same compound for 8 weeks revealed increased alt and decreased ast and total protein. the residual analysis of nickel fish tissues exposed to 1/5 lc50 and 1/10 lc50 of nickel chloride revealed increased residues in gills, liver and muscles in different values depending on the used concentration and time of exposure. the residue of nickel increased in the liver more than gills and muscles. the histopathological investigations of tilapia species exposed to nickel chloride revealed pathological tissue alterations in the gills, liver, kidney, spleen, intestine. the gills showed hyperplasia, edema and complete sloughing of the secondary lamellae. the liver showed congestion of the central vein, vacuolar degeneration of hepatocytes and periductal fibrosis. the kidney showed alternative areas of activation and depletion of hemopoietic elements and condensed glomeruli with edema in bowaman’s capsule. in the spleen, depletion of large area of hemopoietic elements and multiple melanomacrophag cells were encountered while the intestine showed mucus cell metaplasia, submucosal edema and round cell infiltration. it is concluded that important trace metals including nickel altered physiological function results, when one or m0re of these reach sufficiently high concentration in cells.
Introduction
Metals are commonly found in the environment, they are present as a natural elements or as a result of anthropogenic activities in different environmental media such as air, water and soil, which constitute an important factor of exposure to animals and human (Louis, 1993). Heavy metals are considered as one of the most important factors which affect fish population, reducing their growth, reproduction and/or survival rate (Mohamed and Saleh, 1996 and Saeed, 2000). Nickel is one of the microelements which occur in trace amounts in living organisms. It constitutes a potential hazard to the enviornment media (air, water and soil). This is due to its extensive and wide spread utilization in various industries, it is a common by-product of electroplating industries, steel production, metal mining, smelting, refining,ceramic and processing along with fuel combusation, and waste incineration activities. Effluents that spread to streams, rivers and lakes may disrupt the integrity of the aquatic environment. Excess of nickel contamination is a real hazard to aquatic ecosystems due to its persistence and bioaccumulation (Atchison et al., 1987). In recent years, production of electroplating factories contains high concentration of heavy metals, including high concentrations of nickel (Wong and Wong, 1990 and WHO, 1991) was sharply increased.
Environmental exposure to nickel occurs through inhalation, ingestion, and dermal contact. The general population is exposed to high levels of nickel because it is widely present in air, water, food, and consumer products. The general population is exposed to nickel in nickel alloys and nickel-plated materials such as coins, steel, and jewelry, and residual nickel may be found in soaps, fats, and oils (ATSDR 1997). In aquatic systems nickel is adsorbed on clay particles of organic matter (algae, bacteria) and invertebrates. Since invertebrates are major food resource for fish, they constitute an important link in nickel transport chain to fish (Wong et al., 1991).Also it induce decrease in body weight of Oreochromis niloticus fish (El-Saieed and Mekawy 2001).Little studies have investigated nickel uptake in fish through aqueous and dietary exposure. Further research investigating the exposure of fish to dietary nickel which is needed to elucidate the potential impacts of chronic dietary nickelckexposure on natural populations of fresh water fish (Ptashynski and Klaverkamp, 2002). Tilapia fish ere selected as a research fish model because these fish were easily produced and economically important. Fish are known by their tendency to localize significant amounts of metals. They absorb metals from water through gills, skin and digestive tract. Bioconcentration and biomagnificantion for heavy metals were previously reported by many authors (Saeed, 2000). This study aims to describe the clinical signs, postmortem lesions and histopathological changes due to nickel toxicity beside estimation of alteration in hematological, biochemical parameters and bioaccumulation and distribution of nickel in Nile tilapia organs.
MaterialS and Methods
Experimental design
A total number of 120 Tilapia fish (Oreochromis niloticus) were divided into 8 groups kept under optimal environmental condition. The groups were treated as following:
Experiment 1: (short term exposure or acute intoxication)
This group contains 40 fish, divided into two subgroups, one of which kept as control and the other group exposed to 1/5 LC5 (7.2 mg/L) of nickel chloride for one week according to WHO (1991).
Experiment 2: (Long term exposure or sub chronic intoxication)
In this experiment 80 tilapia used where 40 of which were kept as control in 4 glass aquaria and the other fishes were divided into 4 subgroups in 4 glass aquaria containing 1/10 LC50 (3.6 mg/L) of nickel cholride for 8 weeks. The collected fish in both acute and sub chronic toxicity were examined clinically using the methods described by Lucky (1977) and Noga (1996).
II- Laboratory analysis:
a- Hematological investigations
Blood samples were taken from caudal vein of experimental Tilapia (20 fish) after one week by syringe using heparin as anticoagulat (1000 unit/ml blood), from both control and nickel exposed groups for acute and (32 fish) for subchronic intoxication. 8 fish each two weeks (After 2, 4, 6 and 8 week) 0.5 ml blood was used for determination of different blood parameters (erythroyctic count, Hemoglobin concentration, leucocytic count ) (Lied et al., 1975).
b- Serum Biochemical analysis
Blood was collected in plain centrifuge tubes; centrifugated at 3000 r.p.m. for 15 minutes for serum separation for determination of serum transaminases and total protein. Time of collection of serum and number of fish as in hematological investigations
c- Histopathological examination
Specimens from the liver, spleen, gills, kidney and intestine (12 fish) were collected from both control and treated fish one week from acute intoxication. The same samples were taken at 2nd, 4th , 6th, 8th weeks from sub chronic (24 fish) intoxicated fish for the microscopic changes.
The collected Tissue specimens were fixed in 10% buffered formalin solution. Then, dehydrated in ascending concentrations of ethyl alchol, embedded in melted paraffin wax, blocked in hard paraffin, sectioned at 4-5 microns and stained by hematoxylin and eosin stain according to Carleton et al. (1967).
d- Residual analysis
Muscle, gills and liver (5 g dry wt for muscle and gills and 1 g for liver) from twenty eight fish of the control and treated fish after one week and 24 fish for subchronic were kept frozen at -20ºC till analysis. Nickel was extracted according to (Analytical Methods For Atomic Absorption spectrophotometry, 1982).
Statistical analysis
The data obtained in this study were statistically analysed using analysis of SPSS (Independent sample test) according to Snedecor and Cochran (1969) for comparing the different mean values with Duncan´s multiple range test by Duncan´s.
RESULTS AND DISCUSSION
Nickel compounds are known to be human carcinogens based on sufficient evidence of carcinogenicity from studies in humans, including epidemiological and mechanistic information, which indicates a causal relationship between exposure to nickel compounds and human cancer. The findings of increased risk of cancer in exposed workers are supported by evidence from experimental animals that shows that exposure to an assortment of nickel compounds by multiple routes causes malignant tumors to form at various sites in multiple species of experimental animals (Tenth Report on Carcinogens 2002).
Clinical signs and post-mortem findings
The short term exposure of the fish to nickel chloride (7.2 mg/l) for one week the exposed fish showed changes in their behavior as the fish were immobile, gathered near the bottom and delayed reactions to light and sound. In the group exposed to 1/5 lc50 (3.6 mg/l) of nickel chloride along two months showed respiratory manifestation characterized by surface swimming and white discoloration of the skin (photo 1) and gasping. The Post-mortem examination revealed paleness of the gills, and kidney while congestion in liver and distended gall bladder was apparent. The above-mentioned clinical signs and post-mortem finding were more severe and prominent in fish exposed to 1/10 Lc50 (3.6 mg/L) of nickel chloride for two months (photo 2).
The clinical symptoms recorded during our study were manifested by respiratory disorders in fish exposed to nickel chloride along 2 months especially after 6th and 8th weeks. This could be attributed to the nickel nature as a respiratory toxicant, causing decrease in arterial oxygen tension, an increase in arterial carbon dioxide tension and a subsequent respiratory acidosis. The white skin lesion in the present work is similar to these results observed by El-Saieed and Mekawy (2001). This pattern may be attributed to nickel and their water-soluble salts which are potent skin senstizers induce skin irritation as studied by Menne, et al. (1982).
In the present study, the haemoglobin (Hb) value in fish exposed to 1/5 Lc50 (7.2 mg/ L) for one week was decrease nonsignificantly and in fish exposed to 1/10 Lc50 (3.6 mg/L) of nickel chloride decrease significantly in 2nd week and increase non significantly in 4th, 6th and 8th weeks comparing with control (Table 1).
Table 1. Effect of nickel chloride on heamoglobin, erythrocyte count (RBCs) of Tilapia fish after exposure to nickel chloride acute and subchronic for 8 weeks (Mean ± SE).
Time of exposure / No. of fish group / Hb g/dl / RBCs counts x 106 µcontrol / Acute / Sub
chronic / control / Acute / Sub
chronic
1st week / 20 / 7.5±0.06 a / 6.3±0.03 a / 1.193±23.4 a / 1.373±62.6 a
2nd week / 10 / 7.63±0.32a / 6.5±0.05 b / 1.26±11.6 a / 1.66±23.01 a
4th week / 10 / 7.5±0.31 a / 7.66±0.40 a / 1.47±10.7 b / 2.27±8.83 a
6th week / 10 / 8.06±0.19 a / 8.5±0.41 a / 1.14±9.06 b / 1.61±8.46 a
8th week / 10 / 8.4±0.1 a / 9.14±0.49 a / 1.69±0.33 a / 1.96±11.83 a
Means in the same row carrying different superscript are significant at P0.05. a :increase, b:decreased
These results agree with that obtained by Sobecka (2001). The decrease of hemoglobin may be attributed to the destructive influence of nickel on the cell membranes of erythrocytes through binding of the toxicants with immunoglobulins or through disturbance of the activity of erythrocyte enzymes, especially those responsible for reduction of glutathione and thiol groups of proteins (Sun et al. 1985). According to Kleczkowski et al. (1998), the excessive loss of glutathione, increased release of iron to intracellular spaces, peroxidation, destruction of cell membranes, and release of metal ions to the surrounding tissues should be attributed to free oxygen radicals. The effect of the described processes is instability of hemoglobin, structural changes in erythrocytes and increased susceptibility to hemolysis. Consequently, the pool of the serum iorn from disintegrating erythrocytes increases,while the iron content in the spleen decreases. In contrast to our results, Ptashynski and J. F. Klaverkamp (2002) observed that, concentration of hemoglobin, value was unaffected between control and treated lake white fish “Coregonus clupeaformis” and lake trout “salvelinus namaycush” by exposure to nickel in diet 0, 10, 100 and 1000 µg for 10, 51, 104 days.
Agrawal et al. (1979) studied that, colisa fasciatus, a fresh water teleost, were exposed for 90 hrs to 45 p.p.m nickel sulphate under static test conditions. The treatment resulted in increase in hemoglobin value, this difference may be due to variation in dose, and fish species and duration of treatment and time of adimstration.
The RBCs count is highly affected by high concentrations and long time of exposure to nickel. The increase of RBCS count started from the 1st week of exposure till the end of the exposure time (Table 1).These results agree with that obtained by Agrawal et al., (1979). This result may be attributed to that nickel induced hypoxia. These finding are consistent with the mechanism of adenergically stimulated splenic contraction to release supplemental erythrocytes into the circulatory system to increase oxygen carrying capacity. This explanation agrees with Perry and Wood (1989).
The calculated blood indices MCH have a particular importance in describing anemia in most animals (Coles, 1986). The decrease of MCH along the experimental periods (Table 3) may be attributed to the disturbances in RBCS count and Hb content and also to the exaggerated disturbances that occurred in both the metabolic and hemopoietic activities of fish exposed to sublethal concentrations of pollutants (Mousa, 1996).
Table 2. Mean cells hemoglobin, leucocytic count (WBCs) of Tilapia fish after exposure to nickel chloride either acute 7.2 mg/L (1/5 LC50) for one week or subchronic 3.6 mg/L (1/10 LC50) for 8weeks (Mean ± SE).
Time of exposure / No. of fish group / MCH (pg/cell) / WBCs counts x 103 µcontrol / Acute / Sub
chronic / control / Acute / Sub
chronic
1st week / 20 / 51.092±12.34a / 38.8±0.519a / 33.66±5.811a / 30.33±6.06a
2nd week / 10 / 54.49±7.925a / 24.31±0.76b / 35.00±6.928a / 24.66±4.66a
4th week / 10 / 52.55±4.67a / 45.22±5.921a / 34.66 ±6.35a / 25.66±2.96a
6th week / 10 / 70.133±13.39a / 40.63±4.184a / 36.33±6.64a / 24.66±7.7 a
8th week / 10 / 31.87±0.274a / 31.6±3.62a / 36.33±6.38a / 31.33±11.62 a
Means in the same row carrying different superscript are significant at P0.05. a :increase, b:decreased