Healthy specimens of African catfish Clarias gariepinus were obtained from a fish farm in Ondo State, Nigeria. The choice of Clarias gariepinus was informed by its ability to withstand stress and its high commercial value in Nigeria. In the laboratory, fishes were kept in large plastic bowls of 120L capacity containing 60L of clean tap water and acclimatized for 6 weeks to the laboratory conditions, during which time they were provided with artificial feed (grower's mash) and ground shrimps obtained locally to avoid possible effects of starvation on any of the haematological parameters of the fish. The size of the fish varied from 18.1 – 22.7cm in standard length and 50.6 - 97.4g in weight. Fish of both sexes were used without discrimination. The fish were inspected for disease conditions and general fitness.

Water was changed every other day. The mean temperature, pH, alkalinity, hardness and dissolved oxygen of the water used were 27.4⁰C, 6.51, 193.3 mg/L (as HCO₃ ^-), 227.5mg/L as CaCO₃ and 6.56 mgO₂/L respectively. Ten fingerlings were kept per bowl. There were five different treatment groups and each had three replicates. The fish were fed three times daily. Feeding was discontinued while aeration continued during the 96-hr test period.

Stock solution of the test metal compound, a chemically pure nickel tetraoxosulphate IV hexahydrate (NiSO_4.6H₂O) was prepared by dissolving 4.5g of Merck grade reagent equivalent to 1 g of nickel in 1000 ml distilled water at concentration of 1000 mg/L. From the stock solutions, different concentrations required were prepared after a range – finding test using a screening procedure. The 96-hr LC₅₀ was found to be 8.87mg/L in C.gariepinus using the probit analysis method (Finney, 1971). The concentrations prepared for the experiment were: 4, 6,8,10 and 12 mg/L.

Five sets of ten fish each were subjected to serial dilutions of the stock solution of Ni (from 4mg/L – 12mg/L) in triplicates. Two sets of control (each consisting 10 fishes) which contains no toxicants were also set up. The test was performed by the semistic (renewal) bioassay method in which the exposure medium was exchanged every 24-hr to maintain toxicant strength and level of dissolved oxygen as well as minimizing the ammonia excretion levels during this experiment. Initially, the fish were observed at 1-hr intervals for the first 6-hr after which they were observed at 2-hr intervals. Dead fish were identified by an absolute lack of movement. They were removed as soon as this was noticed. No mortality was observed among control fish. The toxicity of the test chemical was determined using the logarithmic method of analysis (Litchfield and Wilcoxon, 1949).

The results of the physico-chemical parameters (mean values) measured are given in Table 1. Table 2 represents detail of mortality as recorded during the study. The estimation of the lethal concentration values (LC₅₀) was carried out using the Logarithmic method (Litchfield and Wilcoxon, 1949). The 96-hr LC₅₀ values were determined from the graph (Fig.1) to be 8.87mg/L. Fig.1 depicts the percentage mortality for different exposure periods at different concentrations of nickel sulphate (4.0 ppm - 12.0 ppm). The LC₅₀ value of NiSO₄.6H₂O for the fish C. garienpinus was determined by the simple graphic method (Fig.2). The calculated average LC₅₀ is 8.87mg/L. The equation for the dose-mortality regression line was found to be Y = 174.74X – 97.71. In the present study of C. garienpinus, it was observed that the behaviour and mortality rate was dependent on both duration of exposure and concentration of the toxicant. Even with the renewal bioassay method of study, using medium of equal physico-chemical characteristics and fishes of approximately similar sizes under the same experimental atmosphere, significant changes were observed behaviour of fishes.

Figure 1

Table 1: The physico-chemical characteristics of the water used

Figure 2

Table 2: Variable Zinc Concentrations and Mortality (N = 20)

Figure 3

Figure 1: Trend in fish mortality with duration of exposure to zinc sulphate.

Figure 4

Figure 2: Logarithmic graph of mortality pattern

A regular trend was generally observed in the mortality rate which increases with increased concentration. At the early stage (i.e. the first 24hr) of the toxicants introduction, all the fishes survive initial attack. This may be due to their protective adaptations and the hardy nature of C. gariepinus. During the second renewal (48-hr exposure) some damages or injuries were noticeable particularly amongst some fishes in the highest concentration (10ppm and 12ppm). These injuries are believed to weaken the organisms’ resistance to toxins and consequently resulting to significant death of 50% within the highest concentration. With progressive exposure spanny 72-hr, deaths becomes inevitable even at lower concentrations. This could be due to stress and cumulative impact of Ni-toxicity. Apart from least concentration (4ppm), death, though at different rates, were recorded at every other concentration.

Nickel toxicity to aquatic life depends on the species, pH, water hardness and other environmental factors (Blaylock and Frank, 1979). The water pH and hardness which increases with increased concentration of toxicants showed significant direct relationships with 96-hr LC₅₀ concentration of the fish. Skin damage i.e. body lesions associated with red spot disease as noticed especially after 72-hr with fishes within 8.0 – 12.0ppm is indicative of pH stress. It shows further that fish like C. gariepinus has limited tolerance to abnormal pH changes. The dissolved oxygen of the test medium decreased especially at high levels of toxicants. At higher 96-hr LC₅₀ values of Ni, dissolved oxygen decreased significantly in the test medium. According to Nebeker et al., (1985), nickel has been shown as moderately toxic to fish and aquatic invertebrates when compared to other metals. Newly fertilized rainbow trout eggs, exposed to 0.035 mg/L nickel, produced smaller larvae than when eyed eggs or pre-swim up larvae were exposed even up to 0.134 mg/L

Figure 5

Figure 3 : A typical behavioural changes in the exposed fish. Curling of Spine and vertical movement of the fish due to loss of equilibrium is depicted. Due to complete loss of equilibrium, fish turned upside down and finally died as can be noticed in the picture.

Efforts were made to carefully observe the behaviour of the fishes during the 96-hr study. Behavioural functions are generally quite vulnerable to contaminant exposures, and fish often exhibited these responses first when exposed to pollutants (Little et al., 1993). Behavioral changes such as curling of spine and vertical movement (Fig.4.3) of the fish was observed. This may be due to loss of equilibrium at high intoxication which makes the fish to turn upside down and finally died. Thus, swimming performance is considered one of the measures which could serve as possible sensitive indicator of sub-lethal toxic exposure. Various methods have been used to quantify the effects of toxics on an organism’s swimming performance (Rose et al., 1993). This kind of behavioural abnormality has been reported in various fish species on exposure to heavy metals (Little et al., 1993). Frequent surfacing with irregular opercular movement and loss of equilibrium in Tilapia mossambica has been reported when exposed to cadmium (Ghatak and Konar, 1990). Similarly, hyperactivity, erratic swimming, and loss of equilibrium in brook trout, Salvalinus fontinalis, in response to lead treatment have been reported (Holcombe et al., 1976). In addition, Singh and Reddy (1990) in their study on Heteropneustes fossilis, had reported lethargy response and frequent surfacing along with gulping of air in exposure to just 0.25ppm copper. According to Little et al. (1993) behavioural measurements may be useful indicators of sub- lethal contamination due to concentrations even being lower than those that affect growth. Behavioural changes usually occur much earlier than mortality.

Several factors have been attributed to behavioral changes/abnormalities in fish exposed to heavy metals like Ni (U.S. EPA,1986). These include nervous impairment due to blockage of nervous transmission between the nervous system and various effector sites, paralysis and depression of respiratory centre due to enzyme dysfunction, and alteration of energy pathway which results in energy depletion (Singh and Reddy,1990).

Bioaccumulation is not a valid criterion for judging the ecotoxicity of nickel substances because nickel is an essential element for many organisms and these organisms would suffer if they did not have the ability to accumulate and utilize nickel. Additionally, as a naturally occurring element, many organisms have mechanisms for detoxifying Ni through sequestration, thereby accumulating Ni in a non-toxic form. However, while the fish physiologically adapted to this environmental stressor, this trend does not always reflect a state of normality. The mortality recorded in the study is considered a consequence of stress induced on the immune system of fish. Thus, slow toxic progress and long continuance can result into chronic toxic response.