MATERIALS AND METHODS

Analytical Methods
The 96 h median lethal concentration (96 h LC₅₀) of Cu, Zn and Pb were prepared prior to laboratory experiment. The 96h LC₅₀ values obtained (1.819 mg L^-1 for Cu, 5.906 mg L^-1 for Zn and 2.731 mg L^-1 for Pb) were then used to design a sub-lethal concentration whereby the 96 h LC₅₀ value was reduced to 25% from highest concentration towards the lowest concentration. A flow through system was applied with 15 fingerlings of tilapia species have been exposed in each tank with tri-replicate for 21 days. At interval 7, 14 and 21 days, fishes were harvested for heavy metal analysis.

Open Acid Digestion
An Inductively-Coupled Plasma Mass Spectrometer (ICP-MS) was used for the quick and precise determinations of Cu, Zn and Pb in the digested fish gills (Vijayendran et al., 2006). Briefly, the killed fishes were first cleaned with de-ionized distilled water and the total length and body weight were then measured. The gills obtained from the fish were vigorously rinsed in deionised water, bottled and immediately stored at –70°C to avoid any contamination. The crushed dry tissue were placed in a mixture of 1 mL concentrated HCI and 1 mL 65% HNO₃ and left to preliminary digestion for 3 h in the teflon beaker. The samples mixture were then heated on the hot plate until completely digested. One milliliter of H₂O₂ was added to the sample in order to break down any recalcitrant lipid material. After cooling to the room temperature, the content of the vessel was thoroughly transferred into a 50 mL polypropylene test tube with deionized double distilled water. A clear solution with no residue was obtained at this stage. The precision assessed by replicate analyses was within 3%. The accuracy was also examined by analyzing, in duplicate a dogfish muscle tissue (DOLT-3) and the results coincided with the certified values within a difference of ±3%.

Statistical Analysis
Statistical analysis of data was carried out using Microsoft Excel programme. Data obtained from the ICPMS reading were plotted on graphs to check the patterns of different heavy metal accumulation in fingerlings. Normal distribution or close to normal distribution was observed and hence, no transformation were done for statistical analysis. The obtained data were also compared by two ways Analysis of Variance (ANOVA) with blocking.

RESULTS

This experiment was designed to measure the accumulation of lead, copper and zinc in the tilapia fish gills after exposure for 21 days. Analysis of the certified materials (National Research Council Canada) of all 3 heavy metals was found to be within 10-25% of expected values (75-90% recovery). For the Cu, there was no much differences between mean metal accumulation at 7 and 14 days. The accumulation rate was between 0.01 to 0.02 μg kg^-1 at the highest concentration. However, there was a steady increase occurred when exposure period was extended for another 7 days, whereby at 21 days, 0.06 μg kg^-1 of Cu was detected in the gills of the fish (Fig. 1).

Although, there was a steady increase in the accumulation level of Zn noted at all the time between 7 and 14 days (the accumulation rate was 500 μg kg^-1), dramatic increase of Zn was noted at 21 days of exposure (3000 μg kg^-1) (Fig. 2).

The fish had accumulated up to 142 μg kg^-1 of Pb at the highest concentration in 30 days of exposure. There is no much differences between the metal uptake of tilapia fish at the highest concentration in 10 days and 20 days whereby 50 μg kg^-1 of lead was reported at day 10 and it increased only 2 to 52 μg kg^-1 at 20 days of exposure. However, there was an elevated increase in metal uptake reported at the days of 30 where the concentration has increased nearly 3 folds upto 142 μg kg^-1 (Fig. 3).

Fig. 1:	Mean metal concentration of Cu in gills of Tilapia fingerlings during different days of exposure

Fig. 2:	Mean metal concentration of Zn in gills of Tilapia fingerlings during different days of exposure

Fig. 3:	Mean metal concentration of Pb in gills of Tilapia fingerlings during different days of exposure

Overall, there was no statistically significant correlations (p>0.05) between days of exposure and the bioaccumulation of metal in the body of the fish but there were statically significant statistical differences was observed when different concentration of toxicant was exposed to the fish.

DISCUSSION

It was observed that fish responded more towards the increased concentration of selected heavy metals than the time frame of exposure. It might be due to, when a toxic substance is introduced in to the environment, 3 major steps can be identified before the fish start to show response to the pollutant. They were:

So, based on these facts, it was found that, the metal might be transported to other parts of the organ in its body and deposited there. There is in-toxic mechanism practiced by the fish to defend itself from the toxicity of the pollutants. Bervoets et al. (2001) reported that gill accumulate more concentration of heavymetal than liver followed by muscle and gut region for short time exposure. However, for long term exposure, the heavy metal level was in this decreasing order gut>kidney>liver> gills.

Knowledge of heavy metal concentrations in fish is important with respect to nature of management and human consumption of fish. In the study, heavy metal concentrations in the tissue of freshwater fish vary considerably among different studies (Hayat et al., 2007; Chattopadhyay et al., 2002; Papagiannis et al., 2004), probably due to differences in metal concentrations and chemical characteristics of water from which fishes were sampled. In river, fishes are often at the top of the food chain and have the tendency to concentrate heavy metals from water at higher rate (Mansour and Sidky, 2002). Therefore, bioaccumulation of metals in fish can be considered as an index of metal pollution in the aquatic bodies (Javed, 2005; Tawari-Fufeyin and Ekaye, 2007; Karadede-Akin and Unlu, 2007) that could be a useful tool to study the biological role of metals present at higher concentrations in fish (Dural et al., 2007). Therefore, the toxicity tests (Bioassay) are necessary in water pollution evaluation because chemical and physical measurements alone are not sufficient to assess potential effects on aquatic biota (Tarzwell, 1971).

CONCLUSION

Prediction of accumulated levels of any metal in tissues is possible only when the relative importance of each exposure route is known from the environment. In this study, it was clearly shown that the heavy metal exposure via water borne has a significant impact on the gills of the fish. It was also apparent that fishes exposed to longer period with minimal concentration tend to accumulate less heavy metals in gills as they are transported to other parts of the body. Knowledge of these outputs made a possible to give a better description of the variation and the pattern of metal accumulation in the tissues. Detailed study need to be carried out to know the various physiological changes undergone by the fish during different concentration of heavy metal exposure.

ACKNOWLEDGMENTS

Authors express their sincere gratitude to the International Islamic University Malaysia for providing infrastructure facilities.