In vitro bioassays as tools for evaluating toxicity of acidic drainage from a coal mine in Mpumalanga, South Africa

Abstract

Coal mining and coal utilization in Mpumalanga have increased over the years due to national reliance on coal as a source of power generation. In general, this has caused significant deterioration of water quality wherever streams are impacted by acid mine drainage (AMD). The aim of this research was to assess the use of in vitro bioassays as a complement to, or potential future replacement of, waste effluent testing in whole animals from AMD impacted watersheds subjected to passive and active treatment, correlating observed changes with water chemistry analysis. To accomplish this goal, water samples were collected and in vitro bioassays carried out to investigate generation of reactive oxygen species by the water samples and cytotoxicity against Vero kidney cells, C3A liver cells and trout RTgill-W1 cells. Primary fish gill cultures were established and used as sensitive in vitro models for assessing possible contaminants in water, measuring the induction of cytochrome P450 (CYP) 1A and resultant increase in 7-ethoxyresorufin-o-deethylase activity as a potential biomarker in fish gill cells exposed to polycyclic aromatic hydrocarbons. The genotoxic potential of AMD water on commercially available cell lines was also determined.

The study site was an impacted stream located downstream of a coal mine discharge point whose effluent flowed away from the mine. Water chemistry results suggested high AMD impact evidenced by acidity, elevated sulphates, increased conductivity and presence of heavy metals. Al, Fe, Zn, Mn and Si were the major metals of potential concern in the AMD impacted stream; sulphates and major ions like Ca, K, Na and Mg were present at levels above target water quality range (TWQR) for effluents in receiving stream. The AMD impacted stream caused increased generation of reactive oxygen species (ROS) detectable in vitro in selected cell lines (Vero, C3A and RTgill-W1 cell lines), an indication of oxidative stress. In-stream, active treatment with caustic soda was efficient at reducing metal burden, with subsequent reduction in ROS generation in fish gill cell lines. For in vitro cytotoxicity tests, passive and active treated AMD water was cytotoxic to cell lines (Vero and RTgill-W1), with the fish RTgill-W1 cells exhibiting greater sensitivity compared to the mammalian Vero cells. Mitochondria played a larger role in observed loss in cellular viability (increased vacuolization, mitochondrial membrane swelling and damage), which was detected using mitochondrial specific stains, and by transmission electron microscopy (TEM). Increased dose- dependent cytotoxicity was observed in the fish gill and mammalian cell lines. Cells exposed to water samples (AMD and reference sites) revealed significant differences (p < vi 0.05) between the AMD impacted watershed and a relatively pristine site (reference site) where exposure to the same cells maintained approximately 100% viability at all concentrations for up to 72h exposure. The observed differences in effect in this study demonstrate that the effluent from the coal mine negatively impacted surface water quality, resulting in toxicity to cell lines, therefore creating an environment that would not be conducive for the survival of biological aquatic communities and potentially of concern for downstream human end users.

The induction of cytochrome P450 (CYP) 1A and resultant increase in 7- ethoxyresorufin-o-deethylase activity in primary fish gill cultures exposed to polycyclic aromatic hydrocarbons B[a]P, a known AhR agonist contaminant associated with coal mining, showed that there was as increase in EROD activity which was not observed using the RTgill-W1 cell lines. Gill epithelial cells isolated from the gills of Tilapia fish (Oreochromis mossambicus) bear close similarities to fish gills in vivo and their capacity to respond to the presence of AhR indicates that they may serve as a simple, cost-effect screening tool for assessing PAHs and dioxin-like compounds in fresh water.

For genotoxicity evaluation, the Ames test performed without metabolic activation using bacterium Salmonella typhimurium TA98 and TA100 strains revealed no indication of genotoxic activity in any of the water samples. Genotoxicity assessment of all water samples using the comet assay however exposed DNA damage to Vero and RTgill-W1 cell lines. A significant reduction in DNA damage was observed following active treatment. The results suggest that neither treatment technologies employed were efficient at removing all potential genotoxicants so further improvements are required. The comet assay proved sensitive enough to detect genotoxicity in reference water samples despite no known untoward effluent inputs at the site, suggesting potential for this assay to be integrated into an environmental monitoring framework.

The results obtained support the use of in vitro bioassays for evaluating toxicity of industrial effluent through biological responses in test systems elicited following exposure, improving ability to detect AMD polluted water. This could be beneficial when assessing the degree and extent of impact of AMD in natural water sources, and the possible environmental impact resulting from hazardous elements present in effluent water. In conclusion, these results suggest that in vitro techniques involving cell lines and primary cultures from fish may serve vii as simple, rapid and cost-effective tools for assessing risk and potential toxic effects of contaminants in AMD waters.