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Coal fly ash is a residue of coal-fired thermoelectric power plants (TPPs) and is mostly dumped in ash ponds or landfill sites, even though it potentially contains significant amounts of water-soluble hazardous contaminants. Bioassay using the bioluminescent bacterium *Vibrio fischeri* is known to be applicable for assessing the short-term and sublethal toxicity of complex mixtures without the need for precise chemical characterization. However, this type of bioassay is potentially adversely influenced by the pH-induced protein denaturation of cells. Because coal fly ash leachates often have alkaline or acidic properties, when applying the *V. fischeri*-based bioassay to the samples, we need to know potential effect of the leachates on the bioluminescence of the bacteria. This study accordingly aimed to investigate the feasibility of applying the *V. fischeri* bioassay to coal fly ash leachate as a screening method. Fly ash samples were collected from 12 TPPs located in three East European countries: Bulgaria, Greece, and Serbia. The fly ash samples were prepared in sterilized distilled water by ultrasonic extraction and filtration using 0.45-MGRmPHI membrane filters. The filtrates were then mixed with a solution of the test bacterium. The bioluminescence intensity was measured using a luminometer. The results showed the ostensible influence of pH on bioluminescence intensity pronounced when following the typical protocol using a 5.0-g/L solid:liquid ratio. Accordingly, the pH of water extracts should be adjusted to within a range of 6 to 9 by dilution to observe the inhibition of bioluminescence by coal fly ash leachate as the objective endpoint.

Effect of Coal Fly Ash Leachate on the Bioluminescence Intensity of Vibrio fischeri