A Colorimetric Bioassay for Perchlorate

Recognition of perchlorate (ClO4-) as a widespread contaminant across the United States and its potential adverse affects towards human health has motivated the EPA to place ClO4- on its contaminant candidate list for drinking water supplies. While a federal MCL has not yet been set, a recommended public health goal of 1 ppb (μg.L-1) was established by the US EPA in 2002. To date, methods of detection require use of sensitive ion chromatographic equipment that are expensive, time consuming, and require highly trained personnel for use. Our studies are focused on the development of a highly sensitive, simple, and robust colorimetric bioassay based on the primary enzyme involved in microbial ClO4- reduction, the perchlorate reductase (Pcr). A previously published assay used reduced methyl viologen (MV, the dye is reduced with sodium hydrosulfite) as an electron donor to demonstrate Pcr activity. The assay directly correlates the amount of MV oxidized with the amount of ClO4- reduced by assuming a transfer of four electrons. To test this assumption, we compared actual concentrations of MV oxidized to ClO4- reduced in this assay. ClO4- concentrations were determined using a Dionex ICS-500 ion chromatography system, while MV concentrations were determined using a standard curve generated at 578 nm. Comparisons between the two revealed that twelve molecules of MV were oxidized for each molecule of ClO4- reduced. The oxidation of these additional eight MV molecules is explained by the interaction of the dye with chlorite (the product of the Pcr reaction) and other contaminants that could be present in the enzyme prep. This unsettling result indicated this assay would be problematic for the detection of ClO4- in soil, which has many chemicals that could react with MV. To improve upon this assay, we have tried to reduce ClO4- using less reactive dyes and reductants. The reductants ascorbic acid, NADH, and dithiothreitol drive Pcr catalyzed ClO4- reduction, however, they are not effective unless an accompanying dye is used as a shuttle. N-methylphenazinium methosulfate (PMS) was selected as the most suitable dye because of its interaction with NADH, an oxygen stable reductant. In addition, the positive redox potential of PMS (E°' = +80 mV), makes it significantly less reactive than MV (E°' = -450 mV). A comparison of actual concentrations of ClO4- reduced vs. NADH oxidized show exactly four molecules of NADH oxidized for each molecule of ClO4- that is reduced (8 electrons). These studies have resulted in the successful development of a method that can accurately determine ClO4- concentrations with a small error using the enzyme Pcr and indicate the great potential for the ultimate development of a simple, robust, and highly sensitive colorimetric bioassay for perchlorate that can be widely used to screen laboratory and environmental samples .