Ammonium perchlorate represents 90% of all perchlorate salts manufactured and is used as an energetics booster or oxidant in solid rocket fuels. Its presence in the environment from legal historical discharge poses a significant health threat. Current analytical technologies for identifying and quantifying perchlorate at low levels are based on ion chromatography with conductivity or mass spectrometry detection. Although sensitive, these methods are inefficient as they are slow and arduous, exceptionally expensive, and require significant sample preparation by highly trained personnel in specialized laboratories. As such, their application for the rapid delineation of contamination zones in an environment is neither time nor cost effective.

The objective of this study was to develop a highly sensitive and specific analytical colorimetric assay for the rapid determination of perchlorate in environmental samples.

The original project concept was based on recent advances in understanding of the biochemistry and genetics of microbial perchlorate reduction. The goal was to take advantage of the activity of the primary enzyme involved in the biochemical pathway of perchlorate reduction, the perchlorate reductase, which quantitatively reduces perchlorate to chlorite. The project team proposed to couple the activity of the perchlorate reductase enzyme to a bioassay previously developed for chlorite. The purified perchlorate reductase stoichiometrically reduces any perchlorate in the sample to chlorite and the chlorite can then be quantified by the chlorite assay producing a readily measurable yellow color. Unfortunately, due to an unpredicted chemical reaction between the electron donor of the perchlorate reductase and the chlorite formed by the enzyme, this approach was unlikely to prove successful. However, the reactivity of the chlorite with the primary electron donor may yield an unforeseen advantage. If transformation of the electron donor (e.g., dithionite; S₂O₄^2-) is monitored, the abiotic reaction between the chlorite formed and the residual electron donor available would result in an inherent signal amplification in the assay, which should significantly enhance the assay sensitivity for perchlorate.

This new conceptual direction of the project did not alter the original tasks performed. Under the revised conceptual plan, the perchlorate bioassay was based on a three-step process: (1) extraction of the perchlorate; (2) reaction of the perchlorate with the perchlorate reductase using a suitable electron donor; and (3) quantification of the electron donor consumed by the perchlorate reductase.

The developed bioassay uses the partially purified perchlorate reductase enzyme from Dechloromonas agitata to detect perchlorate with the redox active dye phenazine methosulfate (PMS) and nicotine adenine dinucleotide (NADH). By using a specific addition scheme and covering all reactions with mineral oil, the reaction can be performed on the benchtop with a lower detection limit of 2 ppb when combined with perchlorate purification and concentration by solid phase extraction (SPE). Perchlorate concentrations (0-17,000 ppb) were accurately analyzed using the bioassay in the presence of a range of ions (nitrate, phosphate, sulfate, iron, chloride) at a concentration of 100 ppm.

The bioassay offers a rapid, sensitive, specific, and cost-effective solution that can be performed onsite with minimal training utilizing robust ubiquitous laboratory equipment. The cost of an ion chromatography system with conductivity detection can reach $50,000 ($500,000 with mass spectrometry detection) and has a consumables charge of  more than $1 per sample. The bioassay has a much lower instrument and materials cost (handheld spectrophotometer approximately $300, reusable SPE columns $250) with consumables of $0.13 per sample. Furthermore, in contrast to an ion chromatograph, the bioassay allows for multiple samples to be assayed simultaneously while achieving a minimum detection limit of 2 ppb. The assay is currently being demonstrated and validated under ESTCP project ER-201030.