The bioassay for low-level perchlorate determination (range approximately 6 to 40 µg/L), which was the focus of the project, is a benchtop enzyme assay combined with a perchlorate concentration and purification step using solid phase extraction (SPE) cartridges (Heinnickel et al., 2011). It is based on perchlorate reductase activity and couples NADH oxidation to perchlorate reduction, with phenazine methosulfate (PMS) as an electron shuttle. Hence the amount of perchlorate in an aqueous sample can be determined enzymatically by monitoring the amount of NADH oxidized spectrophotometrically (i.e., by measuring the decrease in absorbance at 340 nm). The instrumentation for the bioassay is an ultraviolet (UV) spectrophotometer. The equipment and materials are relatively inexpensive, and the bioassay potentially may be performed by less highly trained personnel than required by ion chromatography (IC) or liquid chromatography-mass spectrometry (LC/MS) analytical methods. Thus it was thought likely that the benchtop bioassay might supplement more expensive and time-consuming analytical procedures as a screening test for perchlorate in groundwater to facilitate tasks such as mapping plumes and monitoring perchlorate levels during remediation.

The objectives of this project were to: (1) Compare benchtop bioassay results from testing in the laboratory to results from a reference analytical method performed by a commercial laboratory; (2) Compare results with a benchtop bioassay kit format tested by site field personnel to the site’s routine perchlorate detection method; and (3) Evaluate the ease of use of the benchtop bioassay kit format.

Technology Description

The enzymatic bioassay for perchlorate was developed under SERDP project ER-1530. It makes use of SPE cartridges to concentrate and purify groundwater based on the procedure developed by Thorne, 2004. Since different groundwater samples vary in the efficiency of perchlorate recovery at the SPE step, subsamples are spiked with perchlorate according to the standard additions method for quantitating an analyte. After applying perchlorate-spiked groundwater to SPE cartridges, the cartridges are rinsed, and then perchlorate is eluted with an alkaline solution (pH >12) of morpholinepropane sulfonic acid (MOPS) and sodium chloride (NaCl). The eluates are neutralized to about pH 7.2 and treated non-enzymatically to remove oxygen prior to enzyme analysis. Addition of enzyme initiates the enzyme reaction. An extract of the perchlorate reducing bacterium Dechloromonas agitata strain CKB, which contains the enzyme perchlorate reductase, provides the enzyme source for the assay.

Demonstration Results

Six groundwater sources were obtained for the project. Bioassay results with groundwater collected from three different locations at one site were in good agreement with results obtained by a reference IC method. This testing was performed in a 96 well microplate using a UV spectrophotometric microplate reader inside of an anaerobic chamber with an atmosphere of nitrogen and hydrogen. In addition, a benchtop version of the bioassay was developed, using quartz cuvettes, and a UV spectrophotometer to be performed in ambient laboratory conditions. The benchtop bioassay format performed as well as the microplate reader format with perchlorate standards. Success criteria were not met for four other groundwater sources.

The benchtop bioassay was deemed not suitable in its current format as a screening test for perchlorate in groundwater. In depth analysis indicated that oxygen contamination of the benchtop assay, even in the face of several oxygen barriers and an active chemical reductant, was the culprit for the unpredictability and poor performance of the bioassay comparison under objective 1. Retesting of the water samples with the bioassay in an anaerobic chamber with a nitrogen/hydrogen atmosphere confirmed this finding and provided a different outcome. In this instance, the bioassay accuracy met all performance criteria for groundwater eluates amenable to quantitative testing. However, this requirement defeats the original purpose of the bioassay as a robust field applicable tool. Since performance of the bioassay was not high enough to warrant proceeding with development of a kit format for the benchtop bioassay, no testing was done relative to performance objectives 2 and 3.

Implementation Issues

The benchtop bioassay was deemed not suitable in its current format as a screening test for perchlorate in groundwater.