Evaluation of multiple laboratory performance and variability in analysis of recreational freshwaters by a rapid Escherichia coli qPCR method (Draft Method C).

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to "new" water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the "new" laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials.

Standardized data quality acceptance criteria for a rapid Escherichia coli qPCR method (Draft Method C) for water quality monitoring at recreational beaches.

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made.

Environmental analysis of groundwater in Mecosta County, Michigan.

Groundwater withdrawal has major economic, social, and environmental implications. In Michigan, recent legislative activity has begun to address the issue of groundwater sustainability. However, more hydrologic data are needed to help inform policy and legislation. A study was conducted in Mecosta County, Michigan to: (1) determine if a relationship could be established between land use/land cover and groundwater quality; and (2) develop a conceptual model for the shallow groundwater system of the study region. In general, groundwater quality was good, with below detection levels of E. coli, low total bacterial counts, and relatively low nutrient concentrations. No statistically significant associations were found between the bacterial numbers and either land use or the physical/chemical attributes measured, which may be because the scale of our spatial analysis was too coarse to detect patterns. Finer-scale, localized processes may have a greater influence on microorganism growth and abundance than coarser-scale, regional processes in this area. Our groundwater analysis suggested that shallow groundwater flow paths are generally consistent with regional surface water flow networks, and that shallow groundwater levels in most of the region have fluctuated within 1-2 m over the past 30 years, with no obvious increasing or decreasing trend.

The reduction of internal phosphorus loading using alum in Spring Lake, Michigan.

The release of P from lake sediments, which occurs as a part of internal loading, may contribute a significant portion of the total P load to a lake. Phosphorus release rates from sediments in Spring Lake, Michigan, and the degree to which alum reduces P release from these sediments, were investigated during the summer of 2003. Triplicate sediment cores were sampled from four sites in the lake, and exposed to one of four treatments in the laboratory: (i) aerobic water column/alum, (ii) aerobic water column/no alum, (iii) anaerobic water column/alum, or (iv) anaerobic water column/no alum. Total P (TP) release rates were virtually undetectable in the alum treatments (both aerobic and anaerobic). Low, but detectable, release rates were measured in the aerobic/no alum treatment. The highest release rates were measured in the anaerobic/no alum treatments, and ranged from 1.6 to 29.5 mg P m(-2) d(-1) depending on how the calculations were derived. These fluxes translated to mean internal loads that ranged between 2.7 (low range) and 6.4 (high range) Mg yr(-1) when extrapolated to a whole-lake basis. Internal P loads accounted for between 55 and 65% of the total P load to Spring Lake. Although alum is a potentially effective means of reducing the sediment source of P, there is considerable uncertainty in how long an alum treatment would remain effective in this system given the current rates of external loading and the lack of information on wind-wave action and bioturbation in Spring Lake.