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Development of Quantitative Quality Control (QC) Criteria for US EPA 1600-Series Methods for the Determination of Fecal Coliforms and Salmonella in Biosolids

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In the United States, the use and disposal of biosolids are regulated under 40 CFR part 503. Subpart D of this regulation protects public health and the environment through requirements designed to reduce the potential for contact with pathogens in biosolids applied to land or placed in a surface disposal site. For Class A biosolids, the 503 regulations require evaluation of either fecal coliforms or Salmonella, and may include enumeration of enteric viruses, or viable helminth ova. In order to demonstrate compliance, precise measurement of microbial contaminants is a necessity. However, interlaboratory comparisons of standard protocols were not available when the 503 regulations were promulgated.

Studies evaluating fecal coliform (LTB/EC and A-1) and Salmonella [Modified Semisolid Rappaport-Vassiliadis (MSRV)] methods were conducted to assess method precision and recovery, develop quantitative quality control (QC) acceptance criteria for initial and ongoing method/laboratory performance assessments, and establish quantitative QC criteria for matrix spikes (MS). This presentation will be limited to development of quantitative QC criteria. One referee lab (EPA) and 12 participant laboratories familiar with the microbiological analysis of biosolids participated in both studies. The fecal coliform methods were used to analyze three Class A matrices (composted, heat dried, and thermophilically digested) and two Class B matrices (anaerobically digested and aerobically digested). Five Class A biosolid matrices were evaluated during the Method 1682 study: composted, heat-dried, thermophilically digested liquid, thermophilically digested solid, and alkaline stabilized. Milorganite®, a heat dried biosolid available at most nurseries, served as the reference matrix for both studies.

Quantitative QC criteria for initial and ongoing method/laboratory performance were developed based on each laboratory's analysis of four spiked replicates of Milorganite® and quantitative MS criteria were developed based on each laboratory's analysis of two spiked replicates of each matrix. Samples were spiked with either Esherichia coli or Salmonella typhimurium, as appropriate based on the method being evaluated. QC criteria were developed for both analytes based on results from samples spiked with laboratory-prepared spiking suspensions. Additional QC criteria were developed for the MSRV procedure (Salmonella) based on results of samples spiked with BioBalls, a pre-packaged, pre-enumerated product from BTF (Sydney, Australia).

Quantitative QC criteria were calculated for the LTB/EC, A-1, and MSRV procedures using within-laboratory, between-laboratory and between-matrix variance components calculated using Maximum Likelihood Estimation. Final QC criteria were calculated as prediction intervals for individual or mean recovery, and as upper prediction limits for relative standard deviation (RSD) or relative percent difference (RPD). Prior to analyses, two different types of outlier tests were performed to remove datathat were not representative of the overall labaratory performance observed during the studies, in aordance with ASTM protocol.

Microbiological method performance generally is difficult to assess. Method recoveries can be problematic due to uneven distribution of organisms in suspension and densitiesof organisms can change between enumeration and spiking. The QC criteria developed for the fecal coliform and Salmonella methods will allow laboratories to assess laboratory and method performance, identify and solve problems, and assess quality of occurrence results.
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Document Type: Research Article

Publication date: 2005-01-01

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