This method describes multiple-tube fermentation procedures (also called the most probable number (MPN) procedure) for the detection and enumeration of fecal coliform bacteria in biosolids. These methods use culture-specific media and elevated temperature to isolate and enumerate fecal coliform organisms. Fecal coliform bacteria, including Escherichia coli (E. coli), are commonly found in the feces of humans and other warm-blooded animals, and indicate the potential presence of other bacterial and viral pathogens. This method is adapted from methods 9221E in Standard Methods for the Examination of Water and Wastewater, 20th Edition, for the determination of fecal coliform bacteria in a variety of matrices. This method is designed to meet the survey and monitoring requirements of the U.S. Environmental Protection Agency (EPA) in regulating the use and disposal of biosolids under 40 CFR Part 503. Subpart D of the 503 regulations protects public health and the environment through requirements designed to reduce the potential for contact with disease-bearing microorganisms (pathogens) in biosolids applied to land or placed on a surface disposal site. Fecal coliform density is expected to correlate with the probability of pathogens present and document process performance (vector attraction reduction). This method may be used to determine the density of fecal coliform bacteria in Class A and Class B biosolids to satisfy the pathogen reduction requirements of Subpart D of Part 503. A biosolid sample is classified as Class A if it contains a fecal coliform density below 1,000 MPN / g of total solids (dry weight basis). A biosolid sample is classified as Class B if the geometric mean fecal coliform density is less than 2 x 10 (sub 6) MPN/g of total solids (dry weight basis). To satisfy the pathogen reduction monitoring alternatives for Class B biosolids, seven samples of treated biosolids are collected at the time of use or disposal and the geometric mean fecal coliform
bacterial density of these samples is confirmed not to exceed 2 x 10 (sub 6) MPN g of total solids (dry weight basis). Although the Part 503 regulation does not specify the total number of samples for Class A biosolids, it is recommended that a sampling event extend over two weeks, and that at least seven samples be tested to confirm that the mean bacterial density of the samples is below 1,000 MPN/g of total solids (dry weight basis). The analysis of seven samples increases the method precision by reducing the standard error caused by inherent variations in biosolid quality. The presence of fecal coliforms may be determined in both Class A and Class B biosolids using the MPN procedure. Any modification of the method beyond those expressly permitted is subject to the application and approval of alternative test procedures under 40 CFR Parts 136.4 and 136.5. Method 1681 was submitted to interlaboratory validation in Class A and Class B biosolid matrices. A summary of method performance results from this validation study are provided. A comprehensive evaluation of the study results is presented in the validation study report. Based on these validation study results, Method 1681 is proposed for approval for the analysis of fecal coliforms in Class A and Class B biosolid matrices.