EPA-821 -R-04-008 vxEPA United States Environmental Protection Agency Results of the Interlaboratory Validation of EPA Method 1600 (mEI) for Enterococci in Wastewater Effluent February 2004 ------- U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460 EPA-821-R-04-008 U.S. Environmental Protection Region 5, Library (PL-12J) 77 West Jackson Boulevard, 12th Floor Chicago, IL 60604-3590 ------- Acknowledgments This report was prepared by the DynCorp/CSC Biology Studies Group under the direction of Robin K. Oshiro, of the Office of Science and Technology's Engineering and Analysis Division (HAD) within the U.S. Environmental Protection Agency (EPA's) Office of Water. The contributions of the following persons and organizations to this study are gratefully acknowledged: Volunteer Research Laboratory EPA Office of Research and Development, National Risk Management Research Lab: Mark C. Meckes Volunteer Participant Laboratories City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda, and Zora Bahariance County Sanitation Districts of Los Angeles County, JWPCP: Kathy Walker, Michele Padilla, and Albert Soof County Sanitation Districts of Los Angeles County, SJC: Shawn Thompson and Julie Millenbach Environmental Associates (EA): Susan Boutros and John Chandler Hampton Roads Sanitation District (HRSD): Anna Rule, Paula Hogg, and Bob Maunz • Hoosier Microbiological Laboratories (HML): Don Hendrickson, Katy Bilger, and Lindsey Shelton Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofhsteyn North Shore Sanitation District (NSSD): Robert Flood Texas A&M University: Suresh Pillai and Reema Singh • University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord • Wisconsin State Laboratory of Hygiene (WSLH): Jon Standridge, Sharon Kluender, Linda Peterson, and Jeremy Olstadt Utah Department of Health: Sanwat Chaudhuri and Devon Cole Volunteer Verification Laboratory City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda, and Zora Bahariance ------- Disclaimer This document has been reviewed and approved by the EPA/EAD. Mention of company names, trade names, or commercial products does not constitute endorsement or recommendation for use. ------- Questions concerning this report should be addressed to: Robin K. Oshiro Engineering and Analysis Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsylvania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov 202.566.1075 202.566.1053 (facsimile) Requests for additional copies of this publication should be directed to: Water Resource Center Mail Code RC-4100 1200 Pennsylvania Avenue, NW Washington, DC 20460 202.566.1729 or 202.566.1730 ------- Table of Contents Section 1 .0 Background 1.1 Summary of the Method Section 2.0 Study Objectives and Study Design ............... . ................................... 2 2.1 Study Objectives [[[ 2 2.2 Phase 1 Technical Approach: Identification of Laboratories ................................ 3 2.2.1 Research Laboratory [[[ 3 2.2.2 Participant Laboratory [[[ 3 2.2.3 Verification Laboratory [[[ 3 2.3 Phase 2 Technical Approach: BioBall™ Spikes ......................................... 3 2.4 Phase 3 Technical Approach: Lab-Prepared Spiking Suspensions ........................... 4 2.5 Phase 4 Technical Approach: Sample Analysis .......................................... 4 2.5.1 Range-finding Analyses [[[ 5 2.5.2 Assessment of Method Sensitivity and Specificity ................................. 5 2.5.3 Assessment of Method Accuracy (Precision and Recovery) .......................... 6 2.5.4 Development of Quantitative QC Criteria for Initial (IPR) and Ongoing (OPR) Method/Laboratory Performance Assessments .................................... 6 2.5.5 Development of Quantitative QC Criteria for Matrix Spikes (MS) .................... 7 2.5.6 Quality Control (QC) Analyses ................................................ 7 2.5.7 Minimum Validation Study Requirements ....................................... 7 Section 3.0 Study Implementation [[[ 8 3.1 Study Management [[[ 8 3.2 Schedule [[[ 9 3.3 Research, Participant, and Verification Laboratories ...................................... 9 Section 4.0 Data Reporting and Validation [[[ 10 4.1 Data Reporting [[[ 10 4.2 Data Validation [[[ 10 4.3 Censored Data .................................. . ............................ ... 1 1 Section 5.0 Results ......... . [[[ 12 5.1 Unspiked Sample Results [[[ 12 5.2 Spiked Disinfected Sample Results .................................................. 15 5.3 Spiked PBS Results [[[ 17 Section 6.0 Development of QC Acceptance Criteria .............................................. 19 ------- List of Tables Table 1. Method 1600 Validation Study Analyses Performed by Each Laboratory 5 Table 2. Comparison of ASTM Recommendations and the Method 1600 Study 7 Table 3. Laboratories Participating in the Interlaboratory Validation of Method 1600 9 Table 4. Summary of Enterococci Results from Unspiked Disinfected Wastewater Samples 13 Table 5. Summary of False Positive and False Negative Rates Associated with Unspiked Disinfected and Unspiked Secondary Wastewater Effluents 13 Table 6. Laboratory-Specific False Positive and False Negative Rates Associated with Unspiked Wastewater Effluents 14 Table 7. Summary of Enterococci Results from Disinfected Samples Spiked with BioBalls™ 15 Table 8. Summary Enterococci Results from Disinfected Samples Spiked with Laboratory-Prepared Spiking Suspensions 16 Table 9. Summary of Enterococci Results from PBS Samples Spiked with BioBalls™ 17 Table 10. Summary of Enterococci Results from PBS Samples Spiked with Laboratory-Prepared Spiking Suspensions 18 Table 11. Calculated Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria 22 Table 12. Calculated Matrix Spike Precision and Recovery Acceptance Criteria 24 ------- List of Appendices Appendix A: Method 1600 Spiking Protocol A-1 Appendix B: Wastewater Laboratory Capabilities Checklist B-l VI ------- Executive Summary This report presents the results of the U.S. Environmental Protection Agency's (EPA's) interlaboratory validation study (the "Study") of a membrane filtration procedure for the analysis of enterococcus in wastewater: EPA Method 1600 which uses membrane-enterococcus indoxyl-|3-D-glucoside agar medium (mEI). The September 2002 version of the Method (EPA-821-R-02-022) was followed during the Study. The purposes of the Study were to characterize method performance (sensitivity, specificity, precision, and recovery) across multiple laboratories and disinfected wastewater matrices and to develop quantitative quality control (QC) acceptance criteria. Twelve volunteer participant laboratories, an enterococci verification laboratory, and a research laboratory participated in the Study which was conducted during the week of September 22,2003. Each laboratory spiked samples with BioBall™ Enterococcus faecalis (E.faecalis) spikes and laboratory-prepared E.faecalis spikes. Samples were spiked in accordance with the Method 1600 spiking protocol (the "Spiking Protocol," Appendix A). Results from samples spiked with BioBall™ spikes were used to assess method performance and for the development of (QC) acceptance criteria to support future assessments of method and laboratory performance for disinfected wastewater matrices. QC criteria were also developed based on results of the laboratory-prepared spikes (in addition to criteria developed based on BioBalls™) to ensure that QC criteria will be available for these methods, if BioBalls™ become unavailable. Results submitted by laboratories were validated using a standardized data review process to confirm that results were generated in accordance with study-specific instructions and the September 2002 version of EPA Method 1600. Method 1600 recovery of enterococci was acceptable, with mean laboratory-specific recoveries of enterococci from disinfected wastewater samples spiked with BioBalls™ ranging from 77.1% to 114.9%, with an overall mean recovery of 90.8%. Laboratory-specific relative standard deviations (RSDs) ranged from 0% to 69.5%, with an overall pooled, within-laboratory RSD of 22.6%. False positive rates were also acceptable, with laboratory-specific false positive rates for unspiked disinfected/secondary results combined, ranging from 0% - 27.8%. For secondary wastewater (excluding disinfected results), only 11 of 132 typical colonies submitted to verification were non-enterococci, resulting in a false positive rate of 8.3% for secondary wastewater. For disinfected wastewater (excluding secondary results), only three of 69 typical colonies submitted to verification were non-enterococci, resulting in a false positive rate of 4.3% for disinfected wastewater. In contrast, laboratory-specific false negative rates for unspiked disinfected/secondary results combined, ranged from 13.3% - 100.0%. For secondary wastewater (excluding disinfected results), 62 of 118 atypical colonies submitted to verification were identified as enterococci, resulting in a false negative rate of 52.5% for secondary wastewater. For disinfected wastewater (excluding secondary results), eight of 12 atypical colonies submitted to verification were identified as enterococci, resulting in a false negative rate of 66.7% for disinfected wastewater. Results of this study indicate that Method 1600 precision, recovery, and false positive rates are acceptable for the determination of enterococci in disinfected wastewater. However, false negative rates observed during this study were high and should be taken into consideration when using results from this method. When evaluating wastewater using Method 1600, it is recommended that the false negative rate for each matrix be evaluated through biochemical confirmation and results adjusted accordingly, especially if large numbers of atypical colonies are observed in a particular matrix. If very few atypical colonies are observed in samples for a particular matrix, the high false negative rates observed during this study may be less of a concern. VII ------- EPA Method 1600 Validation Study Results SECTION 1.0 BACKGROUND The enterococci test is recommended as a measure of recreational water quality. Epidemiological studies have led to the development of criteria which can be used to promulgate recreational water standards based on established relationships between health effects and water quality. Method 1600 was recently approved for monitoring ambient waters for enterococci (68 FR 43272, July 21,2003). Prior to this study, none of the approved enterococci methods had been validated for wastewater analyses. National Pollutant Discharge Elimination System (NPDES) permit holders and others have requested that EPA validate one or more enterococci methods for evaluation of wastewater effluents. 1.1 Summary of the Method Method 1600 is a membrane filtration procedure for the detection of enterococci in water samples. In Method 1600, a water sample is filtered through a membrane (0.45 um pore-size), which retains the bacteria. After filtration, the membrane containing the bacteria is placed on a selective medium, mEI agar, and incubated at 41°C ± 0.5°C for 24 hours. All colonies that produce a blue halo (regardless of color) are considered enterococci. February 2004 ------- EPA Method 1600 Validation Study Results SECTION 2.0 STUDY OBJECTIVES AND STUDY DESIGN 2.1 Study Objectives The following objectives were established for the Study: Generate at least six sets of useable, valid data to characterize method performance • Characterize Method 1600 sensitivity and specificity across multiple laboratories and disinfected wastewater matrices through the assessment of false positive and negative rates • Characterize Method 1600 accuracy (recovery and precision) across multiple laboratories and disinfected wastewater effluents • Establish Method 1600 quantitative Quality Control (QC) acceptance criteria for initial and ongoing laboratory and method performance assessments Establish Method 1600 quantitative QC acceptance criteria for matrix spikes To accomplish these objectives, this Study was conducted in five phases (the technical approach for each phase is described below): • Phase 1 involved identification of qualified laboratories to participate in the Study, including a research laboratory to confirm that the spiking approach for the Study was acceptable; participant laboratories to analyze both BioBall™ spikes and laboratory-prepared spiking suspensions; and a verification laboratory to speciate colonies from the participant laboratories for the assessment of false positive and negative rates. • Phase 2 involved enumeration and use of BioBall™ (BTF Pty Ltd, Sydney, Australia) spikes ofE.faecalis ATCC #19433 (Manassas, VA) at each of the participant laboratories. BioBalls are pre-packaged, water- soluble balls containing a precise number of bacteria. BioBall™ products are freeze-dried and have a shelf life of 6 months when stored at -20°C. BioBalls™ were selected for this study because they minimize the burden on the participant laboratories and because BioBalls™ are typically very precise spikes. • Phase 3 involved enumeration and preparation of a laboratory-prepared spiking suspension ofE.faecalis ATCC #19433 at each of the participant laboratories. • Phase 4 involved the analysis of unspiked/spiked wastewater samples and unspiked/spiked sterile phosphate buffered saline (PBS) samples at the participant laboratories. The following data quality objective was established for this Study: Data produced under this Study were required to be generated according to the analytical and quality assurance (QA)/QC procedures in the September 2002 version of Method 1600 (EPA-821-R-02-022) or approved changes to these procedures as provided to participant laboratories during the course of the Study. This data quality objective was developed to ensure that data were of known and reliable quality, thereby allowing EPA to use the results of the Study to identify the need for further revision of the method. February 2004 ------- EPA Method 1600 Validation Study Results 2.2 Phase 1 Technical Approach: identification of Laboratories The Study required three types of laboratories: a research laboratory, participant laboratories, and a centralized verification (identification) laboratory. 2.2.1 Research Laboratory EPA's National Risk Management Research Laboratory (NRMRL) in Cincinnati, OH, served as the research laboratory for this Study. Prior to sample analysis at the participant laboratories, the research laboratory evaluated the procedure for preparation of the E.faecalis spiking suspensions that would be used as the laboratory-prepared spiking suspensions during the Study. The research laboratory (Section 3.1) confirmed that appropriate spike levels could be obtained by growing E.faecalis in 1% azide dextrose broth as specified in the Method 1600 Spiking Protocol (Appendix A). 2.2.2 Participant Laboratories Participant laboratories analyzed samples to provide EPA with the data necessary to assess method performance and develop QC acceptance criteria. The participant laboratories also provided typical and atypical colonies to the verification laboratory for identification. Participant laboratories (Section 3.3) were representative of the general user community, with experience analyzing wastewater or ambient water samples for enterococci using membrane filtration techniques. Laboratory availability was also considered. A detailed Wastewater Laboratory Capabilities Checklist (Appendix B) was used to collect information from laboratories and screen potential participants to ensure that laboratories were qualified. Participants also needed to have access to representative disinfected and secondary treated wastewater effluents from the same facility. Qualified volunteer laboratories were recruited in an effort to reduce costs. To reduce the burden on participant laboratories, EPA provided the media, reagents, and supplies necessary for the Study. 2.2.3 Verification Laboratory The City of Los Angeles Bureau of Sanitation Microbiology Laboratory in Playa del Rey, CA served as the centralized verification laboratory. To assess false positive and negative rates, the verification laboratory speciated all typical and atypical colonies submitted by the participant laboratories. Colonies were identified using the Vitek® automated identification system. A detailed Wastewater Laboratory Capabilities Checklist (Appendix B) was used to collect information from laboratories and screen potential verification laboratories to ensure the centralized verification laboratory was qualified. The verification laboratory was also recruited as a volunteer in an effort to reduce costs. To reduce the burden on the verification laboratory, EPA provided all necessary verification media and supplies. 2.3 Phase 2 Technical Approach: BioBall™ Spikes Phase 2 involved enumeration and preparation of BioBall™ spikes of E.faecalis ATCC #19433 according to the Method 1600 Spiking Protocol (Appendix A). Results from samples spiked with BioBalls™ were used to assess inter- and intra-laboratory precision and recovery (method performance) and to develop QC acceptance criteria. The "lot mean value" provided by the manufacturer was used as the "true spike concentration." In addition participant laboratories enumerated BioBalls™ on the day of analysis in triplicate, using the spread plate 3 February 2004 ------- EPA Method 1600 Validation Study Results technique and tryptic soy agar (TSA) as described in the Method 1600 Spiking Protocol (Appendix A), to confirm stability of the BioBalls™. 2.4 Phase 3 Technical Approach: Lab-Prepared Spiking Suspensions Phase 3 involved each laboratory preparing and enumerating spiking suspensions ofE.faecalis ATCC #19433 according to the Method 1600 Spiking Protocol (Appendix A). To ensure that QC criteria are available for this method if BioBalls™ become unavailable, QC criteria were also developed based on results of the laboratory- prepared spikes. Results from samples spiked with laboratory-prepared spiking suspensions were not used to assess method performance. Spiking suspensions were enumerated in triplicate, using the spread plate technique and TSA as described in the Method 1600 Spiking Protocol (Appendix A). To estimate the "true spike concentration," the participant laboratories enumerated the laboratory-prepared spiking suspensions on the same day that the validation study samples were spiked and analyzed. 2.5 Phase 4 Technical Approach: Sample Analysis Phase 4 entailed the use of Method 1600 at multiple laboratories to analyze unspiked/spiked wastewater samples and PBS samples farE.faecalis. The following objectives were established for Phase 4: • Generate false positive and negative rate data for Method 1600 in disinfected wastewater effluents for the assessment of sensitivity and specificity. It should be noted that while the objective of the Study was to assess sensitivity and specificity for Method 1600 in disinfected wastewater, this was not possible because of the low numbers of colonies from disinfected wastewater samples. As a result, colonies from secondarily treated wastewater were also used to assess sensitivity and specificity of the Method. It was not possible to assess sensitivity/specificity solely in the matrix of interest (disinfected wastewater) during this study. • Generate precision and recovery data for Method 1600 in disinfected wastewater effluents • Develop quantitative QC acceptance criteria for Method 1600 in sterile PBS to support future assessments of laboratory performance • Develop quantitative QC acceptance criteria for Method 1600 in the matrix of interest (disinfected wastewater) to support future assessments of method performance • Generate a minimum of six sets of useable (as recommended by ASTM for method validation), valid data from the interlaboratory validation study to characterize method performance Table 1 summarizes the number of samples that were evaluated to meet the objectives listed above. A detailed discussion is included in Sections 2.5.1 through 2.5.5 below. February 2004 ------- EPA Method 1600 Validation Study Results Table 1. Method 1600 Validation Study Analyses Performed by Each Laboratory Matrix Disinfected wastewater Secondary Disinfected wastewater Secondary a Disinfected wastewater Disinfected wastewater Sterile PBS Sterile PBS Spiking Description Unspiked Unspiked Unspiked Unspiked BioBalls™ Lab-prepared BioBalls™ Lab-prepared Sample Number N/A N/A 1-4 5-6 7-8 9-10 11-14 15-18 Verification N/A" N/A 5 typical and 5 atypical 5 typical and 5 atypical N/A N/A N/A N/A Purpose of Analysis Range-finding (Section 2.5.1) Range-finding (Section 2.5.1) Evaluation of ambient enterococci concentrations (Section 2.5.2) False positive and negative rates (Section 2.5.2) False positive and negative rates (Section 2.5.2) Assessment of method accuracy (Section 2.5.3) Assessment of matrix spike QC criteria (Section 2.5.5) Assessment of matrix spike QC criteria (Section 2.5.5) Develop quantitative QC criteria for IPR c and OPR " (Section 2.5.4) Develop quantitative QC criteria for IPR and OPR (Section 2.5.4) Results Provided in the Following Tables N/A N/A 4 5 and 6 5 and 6 7 8 9 10 1 Colonies from these samples were only submitted to verification when a sufficient number of colonies from disinfected samples were not available b N/A: Not applicable c IPR: Initial Method/Laboratory Performance Assessment " OPR: Ongoing Method/Laboratory Performance Assessment 2.5.1 Range-finding Analyses Range-finding analyses were conducted on the secondary and disinfected wastewater effluents during the week of validation study analyses. These analyses were conducted one day prior to the analysis of validation study samples to determine the filtration volume(s) necessary to obtain plates within the optimum counting range for the method. 2.5.2 Assessment of Method Sensitivity and Specificity Sensitivity and specificity of Method 1600 was assessed through the evaluation of false positive and false negative rates. Each of the 12 participant laboratories evaluated four unspiked disinfected wastewater samples for false positive/negative results by submitting five typical and five atypical colonies from each of the four disinfected wastewater samples to verification through biochemical evaluation. Because very few colonies were expected to be available for verification from the unspiked disinfected samples, two unspiked secondary effluent samples were also filtered to ensure that a sufficient number of colonies were available for verification during the February 2004 ------- EPA Method 1600 Validation Study Results Study. Colonies from the secondary samples were only submitted to verification when a sufficient number of colonies from disinfected samples were not available. As a result, the study was designed to verify a total of 240 typical and 240 atypical colonies (including colonies from disinfected and secondary samples). For each colony submitted to verification, the laboratory streaked the colony onto an mEI agar plate for isolation, inverted the plate, and incubated at 41°C ± 0.5°C for 24 ±2 hours. Plates were labeled with sample identification information and colony type. To prepare plates for shipping, the laboratory wrapped the edges of the plates with parafilm, wrapped the stack of plates associated with each sample with bubble wrap, placed the plates into a cooler lined with a trash bag, and surrounded the plates with ice packs. Plates were shipped to the verification laboratory via Federal Express Priority Overnight Service. Upon receipt at the verification laboratory, a single isolated colony was picked from each mEI plate and streaked for growth onto a tryptic soy agar (TSA) plate with 5% sheep blood (one TSA plate for every mEI plate) and incubated at 35°C ± 0.5°C for 18 to 24 hours. A suspension was prepared by placing growth from the blood agar plates in 3 mL of physiological saline. The suspensions were then evaluated using a Vitek® (bioMerieux, Hazelwood, Missouri), which is an automated biochemical identification system that utilizes test cards with either 30 or 45 microwells containing identification substrates and antimicrobials. For identification of enterococci during the Study, gram positive identification (GPI) cards were filled with the suspension using the Vitek®'s automated card filler and placed into the card reader/incubator. Readings were taken by the Vitek® every 15 minutes until identification (speciation) was complete. 2.5.3 Assessment of Method Accuracy (Precision and Recovery) Method bias was evaluated through the analysis of two disinfected wastewater samples spiked with BioBalls™ at each of the participant laboratories. Each sample was spiked with a single BioBall™ containing a lot mean value (provided by the manufacturer) of 32.1 E.faecalis ATCC #19433 colony forming units (CFU). Recovery was assessed by comparing E.faecalis concentrations in the spiked samples (minus the ambient concentrations assessed from the analysis of the unspiked disinfected samples from Section 2.5.2) to the BioBall™ lot mean value. Precision was assessed based on the relative standard deviation of the two replicate recoveries. 2.5.4 Development of Quantitative QC Criteria for Initial (IPR) and Ongoing (OPR) Method/Laboratory Performance Assessments To collect the data necessary to develop quantitative QC recovery and precision criteria for use in initial and ongoing method and laboratory performance, each participant laboratory analyzed four sterile 100-mL PBS samples spiked with BioBalls™ and four sterile 100-mL PBS samples spiked with laboratory-prepared spiking suspensions using ATCC #19433 in both cases. Samples spiked with BioBalls™ were spiked with approximately 32.1 CFU per sample and samples spiked with laboratory-prepared spiking suspensions were spiked with approximately 68.5 CFU per sample. QC criteria were developed based on results of the laboratory-prepared spikes, in addition to developing criteria based on BioBalls™, to ensure that QC criteria are available for Method 1600, if BioBalls™ become unavailable. February 2004 ------- EPA Method 1600 Validation Study Results 2.5.5 Development of Quantitative QC Criteria for Matrix Spikes (MS) Quantitative QC criteria for matrix spikes were developed for use in assessing matrix interferences, should the methods be implemented for use in disinfected wastewater by EPA. To collect the data necessary to develop these criteria, each participant laboratory analyzed two disinfected wastewater samples spiked with BioBalls™ and two disinfected wastewater samples spiked with laboratory-prepared spiking suspensions using ATCC #19433 in both cases. Samples spiked with BioBalls™ were spiked with approximately 32.1 CPU per aliquot filtered and samples spiked with laboratory-prepared spiking suspensions were spiked with approximately 68.5 CPU per aliquot filtered. It should be noted that several laboratories were instructed to spike less than 100 mL of disinfected wastewater because of either turbidity or high enterococci concentrations in the samples (see Section 4.1). It should be noted that the same two disinfected wastewater samples spiked with BioBalls™ were used to assess method accuracy (Section 2.5.3) and to develop quantitative QC criteria for matrix spikes. 2.5.6 Quality Control (QC) Analyses Participating laboratories completed the following QC requirements: media sterility checks, dilution water sterility checks, method blanks (sterile unspiked PBS), filtration blanks, positive controls, and negative controls. E.faecalis (ATCC #19433) served as the positive control and E. coll (ATCC #11775) as the negative control. 2.5.7 Minimum Validation Study Requirements The Study met or exceeded the ASTM D2777-98 (Reference 10.2) method validation recommendations with every respect except number of concentrations. Only one spike concentration (instead of three) was evaluated because the Study was designed to evaluate samples spiked at levels similar to what was expected to be observed in disinfected wastewater samples at most laboratories. Table 2 presents a comparison of the Study with ASTM D2777-98 validation study requirements. Table 2. Comparison of ASTM Recommendations and the Method 1600 Study Minimum ASTM Recommendations * 6 participant laboratories 1 matrix type plus reference matrix (typically reagent water) 1 matrix type plus reference matrix (typically reagent water) -3 concentrations 36 spiked reagent water samples (6 samples at 6 laboratories) 36 spiked matrix samples (6 samples at 6 laboratories) Method 1600 Study 12 participant laboratories 1 matrix type plus reference matrix (PBS) Samples from a total of 12 facilities plus reference matrix (PBS) 1 concentration 96 IPR samples (for each of 12 participants, 4 replicate PBS samples spiked with BioBalls™ and 4 replicate samples spiked with lab-prepared spikes) 48 MS samples (for each of 12 participants, 2 duplicate MS samples spiked with BioBalls™ and 2 duplicate MS samples spiked with lab-prepared spikes) ASTM. Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D-19 on Water (ASTM 02777-98), October 1998. February 2004 ------- EPA Method 1600 Validation Stvdy Results SECTION 3.0 STUDY IMPLEMENTATION 3.1 Study Management This study was designed under the direction of the Office of Science ana Technology, Engineering and Analysis Division within the U.S. Environmental Protection Agency's (EPA's) Office of Water (OW). The EPA technical lead was Robin K. Oshiro. Coordination of activities for the Study were performed by DynCorp/CSC Biology Studies Group. Evaluation of the 1% azide dextrose broth and Enterococcus faecalis (ATCC #19433) following the Method 1600 Spiking Protocol (Appendix A) was performed by the EPA's Office of Research and Development (ORD), National Risk Management Laboratory (NRMRL). 3.2 Schedule The Study schedule was as follows: practice analyses occurred the week of August 25,2003 and the actual validation study analyses occurred the week of September 22,2003. Range-finding analyses were conducted on Monday, September 22nd. Isolates were received at the verification laboratory on September 26, 2003. Verifications were started on October 2,2003 and completed on October 30,2003. February 2004 ------- EPA Method 1600 Validation Study Results 3.3 Research, Participant, and Verification Laboratories The 12 participating laboratories, verification laboratory, and research laboratory involved in the Study are shown in Table 3. Table 3. Laboratories Participating in the Intel-laboratory Validation of Method 1600 * City of Los Angeles Bureau of Sanitation Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda, and Zora Bahariance Hyperion Treatment Plant 12000 Vista del Mar, Playa del Rey, CA 90293 County Sanitation Districts of L.A. County (JWPCP) Kathy Walker, Michele Padilla, and Albert Soof 24501 South Figueroa Street, Carson, CA 90745 County Sanitation Districts of L.A. County (SJC) Shawn Thompson and Julie Millenbach 1965 South Workman Mill Road, Whittier, CA 90601 Environmental Associates Ltd. Susan Boutros and John Chandler 24 Oakbrook Drive, Ithaca, NY 14850 Hampton Roads Sanitation District Anna Rule, Paula Hogg, and Bob Maunz 1432 Air Rail Avenue, Virginia Beach, VA 23471 Hoosier Microbiological Laboratories Don Hendrickson, Katy Bilger and Lindsey Shelton 912 West McGalliard, Muncie, IN 47303 Massachusetts Water Resources Authority Steve Rhode and Mariya Gofhsteyn 190 Tafts Avenue, Winthrop, MA 02152 North Shore Sanitation District Robert Flood William Koespel Drive, Guemee, IL 60031 Texas A&M University Suresh Pillai and Reema Singh 4180 Kleberg Center, 2472 TAMUS, College Station, TX 77843 University of Iowa, Hygienic Laboratory Nancy Hall and Cathy Lord Oakdate Campus # H101 OH, Iowa City, IA 52242 Wisconsin State Laboratory of Hygiene Jon Standridge, Sharon Kluender, Linda Peterson, and Jeremy Olstadt 2601 Agriculture Drive, Madison, Wl 53718 Utah Department of Health Sanwat Chaudhuri and Devon Cole 46 North Medical Drive, Salt Lake City, UT 841 13 Verification laboratory: City of Los Angeles Bureau of Sanitation, Microbiology Laboratory Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda, and Zora Bahariance Hyperion Treatment Plant, 12000 Vista del Mar, Playa del Rey, CA 90293 Research laboratory: EPA Office of Research and Development, National Risk Management Research Lab Mark C. Meckes 26 West Martin Luther King Dr., Cincinnati, OH 45268-1320 No endorsement of these laboratories is implied, nor should any be inferred. Participant laboratories have been randomly assigned numbers for purposes of presenting data in this report. February 2004 ------- EPA Method 1600 Validation Study Results SECTION 4.0 DATA REPORTING AND VALIDATION 4.1 Data Reporting Laboratories submitted the following data to DynCorp for review and validation: • Completed cover sheet with sample collection and QC information • Completed sample-specific reporting forms • Documentation of any additional information that would assist in evaluating the data 4.2 Data Validation DynCorp used data review checklists to ensure that each data package was complete and to ensure that each sample result met the study-specific and method-specific requirements. Items reviewed for each sample included the following: • Confirmation that original forms were submitted • Confirmation that incubation times were met • Confirmation that incubation temperatures were met • Confirmation that pre-filtration blank and phosphate buffer blank tested negative for enterococci • Confirmation that media sterility checks were performed and acceptable • Confirmation that positive and negative controls were performed and exhibited the appropriate response • Confirmation that samples were spiked with the appropriate dilution • Confirmation that calculations were correct This process was performed independently by two data reviewers, each of whom entered the results into separate spreadsheets designed for data review and validation for this study. The results were compared to verify consistency and identify potential data entry errors. Based on data review, the data from Laboratory 2 were considered invalid and unacceptable for inclusion in subsequent data analysis because the laboratory did not perform all of the required quality control checks (pre- filtration blank and negative control) during the validation study. It should be noted that several laboratories were instructed to spike less than 100 mL of disinfected wastewater because of either turbidity or high enterococci concentrations in me samples (based on range-finding analyses). Spiking and filtering a smaller volume of sample helped ensure that plates within the optimum counting range were obtained. Adjustments were made as follows: • Laboratory 1: Due to high enterococci concentrations, Laboratory 1 was instructed to spike 1 mL of disinfected wastewater per sample. Laboratory 9: Laboratory 9 spiked 100 mL of disinfected effluent and filtered three volumes (i.e., 50, 30, and 10 mL) to obtain countable plates. • Laboratory 10: Laboratory 10 could not filter volumes greater than 25 mL due to the turbidity of the disinfected effluent samples. As a result, Laboratory 10 was instructed to spike 25 mL of disinfected wastewater per sample. Note/Results from Laboratory 10, Sample 8 (disinfected wastewater spiked with a BioBall™) were not available because of confluent growth. • Laboratories 11 and 12: Due to high enterococci concentrations, Laboratories 11 and 12 were instructed to spike 10 mL of disinfected wastewater per sample. Results from Laboratory 10, Sample 8 (disinfected wastewater spiked with a BioBall™) were not available February 2004 10 ------- EPA Method 1600 Validation Study Results because of confluent growth. 4.3 Censored Data During the evaluation of validation study samples, results below the analytical range of the method (less-than results, also referred to as left-censored) were observed for some of the unspiked disinfected effluent samples. Left-censored results were observed with the following frequency: <1 enterococci per 100 mL, 18 results; <4 enterococci per 100 mL, four results; <10 enterococci per 100 mL, one result; and <100 enterococci per 100 mL, one result. The censor limit was replaced with one half of the "less than" value for subsequent data analyses for these samples. It should be noted that at first glance, replacing the < 100 enterococci per 100 mL observed in Laboratory 1's unspiked disinfected wastewater with 50 enterococci per 100 mL seems unreasonable. However, that laboratory's other results for unspiked disinfected wastewater were 700, 300, and 300 enterococci per 100 mL. Since the other results were much higher than the < 100 value, simply removing the < 100 from the data set for estimating the concentration of background enterococci in the disinfected sample was deemed inappropriate and half the censor limit was used instead. 11 February 2004 ------- EPA Method 1600 Validation Study Results SECTION 5.0 RESULTS All of the results included in this section were considered valid. Please see Section 4 for detailed data invalidation information. Results of unspiked wastewater samples and false positive/negative rates are provided in Section 5.1, results of spiked disinfected wastewater samples are provided in Section 5.2, and results of spiked PBS samples are provided in Section 5.3. Please see Section 6 for the development of QC acceptance criteria and Section 7 for a discussion of method performance. 5.1 Unspiked Sample Results Results from unspiked disinfected wastewater sample analyses are provided in Table 4. These data were used to estimate the background concentration of enterococci in disinfected wastewater samples. Results of the verification analysis (assessment of false positive and negative rates based on unspiked disinfected and secondary confirmations) were used to assess method performance (see discussion in Section 7). Valid verification results from unspiked disinfected and secondary wastewater effluent samples are summarized in Table 5. Valid, laboratory-specific verification results are summarized in Tabk 6. Any typical colony that was identified as non-enterococci by the Vitek® was considered a false positive result. Any atypical colony that was identified as an enterococci by the Vitek® was considered a false negative result. It should be noted that some of the isolates submitted to verification did not exhibit growth on one of the two verification streak plates (mEI or blood agar). Isolates that did not grow on blood agar at the verification laboratory were treated as if they had not been submitted to verification and eliminated from subsequent data analyses. Colonies that did not grow after streaking for isolation on mEI agar plates, were considered to be non-enterococci and included in data analyses as non-enterococci colonies. The decision to include the colonies that exhibited no growth on the mEI as non- enterococci was based on the selective nature of the medium, as the mEI was considered the first step in the verification process. February 2004 12 ------- EPA Method 1600 Validation Study Results Table 4. Summary of Enterococci Results from Unspiked Disinfected Wastewater Samples (see Table 1 for cross-reference to sample number) Lab 1 2 3 4 5 6 7 8 9 10 11 12 Sample number (CPU a/100 mL) 1 <100 ; ^*A^ <1 <1 <1 <1 4 <1 84 <4 30 10 2 700 > "'", '3&K '»"** :' ',&?:" 0.88 0.50 0.63 0.50 6.00 0.50 76.25 2.00 47.50 8.75 43.73 SDb 268.87 I'V- ' ' " ^ 0.75 0.00 0.25 0.00 2.83 0.00 5.44 0.00 22.17 2.50 81.4* Colony forming units " Standard deviation c Data validation discussed in Section 4.2 " Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances Table 5. Summary of False Positive and False Negative Rates Associated with Unspiked Disinfected and Unspiked Secondary Wastewater Effluents Matrix (Sample No.) Unspiked Disinfected (Samples 1 - 4) Unspiked Secondary (Samples 5, 6) Unspiked Disinfected/Secondary (Samples 1-6) False Positive Assessment Typical colonies submitted 69 132 201 Number of false positives 3 11 14 False positive rate (%) 4.3 8.3 7.0 False Negative Assessment Atypical colonies submitted 12 118 130 Number of false negatives 8 62 70 False negative rate (%) 66.7 52.5 53.9 13 February 2004 ------- EPA Method 1600 Validation Study Results Table 6. Laboratory-Specific False Positive and False Negative Rates Associated with Unspiked Wastewater Effluents (Disinfected and Secondary Results Combined, Samples 1-6, see cross- reference in Table 1) Lab 1 2 3 4 5 6 7 8 9 10 11 12 False Positive Assessment Typical colonies submitted 20 14 17 18 20 17 18 20 19 20 18 Number of false positives 0 0 0 0 2 0 5 0 4 3 0 False positive rate(%) 0.0 0.0 0.0 0.0 10.0 0.0 27.8 0.0 21.1 15.0 0.0 False Negative Assessment Atypical colonies submitted 15 Number of false negatives 14 3 6 17 3 6 17 False negative rate (%) 93.3 ;^/'^%?^ 100.0 100.0 100.0 ' - 11 15 18 14 18 13 4 2 8 4 7 5 36.4 13.3 44.4 28.6 38.9 38.5 Data validation discussed in Section 4.2 b Atypical colonies were only observed on filters with colonies that were too numerous to count (TNTC) February 2004 14 ------- EPA Method 1600 Validation Study Results 5.2 Spiked Disinfected Sample Results Results from disinfected wastewater samples spiked with BioBalls™ (Table 7) were used to assess method performance (see discussion in Section 7) and develop QC acceptance criteria for matrix spikes for use in assessing matrix interferences (see discussion in Section 6). Results from disinfected wastewater samples spiked with laboratory-prepared spiking suspensions (Table 8) were used to develop QC acceptance criteria (Section 6, these data were not used to assess method performance). Table 7. Summary of Enterococci Results from Disinfected Samples Spiked with BioBalls™ (see Table 1 for cross-reference to sample number) Lab 1 2 3 4 5 6 7 8 9 10 11 12 Spike Level (CFU/100mL)''b 3210 Percent Recovery by Sample 7 95.4 8 95.4 .iL^iL^v^* ^ ,<:> * "w 32.1 32.1 32.1 32.1 32.1 32.1 32.1 128.4 321 321 87.6 76.3 82.2 95.1 96.6 101.2 58.4 82.6 81.8 93.8 87.6 91.9 91.5 73.2 90.3 85.7 171.3 72.4 97.0 Overall (n = 21) Mean Percent Recovery 95.4 SDe 0.0 «^;:,^;w , ^-''<^*T« 87.6 84.1 86.8 84.1 93.5 93.5 114.9 82.6 77.1 95.4 90.8 0.0 11.0 6.6 15.4 4.4 11.0 79.9 "' " " NtM flS! * , ^^*™! 6.6 2.2 25.2' RSD'(%) 0.0 <;<~'^r^ 0.0 13.1 7.6 18.3 4.7 11.8 69.5 sessed : 8.6 2.3 22.6<> Colony forming units b Spike level is based on the tot mean value provided by the manufacturer and the volume of sample that was spiked (it was necessary to spike <100 mL at some laboratories because of either high turbidity or high background concentrations of enterococci) c Standard deviation d Relative standard deviation " Data review and validation discussed in Section 4.2 ' Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances 8 Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab RSDs 15 February 2004 ------- EPA Method 1600 Validation Study Results Table 8. Summary Enterococci Results from Disinfected Samples Spiked with Laboratory-Prepared Soikinq Suspensions (see Table 1 for cross-reference to sample number) Lab 1 2 3 4 5 6 7 8 9 10 11 12 Spike Level (CFU/100 mL)"* 3440 Percent Recovery by Sample 9 26.5 55.2 64.2 44 193 60.7 62.52 32 114 1080 710 87.2 75.5 105.4 43.8 42.8 90.4 84.6 107.0 74.3 28.3 10 25.1 103.5 81.8 78.1 46.4 41.2 71.2 58.6 86.0 81.7 28.3 Overall (n = 22) Mean Percent Recovery 25.8 95.3 78.7 91.8 45.1 42.0 80.8 71.6 96.5 78.0 28.3 66.7 SDe 1.0 11.5 4.4 19.3 1.8 1.2 13.6 18.4 14.9 5.2 0.0 10.9' RSD"(%) 4.0 «4:ftv«'i-';i^,- VJ, ... , ,„* . 12.1 5.6 21.0 4.1 2.8 16.8 25.7 15.4 6.7 0.0 13.1" Colony forming units b Spike level is based on laboratory enumeration of spiking suspension and the volume of sample that was spiked (it was necessary to spike <100 mL at some laboratories because of either high turbidity or high background concentrations of enterococti) c Standard deviation * Relative standard deviation • Data validation discussed in Section 4.2 ' Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances 8 Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab RSDs February 2004 16 ------- EPA Method 1600 Validation Study Results 5.3 Spiked PBS Results Results from PBS samples spiked with BioBalls™ (Table 9) and PBS samples spiked with laboratory-prepared spiking suspensions (Table 10) were used to develop QC acceptance criteria for use in assessing initial and on- going method/laboratory performance (see discussion in Section 6). Table 9. Summary of Enterococci Results from PBS Samples Spiked with BioBalls™ (see Table 1 for cross-reference to sample number) Lab 1 2 3 4 5 6 7 8 9 10 11 12 Spike Level (CFU/IOOmL)"* 32.1 32.1 32.1 32.1 32.1 32.1 32.1 32.1 32.1 32.1 32.1 Percent Recovery by Sample 11 93.5 99.7 102.8 102.8 90.3 87.2 87.2 99.7 93.5 90.3 96.6 12 99.7 13 96.6 14 102.8 Mean Percent Recovery 98.1 e •' ^Slii^:^^ • ''.:• '"' *:>$!$*$:'' •'-.': 96.6 96.6 102.8 87.2 99.7 87.2 93.5 93.5 87.2 99.7 109.0 90.3 99.7 93.5 71.7 96.6 105.9 96.6 99.7 90.3 93.5 84.1 115.3 96.6 93.5 84.1 77.9 99.7 109.0 102.8 Overall (n = 44) 99.7 93.5 105.1 91.9 88.0 88.8 94.2 95.8 96.6 97.4 95.4 SDe 4.0 t >': •>;;?;• = 6.7 8.0 6.9 4.0 12.0 5.4 12.0 3.0 9.9 5.3 7.6' «-•« 4.1 4 6.8 8.6 6.6 4.4 13.7 6.1 12.8 3.1 10.2 5.5 8.1- Colony forming units " Spike level is based on the tot mean value provided by the manufacturer c Standard deviation " Relative standard deviation 8 Data validation discussed in Section 4.2 ' Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances 9 Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab RSDs 17 February 2004 ------- EPA Method 1600 Validation Study Results Table 10. Summary of Enterococci Results from PBS Samples Spiked with Laboratory-Prepared Spiking Suspensions (see Table 1 for cross-reference to sample number) Lab 1 2 3 4 5 6 7 8 9 10 11 12 Spike Level (CFU/IOOmL)1* 34.4 •*?•', ' 4 Ife-'Jfllfe^SC 55.2 64.2 44 193 60.7 62.5 32 28.5 108 71 Percent Recovery by Sample 15 49.4 90.6 76.3 81.8 46.1 37.9 76.8 93.8 80.7 86.1 42.3 16 26.2 123.2 77.9 86.4 51.8 44.5 120.0 93.8 77.2 80.6 26.8 17 34.9 83.3 84.1 84.1 51.3 34.6 96.0 125.0 98.2 82.4 23.9 18 20.3 Mean Percent Recovery 32.7 65.2 82.6 118.2 43.0 32.9 88.0 81.3 77.2 82.4 36.6 Overall (n = 44) 90.6 80.2 92.6 48.1 37.5 95.2 98.4 83.3 82.9 32.4 70.4 SDe 12.6 RSD-(%) 38.7 24.2 3.7 17.1 4.2 5.1 18.3 18.7 10.1 2.3 8.5 13.4' 26.7 4.6 18.5 8.8 13.6 19.2 19.0 12.1 2.8 26.3 20.0" Colony forming units b Spike level is based on laboratory enumeration of spiking suspension on tryptic soy agar (ISA) plates c Standard deviation d Relative standard deviation ' Data validation discussed in Section 4.2 ' Pooled withln-lab standard deviation was determined by calculating the square root of the mean of the lab variances 9 Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab RSDs February 2004 18 ------- EPA Method 1600 Validation Study Results SECTION 6.0 DEVELOPMENT OF QC ACCEPTANCE CRITERIA All data analyses described below were performed using the results of disinfected wastewater and PBS samples spiked with either BioBalls™ or laboratory-prepared spiking suspensions. Separate QC acceptance criteria were calculated for BioBall™ and laboratory-prepared spike results, to ensure that QC criteria are available for this method if BioBalls™ become unavailable. 6.1 Outlier Analyses Valid results from samples spiked with BioBalls™ and laboratory-prepared spiking suspensions were screened for outliers in accordance with the procedures described in American Society for Testing and Materials (ASTM) guidance D2777-98 (Reference 8.2). Outlying data were identified and removed in two steps: identification of outlying laboratories, followed by identification of individual sample results. First, outlying laboratories were identified using Youden's laboratory ranking test (Reference 8.2). For this test, laboratories were ranked and screened to identify laboratories with significantly higher or lower results than the other laboratories. The second test for identification of outlying data is the Grubbs test (Reference 8.2), which identifies individual sample results for outlying observations. It should be noted that outlier analyses were only performed for development of QC acceptance criteria (Section 6). Outlier analyses were not conducted for the assessment of method performance (Section 7), as all valid data were included in the assessment of method performance. 6.1.1 Youden's Laboratory Ranking Test The valid data were first tested for the presence of an outlying laboratory using Youden's laboratory ranking test. For this test, results were stratified by spike type (BioBalls™ vs laboratory-prepared), using recoveries from both disinfected wastewater and PBS samples. Youden's test was conducted separately by spike type, because separate criteria were to be calculated for BioBall™ and laboratory-enumerated spiked samples. Sample results for each laboratory were sorted based on sample number. Prior to conducting the Youden's laboratory ranking test, one missing result was replaced by the mean of the non-missing results for the given spike type, matrix, and laboratory. This replacement value was only used for the Youden test and was not used in any other data analyses. Based on the Youden's test, two laboratories were removed for laboratory-prepared sample analyses. These two laboratories (Laboratories 1 and 12) were both biased low compared to the remaining laboratories, and were not used in the development of QC acceptance criteria. (The low recoveries from the laboratory- prepared spiking suspensions may be related to spiking suspensions not being sufficiently homogenized prior to spiking samples or enumerating the spiking suspensions.) No laboratories were removed for BioBall™ spiked sample analyses. 6.1 .2 Grubbs Test for Individual Outlying Sample Results After removing outlying laboratories identified using Youden's lab ranking test, the remaining data were then tested for the presence of individual outlying results using Grubbs test. Grubbs test was run separately for each matrix (disinfected wastewater and PBS) and spike type. Grubbs test was run without performing any data transformations. This was because statistical and graphical evaluations did not reveal any major departures from the assumption of the data following a Normal distribution. Application of Grubbs test resulted in the removal of one BioBall™ disinfected wastewater sample result analyzed by Laboratory 9 with a recovery of 171%. This result was not used in the development of QC acceptance criteria. February 2004 ------- EPA Method 1600 Validation Study Results 6.2 Initial Precision and Recovery (IPR) and Ongoing Precision and Recovery (OPR) QC acceptance criteria for initial precision and recovery (DPR) and ongoing precision and recovery (OPR) were developed based on the results from PBS (reference matrix) samples spiked with BioBalls™ and laboratory- prepared spiking suspensions during the Study, as these QC tests will be performed using PBS as the reference matrix by laboratories using the method during monitoring. Again, separate QC acceptance criteria were calculated for BioBall™ and laboratory-prepared spike results The IPR and OPR recovery criteria were calculated based on within and between laboratory variance components. These variance components were calculated with PROC MIXED from the SAS version 8 program using the maximum likelihood method of estimation on the recovery results. Details on the maximum likelihood estimation can be found in the user's guide for this program (Reference 8.3). Estimates of between laboratory variance and within laboratory variance were labeled s2L and s2w, respe. vely. The combined standard deviation for IPR (isj is: Where: L = number of labs for the given spiking procedure n, = number of PBS sample results for laboratory if or the given spiking procedure nT = total number of PBS results from all laboratories for the given spiking procedure Upper and lower limits for IPR samples were then calculated as: y +1 *iv •* Mem - '(0.975; Uf) "c Where idfis calculated using Satterthwaite 's estimate as given below: IY L \ 1"' 1 i '•' i i r" V / 1 *«2 SL 2 \(l 0* *1 \A nj " _ L-\ nT-L February 2004 20 ------- EPA Method 1600 Validation Study Results The combined standard deviation (osc) for OPR is: Where: L — number of labs for the given spiking procedure n, = number of PBS sample results for laboratory if or the given spiking procedure nT = total number of PBS results from all laboratories for the given spiking procedure Upper and lower limits for OPR samples were then calculated as: y + t * s*e •"- Mean — l(0.975;oay) UAc Where odfis calculated using Satterthwaite 's estimate as given below: odf = - OS, '( I > Z-? 1 1 '•' n\ V. ) *s2 •*i 2 \- "M'-1-" V nT) L-l nT- L The precision criterion for IPR samples was calculated as a maximum relative standard deviation (RSD). The RSD for each laboratory and each spiking procedure was calculated by dividing the standard deviation of the recoveries by the mean of the recoveries for that laboratory and procedure. The RSDs were pooled directly, rather than calculating a pooled RSD by pooling standard deviations and dividing by an overall mean, because the pooled RSD calculated using the latter approach was unduly affected by laboratories which exhibited high bias and low variability. The pooled RSD was then calculated as: *SD^- = (n,-l)*RSD? Where: L = number of laboratories for the given procedure n, = number of PBS sample results for laboratory ifor the given spiking procedure nT = total number of PBS sample results for all laboratories for the given spiking procedure RSD, - RSD calculated using the recoveries for laboratory ifor the given spiking procedure 21 February 2004 ------- EPA Method 1600 Validation Study Results The maximum RSD was then calculated as: -Ł) pool Where: nT - total number oflPR and OPR results from all laboratories L = number of laboratories The calculated DPR and OPR QC acceptance criteria are provided in Table 11. Table 11. Calculated Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria Performance test Initial precision and recovery (IPR) Mean percent recovery Precision (as maximum relative standard deviation) Ongoing precision and recovery (OPR) as percent recovery BioBall™ acceptance criteria 85% - 106% 14% 78% -113% Lab-prepared spike acceptance criteria 31% - 127% 28% 27% -131% 6.3 Matrix Spike (MS) and Matrix Spike Duplicate (MSD) Recovery and Precision QC acceptance criteria for matrix spikes (MS) and matrix spike duplicates (MSD) were developed based on data from the spiked disinfected wastewater matrices used in the validation study. Separate QC acceptance criteria were calculated for BioBall™ and laboratory-prepared spike results Recovery criteria were based on estimates of each variance component (between laboratory and within laboratory) and were calculated using PROC MIXED from SAS version 8 using the maximum likelihood method of estimation on the recovery results. Details on the maximum likelihood estimation can be found in the user's guide for this program (Reference 8.3). Between matrix variability could not be separated from between laboratory variability because each laboratory analyzed a different disinfected wastewater sample, and therefore the estimate of between laboratory variance also includes matrix variability. February 2004 22 ------- EPA Method 1600 Validation Study Results Estimates of between laboratory variance and within laboratory variance were labeled s\and s2w, respectively. The combined standard deviation for MS/MSD (sc) is: 1 Where: L = number of labs for the given spiking procedure n, = number of disinfected sample results for laboratory ifor the given spiking procedure nT = total number disinfected sample results from all laboratories for the given spiking procedure Upper and lower limits for MS/MSD samples were then calculated as: 4- / * Mean — ' (0.975;df) Where: Km*™ = tne wea/i recovery of all disinfected wastewater samples, and for the given spiking procedure dfis calculated using Satterthwaite 's estimate as given below: */ T v 2! i . i=l 1+ j nT \ 1 •*s2 SL 2 — j_ - rr i 1+ — *s2 \ nT) L-l nT- L The precision criterion for MS/MSD samples was calculated as a maximum relative percent difference (RPD). The RSD for each laboratory and matrix was calculated by dividing the standard deviation of the two recoveries by the mean of those recoveries for that lab and matrix. The RSDs were pooled directly, rather than calculating a pooled RSD by pooling standard deviations and dividing by an overall mean, because the pooled RSD calculated using the latter approach was unduly affected by laboratories/matrices which exhibited high bias and low variability. 23 February 2004 ------- EPA Method 1600 Validation Study Results The pooled RSD was then calculated as: lv = ,->. RSD? 1=1 Where: L = number of laboratories for the given spiking procedure, and RSD, = RSD calculated using the recoveries for laboratory if or the given spiking procedure The maximum RPD was then calculated as: RSD pool Where: L - 2 = the total number of 'laboratories for the given spiking procedure, and a constant used to convert an RSD to an RPD (when calculated using 2 values, the RSD and RPD differ by a factor of V2 ). The calculated MS/MSD QC acceptance criteria are listed in Table 12. Table 12. Calculated Matrix Spike Precision and Recovery Acceptance Criteria Performance test Mean percent recovery for MS or MS/MSD Precision (as maximum relative percent difference of MS/MSD) BioBall™ acceptance criteria 63% -110% 28% Lab-prepared acceptance criteria 29% - 122% 47% February 2004 24 ------- EPA Method 1600 Validation Study Results SECTION 7.0 ASSESSMENT OF METHOD PERFORMANCE: DISCUSSION AND CONCLUSIONS Results of this Study enabled assessment of the Method's performance in PBS and disinfected wastewater, and enabled development of quality control (QC) acceptance criteria that can be used to confirm acceptable laboratory and method performance on an ongoing basis in disinfected wastewater monitoring surveys and other studies. Method performance was evaluated through the assessment of false positive and negative rates in unspiked disinfected and unspiked secondary wastewater samples and through the evaluation of precision and recovery in disinfected wastewater samples spiked with BioBalls™. (Results from samples spiked with laboratory-prepared spiking suspensions were not used to assess method performance.) It should be noted that outlier analyses were not conducted for the assessment of method performance, as all valid data were included in the assessment of method performance. Results from the laboratory enumerations of the BioBalls™ indicate that BioBall™ spikes ofE.faecalis (ATCC #19433) were stable and precise, as laboratory enumeration of the BioBalls™ estimated a mean of 29.9 CPU per BioBall™ and a pooled within-laboratory RSD of 10.5%, compared to the manufacturer's lot mean value of 32.1 CPU per BioBall™ and RSD of 7.8%. In contrast, the laboratory-prepared spiking suspensions (used for development of QC acceptance criteria) were significantly less precise (based on an F-test comparing the pooled within-laboratory RSDs at a=0.05), with a pooled within-laboratory RSD of 24.0%. Method 1600 recovery of enterococci was acceptable, with mean laboratory-specific recoveries of enterococci from disinfected wastewater samples spiked with BioBalls™ ranging from 77.1% to 114.9%, with an overall mean recovery of 90.8%. Laboratory-specific RSDs ranged from 0% to 69.5%, with a pooled, within-laboratory RSD of 22.6%. False positive rates were also acceptable, with laboratory-specific false positive rates for unspiked disinfected/secondary results combined, ranging from 0% - 27.8%. For secondary wastewater (excluding disinfected results), only 11 of 132 typical colonies submitted to verification were non-enterococci, resulting in a false positive rate of 8.3% for secondary wastewater. For disinfected wastewater (excluding secondary results), only three of 69 typical colonies submitted to verification were non-enterococci, resulting in a false positive rate of 4.3% for disinfected wastewater. In contrast, laboratory-specific false negative rates for unspiked disinfected/secondary results combined, ranged from 13.3% - 100.0%. For secondary wastewater (excluding disinfected results), only 62 of 118 atypical colonies submitted to verification were identified as enterococci, resulting in a false negative rate of 52.5% for secondary wastewater. For disinfected wastewater (excluding secondary results), eight of 12 atypical colonies submitted to verification were identified as enterococci, resulting in a false negative rate of 66.7% for disinfected wastewater. Results of this study indicate that Method 1600 precision, recovery, and false positive rates are acceptable for the determination of enterococci in disinfected wastewater. However, false negative rates observed during this study were high and should be taken into consideration when using results from this method. When evaluating wastewater using Method 1600, it is recommended that the false negative rate for each matrix be evaluated through biochemical confirmation and results adjusted accordingly, especially if large numbers of atypical colonies are observed in a particular matrix. If very few atypical colonies are observed in samples for a particular matrix, the high false negative rates observed during this study may be less of a concern. 25 February 2004 ------- EPA Method 1600 Validation Study Results SECTION 8.0 REFERENCES 8.1 American Public Health Association, American Water Works Association, and Water Environment Federation. 1995. Standard Methods for Water and Wastewater. 20th Edition. Sections: 9020, 9221, 9222. 8.2 American Society for Testing and Materials. 1998. Annual Book of ASTM Standards, Vol. 11.01. Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D-19 on Water, ASTM D2777-98, October 1998. 8.3 SAS Institute Inc. 1994. SAS/STAT User's Guide, Volume 2, GLM-VARCOMP. Version 6,4th Edition, June 1994. 8.4 USEPA. 2002. EPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane- Enterococcus Indoxyl-p-D Glucoside Agar (mEI), EPA-821-R-02-022, September 2002. February 2004 26 ------- EPA Method 1600 Validation Study Results SECTION 9.0 ACRONYMS CPU Colony forming unit IPR Initial precision and recovery MS Matrix spike MSD Matrix spike duplicate OPR Ongoing precision and recovery QA Quality assurance QC Quality control RPD Relative percent difference RSD Relative standard deviation SAS Statistical analysis software SD Standard deviation 27 February 2004 ------- ------- Appendix A: Method 1600 Spiking Protocol ------- ------- EPA Method 1600 Validation Study Results Enterococci Spiking Protocol Interlaboratory Wastewater Validation Study of Method 1600 (August 20,2003) The purpose of this protocol is to provide laboratories with enterococci spiking procedures for the interlaboratory wastewater validation study of Method 1600. During this study, laboratories will spike samples using laboratory- prepared spiking solutions and with BioBalls™ (a commercially available product from BioTechnology Frontiers, Sydney, Australia). The following sections are included in this protocol: Laboratory-Prepared Spiking Solutions Section 1: Preparation of Laboratory-Prepared Spiking Suspensions Section 2: Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration Section 3: Calculation of Laboratory-Prepared Spike Percent Recovery BioBalls™ Section 4: BioBall™ Sample Spiking and BioBall™ Enumeration Section 5: Calculation of BioBall™ Spike Percent Recovery 1.0 Preparation of Laboratory-Prepared Spiking Suspensions 1.1 Stock Culture. Prepare a stock culture by inoculating a trypticase soy agar (TSA) slant (or other non- selective media) with Enterococcus faecalis ATCC #19433 and incubating at 35°C ± 3°C for 20 ± 4 hours. This stock culture may be stored in the dark at room temperature for up to 30 days. 1.2 1% Azide Dextrose Broth. Prepare a 1% solution of azide dextrose broth by combining 99 mL of sterile phosphate buffered saline (Method 1600, Section 7.4) and 1 mL of sterile single strength azide dextrose broth in a sterile screw cap bottle or re-sealable dilution water container. Shake to mix. 1.3 Spiking Suspension (Undiluted). From the stock culture of Enterococcus faecalis ATCC #19433 in Section 1.1, transfer a small loopful of growth to the 1% Azide dextrose broth solution and vigorously shake a minimum of 25 times. Incubate at 35°C ± 3°C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0 * 106 to 1.0 x 107 enterococci colony forming units (CFU) per mL. This is referred to as the "undiluted spiking suspension." Note: During the Method 1600 validation study, growth of spiking suspensions will begin on Monday, so the spiking suspensions are ready to be spiked into the samples on Tuesday. 1.4 Proceed to Section 2.0 for sample spiking and enumeration of spiking suspension. 2.0 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration Since the objective of spiking the sample is to establish percent recovery, it is necessary to determine the number of enterococci in the undiluted spiking suspension prepared in Section 1.3. This section provides instructions for sample spiking (Section 2.1) and spiking suspension enumeration (2.2). A-1 February 2004 ------- EPA Method 1600 Validation Study Results 2.1 Sample spiking 2.1.1 Dilute spiking suspension 2.1.1.1 Mix the spiking suspension by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the undiluted spiking suspension (from Section 1.3 above) to 99 mL of sterile phosphate buffered saline (Method 1600, Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution "A". A 1.0-mL volume of dilution "A" is 10"2 mL of the original undiluted spiking suspension. 2.1.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution "A" (from Section 2.1.1.1 above) to 99 mL of sterile phosphate buffered saline (Method 1600, Section 7.4), cap, and mix by vigorously snaking the bottle a minimum of 25 times. This is spiking suspension dilution "B". A 1.0-mL volume of dilution "B" is 10"4 mL of the original undiluted spiking suspension. 2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" (from Section 2.1.1.2 above) to 99 mL of sterile phosphate buffered saline (Method 1600, Section 7.4), cap, and mix by vigorously snaking the bottle a minimum of 25 times. This is spiking suspension dilution "C". A 1.0-mL volume of dilution "C" is 10"5 mL of the original undiluted spiking suspension. 2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C' (from Section 2.1.1.3 above) to 99 mL of sterile phosphate buffered saline (Method 1600, Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution "D". A 1.0-mL volume of dilution "D" is 10"6 mL of the original undiluted spiking suspension. 2.1.2 Spike samplers') 2.1.2.1 To spike sample, add 3.0 mL of spiking suspen. n dilution "D" (from Section 2.1.1.4 above) to 100-mL of unspiked sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the "spiked" sample. The volume (mL) of undiluted spiking suspension added to each 100 mL of sample is 3.0 x 10"* mL per 100 mL [(3.0 mL x 10"6 mL) per 100 mL of sample], which is referred to as V^^ ,„ 100 ^ ^^ in Section 3.2 below. This is the "spiked" sample. Analyze the spiked sample according to the instructions provided in Method 1600, Section 11.0. 2.2 Enumeration of undiluted spiking suspension (prepared in Section 1.3) 2.2.1 Prepare trypticase soy agar (TSA) according to manufacturer's directions, add 10-15 mL of TSA per 100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note: Agar plates must be dry and free from condensation prior to use. To ensure that th^ agar surface is dry, plates should be made several days in advance and stored inverted at re n temperature or dried using a laminar-flow hood. 2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread plates: • Pipet 0.1 mL of dilution "B" (Section 2.1.1.2) onto surface of pre-dried TSA plate [ 10'5 mL (0.00001) of the original spiking suspension]. • Pipet 0.1 mL of dilution "C" (Section 2.1.1.3) onto surface of pre-dried TSA plate [W6 mL (0.00000 l)of the original spiking suspension]. • Pipet 0.1 mL of dilution "D" (Section 2.1.1.4) onto surface of pre-dried TSA plate [ 10"7 mL (0.000000 l)of the. original spiking suspension]. 2.2.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum over the surface of medium by rotating the dish by hand or on a turntable. Note: Please ensure that the February 2004 A^2 ------- EPA Method 1600 Validation Study Results inoculum is evenly distributed over the entire surface of the plate. 2.2.4 Allow inoculum to absorb into the medium completely. 2.2.5 Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours. 2.2.6 Count and record number of colonies per plate. Refer to Section 3.0 for calculation of spiking suspension concentration. 3.0 Calculation of Spiked Enterococci Percent Recovery Spiked enterococci percent recovery will be calculated in three steps as indicated in Sections 3.1 through 3.3 below. Note: The example calculated numbers provided in the tables below have been rounded at the end of each step. If your laboratory recalculates the examples using a spreadsheet and rounds only after the final calculation (Step 3), the percent recoveries may be slightly different. 3.1 Step 1: Calculate Concentration of Enterococci (CPU / mL) in Undiluted Spiking Suspension 3.1.1 The number of enterococci (CFU / mL) in the undiluted spiking suspension (prepared in Section 1.3 above) will be calculated using all TSA plates from Section 2.2 yielding counts within the ideal range of 30 to 300 CFU per plate. 3.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if the colonies are not discrete, results should be recorded as "too numerous to count" (TNTC). 3.1.3 Calculate the concentration of enterococci (CFU / mL) in the undiluted spiking suspension according to the following equation. (Example calculations are provided in Table 1 below.) Enterococci undHutedspike = (CFU, + CFU2 + ... + CFUn) / (V, + V2 + ... + Vn) Where, Enterococci ^^^^ ^1*,, = Enterococci (CFU / mL) in undiluted spiking suspension CFU = Number of colony forming units from TSA plates yielding counts within the ideal range of 30 to 300 CFU per plate V = Volume of undiluted sample on each TSA plate yielding counts within the ideal range of 30 to 300 CFU per plate n = Number of plates with counts within the ideal range A-3 February 2004 ------- EPA Method 1600 Validation Study Results TABLE 1. EXAMPLE CALCULATIONS OF ENTEROCOCCI SPIKING SUSPENSION CONCENTRATION Examples Example 1 Example 2 CPU / plate (triplicate analyses) from TSA plates in Section 2.2.5 10"5 mL plates 94, 106, 89 32, 55, 72 tO"6 mL plates 9,11,28 8,5,3 10-7mL plates 1,0.4 0,0,0 Enterococci CFU / mL in undiluted spiking suspension (Enterococci ondllutad ^J* (94+106+89) /(lO^+IO^+KT5) = 289 / (3.0 x 10*) = 9,633,333 = 9.6x106CFU/mL (32+55+72) / (10*HO*+10*) = 159 / (3.0 x 10*) =5,300,000 = 5.3 x 10* CFU / mL •Enterococci und,^,,*. is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate 3.2 Step 2: Calculate "True" Spiked Enterococci (CFU /100 mL) 3.2.1 Calculate true concentration of spiked enterococci (CFU /100 mL) according to the following equation. Example calculations are provided in Table 2 below. 'spiked Enterococci = (EnteFOCOCCI undj|utec| spike) X ( * spiked per 100 mL sample) Where, ^ Spiked Enterococci Enterococci spiked perl 00 mL sample Number of spiked Enterococci (CFU /100 mL) Enterococci (CFU / mL) in undiluted spiking suspension (calculated in Section 3.1.3) mL of undiluted spiking suspension per 100 mL sample (Section 2.1.2.1) Table 2. Example Calculations of Spiked Enterococci Enterococci „„»,„*«, „*„ (Table 1 above) 9.6x106CFU/mL 5.3 x 106 CFU / ml • spHwd par 100 mL nmpte (Section 2.1.2.1 above) 3.0 XIO^mL per 100mL of sample S.OXIO^mLperlOOmL of sample • SplkKl Enterococci (9.6 x 106 CFU / mL) x (3.0 x 10* ml / 100 mL) = 28.8 CFU /1 00 mL (2.8 x 106 CFU / mL) x (3.0 x 10"6 mL / 100 mL) = 8.4 CFU / 100 mL February 2004 A-4 ------- EPA Method 1600 Validation Study Results 3.3 Step 3: Calculate Percent Recovery 3.3.1 Calculate percent recovery (R) using the following equation. Where, R N, N_ 1 Spiked Enterococci Percent recovery Enterococci (CFU /100 mL) in the spiked sample (Method 1600, Section 12) Enterococci (CFU /100 mL) in the unspiked sample (Method 1600, Section 12) True spiked Enterococci (CFU / 100 mL) in spiked sample (Section 3.2, above) Note: During the validation study, Na (unspiked sample) is the mean enterococci (CFU /100 mL) of the 4 unspiked disinfected wastewater samples. 3.3.2 Example percent recovery calculations are provided in Table 3. Table 3. Example Percent Recovery Calculations N, (CFU/100mL) 42 34 16 10 Nu (CFU / 100 mL) <1 10 <1 <1 Ts^edE-^occcc, (CFU/ 100 mL) 28.8 28.8 8.4 8.4 Percent recovery (R) 100 x (42 -1)7 28.8 = 142% 100 x (34-10)728.8 = 83% 100 x (16-1) 78.4 = 179% 100 x (10 -1)7 8.4 = 107% 4.0 BioBall™ Sample Spiking and Enumeration During the validation study, each laboratory will enumerate enterococci in the BioBalls™ so that percent recovery can be evaluated. This section provides instructions for sample spiking (Section 4.1) and spiking suspension enumeration (4.2). After receipt at your laboratory, the BioBalls™ should be stored at -20°C. Preparation of the BioBalls™ prior to spiking is not necessary, as they can be spiked directly into the sample once the vial is opened. A-5 February 2004 ------- EPA Method 1600 Validation Study Results 4.1 Sample spiking 4.1.1 Open BioBall™ vial by removing the crimp and cap. To spike a sample, aseptically add 1 BioBall™ to 100 mL of unspiked sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the "spiked" sample. Analyze the spiked sample according to the instructions provided in Method 1600, Section 11.0. 4.2 Enumeration of BioBall™ (used in Section 4.1 above) 4.2.1 Prepare trypticase soy agar (TSA) according to manufacturer's instructions, add 10-15 mL of TSA per 100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note: Agar plates must be dry and free from condensation prior to use. To ensure that the agar surface is dry, plates should be made several days in advance and stored inverted at room temperature or dried using a laminar-flow hood. 4.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of three spread plates: • Open BioBall™ vial by removing the crimp and cap. Aseptically place one BioBall™ onto the center of each pre-dried TSA plate by tipping the vial over the medium. Immediately pipette 100 ul of sterile phosphate buffered saline solution (Method 1600, Section 7.4) directly onto the BioBall™. Allow the BioBall™ to dissolve. 4.2.3 For each spread plate, using a sterile bent glass rod or spreader, distribute the BioBall™ inoculum over surface of medium by rotating the dish by hand or on a turntable. Note: Please ensure that the inoculum is evenly distributed over the entire surface of the plate. 4.2.4 Allow inoculum to absorb into the medium completely. 4.2.5 Invert plates and incubate at 35°C ± 3°C for 20 ± 4 hours. 4.2.6 Count and record number of colonies per plate. Refer to Section 5.0 for calculation of the concentration of enterococci in the BioBall™. 5.0 Calculation of BioBall™ Spike Percent Recovery Spiked BioBall™ percent recovery will be calculated following the steps indicated below. Note: The example calculated numbers provided in the tables below have been rounded at the end of each step. If your laboratory recalculates the examples using a spreadsheet and rounds only after the final calculation (Step 3), the percent recoveries may be slightly different. 5.1 Step 1: Calculate Mean Enterococci per BioBall™ and "True" Spiked Enterococci (CPU / 100mL) The mean concentration of enterococci (CPU) in the BioBalls™ will be calculated using all three TSA plates from Section 4.2. Since one BioBall™ is spiked per 100 mL sample, use the mean number of enterococci per BioBall™ as the "true" spiked enterococci per 100 mL sample. For example, Tspiked Enterococci (CFU /100 mL) = (30 + 38 + 28) / 3 = 32 per 100 mL Where, Tspikcd Enterococci = True spiked Enterococci (CFU /100 mL) in spiked sample February 2004 A-6 ------- EPA Method 1600 Validation Study Results 5.2 Step 2: Calculate Percent Recovery Calculate percent recovery (R) using the following equation. R=100x (N.-NJ Where, R N, N,, T x Spiked Enterococci Percent recovery Enterococci (CFU /100 mL) in the spiked sample (Method 1600, Section 12) Enterococci (CFU /100 mL) in the unspiked sample (Method 1600, Section 12) True spiked Enterococci (CFU / 100 mL) in spiked sample (Section 5.2, above) Note: During the validation study, Na (unspiked sample) is the mean enterococci (CFU /100 mL) of the 4 unspiked disinfected wastewater samples. Example percent recovery calculations are provided in Table 4. Table 4. Example Percent Recovery Calculations N, (CFU / 100 mL) 24 36 Nu(CFU/100mL) <1 10 T(CFU/100mL) 32 32 Percent recovery (R) 100 x (24 -1)/ 32 = 72% 100 x (36 -10) 732 = 81% A-7 February 2004 ------- ------- Appendix B: Wastewater Laboratory Capabilities Checklist ------- ------- EPA Method 1600 Validation Study Results Wastewater Laboratory Capabilities Checklist (June 17, 2003) EPA plans to invite 11 laboratories (10 participants and 1 referee) to participate in a study to validate the modified mTEC (E. coif) and mEI (enterococci) methods in wastewater effluent. EPA will provide all media and disposable materials for the study and will also cover all shipping costs. Volunteers will be acknowledged in both the method and validation study reports. The schedule will include two weeks (range-finding and validation study) of analyses per method with an additional week of practice analyses prior to the study. The study is tentatively scheduled to begin in September, with some potential analyses being conducted by the referee laboratory in August. If your laboratory is interested in participating in the validation study as either a participant laboratory or the referee, please provide the requested information below and fax the signed, completed checklist to Mike Chicoine at 703.461.8056 by Thursday July 3. In addition, please send the form electronically to Mike Chicoine at mike.chicoine(a),dvncorp.com.. Mike will confirm receipt of the checklist. If you have any questions pertaining to the information requested below or the validation study, please do not hesitate to contact Yildiz Chambers at 703.461.2165 or vildiz.chambers@.dvncorr).coni. Section 1. Laboratory Capabilities and Experience a. Please complete the requested capabilities and experience information below. The information requested in Table 1 pertains to experience with a given method, regardless of matrix (i.e., surface water, wastewater) analyzed. Table 1. Analyst Experience Analyst Years of experience or estimated number of samples analyzed Methods to be validated modified mTEC mEI Other membrane filter methods mEndo or LESEndo mFC NA-MUG mTEC mE/EIA b. Primary analyst's name: c. Primary analyst's years of experience performing wastewater analyses: d. What certifications does your laboratory have for microbial analyses? e. Additional comments: Section 2. Background Information B-l February 2004 ------- EPA Method 1600 Validation Study Results a. Does your laboratory have access to wastewater samples? Yes No If yes, please indicate in Table 2 or Table 3, as appropriate, the types ofwastewater to which you have access. b. If your laboratory has experience analyzing wastewater samples for E. coli and/or enterococci, in Table 2, place a check " Sn next to the wastewater type(s) to which you have access and indicate the method(s) used for analysis and typical concentrations of each analyte. If your laboratory does not have experience analyzing wastewater samples for E. coli and/or enterococci, please complete Table 3. Table 2. E. coli dnd Enterococci Access? Example / Wastewater type Primary treated Raw Primary treated Secondary treated Tertiary treated Chemically disinfected Monitoring frequency 1 per month E. coli Methods SM9221B/F Typical range 30x10* Enterococci Methods SM 9230B Typical range 12 x 10* c. If your laboratory has experience analyzing wastewater samples for total coliforms, fecal coliforms, fecal streptococci, or other indicator organisms, in Table 3, place a check " /" next to the wastewater type(s) to which you have access and indicate the method(s) used for analysis and typical ranges. Table 3. Other Indicator Organisms Access ? Wastewater type Raw Primary treated Secondary treated Tertiary treated Chemically disinfected Monitoring frequency Other indicator organisms Organism(s) Methods Typical range February 2004 B-2 ------- EPA Method 1600 Validation Study Results d. How many membrane filtration funnels will be available for use during the study? e. How many funnels may be used at one time (i.e., the size of the manifold that will be used to analyze samples 3, 6, etc.)? f. In Table 3, below, please indicate the medium used for isolation prior to inoculation of each type of verification procedure used in your laboratory. Table 3. Verification Procedures Verification procedure API 20E® VITEK® BIOLOG BBL Crystal™ Other (please describe below)* Isolation medium *If other, please describe: g. Is your laboratory potentially interested in verifying isolates from other laboratories? Yes No Section 3. Referee Laboratory If your laboratory is interested in participating in this study as the referee laboratory, please respond to the following questions. a. Has your laboratory served as a referee laboratory for other studies? If yes, please briefly describe the study or studies. b. Does your laboratory have experience preparing and enumerating spiking suspensions? (Please provide a brief description including organism(s) and associated study.) February 2004 ------- EPA Method 1600 Validation Study Results b. Does your laboratory have experience isolating and propagating environmental (wild type) strains from environmental samples? (Please provide a brief description including organism(s) and associated sample(s). c. Is your laboratory's shipping and receiving department familiar with the shipment of dangerous goods? (Dangerous goods shipping containers and associated shipping documentation will be provided.) d. If preparation of spiking suspensions is necessary, does your laboratory have sufficient personnel, supplies (e.g., flasks, etc.), and equipment (e.g., incubator space) to propagate spiking suspensions (E. coli and enterococci) and ship the suspensions to the participant laboratories? e. If the participant laboratories spike samples with wastewater from their own facility, does your laboratory have sufficient personnel, supplies and equipment to enumerate spikes in triplicate for each participant? f. Additional comments: I certify that the information provided above is accurate and complete: Primary Analyst or Lab Manager: Laboratory name: Signature: _____ Date: February 2004 B-4 ------- |