vvEPA United States Environmental Protection Agency EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking Water, Ambient Water, Wastewater, and Sewage Sludge Monitoring Methods September 2010 ------- U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460 EPA-821-B-10-001 ------- DISCLAIMER Neither the United States Government nor any of its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use of or the results of such use of any information, apparatus, product, or process discussed in this report, or represents that its use by such party would not infringe on privately owned rights. Mention of company names, trade names, or commercial products in this protocol 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 or OSTCWAMethods@epa.gov ------- FOREWORD This document describes a process for seeking EPA approval of microbiological alternate test procedures (ATPs) or new methods for use in monitoring drinking water, ambient water, wastewater, and sewage sludge (biosolids). This document serves as a supplement to the ATP guidelines at 40 CFR 136.4, 136.5, and 141.27. This ATP protocol describes a process for conducting side-by-side method comparisons and for conducting quality control (QC) acceptance criteria-based method studies for EPA-approved reference methods with QC acceptance criteria. Additionally, in some cases the revised protocol provides applicants an opportunity to demonstrate comparability by meeting QC acceptance criteria associated with the EPA-approved reference methods for different combinations of analyte and determinative technique. Under EPA's ATP program, any person may apply for approval of the use of an ATP or new method to test for a regulated analyte. EPA anticipates that the standardized procedures described herein should generally expedite the approval of ATPs, encourage the development of innovative technologies, and enhance the overall utility of the EPA-approved methods for compliance monitoring under the National Pollution Discharge Elimination System (NPDES) permit program and national primary drinking water regulations (NPDWRs). This document is not a legal instrument and does not establish or affect legal obligations under Federal regulations. EPA reserves the right to change this protocol without prior notice. Questions or comments regarding this document should be directed 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(giepa.gov or OSTCWAMethods@epa.gov ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol TABLE OF CONTENTS SECTION 1.0 INTRODUCTION 4 1.1 Background and Objectives 4 1.2 Types of Applications 5 1.2.1 Limited Use 5 1.2.2 Nationwide Use 5 1.3 Types of Studies 6 1.4 Scope of Alternate Test Procedures 6 1.4.1 EPA-Approved Reference Methods 6 1.4.2 Modifications to Sample Preparation Techniques 7 SECTION 2.0 APPLICATION 8 2.1 Submission Addresses and Approval Authority 8 2.2 Application Information 8 2.3 Reason for ATP 10 2.4 Standard EPA Method Format 10 2.5 Method Comparison Table 10 2.6 Method Development Information 10 2.7 Study Plan 11 2.8 Study Report 11 2.9 Proprietary Information in Applications 11 SECTION 3.0 METHOD FORMAT 13 3.1 Scope and Application 13 3.2 Summary of Method 13 3.3 Method Definitions 13 3.4 Interferences 13 3.5 Safety 14 3.6 Equipment and Supplies 14 3.7 Reagents and Standards 14 3.8 Sample Collection, Preservation, and Storage 14 3.9 Quality Control 14 3.10 Calibration and Standardization 14 3.11 Procedure 14 3.12 Data Analysis and Calculations 15 3.13 Method Performance 15 3.14 Pollution Prevention 15 3.15 Waste Management 15 3.16 References 15 3.17 Tables, Diagrams, Flowcharts, and Validation Data 15 SECTION 4.0 STUDY PLAN 16 4.1 Background 16 4.2 Objectives 16 4.3 Study Design 16 4.4 Coordination 17 4.4.1 Study Management 17 4.4.2 Technical Approach 17 4.5 Data Reporting 17 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 5.0 QUALITY ASSURANCE/QUALITY CONTROL 18 5.1 Quality Assurance 18 5.2 Quality Control 18 5.2.1 Analyst Counting Variability 20 5.2.2 Autoclave Sterilization Verification 20 5.2.3 Dilution/Rinse Water Blanks 20 5.2.4 Incubator/Water Bath Temperatures 20 5.2.5 Initial Demonstration of Capability 20 5.2.6 Initial Precision and Recovery 21 5.2.7 Matrix Spike and Matrix Spike Duplicate Samples 21 5.2.8 Media Sterility Checks 21 5.2.9 Method Blank 21 5.2.10 Ongoing Demonstration of Capability (ODC) Samples 22 5.2.11 Ongoing Precision and Recovery (OPR) Samples 22 5.2.12 Positive/Negative Controls 22 5.2.13 Preparation Blanks (PB) 23 5.2.14 Refrigerator/Freezer Temperatures 23 5.2.15 Sample Processing Equipment Sterility Checks 23 SECTION 6.0 STUDY DESIGN 24 6.1 Side-by-Side Comparison Studies 24 6.1.1 Number of Laboratories 24 6.1.2 Number of Samples 24 6.1.3 Verification of Results 27 6.2 QC Acceptance Criteria-Based Comparison Studies 28 6.2.1 Number of Laboratories 28 6.2.2 Number of Matrices 28 6.2.3 Number of Replicates per Matrix 28 SECTION 7.0 SAMPLE PREPARATION AND ANALYSIS 29 7.1 Collection of Samples for Analysis 29 7.1.1 Source Water Characterization 29 7.2 Sample Spiking and "Stressing" Procedures for Bacteriological Methods 29 7.2.1 Drinking Water: Spiking and Chlorine-Stressing 30 7.2.2 Preparation of Enumerated Spiking Suspension 32 7.2.3 Log Phase Growth Curve 33 7.2.4 Commercially Available Enumerated Spikes 34 7.3 Spiking Procedures for Virus Methods 34 7.3.1 Cell Monolayer Propagation 34 7.3.2 Propagation of Virus Stock Suspension 34 7.3.3 Titering of the Virus Stock Suspension 34 7.4 Spiking Procedures for Cryptosporidium andGiardia 34 7.5 Analysis of Samples 35 7.5.1 Side-by-Side Comparison Studies 35 7.5.2 QC Acceptance Criteria-Based Comparison Studies 35 7.6 Verification of Results 36 7.6.1 Verification of Results from Bacteriological Methods 36 7.6.2 Verification of Results from Virus Methods 37 7.6.3 Verification of Results from Cryptosporidium and Giardia Methods 37 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 8.0 REVIEW OF STUDY RESULTS 38 8.1 Assessment of Compliance with Approved Study Plan 38 8.2 Data Review 38 8.3 Data Validation 38 8.4 Development of Descriptive Statistics 38 8.4.1 Mean Recovery 38 8.4.2 Precision 39 8.4.3 False Positive Rates, False Negative Rates, Sensitivity, and Specificity 39 8.5 Statistical Assessment of Method Comparability 41 8.5.1 Presence/Absence Methods 41 8.5.2 Quantitative Methods 42 8.5.3 QC Acceptance Criteria-Based Comparison Studies 47 8.6 Method Recommendation and Approval 49 SECTION 9.0 STUDY REPORT 50 9.1 Background 50 9.2 Study Objectives and Design 51 9.3 Study Implementation 51 9.4 Data Reporting and Validation 51 9.5 Results 51 9.6 Data Analysis and Discussion 52 9.7 Conclusions 52 9.8 Appendix A - Method 52 9.9 Appendix B - Study Plan 52 9.10 Appendix C - Supporting Data 52 9.10.1 Raw Data 52 9.10.2 Electronic Data Reporting 53 9.10.3 Example Calculations 53 9.11 Appendix D - Supporting References 53 SECTION 10.0 REFERENCES 54 APPENDIX A: GLOSSARY 56 APPENDIX B: ATP APPLICATION FORM 67 APPENDIX C: APPLICATION INVENTORY FORM 70 APPENDIX D: DATA ELEMENTS AND EXAMPLE BENCH SHEETS 73 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 1.0 INTRODUCTION 1.1 Background and Objectives In accordance with the Clean Water Act (CWA) and Safe Drinking Water Act (SDWA), the U.S. Environmental Protection Agency (EPA) promulgates guidelines establishing test procedures (analytical methods) for data gathering and compliance monitoring under National Pollution Discharge Elimination System (NPDES) permits and national primary drinking water regulations (NPDWRs). The approved test procedures can be found in the Code of Federal Regulations (CFR) at 40 CFR Part 136 for wastewater and ambient water and 40 CFR Part 141 for drinking water. In addition, EPA's regulations at 40 CFR 136.4, 136.5, and 40 CFR 141.27, allow entities to apply for Agency permission to use an alternate test procedure (ATP) in place of an EPA-approved reference method. Figure 1.1 below summarizes the ATP or new method review process within EPA. These regulations are the basis for the Agency's alternate test procedure (ATP) program for water methods that is administered by the Office of Water, Office of Science and Technology, Director of Analytical Methods. An ATP is a modification of an EPA-approved reference method or a procedure that uses the same determinative technique (i.e., the physical and/or chemical process used to determine the identity and concentration of an analyte) and measures the same analyte(s) of interest as the EPA-approved reference method. The use of a different determinative technique to measure the same analyte(s) of interest as an EPA-approved reference method is considered a new method. Under the ATP program, an organization or individual may apply for approval of an ATP or new method to be used as an alternate to an EPA-approved reference method. The applicant is generally responsible for characterizing method performance of its proposed alternate test procedure prior to submission to the ATP program. EPA can provide assistance to applicants in the development of a study plan to demonstrate comparability with the EPA-approved reference method. Figure 1.1 summarizes the ATP or new method review process within EPA. The Agency reviews the ATP package, approves or disapproves the application, and, for nationwide applications, will generally propose to include successful ATPs in the CFR (unless the ATP is for limited use or constitutes a minor modification - See Appendix A). Figure 1-1 .Summary of the ATP or New Method Review Process ATP application 08 Acronyms: AW = Ambient Water DW = Drinking Water LU = Limited-use NW = Nationwide-use OW= Office of Water RA = Regional Administrator WW= Wastewater d to O OW review ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol The protocol illustrates how applicants can demonstrate comparability by meeting quality control (QC) acceptance criteria associated with EPA-approved reference methods for which those criteria have been developed. The ATP program provides laboratories and regulated facilities with an opportunity to enhance compliance monitoring and encourages the use of innovative technologies. Approval for an ATP or new method may be sought when, for example, the alternate procedure reduces analytical costs, overcomes matrix interferences problems, improves laboratory productivity, or reduces the amount of hazardous materials used and/or produced in the laboratory. Any person or organization may apply to gain approval for the use of an ATP or new method for determination of a specific constituent that is regulated under the NPDES permit program or the NPDWRs. The ATP applicants generally may demonstrate comparability of its proposed ATP or new method with the EPA-approved reference method using the procedures described in this protocol. Other possible method comparison procedures include those provided by organizations such as ASTM (Reference 10.5), AOAC- International (Reference 10.1), and ISO (Reference 10.8). 1.2 Types of Applications The types of applications submitted may depend on the intended use of the ATP. Methods intended for use in demonstrating compliance with the NPDES permit program (wastewater or ambient water ATPs) may be submitted for approval for limited-use (single laboratory) or for nationwide-use (all laboratories). Because only the Administrator has the authority to approve an alternate analytical technique for SWDA purposes, EPA will generally consider proposed methods intended for use in demonstrating compliance with NPDWRs (drinking water ATPs) that are submitted for approval for nationwide-use only. 1.2.1 Limited Use The primary intent of the limited-use ATP is to allow use of an ATP or new method by a single laboratory. Limited-use ATPs can be applied to one or more matrix types, excluding drinking water matrices; limited- use applications generally will not apply to Office of Ground Water and Drinking Water (OGWDW) ATP applications (Reference 10.18). If a method developer intends to apply the method to more than one matrix type, method studies should be conducted on each matrix type. Generally, nine different wastewater types should be analyzed to demonstrate the ATP or new method will be applicable to most other matrix types. If method modifications are within the specified flexibility of the EPA-approved reference method and all QC acceptance criteria are met, it generally will not be necessary to submit the modification to the ATP program. 1.2.2 Nationwide Use The primary intent of a nationwide-use ATP is to allow use of an ATP or new method by all regulated entities and laboratories for one or more matrix types including drinking water. Nationwide-use approval allows vendors to establish that new devices and reagents produce results that are acceptable for compliance monitoring purposes, and allows environmental laboratories across the United States to apply new technologies or modified techniques throughout their chain of laboratories to one or more matrix types. If a method developer intends to apply the method to more than one matrix type, method studies should be conducted on each matrix type. Generally, nine different wastewater types should be analyzed to demonstrate the ATP or new method will be applicable to most other wastewaters. If method modifications are within the specified flexibility of the EPA-approved reference method and all QC acceptance criteria are met, it generally will not be necessary to submit the modification to the ATP program. ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 1.3 Types of Studies The type of study most useful in seeking approval of an ATP or new method generally depends on whether or not the EPA-approved reference methods contain QC acceptance criteria. There are two basic types of studies described in this protocol: • Side-by-Side Method Comparison Study. A side-by-side method comparison study generally consists of parallel testing of an ATP or new method alongside an EPA-approved reference method to determine whether the performance of the new or modified method is acceptable compared to the reference method. • QC Acceptance Criteria-Based Method Comparison Study. For EPA-approved reference methods that contain (or are supplemented with) QC acceptance criteria for most combinations of analyte(s) and determinative technique(s), the goal of the study is for the applicant to demonstrate that its ATP or new method is able to meet the QC acceptance criteria of the EPA-approved reference method (or other EPA-specified document) for the applicable combination of analyte and determinative technique through a QC acceptance criteria-based comparison study. Specific guidelines for the studies can be found in Section 5.0: Quality Control, Section 6.0: Study Design, Section 7.0: Sample Preparation and Analysis, and Section 9.0: Review of Study Results. 1.4 Scope of Alternate Test Procedures This protocol for demonstration of comparability, submission, and approval of an ATP or new method offers flexibility to modify EPA-approved reference methods. Generally, an applicant should demonstrate and document that the modified method produces results better than or equal to those produced by an appropriate EPA-approved reference method for the applicable combination of analyte and determinative technique. 1.4.1 EPA-Approved Reference Methods The ATP process is based on comparing the performance of an ATP or new method to an EPA-approved reference method through a side-by-side comparison study or a QC acceptance criteria-based comparison study. Method comparability is demonstrated when results produced by an ATP meet or exceed the performance criteria associated with the EPA-approved reference method. Table 1-1 below lists the EPA- approved reference methods for the analytes covered by this protocol. This table will be updated as necessary as additional pathogens are added to the list or advances in technology merit a change in the EPA-approved reference method. When performing a study, the applicant should use the reference method that uses the same determinative technique (e.g., MF, MPN) as the ATP for the analyte(s) of interest. If the applicant is validating a new method, which generally will use a determinative technique that is not currently approved for use with the analyte(s) of interest, then the applicant should consult EPA prior to commencing the study to determine the most appropriate reference method. Table 1-1. EPA-Approved Reference Methods Analyte Total coliforms Method Format1 MPN MF Presence/Absence EPA-Approved Reference Method2'3 SM9221B SM 9222B SM9221D 40 CFR Citation 136.3, 141.21, 141.74 141.21 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Analyte Fecal coliforms £. co// HPC Fecal streptococcus Enterococcus Salmonella Enteric virus Helminth ova Aeromonas Coliphage Cryptosporidium Giardia Method Format1 MPN MF MPN MF Pour Plate MPN MF MPN MF MPN Plaque Assay Microscopy MF Plaque Assay Two-Step Enrichment Filtration/IMS/FA Filtration/IMS/FA EPA-Approved Reference Method2'3 USEPA Methods 1680/1681 SM 9222D SM9221F For CWA use: USEPA Method 1603 For SDWA use: mEndo + NA- MUG SM9215B SM 9230B SM 9230C SM 9230B USEPA Method 1600 USEPA Method 1682 EPA Document 4 EPA Document 5 USEPA Method 1605 USEPA Method 1601 USEPA Method 1602 USEPA Method 1622/1623 USEPA Method 1623 40 CFR Citation 136.3, 141.21, 141.74, 503.8(b) 136.3, 141.21, 141.74 141.74 136.3 136.3, 141.402 136.3, 503.8(b) 503.8(b) 503.8(b) 141.40 141.402 136.3, 141.74 MPN = most probable number, MF = membrane filtration, IMS/FA = immunomagnetic separation/fluorescent antibody "SM@ refers to Standard Methods for the Examination of Water and Wastewater. For the edition(s) approved for a specific method, consult the CFR sections referenced in the column headed A40 CFR Citation.@ (References 10.2, 10.3, and 10.4). Not all methods have been approved for use in all matrices. Please see "40 CFR Citation" for specific method approved for specific matrix. See References 10.12 and 10.13 See References 10.12 and 10.19 1.4.2 Modifications to Sample Preparation Techniques A sample preparation technique is any technique in the analytical process conducted at the laboratory that precedes the determinative technique (i.e., the physical and/or chemical process by which measurement of the identity and concentration of an analyte is made). Sample preparation techniques include the procedures, equipment, reagents, etc., that are used in the preparation and cleanup of a sample for analysis. Laboratories generally may modify sample preparation techniques, provided the modification is not explicitly prohibited in the EPA-approved reference method that is being modified and provided the modification can be demonstrated to produce results equal or superior to results produced by an EPA- approved reference method for each combination of analyte and determinative technique. ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 2.0 APPLICATION ATP applications should be submitted in triplicate to EPA to facilitate the review process. The application consists of a completed ATP application form (a sample application form is provided in Appendix D) with any attachments. Electronic submissions are also generally acceptable and often may accelerate the review process. 2.1 Submission Addresses and Approval Authority A summary of ATP submission information and approval authorities is provided in Table 2-1. Table 2-1. Submission of Alternate Test Procedure Applications Level of Use Limited Use for Wastewater or Ambient Water Nationwide Use for Drinking Water, Wastewater, Ambient Water, Sewage Sludge (Biosolids) Applicant EPA Regional laboratories States, commercial laboratories, individual dischargers, or permittees in States that do not have the authority to administer Clean Water Act and Safe Drinking Water Act monitoring programs States, commercial laboratories, individual dischargers, or permittees in States that have the authority to administer Clean Water Act and Safe Drinking Water Act monitoring programs All applicants Submit Application To1 EPA Regional Administrator (Regional ATP Coordinator) EPA Regional Administrator (Regional ATP Coordinator) Director of State Agency issuing the NPDES permit2 ATP Program Coordinator, EPA Headquarters Approval Authority EPA Regional Administrator EPA Administrator See Appendix E for EPA addresses. 2 The Regional Administrator or the Director of State Agency issuing the NPDES permit may choose to forward limited-use applications to the Director of Analytical Methods, Attn: ATP Program Coordinator for an approval recommendation. Generally, the Regional Administrator or the Director of State Agency issuing the NPDES permit will forward a copy of the approval to the Director of Analytical Methods, Attn: ATP Program Coordinator. Generally, upon receipt, the application will be assigned an identification number, and a confirmation letter referencing this identification number will be sent to the applicant. The applicant should use the identification number in all future communications concerning the application. 2.2 Application Information The following information should be provided on the ATP application form (Appendix B): • Name, mailing address, phone number, and email address of the applicant • Date of submission of the application ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol • Method number, title, and revision date of the ATP or new method submitted for review • The analyte(s) included in the ATP or new method submitted for review • The matrix or matrices to which the ATP applies • EPA-approved reference method used for demonstration of comparability • Type of application (i.e., wastewater, drinking water, ambient water, point source categories regulated at 40 CFR Parts 400-499) • The level of use desired (i.e., limited use or nationwide use) • Type of study (side-by-side comparison or QC acceptance criteria-based comparison study) • Applicant's NPDES permit number, the issuing agency, the type of permit and the discharge serial number (if applicable) The following items should be submitted as attachments to the initial application: • Reason for proposing the ATP or new method • The proposed ATP or new method prepared in standardized format (Section 3.0) • A method comparison table that gives a side-by-side comparison of the steps of the proposed ATP or new method and the EPA-approved reference method (Section 2.5) • Method development information including preliminary method performance studies • Study plan for EPA review and comment (Section 4.0) A study plan is generally not needed with the application if an applicant is unsure whether or not a modification is allowed within the method-specified flexibility. In such cases, the applicant may request that EPA determine the usefulness of a full ATP comparability assessment based on the other information submitted with the application. From this information, EPA can determine whether a full ATP assessment will be helpful, whether the proposed modification is considered to be a minor modification (i.e., employs the same chemistry and/or biological principles as the EPA-approved reference method to determine the presence/absence or to quantify the amount of the target organism in a sample), or whether the proposed modification is considered to be within the specified flexibility of the EPA-approved reference method. The elements of a complete application are presented in Table 2-2. A list of the information discussed in detail in Sections 2.3 to 2.8 is provided in Appendix C. EPA will generally seek all application information and attachments before the application is considered complete. Table 2-2. Application Information Application Information Completed application form Reason for ATP Method in EPA format Method comparison table Method development information Study plan (to be approved by EPA before proceeding with study) Study report (final report generally considered part of a complete application) Note: Although the application process generally begins with the initial submission of ATP materials, the application is not usually considered to be complete until the final study report has been submitted, and all EPA questions on the report have been resolved. ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 2.3 Reason for ATP The entity that proposes an ATP should indicate why the ATP is being proposed. Examples include, but are not limited to, the following: • The ATP improves method performance • The ATP provides equivalent method components for a lower cost • The ATP enables laboratories to perform analyses more efficiently • The ATP successfully overcomes some or all of the interferences associated with the EPA-approved reference method • The ATP significantly reduces the amount of hazardous wastes generated by the laboratory • The ATP provides another means for measuring a contaminant (i.e., provides more choices) 2.4 Standard EPA Method Format A method description is needed for all ATPs including minor modifications. In accordance with the standard EPA method description format advocated by EPA's Environmental Monitoring Management Council (EMMC), methods should contain 17 specific topical sections in a designated order. The 17 sections are listed in Section 3.0 of this document. The method description should also contain a date and the ATP case number so that the method version is properly identified. Additional numbered sections may be inserted starting with Section 11.0, Procedure, as appropriate for a particular method. For detailed information on the EPA format for proposed methods, see the Guidelines and Format document (Reference 10.14). 2.5 Method Comparison Table As part of the application, the applicant should provide a two-column table comparing the proposed ATP or new method to the EPA-approved reference method. The two-column method comparison table should include the number and title of each method, the latest revision date of the proposed ATP, and a detailed discussion of each of the 17 topics specified by the standard EPA method format (as applicable). Each topic should be discussed on a separate row in the method comparison table. The applicant should highlight any differences between the proposed ATP and the EPA-approved reference method. 2.6 Method Development Information Before EPA reviews the study plan and works with applicants on ATP studies, the applicant should provide data on performance of the modified or new method in the water matrix for which the ATP or new method is being applied. These data may have been generated during method development by the vendor, or through independent tests by third-party laboratories. Examples include, but are not limited to, replicate spiked reagent water or replicate spiked matrix water tests. It is the responsibility of the applicant to provide sufficient data to demonstrate that the ATP or new method performs sufficiently at a preliminary level in the matrix of interest to merit evaluation of an ATP. If sufficient data is not available, EPA may request additional studies be conducted prior to the review of the ATP study plan. In addition to data, the following descriptive method information will facilitate EPA's evaluation of the ATP application: • The purpose and intended use of the method • The analytical basis for the method, noting any relationship of the method to other existing analytical methods and indicating whether the method is associated with a sampling method 10 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol • Method limitations and an indication of any means of recognizing cases where the method may not be applicable to specific matrix types (e.g., turbidity greater than 50 NTU) • The basic steps involved in performing the test and data analysis • Options to the method, if applicable This information also will aid EPA in preparing the docket and the preamble for the proposed rule that will be published in the Federal Register if EPA proposes to approve the ATP for nationwide use. 2.7 Study Plan Prior to conducting all studies, the applicant should submit a study design for EPA review and comment. A detailed procedure (Section 3.0) for the new method or the modification should be included as an attachment to the study plan. Generally, EPA will evaluate the study plan to ensure that the appropriate data quality objectives identified in this protocol are defined and addressed. EPA comments will be incorporated into the study design and this process will be repeated until EPA has approved the study design. Generally, the study plan should contain the elements listed below: • Background • Objectives • Study Design • Coordination • Data Reporting These elements are further described in Section 4.0. 2.8 Study Report The applicant should conduct a study and provide a comprehensive study report with the ATP or new method application. The study report should include the following elements: • Background • Study Objectives and Design • Study Implementation • Data Reporting and Validation • Results • Data Analysis and Discussion • Conclusions • Appendix A - Method • Appendix B - Study Plan • Appendix C - Supporting Data • Appendix D - Supporting References These elements are further described in Section 9.0. 2.9 Proprietary Information in Applications All information provided to the Federal government is subject to the requirements of the Freedom of Information Act. Therefore, any proprietary information submitted with the proposed ATP application should be marked as confidential. However, EPA prefers that supporting documentation labeled as confidential business information not be submitted as part of the ATP application. If proprietary 11 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol information is determined to be essential to the application, EPA staff will request the information and will handle such information according to the regulations in subparts A and B of 40 CFR Part 2. Specifically, in accordance with 40 CFR §2.203, a business that submits information to EPA may assert a business confidentiality claim covering the information by placing on (or attaching to) the information at the time it is submitted to EPA, a cover sheet, stamped or typed legend, or other suitable form of notice employing language such as trade secret, proprietary, or company confidential. Allegedly confidential portions of otherwise non-confidential documents should be clearly identified by the business, and may be submitted separately to facilitate identification and handling by EPA. If the business desires confidential treatment only until a certain date or until the occurrence of a certain event, the notice should so state. Please be advised, however, that any methods proposed in the Federal Register cannot be claimed as confidential business information. If a claim of business confidentiality is not made at the time of submission, EPA will make such efforts as are administratively practicable to associate a late claim with copies of previously submitted information in EPA files. However, EPA cannot ensure that such efforts will be effective due to the nature of application review that may already be in progress. ATP study designs, ATP study data and method performance characteristics developed from ATP study data are not confidential business information, and may become publically available. 12 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 3.0 METHOD FORMAT Because alternate test procedures may be approved by EPA as comparable to the reference methods, and may be implemented by multiple laboratories, it is important that the written procedures include all of the information necessary to use the technique in the laboratory, including but not limited to: reagents and equipment, sample collection and preservation procedures, quality control, and a detailed description of the procedure. Updated versions of this information are also needed for minor method modifications. The information described below should be provided in the method. In addition, EPA recommends the Environmental Monitoring Methods Council (EMMC) format described below be used for all ATP applications (i.e., including minor method modifications). Sections 3.1 through 3.17 provide a list of EMMC method sections and a general description of the type of information that should be included in each section. The date and revision number and ATP case number of the method should be included on the cover page. In addition, the date should be included as a footer on each page of the method. A detailed description of method format guidelines, as well as an example of a formatted method, is provided in Reference 10.14. The detailed information in Reference 10.14 is provided as guidance for the method write-up and as such, specific suggestions for font size, margins, etc. are optional. 3.1 Scope and Application Include a list of target organisms (by common name), taxonomic group and their CAS registry numbers or other accepted numbering systems (if available), the matrices to which the method applies, a generic description of method sensitivity (the minimum number of organisms the method can detect per unit volume or mass, if known), and the data quality objectives that the method is designed to meet or monitoring programs for which the method was designed to support. 3.2 Summary of Method Summarize the method in a few paragraphs. The purpose of the summary is to provide a succinct overview of the method procedure to aid the reviewer or data user in understanding the method and how the results are generated. Include a general description of the method procedure, sample volume, type of media used, preparation steps, incubation time and temperatures, and the techniques used for qualitative or quantitative determinations. 3.3 Method Definitions Provide definitions of terms that are necessary to understand how the method is used or what the results represent. This should include a definition of the target organism or group of organisms, relative to the determinative step of the method. For extensive lists of definitions, this section may simply refer to a glossary attached at the end of the method document. 3.4 Interferences This section should discuss any known method interferences such as toxic materials, particulates, non- target organisms, etc. If known interferences in the reference method are not interferences in the alternate method, this also should be clearly stated. 13 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 3.5 Safety This section should discuss only those safety issues specific to the method and beyond the scope of routine laboratory practices. Target analytes or reagents that pose specific health, toxicity, or safety issues should be addressed in this section. 3.6 Equipment and Supplies For critical equipment that may affect the performance of the method, cite the manufacturer, model name, and catalog or product number of the equipment that was used to develop or validate the method; note that equivalent equipment can be used, if applicable. Use generic language for standard laboratory glassware and disposables. 3.7 Reagents and Standards Provide sufficient details on the concentration and preparation of reagents and standards to allow the work to be duplicated, but avoid lengthy discussions of common procedures. If only pre-prepared proprietary reagents can be used, specify this. Include catalog and/or product numbers where appropriate. Indicate shelf life of packaged materials and special storage specifications. 3.8 Sample Collection, Preservation, and Storage Provide information on sample collection, preservation, shipment, storage conditions, and holding times. If effects of holding time were specifically evaluated, provide reference to relevant data. 3.9 Quality Control Describe specific quality control (QC) measures that enable one to establish the sensitivity, specificity, false positive rates, false negative rates, bias, and precision of measurements using the method, and that the measurements are free from contamination. Specific QC measures may include positive and negative controls, duplicate samples, method blanks, and media sterility checks. Indicate which QC measures are appropriate initially, before a laboratory uses the method, and which are appropriate on an ongoing basis. Indicate frequencies for each QC measure and list minimum specifications or acceptance ranges (see Section 5.0). Indicate corrective actions that should be taken when QC measures are not met. Define all terms in method definitions section. 3.10 Calibration and Standardization Discuss initial calibration specifications for instruments used in the method (e.g., water baths, refrigerators, thermometers, balances, pH meters, microscopes, etc.). Indicate frequency of such calibrations; refer to performance specifications; and indicate corrective actions that should be taken when performance specifications are not met. This section may also include procedures for calibration, verification, or continuing calibration, or these steps may be included in the procedure section. 3.11 Procedure Provide a detailed description of the sample processing and analysis steps. Avoid unnecessarily restrictive instructions, but provide sufficient detail for manual procedures so that analysts in other laboratories perform the method consistently. Ranges should be provided for temperature requirements, time requirements, etc. 14 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 3.12 Data Analysis and Calculations Identify qualitative and quantitative aspects of the method. List criteria for the identification of target organism(s) and interpretation of results for all steps of the method, including criteria for presumptive and confirmed results. Provide equations used to derive final sample results. Provide discussion of estimating detection limits, recoveries, specificity, false positive/false negative rates, etc., if appropriate. 3.13 Method Performance Provide detailed information on method performance, including data on precision, bias (for quantitative methods), specificity, detection limits (including the method by which they were determined and matrices to which they apply), and statistical procedures used to develop performance specifications (i.e., recovery, precision, specificity, false positive/false negative rates, etc.). Where performance is tested relative to the reference method, provide a summary of the side-by-side comparison of performance versus reference method specifications. 3.14 Pollution Prevention Describe aspects of this method that minimize or prevent pollution that may be attributable to the reference method. 3.15 Waste Management Cite how waste is minimized and the proper disposal of samples and waste. 3.16 References Include source documents, publications, etc. 3.17 Tables, Diagrams, Flowcharts, and Validation Data Additional information may be presented at the end of the method. Lengthy tables may be included here and referred to elsewhere in the text by number. Diagrams should only include new or unusual equipment or aspects of the method. 15 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 4.0 STUDY PLAN Applicants should submit a study design for EPA review, comment, and approval prior to conducting the side-by-side comparison study or the QC acceptance criteria-based comparison study. This process protects the applicant by providing written approval of the study design before resources are spent to conduct the study. The ATP program is intended to be flexible, and thus EPA may modify the study design for a particular proposed method. Data from studies conducted without EPA review and approval may not meet EPA's criteria, and may not adequately address the applicant's study objectives. A detailed procedure (Section 3.0) for the ATP or new method should be included as an attachment to the study plan. EPA will generally evaluate the study plan to verify that the appropriate data quality objectives identified in this protocol are defined and addressed. EPA comments are incorporated into the study design. This review/revision process is repeated until EPA has approved the study design. Generally, the study design should include the information described in Sections 4.1 through 4.5. 4.1 Background This section of the study plan should include the following information: • A statement identifying the ATP as a new method or a modification of a reference method • The EPA program(s) to which the ATP or new method applies (e.g., drinking water, wastewater, ambient water, point source categories regulated at 40 CFR Parts 400-499, etc.) • A short (one paragraph) summary of the ATP or new method • The organization and method number of the reference method if applicable • A description of the reasons for the extent of the modification, the logic behind the technical approach to the modification, and the result of the modification • The matrices (e.g., finished water, wastewater, ambient water, etc.), matrix types (e.g., turbidity greater than 10 NTU, etc.), and/or media to which the ATP or new method is believed to be applicable • A list of the analytes measured by the ATP or new method, including the corresponding CAS registry number (if available) or other identification number 4.2 Objectives Include a description of the new method or modification, describe the goals of the study, and define data quality objectives. 4.3 Study Design The following information should be included in the study design: • Laboratories that will participate in the study (Sections 5.0 and 6.0) • Number and type of samples to be analyzed (Section 6.0) • Description of the matrices that will be used (Sections 6.0 and 7.0) • Description of the spikes that will be used (Section 7.0) • Description of the spiking procedure (Section 7.0) • Positive and negative control organisms (Section 5.0) • Quality control procedures that will be followed (Section 5.0) 16 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 4.4 Coordination Describe how the study will be coordinated, how the spikes will be shipped to the laboratories, and who will compile the data for submission. Data compilation should not be performed by any of the analysts conducting the sample analyses. 4.4.1 Study Management This section of the study plan should include the following information: • The organization responsible for managing the study • The laboratories, facilities, and other organizations that will participate in the study • A delineated study schedule including, but not limited to, sample collection, start of sample analysis, interpretation of sample results, completion of study, etc. 4.4.2 Technical Approach This section of the study plan should include the following: • A description of how sample matrices and participating laboratories will be selected • A description of how samples will be collected and distributed • The numbers and types of analyses to be performed by the participating laboratories • A description of sample spiking procedures • A description of how analyses are to be performed 4.5 Data Reporting List the data elements that will be collected and provide sample bench sheets (see Appendix D) that will be used to record raw data during the study. Raw data should be submitted as an attachment to the Study Report (Section 9.0). A list of the supporting data that should be included as Appendix C of the study report is given in Section 9.10.1. This supporting information includes QC data, instrument logs, media preparation records, and other parameters. Address the statistical analysis of the study results that will be performed, if the statistical analyses will differ from those described in Section 8.0. Please note, however, that EPA's evaluation of method performance will generally be based on the statistical analyses described in Section 8.0. 17 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 5.0 QUALITY ASSURANCE/QUALITY CONTROL For side-by-side comparison studies in which only one laboratory is performing analyses, the comparability study should be conducted at an independent laboratory and the laboratory should be certified to perform microbiological analyses under EPA's drinking water laboratory certification program. A laboratory with a vested interest in the method, instrumentation, apparatus, reagents, media, or associated kits may not participate in the side-by-side comparison study. For QC acceptance criteria-based comparison studies, at least three independent laboratories should participate. A laboratory with a vested interest in the ATP also may participate in the study, but in such instances there should be at least three independent laboratories participate and the majority of the laboratories participating are independent laboratories. All laboratories should be certified to perform microbiological analyses under EPA's drinking water certification program, or a comparable certification program, if a laboratory located outside of the U.S. is included. At least three independent laboratories participating in the study should to be certified for microbiological analyses under EPA's drinking water certification program. If more than three laboratories participate, the majority of the laboratories should be certified for microbiological analyses under EPA's drinking water certification program. 5.1 Quality Assurance The laboratory should have a comprehensive quality assurance (QA) program in place and operating at all times during the performance of the comparability study. General QA program guidance is provided at http://www.epa.gov/quality/qs-docs/r2-final.pdf (Reference 10.11). The laboratory should adhere closely to all QA and quality control (QC) measures in this protocol as well as the QC measures in the method(s). Laboratory QA/QC criteria for facilities, personnel, and laboratory equipment are included in Standard Methods 9020-Quality Assurance (Reference 10.4) and the U.S. EPAManualfor the Certification of Laboratories Analyzing Drinking Water, Fourth Edition (March 1997) (Reference 10.13). The laboratory should adhere to standard laboratory practices for cleanliness and environment, and to the methods for glassware and apparatus, reagents, solvents, and safety. Additional guidelines regarding general laboratory procedures should generally be followed, as specified in Sections 4 and 5 of the Handbook for Analytical Quality Control in Water and Wastewater Laboratories, EPA-600/4-79-019 (Reference 10.19). 5.2 Quality Control Laboratories participating in a comparability study should perform all QC procedures specified in the methods, except where explicitly stated in the approved study plan. The QC procedures listed in Table 5-1 and described below should be performed, as appropriate, based on the technique used in the method. Other QC procedures may be necessary, based on the approved study plan. Laboratories participating in side-by-side comparison studies or QC acceptance criteria-based comparison studies should perform all QC procedures specified in the methods, except where explicitly stated in the approved study plan. The laboratory should maintain records to define the quality of data that are generated. The laboratory should maintain a record of the date and results of all QC sample analyses. Laboratories should maintain reagent and material lot numbers along with samples analyzed using each of the lots. Laboratories should also maintain media preparation records. Table 5-1 lists quality control measures for each laboratory participating in side-by-side comparison studies and QC acceptance criteria-based comparison studies. Detailed descriptions of the QC measures are provided in Sections 5.2.1 to 5.2.15. If contamination is detected in any of the blanks or sterility checks described below, the source of contamination should be identified and corrected. The blank/sterility check 7s ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol and all samples associated with that contaminated blank/sterility check should be re-prepared and reanalyzed. Measures taken to eliminate contamination should be reported. Table 5-1. Quality control measures for each laboratory involved in the study Quality Control Measure Analyst counting variability (Section 5.2.1) Autoclave sterilization verification (Section 5.2.2) Dilution/rinse water blanks (Section 5.2.3) Incubator/water bath temperatures (Section 5.2.4) Initial demonstration of capability (IDC) (Section 5.2.5) or Initial precision and recovery (IPR) (Section 5.2.6) Matrix spike/matrix spike duplicate (Section 5.2.7) Media sterility checks (Section 5.2.8) Method blank (Section 5.2.9) Ongoing demonstration of capability (ODC) (Section 5.2.10) or Ongoing precision and recovery (OPR) (Section 5.2.11) Positive and negative controls (Section 5.2.12) Preparation blanks (Section 5.2.13) Refrigerator/freezer temperatures (Section 5.2.14) Sample processing equipment sterility checks (Section 5.2.15) Frequency 2 samples per study Within one week prior to the start of the study 1 per every 20 samples or 1 per day of study, whichever is greater 2 times per day when used in study Each laboratory participating in the study should generate acceptable IDC/IPR data. Per approved study plan 1 per each batch of media used in the study or per each test run, whichever is greater Per approved study plan Included as part of the study; not needed as separate QC. 1 positive control and 1 negative control for each media/stain used in the study. See 5.2.12 below for more details. Frequency depends on the type of method. See 5.2.13 below for more details. Once per day when used in study Prior to the analysis of samples. See 5.2.15 below for more details. Suggested for Study Side-by-Side Comparison Study / / / / / / / / / / QC acceptance criteria-based Comparison Study / / / / / / / / Optional / / / / 19 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.1 Analyst Counting Variability If the laboratory has two or more analysts, each should count colonies, plaques, or positive wells on the same plate/tray from one positive field sample per month. Compare each analyst's count of the colonies, plaques, or wells. Counts should fall within 10% between analysts. If counts fail to fall within 10% of each other, analysts should perform additional sets of counts, until the number of target colonies, plaques, or positive wells counted fall within 10% between analysts for at least three consecutive samples. If there is only one analyst replicate counts should be done and be within 5% of original counts. 5.2.2 Autoclave Sterilization Verification Autoclave sterilization verification should be performed within one week of the start of the study by placing Bacillus stearothermophilus spore suspensions or strips inside glassware. Autoclave at 121°C for 15 minutes. Place Bacillus stearothermophilus spore suspensions in trypticase soy broth tubes and incubate at 55°C for 48 hours. Check for growth to verify that sterilization was adequate. If sterilization was inadequate, determine appropriate time for autoclave sterilization. Repeat spore test. The laboratory should have historical data verifying that at a minimum, autoclave sterilization is performed on a monthly basis. 5.2.3 Dilution/Rinse Water Blanks The laboratory should analyze dilution/rinse water blanks to demonstrate freedom from contamination. An aliquot of dilution/rinse water which is analyzed exactly like a field sample should be analyzed for every day of the study or for every 20 samples, whichever is more frequent, and observed for contamination with agent of interest. 5.2.4 Incubator/Water Bath Temperatures Incubator or water bath temperatures should be measured and recorded two times per day when in use. Temperatures should be taken at least 4 hours apart and should be within the range of the desired temperature as specified in each method. Thermometers used to measure "in-use" temperatures should be calibrated yearly against an NIST traceable thermometer. 5.2.5 Initial Demonstration of Capability Laboratories participating in a QC acceptance criteria-based comparison study should have successfully performed an initial demonstration of capability (IDC) test for the new or modified method under evaluation in the study. An IDC test is performed when QC acceptance criteria are available for the evaluation of precision or recovery, but not both. The laboratory should perform an IDC as specified in the method to demonstrate acceptable performance. The laboratory should complete any additional analyses as specified in the study plan. For the IDC test, the laboratory spikes and analyzes reference matrix (e.g., reagent water, buffered water, etc.) samples to demonstrate acceptable performance with the method prior to the analysis of field samples. The number of samples involved in the IDC varies by method. If the results of the IDC test meets all IDC acceptance criteria (e.g., RSD, minimum number of samples positive, etc.), system performance will generally be acceptable. If any of the IDC test results fail to meet the acceptance criteria, system performance will generally be unacceptable. In this event, the laboratory should identify and correct the problem and repeat the test. IDC tests should be accompanied by a method blank (Section 5.2.9). 20 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.6 Initial Precision and Recovery Laboratories participating in a QC acceptance criteria-based comparison study should have successfully performed an initial precision and recovery (IPR) test for the method using the modified version of the method under evaluation in the study. An IPR test is performed when QC acceptance criteria are available for the evaluation of both precision and recovery. The laboratory should perform an IPR as specified in the method to demonstrate acceptable performance. The laboratory should complete any additional analyses as specified in the study plan. For the IPR test, the laboratory spikes and analyzes reference matrix (e.g., reagent water, buffered water, etc.) samples to establish the laboratory's ability to generate acceptable precision and recovery prior to the analysis of field samples. Using results of the analyses, the laboratory calculates mean percent recovery and relative standard deviation (RSD) of the recoveries for the analyte(s) and compares them with the corresponding limits for the IPR test criteria in the method. If the RSD and the mean percent recovery meet the acceptance criteria, system performance is acceptable and analysis of samples may begin. If the RSD or the mean percent recovery is unacceptable, system performance will generally be unacceptable. In this event, the laboratory should identify and correct the problem and repeat the test. IPR tests should be accompanied by method blank tests (Section 5.2.9). 5.2.7 Matrix Spike and Matrix Spike Duplicate Samples Matrix spike (MS) and matrix spike duplicate (MSD) samples are spiked matrix water samples analyzed by the laboratory to verify acceptable method performance in the matrix being monitored. During routine performance of the method, MS/MSD samples are analyzed by the laboratory for the first sample of any new matrix that will be monitored, and on the 21st sample thereafter. During a QC acceptance criteria- based comparison study for some methods (e.g., Cryptosporidium and Giardia methods), the laboratory should analyze MS samples as part of routine laboratory QC, however these analyses may not be necessary during this study because MS/MSD samples using blinded spiking suspensions distributed by the study coordinator will be used for the study. 5.2.8 Media Sterility Checks Before using newly prepared media, a representative portion of each media batch needs to be checked for sterility. The laboratory should test media sterility by incubating one unit (tube or plate) from each batch of medium specified in the method or per each test run, whichever is more frequent, at the appropriate temperature for the length of the method-specified incubation time and observing for growth. 5.2.9 Method Blank Method blanks are reagent water blanks or other blanks including but not limited to buffered water, tap water, etc., depending on the method analyzed to demonstrate freedom from contamination. Method blanks should be analyzed at the frequency specified in the approved study plan. For QC acceptance criteria-based comparison studies, the laboratory needs to analyze a method blank with the IPR and IDC tests. During routine performance of the method, the laboratory should analyze at least one method blank per every 20 test samples or per every week. During a QC acceptance criteria-based comparison study for some methods (e.g., Cryptosporidium and Giardia methods), the laboratory should analyze method blanks as part of routine laboratory QC, but method blanks also should be shipped to the laboratory as double- blind samples by the study coordinator. 21 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.10 Ongoing Demonstration of Capability (ODC) Samples Ongoing demonstration of capability samples are spiked reference matrix (e.g., reagent water, buffered water, etc.) samples that are analyzed to demonstrate that the analytical system is in control on an ongoing basis. 5.2.11 Ongoing Precision and Recovery (OPR) Samples Ongoing precision and recovery samples are spiked reference matrix (e.g., reagent water, buffered water, etc.) samples that are analyzed by the laboratory to verify that method performance criteria are being met. During a performance based comparison study for some methods (e.g., Cryptosporidium and Giardia methods), the laboratory should analyze OPR samples as part of routine laboratory QC because generally OPR samples using blinded spiking suspensions distributed by the spiking coordinator will be used for the study. 5.2.12 Positive/Negative Controls Positive and negative controls are target and non-target organisms processed to ensure the laboratories are familiar with the identification of the target organism and to ensure that confirmation test results are appropriate. One hundred (each) positive and negative control tests should be conducted. These results should be obtained from completed tests (e.g., obtaining a sample at 12 hours from a test that takes 24 hours to complete is unacceptable). 5.2.12.1 Positive/Negative Culture Controls (Culture-Based Methods) Positive and negative culture controls refer to cultures that, when analyzed exactly like field samples, produce a known positive or a known negative result, respectively, for a given type of media. One positive culture control and one negative culture control should be prepared and analyzed for every media (including confirmation media) used in the method whenever a new batch of medium or reagents is used, every day of the study, or every 20 samples, or as specified in the method, whichever is more frequent. Each control should be carried through the entire procedure and should exhibit the expected positive or negative result. 5.2.12.2 Positive/Negative Staining Controls (Cryptosporidium and Giardia Methods) A positive staining control for Cryptosporidium and Giardia methods is a slide containing positive antigen or intact Cryptosporidium oocysts and Giardia cysts, and that is stained using the same procedure as used for field samples or test samples. A negative staining control is a slide containing only phosphate buffered saline (PBS) that is stained using the same procedure as used for field samples or test samples. The laboratory should prepare and examine positive and negative staining controls with each batch of slides the laboratory prepares during the study. Positive staining controls should exhibit acceptable fluorescence and negative staining controls should not exhibit fluorescence. 5.2.12.3 Positive/Negative Staining Controls (Other) At a minimum, the laboratory should prepare and examine a positive and negative control using the same procedure as used for field or test samples whenever a new batch of media or reagents is used every day of the study or every 20 samples, whichever is more frequent. Each control should be carried through the entire procedure and should exhibit the expected positive or negative result. 22 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.13 Preparation Blanks (PB) 5.2.13.1 Membrane Filter Preparation Blank (PB-MF) If membrane filtration is used, at the beginning and the end of each filtration series, a PB-MF is performed by filtering 20-30 mL of dilution water through the membrane filter and testing for growth. If the control indicates contamination with the target organism, all data from affected samples should be rejected. A filtration series ends when 30 minutes or more elapse between sample filtrations. 5.2.13.2 Multiple-Tube Fermentation Test Preparation Blank (PB-MTF) If a multiple-tube fermentation test is used, a volume of sterile buffered water that is analyzed exactly like a field sample should be analyzed for every day of the study or every 20 samples, whichever is more frequent. The preparation blank should be incubated with the sample batch and observed for growth of the target organism. If the control indicates contamination with the target organism, all data from affected samples should be rejected. If buffered water is not used for dilutions, only the multiple-tube fermentation media should be included. 5.2.13.3 Other Preparation Blank (PB-Other) A volume of sterilized water (e.g., reagent grade as defined in Specification D 1193, Annual Book of ASTM Standards) that is analyzed exactly like a field sample should be analyzed for every day of the study or every 20 samples, whichever is more frequent. The preparation blank should be incubated with the sample batch and observed for growth of the target organism. If the control indicates contamination with the target organism, all data from affected samples should be rejected. 5.2.14 Refrigerator/Freezer Temperatures Refrigerator and freezer temperatures should be measured and recorded once per day when in use. Refrigerator temperature should be maintained at 1°C to 4°C. Freezer temperatures should be maintained at -15°C to -20°C. Special freezers capable of long-term storage of cultures or virus should be maintained at -70°C to -80°C. Thermometers used to measure "in-use" temperatures should be calibrated yearly against an NIST traceable thermometer. 5.2.15 Sample Processing Equipment Sterility Checks A representative portion of non-disposable items such as sample containers, blender jars, etc., used to collect or process samples should be checked for sterility prior to use in analyses. To test for sterility add approximately 500 mL (or appropriate volume based on the size of the equipment being used) of a sterile non-selective broth (e.g., tryptic soy, trypticase soy, or try tone broth) to the non-disposable item and incubate at 35°C ± 0.5°C for 24 hours and check for growth. Depending on the incubation times specified in the method, the length of the incubation time for the sample processing equipment sterility check may be increased. 23 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 6.0 STUDY DESIGN This section provides a description of the study design that is important for assessing comparability using either side-by-side method comparison studies (Section 6.1) or QC acceptance criteria-based studies (Section 6.2). The sections below address the number of laboratories, number of matrices, and the number of samples that should be analyzed in the studies. 6.1 Side-by-Side Comparison Studies The reference method to be compared to the proposed method for drinking water presence/absence ATPs are LTB/BGLB for total coliforms and LTB/EC-MUG for E. coli. For drinking water membrane filter methods, the reference method for total coliforms is m-Endo and for E. coli, it is nutrient agar with MUG. Methods are compared using the following parameters: • Recovery. Does the new method have similar, better or worse recoveries of the target organism as the reference method? • Precision. Are the recoveries by the new method significantly less or more variable than the reference method? • False positive rate/specificity. Is the new method significantly more likely or less likely to detect non-target organisms or other sample constituents that would be reported as the target organism by the analyst when compared to the reference method? • False negative rate/sensitivity. Is the new method significantly more likely or less likely to exhibit non-detects for samples with the target organism or to exhibit results that are biased low when compared to the reference method? To generate these parameters, samples are analyzed by a single laboratory (6.1.1). The number of samples (and matrix types) used in the study are determined using a historical EPA standard (6.1.2.1) 6.1.1 Number of Laboratories A single laboratory should be used for a side-by-side comparability study. Since the study should be conducted in a single independent laboratory with no conflict of interest; the laboratory selected cannot be the method developer's laboratory and cannot be affiliated with the method developer. 6.1.2 Number of Samples The following standards generally provide the minimum number of samples that should be analyzed. Additional data are generally acceptable and may be very helpful when reviewing an ATP or new method. 6.1.2.1 Method Comparison Study Design Summary Table 6.1 provides a summary of the method comparability requirements for nationwide microbiological ATPs and new methods. 24 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Table t -1 Summary of Side-by-Side Method Comparison Study Design for Nationwide Microbiological Alternate Test Procedures Regulatory Information RtfafPragram National Pollutant Dtechargs Elimination Program (NPOES) GuWellrss Establishing Test Procedure* For the Analyala or Pollutants; Analytical Msinoas Tor Biological Pollutants In Wastewater and Sewage Sludge Gulaellnes Establishing Teet Procedures for the Analysis of Pollutante: Analytical Methods for HofogHHI Pollutante In AniBlsnt water Total Conform Rule surface water Treatment Rule Long Term Surface Water Traafnunt Kite «CFR CItanon 136.3 136.3 136.3 14121 141.74 1417D4 Matrix vvastewata- VVaslewater Swage siuflge jBOSOIlQE) Al REcrsaltoral iVas-5 Fresh REcraattonal Waters Maine Recraaloral Watot DrlrKInc W3»- Ourrace water OurtKe water Type of Teat Qsantnaflve Qjartltaive OuantnaDfe Quartraive PreBeroe Msents OjartTHlve diantiatr.^ Regulated Analytee" Tctacamxni Feaa Confoc™ e.«« EntertKoccss Fecacarnmi Sanona'a Feca carorr EHJrt fifl^DOKCOS EnJWTKOCCJS TMacoHTDnn Feca conform ECM Total ca iron! FeeaColirorm Haie«ajDc«iE Sjserj QjpfoaMrOliim Eear TechnkpiBB1 k*N Iff wm P/F WF WF M=N Mm WF WF MF MPN HP Enzyme BuKiras Mm HT MM MF Enzyme Substrate WPN MF Enzyire suDslrate WPN MF =>our Plate FirjajarV'lMOFA um MF Enzyme SiCElrate Comparability Study1 Sample Source Typs WaEtEwatsr1 Waetewater1 vatetOaf H '.ea-eaaaia Waci-E Freai Water Marre Wi^r Drntlrg'.'Jaa lOKHara-Treei Dnntlpgwasr (DKldait-free) Surface 'A jsr MurnOflr 10 10 ID ID ID « 1 1 1 1 j Natural Source of Organisms (For SplKIng Into Sampte &c*jrcs) Type Mi Mi WaEtewaer waaswaer' lV3C2#3tET wastenaaf =t»iuea Surace •AaHT1 Erajneraiea spite'1 W3Se*a1er Kurtitwr Ml r»a 10 1 ID ID in R3pUC3te9 3P 2D in 3D 20 20 215 verification of Reference Method' Typical5 400 400 400 200 2DD 400 iac itypica" MM requires ',3: requrefi NotreqUretf Not required MM requlrett Not required Matraqulreo Minimum Comparability RBSUttB •'.-'-<-- am 200 an 2DC^ 2OD 2TJO 20C Propo&dd 2DO 2CO 200 200 200 2m am Specificfty Study1 Minimum Proposed Method Reruns =ala«.pog 200 200 200 200 100 200 200 Fa=e r*9g 20C 2DD 2D: 2O: m 200 2D: 25 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Ground Waiter Rute •-M-:. and •41 .in Ground waer •-ijTvjr.j EeUl EnBQHXriC caiphage WFN NF Ertzyma sustrae WPN hr Enzyire Substrate PQuratate MrtmgWasr (otuam-lree) i Wastptaar waatewaer iVastewaer 13 2C a Not requires an 20C 2K aw 1) For sfjdies conducted in drying a-d s-raee vrale" matrees "'or d-inki-g water purposes, the comparanlity study i-oud ne ccnducted in 3 lacoraKry csdified u-d*- tfie dr -fcng i'.3tef larofaiofy cenf cat en pfDgran. A rTflhcc Kv^ccer ha^i-g 3 vestsa i rn^hod, rstnjnentaton, 3ppaT3tus. reagenE meda cr asBodated kits s.'xxJc rxS perfonr the conr>araality study analyses r the appJicart's laixxatory 2) The specifidty sfjdy -nay De conducted in r* method de'neoper's aberatory. Spec-city data s not requiec fof the reference -netrod. 3) For wastewater vaiidatkxi sludes unsp k^d •rt3s1e¥^3f!er sarrpes should be teslec No addtionai n3tural sources O"' organ&Tts are required. 4) For coilbiTn methods, a s "gte dnnki1^ water sample source is spikec v/1h 10 d'^fepsnt wastewoters ID create "0 diferem lest samples- For HFC methods a sigle c"inkhg water source sample is spiked wth 10 different surface waters (e.g., ambient water, recevtig water, source wader) to create 'Q different lest sampSes- 5) For quarrtitatwe rererence melhoos, ' Q typcal oocnes (sheen cdonesi must be verifed per lepicate for a total of 4DO colony vefrcations CO samples x 4 random^ chosen out of 20 replicates x 10 sheen colonies}. For presence'abserce tests, all postive samples must be ver-Ted ;up to 200 samptes). 6) For presence'abseoce reference methods. ver-Tcatiwi «r atypical colonies s rex requirec Methoo developers may uerfy atjoical colonies to prowoe addrtional cata 7j Verificabcri of HDpossl met-od 15 "ct r=qu nsd. 6) Readers sroocJefer to Section 1.4.1 of the document for a list of approved reference mettxxjs. 9) Where appfcaUe (e.g., Oass A matices) 10} The eomparsor stjdy may also appiy to analytes not carefitly regulateo iby the Agency n standards -'twere are appnraned methods 'or that analyte rune specific rratra. For example. £ cali and entenxoeci r wastevjater and fecal cofiforms and Salmonella ii sewage skxlge (biosolds; Interested ertfes s.--oud nqLire accut other anaiytes if there are apu-oved methoos purlisheo for trerr in Fede*al Fegsier f na r^lirtjs i4; C"R cons 136 -=r 141 j 1! I Enumerated spiking suspensions c^' Cvyptecpood'um are cerrmerciaSy availade fkw eytDmeler sorted spiking susperaons vrich ,"^ve lower tfarabiity than maruaTy preparec spikes. 26 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Number of Matrices Ten different water/wastewater matrices from ten (10) different geographically diverse locations should be included to obtain, as much as is practical, a good presentation of the wide range of water types with an even wider range of target organisms to which the method should appropriately respond. Each water or wastewater sample should be collected in sufficient volume to complete all replicate analyses of sample or dilution volumes by both the ATP or new method and the EPA-approved reference method. For ambient water studies, the turbidity of at least one matrix should be greater than 10 NTU. Generally, for matrices other than finished drinking water or ambient water, matrix composition will be addressed on a study- specific basis. Number of Samples and Replicates Twenty replicate analyses should be performed by each method for each of the 10 matrices for a total of 200 replicate analyses per method. The replicate analyses should be performed on the same day for both the proposed and reference methods. Verification of Results For quantitative reference methods, verify 10 typical colonies from 4 randomly chosen replicates (of the 20 replicates) of each of the 10 samples for a total of 400 colony verifications. For presence/absence tests, all positive samples should be verified for up to 200 samples. See Section 6.1.3 for additional information pertaining to the verification of results. Calculations for false positive rates and false negative rates are described in Section 8.4.3. 6.1.3 Verification of Results 6.1.3.1 False Positives To assess whether the false positive rates are significantly different between methods, replicates known to contain non-target organisms that could be falsely identified as the target organism should be analyzed by both the ATP and new method. The determination that the samples do not contain the target organism should be based on a third independent standard method (see Section 7.6) rather than by the EPA- approved reference method being used in the comparison. This is because it should not be assumed that the accepted method has a false positive or negative rate of zero. For drinking water, total coliform specificity is determined by reaction with LTB/BGLB media in accordance with the method defined definition described in Standard Methods. For side-by-side comparison studies, 100 presence/absence broth cultures which are positive for total coliforms by the proposed method must be tested for specificity with LTB/BGLB media. Side-by-side comparisons of membrane filter methods must test 200 colonies determined to be total coliforms by the proposed method for their reaction with LTB/BGLB media. Presence/absence drinking water methods must be tested for positive E. coll reactions by testing 100 positive proposed ATP method cultures for their reaction in LTB/EC-MUG media. Membrane filter methods must determine positive specificity by testing 200 isolated colonies which the proposed method identifies as E. coli positive for reaction in LTB/EC-MUG media. 6.1.3.2 False Negatives To assess whether the false negative rates are significantly different between methods, replicates known to contain target organisms should be analyzed by both the ATP and new method. The determination that the samples do not contain the target organism should be based on a third independent standard method (see 27 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Section 7.6) rather than by the EPA-approved reference method being used in the comparison. This is because it should not be assumed that the accepted method has a false positive or negative rate of zero. For total coliforms in drinking water, LTB/BGLB media are used to test cultures which are negative by the proposed method. For side-by-side comparison studies, 100 presence/absence broth cultures which are negative for total coliforms by the proposed method must be tested for specificity with LTB/BGLB media. Side-by-side comparisons of membrane filter methods must test 200 atypical colonies determined to not be total coliforms by the proposed method for their reaction with LTB/BGLB media. Presence/absence drinking water methods must be tested for negative E. coli reactions by testing 100 negative proposed ATP method cultures for their reaction in LTB/EC-MUG media. Membrane filter methods must determine false negative specificity by testing 200 isolated atypical colonies which the proposed method identifies as E. coli negative for reaction in LTB/EC-MUG media. 6.2 QC Acceptance Criteria-Based Comparison Studies QC acceptance criteria-based comparison studies are generally conducted to demonstrate that the ATP or new method is able to meet the QC acceptance criteria of the EPA-approved reference method. In some instances, the quality control (QC) acceptance criteria specified in a method may not be sufficient to demonstrate comparability between the ATP and the EPA-approved reference method and a side-by-side comparison study should be conducted. Generally, EPA will make this decision based on review of the application materials. 6.2.1 Number of Laboratories A minimum of three laboratories should be used for a QC acceptance criteria-based comparison study. All three laboratories should meet all QC acceptance criteria in the EPA-approved reference method. If more than three laboratories participate, at least 75% of the participating laboratories should meet all QC acceptance criteria in the EPA-approved reference method. 6.2.2 Number of Matrices For all QC acceptance criteria-based comparison studies, there should be one matrix per laboratory. For ambient water studies, the turbidity of at least one matrix should be greater than 10 NTU. Generally, for matrices other than finished drinking water or ambient water, matrix composition should be addressed on a study-specific basis. 6.2.3 Number of Replicates per Matrix The number of replicates per matrix and laboratory should be specific to the QC criteria to which the results will be compared. Generally, the number of reagent results should be 4 and the number of source water results per matrix should be 2. 28 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 7.0 SAMPLE PREPARATION AND ANALYSIS 7.1 Collection of Samples for Analysis Each sample should be collected in sufficient volume to complete all replicate analyses by both the ATP or new method and the EPA-approved reference method. Samples should be spiked (if necessary) and analyzed as soon as possible after collection. 7.1.1 Source Water Characterization Source water characterization information should be collected at the time of sample collection. This information will be useful in characterizing the matrix to identify potential interferences and generally includes, but is not limited to, the following: • Sample collection location • Source of water (e.g., ground water, stream, river, lake, etc.) • Plant treatment processes (if sample is collected from a water treatment facility) • Temperature • pH • Turbidity • Total organic carbon (TOC) • Free and total disinfectant residual at time of sample collection • Heterotrophic plate count (HPC) unless heterotrophic bacteria are the target analytes, in which case total coliforms should be measured In addition to this information, data on the concentration of potential interferences (e.g., competing bacteria, interfering chemicals, etc.) in the water collected for analysis may be necessary. Known interferences should be discussed in the method and addressed in the study plan. The final results, as well as bench sheets, log sheets, instrument printouts, and any associated quality control analyses should be submitted as part of Appendix C of the study report (Section 9.10). 7.2 Sample Spiking and "Stressing" Procedures for Bacteriological Methods Depending on the matrix and the analyte of interest, it may not always be necessary to spike samples prior to analysis. Range finding analyses should be performed to assess the ambient concentration of target organism(s) in the matrix of interest to determine whether sample spiking will be useful. Samples are collected and dilutions are prepared in order to enumerate the number of organisms in the sample without qualifiers (i.e. less than, greater than, or too numerous to count). In addition, range finding can also be used to obtain environmental isolates for use in sample spiking or to determine the concentration of target organism(s) in a spiking suspension or spiked sample. If samples are spiked, environmental isolates should be used, as pure strains may exhibit different recovery and precision characteristics than natural flora. NELAC (Reference 10.9) and ATCC (Reference 10.6) recommend that bacterial cultures be transferred monthly and passed no more than five times before returning to the original culture. Sections 7.2.1 through 7.2.4 below, detail several different procedures for sample spiking and stressing. 29 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 7.2.1 Drinking Water: Spiking and Chlorine-Stressing Drinking water samples should be spiked and stressed with chlorine as described in Sections 7.2.1.1 and 7.2.1.2 below. 7.2.1.1 Drinking Water Spiking Because finished drinking water does not typically contain the target analyte(s) of interest, finished drinking water should be spiked. The drinking water should be oxidant and reductant free. It can be dechlorinated by exposure to UV light, held at room temperature for 24 to 48 hours, or with a Granular Activated Carbon (GAC) filter. Additionally, the drinking water should not contain coliform bacteria or more than 500 CFU/mL of heterotrophic bacteria so that it will not interfere with the determination of the reduction of coliform bacteria during chlorine stressing, or coliform medium reactions during experiments. For the evaluation of drinking water samples, a single finished drinking water matrix is spiked with target organism(s) from other matrices, such as non-chlorinated secondary sewage effluents or polluted surface waters. Sewage effluent generally has the advantage of providing a wide range of strains, whereas surface waters typically have the advantage of providing organisms more variable in quality. Depending on the study design (Section 6.0), the source of spiking suspensions (i.e., wastewaters) should come from geographically dispersed sites. To prepare spiked drinking water samples: (1) Collect at least five liters of each non-chlorinated secondary sewage effluents or polluted surface water to be used as spiking suspensions. (2) Perform range finding analyses as described above on each spiking suspension to determine target organism density as soon as possible after receipt of the sample to ensure that target organism density and diversity are not reduced. Use sterile oxidant and reductant-free drinking water described above as the diluent in range finding studies. (3) For each spiking suspension, spike a sufficient volume of drinking water with a sufficient volume of spiking suspension (based on range finding) to obtain 103-105 target organisms/100 mL. Please note: A single drinking water, with negligible concentrations of oxidants and reductants (e.g., chlorine and sodium thiosulfate, respectively), should be used for the entire study. Refrigerate all spiked drinking waters at 1°C - 4°C for use in preliminary chlorination study and comparability study. High oxidant or reductant levels in the drinking water could interfere with organism stressing. 7.2.1.2 Preliminary Chlorination Study to Determine Appropriate Exposure Time Microorganisms in the spiked drinking water samples (from Section 7.2.1.1) should be stressed by chlorination at ambient temperatures under conditions similar to those in drinking water treatment facilities. The goal of chlorinating the spiked samples is to simulate drinking water treatment by reducing the number of organisms in the spiked drinking water samples from 103-105 target organisms/100 mL to 1-10 chlorine-stressed target organisms/100 mL for most probable number methods or to 20-100 chlorine- stressed target organisms/100 mL for membrane filtration methods (i.e., a 2-4 log removal). After chlorination, no two samples are expected to produce the same levels of injured (stressed) target organisms because the disinfection process is impacted by physical and biological factors. These include: the type of spiked drinking water sample to be disinfected (e.g., spiked with sewage effluent or polluted source water), the initial concentration of the target organism(s), the chlorine demand of the spiked sample, the type and concentration of chlorinating agent, the exposure time, the sample mixing, pH, and Jo ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol temperature. As a result, a preliminary chlorination study should be performed to establish the exposure time necessary to reduce the number of organisms in the spiked drinking water samples from 103-105 target organisms/100 mL to 1-10 chlorine-stressed target organisms/100 mL (or a 2-4 log removal). During this preliminary chlorination exposure time study, the physical and biological parameters in Section 7.1.1 should be carefully monitored and recorded for each sample. The exposure time is directly dependent upon the initial concentration of the target organism present, the matrices' chlorine demand, and the form of chlorine present. For testing, the spiked sample is generally exposed to 2.0-2.5 mg total chlorine/L for over a range of times such as 10, 20, and 30 minutes to reduce the density of target organisms from 103-105 CFU/lOOmLto 1-10 CFU/100 mL sample. However, the period of exposure of a sample with a low chlorine demand may be significantly shorter than 20-30 minutes. Suggested Preliminary Chlorination Study (1) Determine and record the total residual and free residual chlorine concentrations using an EPA- approved N, N diethyl-p-phenylenediamine (DPD) colorimetric method (e.g., Standard Method 4500-C1" G) initially, at midpoint, and at the end of the exposure time just prior to dechlorination. (2) For each exposure time, place 2 L of each spiked drinking water sample (from Section 7.2.1.1) in a glass container. (3) Add an appropriate volume of a diluted solution of reagent grade sodium hypochlorite (e.g., a 1:20 dilution of 5% (w/v) stock solution) to achieve the desired level of chlorinating agent and stir the sample continuously during exposure to chlorination. If the spiked sample has an appreciable chlorine demand (e.g., spiked with a primary effluent or a sewage sample), add dilute sodium hypochlorite solution until a total residual chlorine level between 2.0 and 2.5 mg/L is maintained in the absence of free chlorine. If a sample has a low chlorine demand, avoid over-stressing or killing the organisms by prolonged exposure to free residual chlorine. The free residual chlorine concentration should not exceed 0.5-1.0 mg/L. (4) Stop the chlorine oxidation (dechlorinate) at the end of the exposure period by adding 0.8 mL of a 10% (w/v) sodium thiosulfate solution/L sample. (5) Enumerate the target organism density in an aliquot of the spiked, chlorine-stressed, dechlorinated drinking water sample using the appropriate EPA-approved reference method from Table 1 -1. Different dilutions may be used for total coliform and E. coli enumerations. (6) For each exposure time, repeat Steps 3-5, above. 7.2.1.3 Suggested Procedure for Chlorination and Dilution of Samples for Comparability Study (1) Determine and record the total residual and free residual chlorine concentrations initially, at midpoint, and at the end of the exposure time just prior to dechlorination using an EPA- approved N, N diethyl-p-phenylenediamine (DPD) colorimetric method (e.g., Standard Method 4500-C1" G). (2) Place a sufficient volume of each spiked drinking water sample in a glass container to perform sufficient repeat analyses at multiple dilutions. Use oxidant and reductant-free drinking water described in Section 7.2.1.1 as the diluent 31 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol (3) Immediately prior to chlorination, perform enumeration to determine target organism density as described in 7.2.1.1 (2). This value should be used to determine the log reduction due to chlorination. (4) Add reagent-grade sodium hypochlorite to achieve the same concentration, as in the preliminary chlorination exposure study. (5) To reduce the density of target organisms from 103-105 CFU/lOOmL to 1-10 CFU/100 mL, chlorinate each spiked drinking water sample for the appropriate time, based upon the preliminary chlorination exposure study. Stir the sample continuously during the chlorination. (6) Stop the chlorine oxidation (dechlorinate) at the end of the exposure period by adding 0.8 mL of a 10% (w/v) sodium thiosulfate solution/L sample. (7) Enumerate target organism density in an aliquot of the spiked, chlorine-stressed, dechlorinated drinking water sample using the appropriate EPA-approved reference method from Table 1-1. (8) Refrigerate the spiked, chlorine-stressed, dechlorinated drinking water samples at 1-4°C for use in comparability testing. (9) Read the plates to determine the approximate density of the target organisms. Use these results to estimate the appropriate dilution necessary to reach the target organism density of 1-10 CFU/100 mL. (10) Evaluate three dilutions of each spiked drinking water sample. Different dilutions may be used for total coliform and E. coli enumerations since these likely occur in the spike source at different concentrations. Samples should be diluted with the same, original, oxidant-free and reductant-free drinking water, as necessary to reach a target organism density of 1-10 target organisms/100 mL for most probable number methods or 20-100 target organisms/100 mL for membrane filtration methods. Make the dilution and at least two others that bracket the target density, for example, half and double that dilution. One of these dilutions should contain the desired 1-10 target organisms per 100 mL. Immediately conduct the comparability analyses with each sample using these three dilutions. Please note: For the evaluation of presence/absence methods, the data used for comparison should be from the dilution(s) which produces results closest to an equal number of positive and negative results for the reference method. A 25% - 75% split in responses (in either direction) should be sought. A 100% positive response will be considered unacceptable. For comparability, the evaluated results for the ATP or new method should be from the same dilution as the reference method. If one of the dilutions does not produce an acceptable split in positive and negative results for the reference method, the applicant should return to the original, spiked sample. 7.2.2 Preparation of Enumerated Spiking Suspension This dilution scheme is adapted from Standard Methods for the Examination of Water and Wastewater, 19th Edition, Section 9020 B (Reference 10.3). This entire process should be performed quickly to avoid loss of viable organisms. There should be approximately 1010 organisms per slant. Therefore, dilution bottles "A" through "E," below contain approximately 1010, 10s, 106, 104, and 103 organisms per dilution bottle, respectively. Depending on the growing conditions, these numbers may vary. As a result, until experience has been gained, more dilutions should be filtered to determine the appropriate dilution. 32 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Inoculate bacterial culture onto the entire surface of several nutrient agar slants with a slope approximately 6.3 cm long in a 125 x 16 mm screw-cap tube. Incubate for 24 ± 2 hours at 35°C ± 0.5°C. From the slant that has the best growth, prepare serial dilutions using four dilution bottles with 99 mL of sterile buffered dilution water (bottles A, B, C, and D) and one dilution bottle containing 90 mL of sterile buffered dilution water (bottle E). Pipette 1 mL of buffered dilution water from bottle "A" to one of the slants. Emulsify the growth on the slant by gently rubbing the bacterial film with the pipette, being careful not to tear the agar. Pipette the suspension back into dilution bottle "A." Repeat this procedure a second time to remove any remaining growth on the agar slant, without disturbing the agar. Make serial dilutions as follows: (1) Shake bottle "A" vigorously and pipette 1 mL to bottle "B" containing 99 mL buffer (2) Shake bottle "B" vigorously and pipette 1 mL to bottle "C" containing 99 mL buffer (3) Shake bottle "C" vigorously and pipette 1 mL to bottle "D" containing 99 mL buffer (4) Shake bottle "D" vigorously and pipette 10 mL to bottle "E" containing 90 mL buffer; this should result in a final dilution of approximately 10 organisms/mL. If it is more convenient for your laboratory, an acceptable alternative to the dilution scheme presented for this step is to pipette 11 mL of dilution D into dilution bottle E, which contains 99 mL of dilution water. Filter 1-5 mL portions in triplicate from bottles "D" and "E" according to standard membrane filtration methods to determine the number of CPU in the dilutions. The recommended target dilution and spike volume depends on the method and the target analyte(s). Typically, dilutions should be stored at 1-5°C and may be used throughout the day they are prepared, however, storage conditions should be adjusted as necessary since both storage conditions and viability may vary from organism to organism. 7.2.3 Log Phase Growth Curve Inoculate 100 mL of broth media with a single isolated colony and incubate organisms at optimum temperature. If possible, shake at 200 RPM during incubation. Take optical density (OD) measurements at 550-600 nm at 30 minute intervals for the first 8 hours and record readings. In addition to OD readings at 30 minute intervals, using aseptic technique, remove a 0.1 mL portion of the culture and make a series of 1:10 dilutions in sterile buffer. Initially plate 0.1 mL of the 1.0, 10"1, and 10"2 dilutions in duplicate and incubate overnight at optimum temperature. Count colonies and record data. As the optical density increases, evaluate serial dilutions to accommodate the increased numbers of bacteria (i.e., when the optical density exceeds 1.0 then plate 0.1 mL from dilutions 10'1, ID'2, and 10'3). Based on OD and the CFU/mL results, the OD of fresh cultures (in log-phase growth) can be used to determine the concentration of bacteria in the tubes by a simple graphic representation of the combined OD and CFU/mL results. Commercially available McFarland standards may be used to determine the bacterial density instead of actually doing a growth curve within the lab. In order to determine bacterial densities using McFarland standards, the OD of the standards are compared to the OD of the log phase culture. 33 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 7.2.4 Commercially Available Enumerated Spikes One time use, commercially available spiking suspensions may be obtained from a variety of vendors. Prepare spiking suspensions according to manufacturer's instructions. It should be noted that a different spiking volume than that recommended by the manufacturer may be necessary to achieve the target density. 7.3 Spiking Procedures for Virus Methods 7.3.1 Cell Monolayer Propagation In order to propagate virus stock suspensions cell monolayers should be propagated. The type of assay cytopathic effect (CPE) or plaque assay (PA), being performed determines the size of the flask and the number of days the monolayer incubates prior to use for the assay. Inoculate T75 or T25 flasks with 1x10 cells containing cell specific growth medium. Flasks for CPE should incubate for 5-7 days at 37°C ± 0.5°C with 95% relative humidity (and 5% CO2 concentration, if necessary). One CPE flask should be stained with crystal violet and microscopically checked to ensure a 95% confluent cell monolayer prior to use in the assay. Flasks used for PA incubate for 7-10 days under the same conditions as the monolayers used for CPE. PA flasks should also be checked to ensure 95% confluency before use for assay. 7.3.2 Propagation of Virus Stock Suspension For (CPE) analysis, utilizing the appropriate cell line (e.g., Buffalo green monkey kidney cells; BGMK), inoculate the cell monolayer with virus and incubate culture flasks at 37°C ± 0.5°C until the entire monolayer has been destroyed by virus replication (approximately 72-96 hours). Freeze (to approximately -80°C) and thaw the flasks three times, then pool contents of the flasks and spin at 10,000 x g for 30 minutes. Filter supernatant using a 0.2 (im pore nylon filter to remove any remaining cell debris. The filtrate is the virus stock suspension. 7.3.3 Titering of the Virus Stock Suspension Titerthe virus stock suspension (from Section 7.3.2) by performing a plaque assay. Inoculate monolayers with 0.2 mL of serial diluted viral stock suspension. After rocking and rinsing the monolayers, add the agar overlay to the monolayer. Incubate the culture flasks for seven days while reading the number of plaques each day for the entire seven days. After the viral stock suspension has been titered, appropriate volumes of the suspension can be used to spike test matrices to obtain plaque forming units (PFU)/mL. 7.4 Spiking Procedures for Cryptosporidium and Giardia Enumerated spiking suspensions are needed for initial and ongoing precision and recovery (IPR and OPR) samples (often referred to as positive controls) and matrix spike (MS) samples. Flow cytometer-sorted organisms are necessary for these spiking suspensions, rather than manual techniques. Flow cytometer- sorted spikes generally are characterized by lower variability than manually enumerated spikes. Spiking suspensions should be prepared using unstained organisms that have not been irradiated, heat- fixed or formalin-fixed. Immediately before sorting spiking suspensions, initial calibration of the flow cytometer should be performed by conducting a series of sequential sorts directly onto membranes or well slides. These initial sorts should be stained and counted microscopically to verify the accuracy of the system. When sorting the spiking suspensions, ongoing calibration samples should also be prepared and counted at regular intervals. The mean of the ongoing calibration counts should be used as the estimated spike dose. Flow-cytometer-sorted spiking suspensions should be used by the expiration date noted on the ~4 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol suspension. Flow-cytometer-sorted spiking suspensions containing live organisms should be used within two weeks of the preparation date. General procedures for preparing flow-cytometer-counted spikes for Cryptosporidium and Giardia can be found in EPA Method 1622 and 1623 (April 2001). A potential commercial source of flow-sorted Cryptosporidium and Giardia spiking suspensions is: Wisconsin State Laboratory of Hygiene Flow Cytometry Unit 2601 Agriculture Drive Madison, WI 53718 Phone: (608)224-6260 Fax: (608)224-6213 The Wisconsin State Laboratory of Hygiene prepares and distributes live Cryptosporidium parvum oocysts and Giardia intestinalis cysts that have not been treated to reduce viability. 7.5 Analysis of Samples Samples should be analyzed in accordance with the EPA-approved study plan. Any deviations from the study plan may suggest the need for additional analyses and potentially, rejection of data generated from the study. If any deviations from the approved study plan are necessary prior to or during the study, the applicant should consult EPA and receive approval for the modification to the study plan. Deviations from the approved study plan should be documented in the study report (Section 9.0). Unless otherwise approved in the study plan, the minimum assay size is as specified in the EPA method (100 mL). Example analysis schemes are provided in Sections 7.5.1 and 7.5.2 below. Generally, the analysis schemes will differ depending on the type of organism and the type of method. The study plan (Section 4.0) should detail the order in which samples will be analyzed. 7.5.1 Side-by-Side Comparison Studies for both the reference method and the proposed method Method Blank 1 Sample 1 up to Sample 20 Method Blank 2 Positive Control Negative Control Method Blank 3 Media Sterility Checks 7.5.2 QC Acceptance Criteria-Based Comparison Studies Replicate 1 for IPR Replicate 2 for IPR Replicate 3 for IPR Replicate 4 for IPR Method Blank Matrix Spike Matrix Spike Duplicate Unspiked Matrix Sample J3 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 7.6 Verification of Results The number of positive and negative results to be verified in a side-by-side comparison study is discussed in Section 6.1.3. Sections 7.6.1-7.6.3 below discuss the types of independent standards that may be used for the verification of results from bacteriological methods in Clean Water Act applications, total coliform methods in drinking water applications, virus methods, and Cryptosporidium and Giardia methods, respectively. 7.6.1 Verification of Results from Bacteriological Methods 7.6.1.1 Biochemical Tests that May be Used for Verifications in Clean Water Act Applications Catalase Lysine decarboxylase ONPG Methyl Red and Voges Proskauer (MRVP) Indole Triple sugar iron (TSI) Coagulase Lysine iron agar (LIA) Esculin Urease Citrate Sugars (e.g., Trehalose, lactose, mannitol, and sorbitol) Oxidase It is recommended that multiple biochemical tests be utilized to verify colony identification. The above list of biochemical tests is not exhaustive. Applicants may refer to EPA methods for appropriate verification tests. The choice of which biochemical tests to use is based on type of organism (i.e., gram stain results) being identified/verified. Biochemical tests used for verification should be discussed in the study plan (Section 4.0). It may be appropriate to perform a gram stain prior to biochemical identification. A description of the biochemical tests listed above, as well as additional tests is provided in Standard Methods for the Examination of Water and Wastewater, Method 9225. 7.6.1.2 Commercially Available Biochemical Testing Products Commercial biochemical test systems incorporate multiple biochemical tests to allow for identification to the genus and/or species level, which may be difficult when using individual biochemical tests prepared in house. Commercial biochemical test systems are available in two formats: systems that depend on the analyst to manually interpret the results (e.g., API® strips, BBL crystal, and Enterotube) and systems that automate the interpretation of results (e.g., Vitek® and Biolog). 7.6.1.3 Biochemical Tests for Verification of Total Coliform and E. coli Bacteria in Drinking Water (Total Coliform Rule, Surface Water Treatment Rule, Ground Water Rule) Verify total coliform bacteria by growth/gas production in LTB medium followed by inoculation of positive LTB cultures into BGLB (as described in Standard Methods for the Examination of Water and Wastewater, Method 922 IB). One hundred (100) presence/absence cultures of the proposed method that are positive for total coliform growth must be tested for their reaction in LTB/BGLB. One hundred (100) proposed method cultures that are negative for total coliform growth must be tested in LTB/BGLB for their reaction. One hundred (100) presence/absence cultures that are positive for E. coli must be tested for their reaction in LTB/EC-MUG. One hundred (100) proposed method cultures that are negative for E. coli must be tested for their reaction in LTB/EC-MUG. Two hundred (200) total coliform colonies from the proposed membrane filter method must be tested for their reaction in LTB/BGLB. Two hundred (200) non-total coliform colonies must be picked from proposed membrane filter methods and tested in LTB/BGLB for their reaction. Two hundred (200) E. coli colonies must be picked from the proposed ~6 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol membrane filter methods and be tested for their reaction in LTB/EC-MUG. Two hundred (200) non- E. coll colonies must be picked from the proposed membrane filter method and be tested for their reaction in LTB/EC-MUG. 7.6.2 Verification of Results from Virus Methods Viral protocol testing confirmations tend to be method-specific; therefore a detailed description of the confirmation/verification procedure should be included in the study plan and reviewed on a study-specific basis. 7.6.3 Verification of Results from Cryptosporidium and Giardia Methods Verification of results may not be necessary for Cryptosporidium and Giardia IF A methods beyond the analyst's microscopic examination of the organism. If verification is necessary, a detailed description of the confirmation/verification procedure should be included in the study plan and reviewed on a study- specific basis. 37 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 8.0 REVIEW OF STUDY RESULTS Generally, upon receipt of the applicant's data, EPA will perform the following reviews discussed in Sections 8.1 to 8.5: (1) Assessment of compliance with the approved study plan (2) Data review (3) Data validation (4) Development of descriptive statistics (5) Statistical assessment of method comparability Methods that are deemed acceptable will generally be recommended for approval (Section 8.6). 8.1 Assessment of Compliance with Approved Study Plan Generally, EPA will review the study report and associated data to ensure the study was conducted according to the approved study plan. The applicant should explain and justify (possibly with additional studies) any deviations from the study plan. Deviations from the approved study plan that occur without prior approval from EPA may result in the rejection of some or all study data. 8.2 Data Review Upon receipt of the applicant's data, EPA will generally verify that all raw data described in Section 9.10 are present and complete. Generally, all calculations used in the method will be verified. This may include calculations used for spiking enumeration, preliminary or presumptive stages of the method, and the determination of the final result. 8.3 Data Validation After verifying data completeness and reviewing all calculations, EPA will generally verify that all measurements were performed in accordance with the method. This may include, but is not limited to, the following: • Temperature logs for incubator/water bath/refrigerator • Media preparation records • Sample incubation times • Associated QC samples (as described in Section 5.0) o Method blanks o Preparation blanks o Sterility checks o Positive and negative controls 8.4 Development of Descriptive Statistics 8.4.1 Mean Recovery To determine if matrix characteristics affect method performance, mean recoveries should be calculated separately for each matrix and method. Mean recoveries should also be calculated for each method over all matrices. Methods having an extended read time (e.g. 24 ±2 hours and 48 ± 3 hours for LTB/BGLB) should base mean recoveries on the sum of the confirmed true positives of the replicate cultures which gave positive reactions at anytime during the incubation. Individual positive replicate cultures of the proposed or the reference method may appear positive at earlier read times and negative later due to ~38 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol overgrowth by non-target bacteria, or may appear negative at earlier read times due to a growth lag of the target bacteria. 8.4.2 Precision Precision can be expressed both on an absolute scale (i.e., standard deviation) and on a relative scale (i.e., relative standard deviation). The RSD (sometimes referred to as coefficient of variation) is calculated as the standard deviation divided by the mean, expressed as a percent. For the purpose of summarizing the data, both standard deviations and RSDs should be calculated and the one which is most appropriate for assessing comparability will be used in analyses. Generally, RSDs are most appropriate for summarizing precision when variability increases as concentration increases. To give an indication of the effect of multiple matrices on precision, standard deviations should be calculated separately for each matrix and method. Standard deviations also should be calculated for each method over all matrices. 8.4.3 False Positive Rates, False Negative Rates, Sensitivity, and Specificity False positive (FP) and false negative (FN) rates of approved and reference methods should be evaluated when assessing comparability. An independent standard (described in Section 7.6) may sometimes be necessary to confirm positives and negatives of both the ATP or new method and the EPA reference method. For drinking water method evaluations, confirmed positives and negatives for the proposed method should be determined as described in section 7.6.1.3. The confirmation methods used should be discussed in the validation study plan. False positive rates for methods with extended read times should be based on the sum of the number of confirmed true positives appearing at any time during the incubation period. False negatives should be based on cultures which appeared positive at any time during the incubation period, but never at any time during the extended incubation read time were confirmed as being positive. Generally, performance of the ATP or new method and EPA-approved reference methods will be defined in terms of false positive rates and false negative rates. For the purposes of the ATP protocol, false positive rates and false negative rates are equivalent to (1- Specificity) and (1-Sensitivity), respectively. Specificity is defined as the percent of negative samples correctly identified as negative, and sensitivity is defined as the percent of positive samples correctly identified as positive (see equations on the next page). In order to calculate estimates of false positives, false negatives, sensitivity, and specificity for each method, 2-by-2 tables for each matrix, and over all matrices, should be set up as follows in Table 8-1. 39 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Table 8-1. Standard Format for 2-by-2 Tables ATP or New Method + - Total Independent Standard + TPi FN-i TPi + FNi - FPi TNi FPi + TNi Total TPi + FPi FNi + TNi TP-i + FPi + TNi + FNi EPA- Ap proved Reference Method + - Total Independent Standard + TP2 FN2 TP2 + FN2 - FP2 TN2 FP2 + TN2 Total TP2 + FP2 FN2 + TN2 TP2 + FP2 + TN2 + FN2 Estimates of sensitivity, specificity, false positive rates, and false negative rates as percentages for the two methods should be calculated as follows: TPt Sensitivity, = TP, + FN, * 100 % Specificity, = TN, + FP, * 100 % TN, False positive rate, = TN, + FP, *100%= (1- TN, + FP, )* 100 % = 1 - Specificity, FN, TP, False negative rate, = TP, + FN, *100%= (1- TP, + FN, )* 100% = 1 - Sensitivity, Where F = False N = Negative T = True i = the specified method (i=l for new method, i=2 for reference method) 40 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 8.5 Statistical Assessment of Method Comparability 8.5.1 Presence/Absence Methods For presence/absence methods, the chi-square test and Breslow-Day test will generally be used to compare the percent of false positives and false negatives in the proposed method analyses with the percent of false positives and false negatives in the reference method analyses. Generally, tests for significant differences in false positive and false negative rates between methods will be run using the results summarized in Table 8-1 in Section 8.4. To perform the chi-square and Breslow- Day tests, it may help to first rearrange the data as follows in Tables 8-2 and 8-3: Table 8-2. False Negative Rate Comparison Result True + False - Total Method New TPi FN-i TP-i+FN-i Reference TP2 FN2 TP2 + FN2 Total TPi + TP2 FN-i + FN2 TP-i + TP2 + FN-i + FN2 Table 8-3. False Positive Rate Comparison Result False + True - Total Method New FPi TNi FPi + TNi Reference FP2 TN2 FP2 + TN2 Total FPi + FP2 TNi + TN2 FPi + FP2 + TNi + TN2 Before running the chi-square tests, positives and negatives should be confirmed using the independent standard method. For drinking water methods, positives and negatives should be confirmed as described in section 7.6.1.3. The chi-square test will be used to determine whether the false positive rate and false negative rate have a statistically significant difference between the ATP or new method and the EPA- approved reference method. Methods with an extended read time should statistically compare the positives and negatives for the beginning and end of read times to the number of true and false positives and true and false negatives obtained for the whole incubation period to determine if the full range of read times gives acceptable recovery. 8.5.1.1 Assessing Method Differences (Chi-Square Test) In order to assess whether the false positive and false negative rates differ between methods, chi-square tests should be run over all matrices; additional tests in each matrix also may be necessary depending on the presence of matrix interactions (see Section 8.5.1.2). For false negative rates, the chi-square test(s) indicates whether the proportions of negative samples correctly identified as negative by the two methods are significantly different, and for false positive rates, the chi-square test(s) indicates whether the proportions of true positive results correctly identified as positive for the two methods are significantly different. 41 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 8.5.1.2 Assessing Method/Matrix Interactions (Breslow-Day Test) In order to assess whether there are false positive and false negative rate differences between methods, it is also necessary to establish if there is a matrix effect on these parameters. The effect of matrices on false positive and false negative rate differences between methods is assessed using the Breslow-Day test (Reference 10.7). The Breslow-Day test is used to test whether there is an interaction between matrix and method in terms of the likelihood of a false positive result (for specificity) and in terms of the likelihood of a false negative result (for sensitivity). If a significant interaction is found between method and matrix, then the chi-square tests for a difference between methods for that attribute should be done separately for each matrix. Otherwise, separate chi-square tests for each matrix are not necessary, and a single chi-square test can be run using the data from all matrices. 8.5.1.3 Method Comparability Conclusions A decision on whether the ATP or new method is comparable to the EPA-approved reference method will generally be made based on the results of the false positive and false negative rate comparisons. Generally, the decision on acceptability of the new method should be made based on the chi-square test using data from all matrices. However, if the results of the Breslow-Day test indicate that there is a significant interaction between method and matrix for false positive and/or false negative rates, then further review should be made for those matrices which yielded a higher false positive and/or false negative rate for the proposed method. If the results of the chi-square tests indicate that the false positive and false negative rates of the ATP or new method are not significantly different from the EPA reference method, this generally will be interpreted as not having enough evidence to conclude that the performance of the ATP or new method is worse than the EPA-approved reference method. However, if the results of the chi-square test indicate that the false positive and/or false negative rates of the new method are significantly less than that of the EPA- approved reference method, this will generally be interpreted as worse performance and would lead to rejection of the ATP or new method. 8.5.2 Quantitative Methods 8.5.2.1 Testing for Normality Many of the statistical analyses used to assess differences in recovery and precision between methods require certain assumptions about the data be met. Because the validity of these assumptions affects which statistical tests will be used to assess recovery and precision differences, testing these assumptions is a prerequisite as a first step. There are two assumptions that should be evaluated prior to comparing the two methods statistically. One assumption is that the variability of the replicates is constant for each matrix and method. This assumption is discussed in Section 8.5.2.2. The other main assumption that should be met is that the data follow a normal distribution. An assessment of normality can be done either graphically or with statistical tests, or both. A normal distribution looks like a bell curve (i.e., symmetric around the mean). A graphical assessment can be done using a histogram, stem-and-leaf, or Normal probability plot. Appropriate statistical tests include the Shapiro-Wilk test, D'Agostino test, and Kolmogorov-Smirnov test. These tests are computation intensive, and it may be necessary to use software (e.g., SAS) to run them. The Shapiro-Wilk test is generally inappropriate for testing the normality assumption of a data set with greater than 50 results. For environmental data (especially field data, though sometimes spiked data as well), the distribution of results will often be positively skewed (i.e., with a few unusually high results). A common corrective ~2 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol action of this that can be utilized is the logarithmic transformation, base e (natural log). If the data are positively skewed, this transformation should be attempted, assuming that none of the results are negative or equal to zero. When zero-valued results are present in a skewed data set, a common approach is to add a small constant to every result prior to transformation. However, the arbitrary choice of this constant will have a large effect on the transformed data (the log of 1 and the log of 0.1 are very different, for example), and this approach is not recommended. The uses of non-parametric tests, discussed later in this guidance, are considered to be the appropriate alternatives. If no zero-valued results are present in a positively skewed data set, then the log-transformed data should be tested for normality. If the log-transformed data do follow a Normal distribution, then all analyses should be done using the log-transformed results. 8.5.2.2 Evaluating Precision The decision of what statistical test to use to evaluate precision will generally be made on the results of the normality test described in Section 8.5.2.1. If the assumption of normality is met, then the F-test for differences in variance should be used. If the assumption of normality is not met, a non-parametric test such as the Conover Squared-Rank test should be used. The F-test is based on comparisons of variances (i.e., the squared standard deviations). The Conover Squared-Rank test is based on ranking absolute deviations from the mean. Figure 8-1 provides a summary of the procedure for evaluating precision. Prior to testing whether there is a precision difference between methods, the precision of the different matrices should be compared for each method. If the results of the F-test indicate that variances differ significantly by matrix, or if the results of the Conover Squared-Rank test indicate that absolute deviations from the mean differ significantly by matrix, then comparisons of precision between methods should be done separately for each matrix. Otherwise, a single comparison of method precision can be done using the data from all matrices. If the results of the F- test indicate that there is not enough evidence to conclude that the variances differ significantly by matrix, then the pooled within-matrix variance (i.e., the average of the matrix variances for the given method) should be calculated for each method. The F-test should then be used to compare the pooled within-matrix variance for the two methods. If the Conover Squared-Rank test was used and there was not enough evidence to conclude that the mean absolute deviation differed significantly between matrices, then the Conover Squared-Rank test should be used to compare the mean absolute deviations from the overall method mean for the two methods. 43 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Figure 8-1. Evaluating Precision Compare variances between matrices for each method using F-test. Compare absolute deviations from method mean for two methods using Conover squared rank test. Pool variances over matrices for each method. Compare pooled variances for two methods using the F-test. /Is the nev method .significantly less/ Xorecise?// Yes //Is the neviN, method \, ^significantly less/ precise?// [Failure Evaluate Recovery ,/ls the new method at leastx as precise for at least 80% of , matrices?, rYes Evaluate Recovery Failure 44 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 8.5.2.3 Evaluating Recovery Comparisons of mean recovery of the different methods should be done using either an analysis of variance (ANOVA) model or non-parametric test such as the Wilcoxon-Mann-Whitney (WMW) test. If the assumption of normality is not met, or if the precision differs between methods or matrices based on the precision evaluation described in the previous section, the WMW test should be used. If all assumptions are met, the ANOVA model should be used. Figure 8-2 provides a summary of the procedure for evaluating recovery. If an ANOVA model is used, method-by-matrix interactions should be tested for significance first using the corresponding ANOVA F-test. If the method-by-matrix interaction is significant, it would mean that the difference between methods is not the same for each matrix. In this case, no conclusion regarding an overall difference between the recoveries of methods can be made. If the interaction is not significant, then the test for a significant difference between methods can be run in the ANOVA model, using the F-test for a significant method main effect. If the WMW test is used, between-matrix and between-laboratory variability cannot be separated from replicate variability. Therefore, significant interactions between method and laboratory or matrix cannot be tested when the WMW test is used. Instead the WMW model only tests for an overall difference between methods. The model can be run separately for each matrix, but the diminished power will limit the value of this approach. It is therefore recommended that a single WMW test be run comparing method recoveries, without stratifying by or controlling for matrix type. 45 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Figure 8.2 Evaluating Recovery Run single WMW test to compare method recovery, disregarding matrices. Run ANOVA model testing for method difference, controlling for matrix. Run ANOVA model testing for method difference separately for each matrix. Count number of matrices where the new method is equal to or significantly better. Is recovery significantly lower for the new method? Yes- -NO-, Is recovery significantly lower for the new method? Is recovery \ significantly lower for the new method for at least 20% of all matricies? / Yes- -No Failure Evaluate false positive and false negative rates. Evaluate false positive and false negative rates. Failure Evaluate false positive and false negative rates. / 46 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 8.5.2.4 False Positive Rates, False Negative Rates, Sensitivity, and Specificity Comparisons of false positive rates, false negative rates, sensitivity, and specificity should be conducted according to the methods described in Sections 8.5.1.1 and 8.5.1.2. 8.5.2.5 Method Comparability Conclusions If there is not enough evidence to conclude that there is a significant interaction between method and matrix for recovery, or if there is not enough evidence to conclude that there is a statistically significant difference in precision between matrices for either method, then it can be concluded that any such differences of the method will be consistent. Therefore, lack of sufficient evidence to conclude a significant difference between methods will generally be interpreted as equal or better performance, and might lead to acceptance of the ATP or new method, pending review of false positive and false negative rates. However, a statistically significant test (worse) statistic will be interpreted as worse performance of the ATP or new method, and would generally lead to rejection of the new method. In cases where significant interactions between matrix and method are found when assessing recovery, or in cases where differences in precision between matrices for at least one method (and therefore a comparison of method precision could not be done over all matrices), some judgment will be necessary in deciding whether the proposed method should be deemed acceptable. The decision should be based on the attribute (i.e., recovery or precision) for each matrix. As a general rule, if there was not enough evidence to conclude that the new method was similar or better than the EPA-approved reference method for that attribute for at least 80% of the matrices used in the study, then the new method can generally be recommended for approval, pending review of false positive and false negative rates. If there is not enough evidence to conclude that the new method is worse than the EPA-approved reference method for at least 80% of the matrices for both precision and recovery, the false negative rate and false positive rate should next be compared, based on the methods described in sections 8.5.1.1 and 8.5.1.2. If the results of the chi-square test indicate that there is not enough evidence to conclude that the false negative rate or false positive rate of the ATP or new method is worse than that of the EPA-approved reference method, this will be interpreted as equal or better performance and might lead to acceptance of the new methods. However, if the results of the chi-square test indicate that the false negative rate or false positive rate of the new method is significantly greater than that of the EPA-approved reference method, this will generally be interpreted as worse performance for 100% of the matrices used in the study, and would lead to rejection of the ATP or new method. For example, suppose side-by-side testing is done in one laboratory for ten different matrices. For recovery, there is not enough evidence to conclude that the new method is significantly worse than the EPA-approved reference method for eight of the ten matrices. For precision, there is not enough evidence to conclude a significant matrix effect, and there is not enough evidence to conclude a significant difference between methods. There is not enough evidence to conclude the false negative rate or false positive rate of the ATP or new method is worse than the EPA-approved reference method. Under these circumstances, the ATP or new method may be recommended for approval. However, if there is not enough evidence to conclude that the new method is not worse than the approved method for only seven of ten matrices for recovery, then the ATP or new method will not, generally, be recommended for approval. 8.5.3 QC Acceptance Criteria-Based Comparison Studies For methods with QC acceptance criteria, necessary calculations and QC criteria are typically provided in the method. Generally, QC criteria will include both recovery and precision specifications. Recovery criteria take the form of a recovery interval for either a single result or mean of multiple results. Precision 47 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol criteria will generally be a maximum standard deviation, RSD, or RPD. Details on calculations that may be necessary for the comparisons are described below. 8.5.3.1 Recovery Recovery results will generally be necessary for data spiked into both reagent water (IPR, IDC, etc.) and source water (MS, MSD, etc.). For reagent water, percent recovery should be calculated as follows: Result % Recovery = Spike *100% Where, Result = the amount recovered from the sample after spiking Spike = the estimated amount spiked into the sample. For source water, percent recovery should be calculated as follows: Result - Background % Recovery = Spike *100% Where, Result = the amount recovered from the sample after spiking, Spike = the estimated amount spiked into the sample, and Background = the estimated background amount measured in the sample prior to spiking. For some QC criteria, it may be necessary to calculate the mean of the sample recoveries prior to comparing the results to the criteria. Generally, this will be specified with that given method. 8.5.3.2 Precision Calculation of an RSD, RPD, or standard deviation is generally necessary for precision criteria. For source water data, the calculation should be based on the recovered amount, rather than the percent recovery. This is because the calculated percentage will be inflated if the background amount is large compared to the total amount recovered. For reagent water-spiked data, where the background count is zero, the calculation can be made either using the recovered amount or the percent recovery. Where the precision criteria are to be based on two results, the precision criterion will generally be an RPD. The equation for an RPD is given below: \Amounti - Amount. RPD = * 100 % (Amount! + Amount2) I 2 Where, Amount! andAmount2 = the two recovered amounts. 48 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Where the precision criterion is to be based on more than two results, the precision criterion will generally be either a standard deviation or RSD. An RSD is calculated based on the equation below: SD RSD = * 100 % Mean Where, SD = the standard deviation of all recovered amounts Mean = the mean of all recovered amounts 8.5.3.3 Presence/Absence Criteria QC criteria for presence/absence methods may be defined as false positive and/or false negative rates, sensitivity and/or specificity, or as a specified proportion of positive and/or negative results out of a given number of samples. All calculations should be specific to the given situation and should be defined explicitly therein. 8.6 Method Recommendation and Approval Generally, after completion of the technical and statistical reviews for nationwide-use applications, the Director of Analytical Methods, Attn: ATP Program Coordinator (see Table 2-1 and Appendix E) will prepare a recommendation for approval/disapproval of the ATP or new method and notify the applicant of the recommendation. If the data evaluation demonstrates that the applicant's method performs at least as well as the EPA- approved reference method, the Director of Analytical Methods, Attn: ATP Program Coordinator will generally recommend approval to the Office of Ground Water and Drinking Water (OGWDW) or appropriate office, which begins the regulation development process. Regulation development includes a Federal Register notice proposing to approve an ATP, public comment on the proposed method, and (depending on public comment) a final rule published in the Federal Register that approves the method. Generally, the regulation development process may take one year or more. For limited-use ambient water or wastewater applications the Regional Administrator (see Table 2-1 and Appendix E) will generally prepare a recommendation of approval/disapproval of the ATP or new method and notify the applicant of the recommendation after completion of the technical and statistical reviews. 49 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 9.0 STUDY REPORT Laboratories or other organizations responsible for developing ATPs or new methods should document the results of the side-by-side comparison study or QC acceptance criteria-based comparison study in a formal study report that contains the elements described in this section. The information and supporting data included in the study report should be sufficient to enable EPA to evaluate the performance of the ATP or new method and make a decision on whether it is comparable to the reference method. The applicant is responsible for ensuring that all method-specified criteria are met by the laboratory(ies) involved in the study and that the study report contains all data from the laboratory(ies). A copy of all comparison study data should be maintained at the participant laboratory(ies) or other organization responsible for developing the ATP or new method. Like the study plan developed and approved by EPA before the study was performed, the study report contains background information and describes the study design. However, the study report also details the process and results of the study, provides an analysis and discussion of the results, and presents study conclusions. The approved study report should also identify and discuss any deviations from the study plan that were made in implementing the study, and the study plan should be appended to and referenced in the study report. Significant deviations from the study plan without prior EPA approval could result in the rejection of the study data. The study report should be organized into the following sections: • Background • Study Objectives and Design • Study Implementation • Data Reporting and Validation • Results • Data Analysis and Discussion • Conclusions • Appendix A - Method • Appendix B - Study Plan • Appendix C - Supporting Data • Appendix D - Supporting References Details on the information that should be included in each of these sections are provided below, in Sections 9.1 through 9.11. 9.1 Background This section of the study report should describe the ATP or new method that was tested, and identify the organization responsible for developing the ATP or new method. The background section of the study report should include the following information: • A method summary • The organization, method number, and title for the ATP or new method • The method number or title and publication number (given in 40 CFR parts 136, 141, and 405 -503) for the EPA-approved reference method that is being used for demonstrating method comparability (i.e., the reference method) • A description of the nature of the ATP (e.g., alternate media, alternate concentration technique, etc.) 50 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol • The matrices, matrix types, and/or media to which the ATP or new method is believed to be applicable • The analyte(s) measured by the ATP or new method, including corresponding CAS Registry or other identification numbers, when available 9.2 Study Objectives and Design This section of the study report should identify the overall objectives and data quality objectives of the study and briefly describe the study design. This information should be consistent with the study objectives and design specified in the approved study plan. Any study limitations should be identified. The approved study plan should be appended to the study report. 9.3 Study Implementation This section of the study report should describe the methodology and approach undertaken in the study. This section should include the following information: • The organization that was responsible for managing the study • The laboratories, facilities, and other organizations that participated in the study; describe how participating laboratories were selected; and explain the role of each organization involved in the study • The type of study performed (i.e., side-by-side or QC acceptance criteria-based comparison study) • The study schedule that was followed • A brief description of how sample matrices were chosen, including, for QC acceptance criteria-based comparison studies, a statement of compliance with recommendations for matrix type selection • A description of any preliminary testing conducted prior to the side-by-side or QC acceptance criteria- based comparison study (e.g., method validation, physical and chemical assessment of the matrices, preliminary range-finding analyses) • The numbers and types of analyses performed by the participating laboratories • A description of how samples were collected, distributed, and stored • The source and strain of the organism used for sample spiking • If spikes were quantified, a description of how estimated true spike values were determined and provide all supporting data • The type of water used in the preparation of sample dilutions if not specified by the method (e.g., reagent water, phosphate buffered water, phosphate buffered saline, etc.) • Any problems encountered with samples, spiking organisms, equipment, etc. and their subsequent resolution • Any communications with EPA relevant to the study, such as clarification of the study design or approved changes to the study plan • Any deviations from the study plan and their impact on study performance and/or results 9.4 Data Reporting and Validation This section of the study report should describe the procedures that were used to report and validate study data. While EPA generally does not use a standard format for analytical data submission, a list of necessary data elements and an example bench sheet may be found in Appendix D of this document. 9.5 Results This section of the study report presents the study results. Results may be presented in a summary table that lists the recovery or concentration of each sample, by test method, laboratory, and matrix. 51 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol For QC acceptance criteria-based comparison studies, results should indicate the QC test associated with each sample (e.g. IPR, method blank, MS/MSD, unspiked matrix sample). Raw data and example calculations should be submitted, and should be included in an appendix to the study report (see Section 9.10.1). 9.6 Data Analysis and Discussion This section of the study report provides a statistical analysis and discussion of the study results. Recovery, precision, false positive rates, false negative rates, specificity, and sensitivity, as appropriate, should be calculated by test method, laboratory, and matrix, and summarized in a tabular format that includes the mean, standard deviation, and relative standard deviation. The discussion should address any discrepancies between the results and comparability guidelines, or, for QC acceptance criteria-based comparison studies, any discrepancies between the results and the QC acceptance criteria of the EPA- approved reference method. 9.7 Conclusions This section of the study report should describe the conclusions drawn from the study based on the data analysis discussion. The section should contain a statement(s) regarding achievement of the study objective(s). 9.8 Appendix A - Method The ATP or new method, prepared in accordance with EPA's Guidelines and Format document (Section 3.0 and Reference 10.14), should be appended to the study report. 9.9 Appendix B - Study Plan The study plan approved by EPA (Section 4.0) should be appended to the study report. 9.10 Appendix C - Supporting Data The study report should be accompanied by raw data, quality control information, and example calculations that support the summary results presented in the report. 9.10.1 Raw Data Appendix C of the study report should include sufficient raw data so that an independent reviewer can verify each determination and calculation performed by the laboratory or the study coordinator. This verification consists of tracing all steps of the method to the final result reported. The raw data are, generally, method-specific and may include but are not limited to the following: • Sample numbers or other identifiers used by the laboratory • Sample collection dates and times • Verification that method-specified QC procedures were met for test samples and all associated QC samples • Analysis dates and times for all steps in the method • Sample volume • Any measurements of ancillary parameters (i.e., temperature, pH, turbidity, percent solids, etc.) • Results for all intermediate steps in the method • Preliminary data steps to determine the final result ~2 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol • Final result • If appropriate, quantitation reports, data system outputs, and other data to link the raw data to the results reported • Laboratory bench sheets and copies of all pertinent logbook pages for all sample preparation and cleanup steps, and for all other parts of the determination • Temperature logs for water baths, incubators, refrigerators, etc. • Media preparation information • If appropriate, direct instrument readouts and other data to support the final results Raw data are generally needed for all samples, positive and negative controls, sterility checks, verifications, blanks, matrix spikes and duplicates, and other QC analyses specified in the EPA-approved reference method. Data should be organized so that a microbiologist can clearly understand how the analyses were performed. The names, titles, addresses, and telephone numbers of the analysts who performed the analyses and of the quality assurance officer who verified the analyses should be provided. For instruments involving data systems, raw data on magnetic tape or disk should be made available on request. 9.10.2 Electronic Data Reporting In addition to the hard copy raw data, applicants should also submit data in electronic format (Excel spreadsheet, or equivalent) so that EPA can create a database of study results. EPA anticipates that this database will facilitate automated review and statistical analysis of study results. The information included in electronic format may include: laboratory, analyst, method, sample type, sample number, date and time of analysis, volume analyzed, replicate number, raw data, and calculated results. The applicant should discuss an appropriate electronic format with EPA prior to data submission. 9.10.3 Example Calculations Generally, the study report should provide example calculations that will allow the data reviewer to determine how the laboratory used the raw data to arrive at the final results. Useful examples include both detected analytes and undetected analytes. If the laboratory or the method employs a standardized reporting level for undetected analytes, this should be made clear in the example, as should adjustments for sample volume, etc. 9.11 Appendix D - Supporting References Hard copies of all references and supporting documentation for the ATP or new method should be attached to the study report as an appendix. The list of references may contain links to websites, or documents available on-line. However, a hard copy should be submitted with the final study report. 53 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol SECTION 10.0 REFERENCES 10.1 AOAC. 1999. Qualitative and Quantitative Microbiology Guidelines for Methods Validation, Journal of AOAC International, Vol. 82, No. 2. 10.2 APHA. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition. American Public Health Association. 1015 15th Street, NW, Washington, DC 20005. 10.3 APHA. 1995. Standard Methods for the Examination of Water and Wastewater. 19th Edition. American Public Health Association. 1015 15th Street, NW, Washington, DC 20005. 10.4 APHA. 1992. Standard Methods for the Examination of Water and Wastewater. 18th Edition. American Public Health Association. 1015 15th Street, NW, Washington, DC 20005. 10.5 ASTM. 1999. D4855-91: Standard Practice for Comparing Test Methods, ASTM Standards on Precision and Bias for Various Applications. 1999 Annual Book of ASTM Standards: Water and Environmental Technology, Volume 7.02. 100 Barr Harbor Drive, West Conshohocken, PA 19428. 10.6 ATCC. http://www.atcc.org 10.7 Fleiss, J. F. Statistical Methods for Rates and Proportions, 2nd ed., John Wiley & Sons, New York, NY. 10.8 ISO. 2001. CD17994. Water Quality - Criteria for the Establishment of Equivalence Between Microbiological Methods, Final Version, June 15, 2001. 10.9 National Environmental Laboratory Accreditation Conference. 2001. National Environmental Laboratory Accreditation Conference: Quality Systems. Approved May 25, 2001, effective July 1, 2003 unless otherwise noted. Appendix D - Essential Quality Control Requirements, section D.3, pp D-15 to D-16. 10.10 Title 40, Code of Federal Regulations, Sections 136.4, 136.5, and 141.27. 10.11 USEPA. 2001. EPA Requirements for Quality Management Plans. USEPA Office of Environmental Information. EPA/240/B-01/002. 10.12 USEPA. 1999. Environmental Regulations and Technology Control of Pathogens in Vector Attraction in Sewage Sludge. USEPA Office of Research and Development. EPA/625/R-92/013, revised October 1999. 10.13 USEPA. 2005. Manual for the Certification of Laboratories Analyzing Drinking Water: Criteria and Procedures Quality Assurance, Fifth Edition. USEPA Office of Ground Water and Drinking Water. EPA-815-R-05-004. 10.14 USEPA. 1996. Guidelines and Format for Methods to be Proposed at 40 CFR Part 136 or Part 141. USEPA Office of Science and Technology. EPA-821-B-96-003. 54 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 10.15 USEPA. 1995a. Presence/Absence Membrane Filter Methods for Finished Waters, USEPA Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Drinking Water Regulations, Version 1.2, December 1995. USEPA Office of Research and Development, Cincinnati, OH. 10.16 USEPA. 1995b. Quantitative Membrane Filter Methods, USEPA Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Water and Wastewater Regulations, Version 1.0, December 1995. USEPA Office of Research and Development. Cincinnati, OH. 10.17 USEPA. 1995c. Presence/Absence Liquid Culture Methods for Finished Waters, USEPA Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Drinking Water Regulations, Version 1.2, December 1995. USEPA Office of Research and Development. Cincinnati, OH. 10.18 USEPA 1989. Memorandum: Analytical Methods for Compliance and Limited Alternate Test Procedures Approvals. December 27, 1989. 10.19 USEPA. 1979. Handbook for Analytical Quality Control in Water and Wastewater Laboratories. EPA-600/4-79-019. Environmental Monitoring and Support Laboratory, Cincinnati, OH. March 1979. 55 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol APPENDIX A GLOSSARY 56 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol APPENDIX A: GLOSSARY 40 CFR part 136 — Title 40, part 136 of the Code of Federal Regulations. This part specifies approved test procedures for the analysis of pollutants regulated under the Clean Water Act. 40 CFR part 141 — Title 40, part 141 of the Code of Federal Regulations. This part specifies EPA's N ational P rimary D rinking W ater R egulations pur suant t o t he Safe Drinking Water Act; Subpart C of 40 CFR part 141 lists analytical methods required for monitoring under the Act. 95% confidence interval — A statistical level indicating a 95% probability that the parameter variable is enclosed within the given data interval. Acceptable version — An acceptable version is a method that is either identical to the approved method or exercises the flexibility explicitly allowed in the method. See "minor modification." Accuracy — The degree of agreement between an observed value and an accepted reference value. Accuracy includes random error (precision) and systematic error (recovery) that are caused by sampling and analysis. Aliquot — A representative portion of a sample. Ambient water—Ambient water refers to any fresh, marine, or estuarine surface water used for recreation; propagation offish, shellfish, or wildlife; agriculture; industry; navigation; or as source water for drinking water facilities. Analysis of variance (ANOVA) — A study of the effect of a set of qualitative variables on a quantitative response variable, based on a decomposition of the variance of the response variable. Analyte — The target organism or class of organisms that are measured by the method. Analyte of concern — An analyte designated by EPA to adversely affect or have the potential to adversely affect human health, the environment, aesthetics, or the senses. Analytes of concern are listed in approved methods. Approved method — A testing procedure (analytical method) promulgated at 40 CFR parts 136, 141,405-500, and other parts of the CFR that support EPA's water programs. Average percent recovery — The average of the recovery, expressed as percent. See recovery. Bias — A systematic or persistent distortion of a measurement process that deprives the result of representativeness; i.e., the expected sample measurement is different than the sample's true value. A data quality indicator. (QAMS) Blank— See "method blank." Bulk sample — A large sample that is aliquoted into smaller volumes prior to analyses. 57 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Calibration — The process of establishing the relationship between the concentration or amount of material introduced into an instrument or measurement process and the output signal. Calibration verification — Means of establishing that the instrument performance remains within pre-established limits. Code of Federal Regulations — A codification of the general and permanent rules published in the Federal Register by the Executive departments and agencies of the Federal Government. Comparability test — See side-by-side comparison. Confidence interval — The numerical interval constructed around a point estimated of a population parameter, combined with a probability statement (the confidence coefficient) linking it to the population's true parameter value. If the same confidence interval construction technique and assumptions are used to calculate future intervals, they will include the unknown population parameter with the same specified probability. (EMMC) Confirmed counts — Organism counts that have been verified to ensure proper identification. Conover Squared-Rank test — A nonparametric test for equality of variability, based on the joint squared ranks of deviations from the means. (SPRENT) Contract laboratory — Private, academic, or commercial laboratory under contract to EPA or other organization to perform testing. D'Agostino test — A statistical test for determining whether a given set of results follow a normal or log-normal distribution. Best used for datasets with at least 50 results. (GILBERT) Data quality objective — Qualitative and/or quantitative statement of the overall level of uncertainty that a decision-maker is willing to accept in results or decisions derived from environmental data Data quality objectives provide the statistical framework for planning and managing environmental data operations consistent with the data user's needs. (EMMC) Determinative technique — The physical and/or chemical process by which measurement of the identity and concentration of an analyte is made. Differential medium — A solid culture medium that makes it easier to distinguish colonies of the target organism. Dilution/rinse water blank— An aliquot of dilution/rinse water that is treated exactly like a sample and carried through all portions of the procedure until determined to be negative or positive. The dilution/rinse water blank is used to determine if the sample has become contaminated by the introduction of a foreign microorganism through poor technique. Discharge — Generally, any spilling, leaking, pumping, pouring, emitting, emptying or dumping (40 CFR 109.2; 110.1; 116.3); also, see "discharge of a pollutant" (40 CFR 122.2); the medium that is spilled, leaked, pumped, poured, emitted, emptied, or dumped. Discharge of pollutant — Any addition of any pollutant or combination of pollutants to (1) waters of the U.S. from any point source or (2) to the waters of the contiguous zone or the ocean from any point source other than a vessel or other floating craft which is being used as a means of transportation (40 CFR 122.2; 401.11) 58 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Duplicate — A second sample collected from the same sampling point at the same time the original sample is collected and analyzed exactly like the original sample. Duplicate samples can be used as a measure of sample variability. Effluent — A medium that flows out of a point source, e.g., the discharge from a sewage treatment plant. Enrichment — Using a culture medium to enhance growth of the target organism prior to isolation of that organism. Explicit flexibility — Modifications that are explicitly allowed in an approved method. F distribution — A type of sampling distribution for a random variable. The ratio of 2 chi-square distributions, each divided by their respective degrees of freedom. F-test — In an Analysis of Variance, a test for the equality of factor level means (such as for different methods) or for the presence of an interaction between two factors (such as method and matrix). (RICE) (ASTM) Facility — A plant or group of plants within a single location that is regulated under the CWA and/or SDWA A single facility may have multiple water supplies, discharges, waste streams, or other environmental media that are subject to compliance monitoring. For example, a single facility within the Pulp, Paper, and Paperboard industrial category may have a direct discharge, an indirect discharge, and an in-process waste stream, all of which are subject to compliance monitoring. False negative — A target organism incorrectly identified as a non-target organism or not identified at all using the method of interest. False positive — A non-target organism incorrectly identified as the target organism using the method of interest. False negative error rate — The proportion of target organisms incorrectly identified as a non- target organism or not identified at all using the method of interest, equal to (1 - Sensitivity). In statistical testing, the rate at which one falsely accepts a statistical hypothesis (such as that a difference between methods does not exist) based on a statistical test, when the hypothesis is actually false. Abbreviated as (3, and also referred to as the Type II error rate. (ASTM) False positive error rate — The proportion of non-target organisms incorrectly identified as the target organism using the method of interest, equal to (1 - Specificity). In statistical testing, the rate at which one falsely rejects a statistical hypothesis (such as that a difference between methods does not exist) based on a statistical test, when the hypothesis is actually true. Abbreviated as a, and also referred to as the Type I error rate. (ASTM) Federal Register — A daily publication that provides a uniform system for publishing Presidential and Federal agency documents. Documents published in the Federal Register make changes to the CFR to keep the CFR current. (OFR) 59 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Guidelines and Format — The document titled Guidelines and Format for Methods to be Proposed at 40 CFR Parts 136 and 141; available from the National Technical Information Service (NTIS), U.S. Department of Commerce, Springfield, Virginia, 22161 (703-487-4600) as NTIS publication PB96-2 10448. Histogram — A bar diagram of the distribution of a set of analytical results. The range of values is categorized into sets of subintervals, or bins, and the number of results within each bin are displayed as the height of the bars. (BERRY) Industrial category — A category listed in 40 CFR parts 405-503. Industrial subcategory— A subcategory defined at 40 CFR parts 405-503. Initial demonstration of capability — A test performed to establish the ability to demonstrate control over the analytical system and to demonstrate acceptable performance. Initial precision and recovery — The analysis of a minimum of four spiked reagent water samples under the same conditions as will be used for analysis of environmental samples. The IPR is used to demonstrate that a laboratory is able to produce reliable results with the method prior to analysis of environmental samples. Interaction — The situation where the effect of one variable (such as method type) on a dependent variable (such as recovery) is affected by the value of a third variable (such as matrix). Interference — A positive or negative effect on a measurement caused by a substance other than the one being investigated. (QAD) Intel-laboratory — Occurring in multiple laboratories. Intralaboratory — Occurring within a single laboratory. Kolmogorov-Smirnov test — A statistical test for determining whether a given set of results follow a normal distribution or any other specified distribution. (GILBERT) Limited use — Use of a method by a single regulated entity or laboratory for analysis of one or more matrix types. Log-normal — A distribution of a random variable X such that the natural logarithm of X is normally distributed. Log-phase — Bacterial growth phase in which the logarithm of the bacterial biomass increases linearly with time. Main effect — Situation where a variable (such as method type) has a consistent effect on a dependent variable (such as recovery). Matrix— The component or substrate that contains the analytes of interest. Matrix effect — Variability in the analytical performance of a method that can be attributed to the type of sample analyzed. 60 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Matrix spike — A sample prepared by adding a known mass of target analyte to a specified amount of a sample matrix for which an independent estimate of target analyte concentration is available. A matrix spike is used, for example, to determine the effect of the matrix on a method's recovery efficiency. (QAMS) Matrix spike duplicate — A replicate of the matrix spike to test precision. The MS/MSD is used in combination to test the precision of an analysis. (QAD) Matrix type — A sample medium with common characteristics across a given industrial category or subcategory. For example, C-stage effluents from chlorine bleach mills, effluent from the continuous casting subcategory of the iron and steel industrial category, POTW sludge, and in- process streams in the Atlantic and Gulf Coast Hand-shucked Oyster Processing subcategory are each a matrix type. For the purposes of this initiative all drinking waters constitute a single matrix type. May — This action, activity, or procedural step is neither required nor prohibited. May not — This action, activity, or procedural step is prohibited. Measurement quality — Critical level which, if exceeded, is considered to append objective additional, and possibly unacceptable, measurement uncertainty to the corresponding data. Method — A body of procedures and techniques for performing a task (e.g. sampling, characterization, and quantitation) systematically presented in the order in which they are to be executed. (QAD) Method blank— An aliquot of reagent water or designated matrix that is treated exactly as a sample, including exposure to all glassware, equipment, solvents, and procedures that are used with samples. The method blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus. Method-defined analyte — An analyte without a specific, known composition where the analytical result depends totally on the measurement procedure. Method modification — A change made to an approved method. Method validation — A process by which a laboratory or vendor establishes the performance of a new method or substantiates the performance of a method modification. Methods and Criteria— The document titled: Analysis of Pollutants in Municipal Water and Industrial Wastewater: Test Procedures and Quality Control Acceptance Criteria', available from the National Technical Information Service (NTIS), U.S. Department of Commerce, Springfield, Virginia, 22161 (703-487-4600) as NTIS publication PB 96-210463, and incorporated by reference into this part. Mid-point response factor — The response factor at the concentration at which calibration is verified. Minor modification — A modified method that has been reviewed by EPA and has been determined to be technically equivalent to a method approved for use in compliance monitoring. A minor modification employs the same chemistry and/or biological principles as the approved method to determine the presence/absence or to quantify the amount of the target organism in a 61 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol sample. Supporting data may be necessary to demonstrate that a minor modification will yield results equivalent to those obtained using the approved method but does not require approval as an alternate test procedure through proposal and promulgation in the Federal Register. Modified method — An approved method that has been modified to change a front-end technique. EPA will judge a modified method to be: 1) an acceptable version or minor modification of a previously promulgated method, which does not require approval as an ATP or 2) a significantly different method which requires an application for an ATP approval. Nationwide use — Use of a method by all regulated entities and laboratories for analysis of one or more matrix types. Navigable waters — All waters of the United States, including the territorial seas. (40 CFR 110.1) Negative control — A non-target organism processed to ensure the laboratories are familiar with the identification of the target organism and to ensure that confirmation test results are appropriate. New method — A method that employs a determinative technique for an analyte of concern that differs from determinative techniques employed for 1hat analyte in methods previously approved at 40 CFR part 136 or 141. Nonparametric — A type of statistical analysis for which no assumptions about the underlying distribution of the data are necessary. Non-selective media — An enrichment media that allows most bacteria to grow. Normal probability plot— A graphical depiction of the distribution of a set of analytical results. A normal probability plot is a scatter plot depicting the observed results compared to the expected results based on a normal distribution. If the observed data follow a normal distribution, the graph will display a line at a 45 degree angle from the x-axis. Also known as a Q-Q plot. Ongoing demonstration of capability — The laboratory needs to demonstrate that the analytical system is in control on an ongoing basis through the analysis of ODC samples (positive control/positive control duplicate). Ongoing precision and recovery—A reagent water sample method blank spiked with known quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified within the method for precision and recovery. Other approved methods — Promulgated methods that are not designated as a reference method, but continue to carry the same regulatory status. 62 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Physical phase — The physical phase of a sample matrix (e.g., air, water, soil). Positive control — A target organism that is analyzed to ensure that the laboratory is performing the method acceptably and that the media is providing appropriate results. Power — The probability that a statistical test will conclude that a difference (for example, between methods) exists, when a difference truly does exist. Equal to 1 - (3. Precision — The degree to which a set of observations or measurements of the same property, usually obtained under similar conditions, conform to themselves; a data quality indicator. Precision is usually expressed as standard deviation, variance, or range, in either absolute or relative terms. The precision obtainable from an environmental measurement method may be estimated from replicate analyses of subsamples taken from the same (homogenous) sample. Generally speaking, the more carefully one executes the various steps of a method and controls the variables affecting the method's capability, the more precise will be the results. The use of a non-homogeneous sample will compound the precision estimate with the sample variability. Preparation — Processing performed on a sample prior to analysis, e.g. extraction, concentration, cleanup, etc. Presumptive counts — Numbers of organisms based on results that have not been confirmed or verified. Procedures — A set of systematic instructions for performing an activity. (QAD) Promulgated method — A method that has been published or incorporated by reference into 40 CFR parts 136,141,405-500, or other parts that support EPA's water programs (i.e., an approved method). Promulgation — Publication of a final rule in the FR. Public water system (PWS) — A system for the provision to the public of piped water for human consumption, if such system has at least fifteen service connections or regularly serves an average of at least twenty-five individuals daily at least 60 days out of the year. Such term includes (1) any collection, treatment, storage, and distribution facilities under control of the operator of such system and used primarily in connection with such system, and (2) any collection or pretreatment storage facilities not under such control which are used primarily in connection with such system. A public water system is either a "community water system" or a "non- community water system." Quality assurance — An integrated system of activities involving planning, quality control, quality assessment, reporting, and quality improvement to ensure that a product or service meets defined standards of quality with a stated level of confidence. (QAD) Quality control — The overall system of technical activities whose purpose is to measure and control the quality of a product or service so that it meets the needs of users. The aim is to provide quality that is satisfactory, adequate, dependable, and economical. (QAD) QC acceptance criteria — Performance specifications developed from validation data and used to control the limits within which an analytical method is operated. 63 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol QC acceptance criteria-based comparison study — A study performed to evaluate the performance of a modified method against the quality control acceptance criteria of a reference method. Range finding — Preliminary analyses conducted to assess the ambient concentration of the target organism in a matrix to be used in a study or preliminary analyses of spiked samples involving few, if any, replicates, to assess method performance to identify the spike dose to be used in a study. i i Raw data — Data that have not been processed. Reagent water — Water conforming to Specification D 1193, Annual Book of ASTM Standards, or specifications in Standard Methods 9020B.4.d. Recovery — The total amount of the analyte found in the sample divided by the amount of the analyte added into the sample as a spike. Reference method — A method that serves as a standard against which method modifications can be compared. Regulated entity — Permittees, PWSs, POTWs, and other entities responsible for compliance with provisions of the CWA or SDWA Relative percent difference (RPD) — An estimate of the variability of two numbers expressed in relative terms. Calculated as the absolute value of the difference of the two numbers, divided by their mean: A - B RPD = * 100% 1*04 + 5) Equal to the relative standard deviation of the two numbers multiplied by the square root of 2. Relative standard deviation (RSD) — The standard deviation expressed as a percentage of the mean (lOOo/X); i.e., the coefficient of variation. Replicate — Multiple samples collected from the same sampling point at the same time and analyzed exactly the same way. Replicate samples can be used as a measure of sample variability. Sample matrix— See "matrix." Sample preparation technique — Any technique in the analytical process that precedes the determinative technique, including all procedures, equipment, solvents, etc. that are used in the preparation and cleanup of a sample for analysis. Sample preparation techniques do not include conditions and/or procedures for the collection, preservation, shipment, and storage of the sample. Sensitivity — In presence/absence testing, sensitivity is the proportion of target organisms in the sample that were correctly detected by the method of interest. 64 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Shapiro-Wilk test — A statistical test for determining whether a given set of results follow a normal or log-normal distribution. Best used for datasets with at most 50 results. (GILBERT) Side-by-side comparison — Parallel testing of a new or modified method and a reference method to determine whether the performance of the new or modified method is acceptable compared to the reference method. Specificity — In presence/absence testing, the proportion of non-target organisms in the sample that were correctly identified as not being the target organism by the method of interest. Spike — The process of adding a known amount of target analyte to a sample; used to determine the recovery efficiency of the method. (QAD) Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a concentration appropriate for spiking samples. Standard deviation (o) — The measure of the dispersion of observed values expressed as the positive square root of the sum of the squares of the difference between the individual values of a set and the arithmetic mean of the set, divided by one less than the number of values in the set. For a total of n numbers: Statistical power — See power. Stem-and-leaf plot — A graphical depiction of a set of analytical results that conveys information about the shape of the distribution while retaining the numerical information. The stem-and-leaf plot separates the digits of the values as leaves (the last digit of the values) and stems (the remaining digit of the values). The individual results are grouped according to the stems, and the leaves are listed separately in a format similar to a histogram. (RICE) Stock suspension — A concentrated suspension containing the organism(s) of interest that is obtained from a source that will attest to the host source, purity, authenticity, and viability of the organisms. Study plan — A study design submitted for EPA review, comment, and approval prior to conducting the side-by-side or QC acceptance criteria-based method comparability study. This process protects the applicant by providing written approval of the study design before resources are spent to conduct the study. Data from studies conducted without EPA review and approval may not adequately address the applicant's study objectives. A detailed procedure for the ATP or new method should be included as an attachment to the study plan. EPA will evaluate the study plan to verify that the appropriate data quality objectives identified in this protocol are defined and addressed. EPA comments are incorporated into the study design and this review process is repeated until EPA has approved the study design. Study report — A formal report developed by laboratories or other organizations responsible for developing ATPs or new methods documenting the results of the side-by-side or QC acceptance criteria-based method comparability study. The information and supporting data needed in the study report should be sufficient to enable EPA to evaluate the performance of the ATP or new method and make a decision on whether it is comparable or superior to the reference 65 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol method. The approved study report should also identify and discuss any deviations from the study plan that were made in implementing the study, and the approved study plan should be appended to and referenced in the study report. Summary results — Overall study statistics (not sample-specific results). Target organism — The organism the method is designed to detect. Validate — Reliably assess the performance (bias and precision) of a method in a reference matrix (such as reagent water) and the matrix in which the validated method will be used (such as drinking water, surface water, or municipal wastewater effluent). Validation, single-laboratory — Assessment of method performance (see "validate") in one laboratory. Validation, interlaboratory — Assessment of method performance (see "validate") at multiple laboratories. Variance — A measure of the dispersion of a set of values. The sum of the squares of the difference between the individual values of a set and the arithmetic mean of the set, divided by one less than the number of values in the set. (The square of the sample standard deviation) (QAD) Wilcoxon-Mann-Whitney (WMW) test — A nonparametric analysis used to determine whether the medians from two levels of a given factor (such as method) differ from each other, based on the ranks of all results. (SPRENT) The above definitions are referenced to the following organizations: ASTM ASTM D 4855-9 1. Standard Practice for Comparing Test Methods BERRY Berry, D. A. and B. Lindgren. Statistics Theory and Methods. Wardsworth, Belmonst, CA, 1990. EMMC Environmental Monitoring Management Council GILBERT Gilbert, R. O. Statistical Methods for Environmental Pollution Monitoring. Van NostrandReinhold, New York, 1987. NELAC QS National Environmental Laboratory Accreditation Conference, Quality Systems OFR Office of Federal Register QAD Quality Assurance Division, National Center for Environmental Research and Quality Assurance, Office of Research and Development, USEPA RICE Rice, J. A. Mathematical Statistics and Data Analysis, Second Edition. Wadsworth, Belmont, CA, 1995. SPRENT Sprent, P. Applied Nonparametric Statistical Methods, Second Edition. Chapman & Hall, London, 1993. 66 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol APPENDIX B ATP APPLICATION FORM 67 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol EPA Office of Water Alternate Test Procedure or New Method Preliminary Application Form for Microbiological Analytes Applicant Name and Address: Date Application Submitted: Type of Application: Circle appropriate application type Method Number Title of Method Revision Date EPA-Approved Reference Method: Analyte(s): Applicable Matrices: Circle all that apply Level of Use: Study Design: Circle appropriate study design EPA Use Only ATP Case Number: Alternate Test Procedure New Method Ambient Water Biosolids Drinking Water Wastewater Limited Use Nationwide Side-by-Side Comparison Study QC Acceptance Criteria-Based Comparison Study FOR LIMITED USE APPLICATIONS ONLY ID Number of Existing or Pending Permit: Issuing Agency: Type of Permit: Discharge Serial Number: Attachments Check all that apply D Justification for ATP D Alternate Test Procedure or New Method (in standard D Method Comparison Table D Study Plan D Other: EPA format) Submit Application and Attachments in Triplicate 68 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol EPA Office of Water Alternate Test Procedure or New Method Final Application Form for Microbiological Analytes Applicant Name and Address: Date Application Submitted: Type of Application: Circle appropriate application type Method Number Title of Method Revision Date EPA-Approved Reference Method: Analyte(s): Applicable Matrices: Circle all that apply Level of Use: Study Design: Circle appropriate study design EPA Use Only ATP Case Number: Alternate Test Procedure New Method Ambient Water Biosolids Limited Use Side-by-Side Comparison Study FOR LIMITED USE APPLICATIONS ID Number of Existing or Pending Permit: Issuing Agency: Type of Permit: Discharge Serial Number: Drinking Water Wastewater Nationwide QC Acceptance Criteria-Based Comparison Study ONLY Attachments Check all that apply D Justification for ATP D Alternate Test Procedure or New Method (in standard EPA format) D Method Comparison Table D Study Plan D Study Report D Other: Submit Application and Attachments in Triplicate 69 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol APPENDIX C APPLICATION INVENTORY FORM 70 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 1. Completed application form. Includes the name and address of the applicant; the date of submission of the application; the method number, title, and revision date; the EPA-approved reference method; the analyte(s) for which the ATP or new method is proposed; the type of application; applicable matrices; study design; level of use; NPDES permit information, if applicable; and the attachments submitted with the application. Section 2.2 Appendix C - 2. Justification for ATP or new method. Brief justification for why the ATP or new method is being proposed. Section 2.3 n 3. Method in EPA format. Scope and application Summary of method Definitions of method Interferences Safety Equipment and supplies Reagents and standards Sample collection, preservation, and storage Quality control Calibration and standardization Procedure Data analysis and calculations Method performance Pollution prevention Waste management References Tables, diagrams, flowcharts, and validation data Section 3.1 Section 3.2 Section 3.3 Section 3.4 Section 3.5 Section 3.6 Section 3.7 Section 3.8 Section 3.9 Section 3.10 Section 3.11 Section 3.12 Section 3.13 Section 3.14 Section 3.15 Section 3.16 Section 3.17 n n n n n n n n n n n n n n n n n 4. Method comparison table. A two-column table comparing the proposed ATP or new method with the EPA-approved reference method. This table should include the number and title of each method, the latest revision date of the ATP or new method, and a detailed discussion of each of the method sections listed in Section 3.0. Each topic should be discussed in a separate row of the table and the applicant should highlight any differences between the ATP or new method and the EPA-approved reference method. Section 2.5 - 71 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol 5. Study plan. Background Objectives Study design Coordination Data reporting Section 4.1 Section 4.2 Section 4.3 Section 4.4 Section 4.5 n n n n n 6. Study report. Background Study objectives and design Study implementation Data reporting and validation Results Data analysis and discussion Conclusions Appendix A: Method Appendix B: Approved study plan Appendix C: Supporting data Appendix D: Supporting references Section 9.1 Section 9.2 Section 9.3 Section 9.4 Section 9.5 Section 9.6 Section 9.7 Section 9.8 Section 9.9 Section 9.10 Section 9.11 n n n n n n n n n n n 72 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol APPENDIX D DATA ELEMENTS AND EXAMPLE BENCH SHEETS 73 ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Data Elements The data elements listed below should be reported on the bench sheets or in the lab notebook for each method, as applicable. EPA will review the information during the data validation process to ensure the method-specific QC measures are met, as agreed to in the approved study plan. • Laboratory name • Method number • Media • Procedure • Matrix • Sample collection date/time • Dates and times for all method steps associated with holding times or incubation times • Analyst initials for each processing step in the method • Presumptive results for all applicable media • Confirmed/completed results for all applicable media • All measured volumes • Dilution information • Final result per units of measurement Example Bench sheets Example bench sheets for the following EPA-approved reference methods are included in this appendix: • Aeromonas (USEPA Method 1605) • Cryptosporidium (USEPA Methods 1622 and 1623) • K coli (SM 922 IF, SM 9222G) • Enterococci (SM 9230C) • Fecal coliforms (SM 9221E, 9222D) • Fecal streptococcus (SM 9230B, SM 9230C) • Giardia (USEPA Method 1623) • Total coliforms (SM 922IB, SM 9222B) Note: Additional example bench sheets or electronic copies of the attached bench sheets are available upon request. 74 ------- Laboratory: Sample Collection Date: Multiple-Tube Fermentation: Total Coliform, Fecal Coliform, E. coli (SM 9221B, SM 9221E, SM 9221F) Sample Collection Time: Sampling Point: LTB: Replicate 1 24hr/48hr LTB read Analyst Initials / Read Temp 7 10 mL / 1.0 mL / 0.1 mL / 0.01 mL / 0.001 mL / Please enter date and time for the following: LTB start LTB 24 hr read LTB 48 hr read, BOB 24 hr read (from 24 hr LTB), ECMUG 24 hr read (from 24 hr LTB) BOB 48 hr read (from 24 hr LTB), BOB 24 hr read (from 48 hr LTB), ECMUG 24 hr read (from 48 hr LTB) BOB 48 hr read (from 48 hr LTB) LTB: Replicate 2 24hr/48hr LTB read Analyst Initials Z Read Temp 7 10 mL / 1.0 mL / 0.1 mL / 0.01 mL / 0.001 mL / LTB: Replicate 3 24hr/48hr LTB read Analyst Initials 7 Read Temp 7 10 mL / 1.0 mL / 0.1 mL / 0.01 mL / 0.001 mL / Comments: BGB: Replicate 1 24hr/48hr BGB read (From 24 hr LTB) 24hr/48hr BGB read (From 48 hr LTB) Analyst Initials 7 z Read Temp 7 Z Final tube combination: 10 mL / / 1.0 mL / / 0.1 mL / / 0.01 mL / / 0.001 mL / / Total coliforms/100 mL: EC-MUG: Replicate 1 24 hr fecal read (From 24 hr LTB) 24 hr fecal read (From 48 hr LTB) 24 hr E. coli read (From 24 hr LTB) 24 hr E. coli read (From 48 hr LTB) Analyst Initials Read Temp Fecal tube combination: E. coli tube combination: 10 mL 1.0 mL 0.1 mL 0.01 mL 0.001 mL Fecal/100 mL: E. CO///100 mL: BGB: Replicate 2 24hr/48hr BGB read (From 24 hr LTB) 24hr/48hr BGB read (From 48 hr LTB) Analyst Initials 7 / Read Temp 7 / Final tube combination: 10 mL / / 1.0 mL / / 0.1 mL / / 0.01 mL / / 0.001 mL / / Total coliforms/100 mL: EC-MUG: Replicate 2 24 hr fecal read (From 24 hr LTB) 24 hr fecal read (From 48 hr LTB) 24 hr E. coli read (From 24 hr LTB) 24 hr E. coli read (From 48 hr LTB) Analyst Initials Read Temp Fecal tube combination: E. coli tube combination: 10 mL 1.0 mL 0.1 mL 0.01 mL 0.001 mL Fecal/100 mL: E. CO///100 mL: BGB: Replicate 3 24hr/48hr BGB read (From 24 hr LTB) 24hr/48hr BGB read (From 48 hr LTB) Analyst Initials 7 Z Read Temp 7 Z Final tube combination: 10 mL / / 1.0 mL / / 0.1 mL / / 0.01 mL / / 0.001 mL / / Total coliforms/100 mL: EC-MUG: Replicate 3 24 hr fecal read (From 24 hr LTB) 24 hr fecal read (From 48 hr LTB) 24 hr E. coli read (From 24 hr LTB) 24 hr E. coli read (From 48 hr LTB) Analyst Initials Read Temp Fecal tube combination: E. coli tube combination: 10 mL 1.0 mL 0.1 mL 0.01 mL 0.001 mL Fecal/100 mL: E. co///100 mL: ------- Membrane Filtration: mEndo/NA-MUG (SM 9222B/SM 9222G) Laboratory: Sample collection date: Sample collection time: Sampling point: mEndo incubation start temperature (°C): mEndo incubation end temperature (°C): mEndo incubation start date/time: mEndo incubation end date/time: NA-MUG incubation start temperature (°C): NA-MUG incubation end temperature (°C): NA-MUG incubation start date/time: NA-MUG incubation end date/time: Replicate number 1 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL Total conforms No. colonies per filter Total Conforms per 100 mL £ co// No. colonies per filter £ co// per 100mL Replicate number 2 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL Total coliforms No. colonies per filter Total Coliforms per 100 mL £ co// No. colonies per filter £. co// per 100 mL Replicate number 3 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL Total coliforms No. colonies per filter Total Coliforms per 100 mL £ co// No. colonies per filter £ co// per 100 mL ------- Membrane Filtration: mFC/NA-MUG (SM 9222D/SM 9222G) Laboratory: Sample collection date: Sample collection time: Sampling point: mFC incubation start temperature (°C): mFC incubation end temperature (°C): NA-MUG incubation start temperature (°C): NA-MUG incubation end temperature (°C): mFC incubation start date/time: mFC incubation end date/time: NA-MUG incubation start date/time: NA-MUG incubation end date/time: Replicate number 1 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL Fecal coliforms No. colonies per filter Fecal coliforms per 100 mL £ co// No. colonies per filter £ co// per 100 mL Replicate number 2 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL Fecal coliforms No. colonies per filter Fecal coliforms per 100 mL £ co// No. colonies per filter £. co// per 100 mL Replicate number 3 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL Fecal coliforms No. colonies per filter Fecal coliforms per 100 mL £ co// No. colonies per filter £ co// per 100mL ------- E. co// Membrane Filtration: mTEC (EPA 1103.1, SM 9213D) Laboratory: Sample collection date: Sample collection time: Sampling point: mTEC incubation start temperature (°C): mTEC incubation end temperature (°C): Urease substrate incubation start date/time: mTEC incubation start date/time: mTEC incubation end date/time: Urease substrate incubation end date/time: Replicate number 1 | Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL E. coll No. colonies per filter £. co// per 100 mL Replicate number 2 Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL E. coll No. colonies per filter £. coll per 100 mL Replicate number 3 Analyst initials Sample volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL £. coll No. colonies per filter £. co// per 100 mL ------- E. co// Membrane Filtration: Modified mTEC (EPA 1603) Laboratory: Sample collection date: Sample collection time: Sampling point: modified mTEC incubation start temperature (°C): modified mTEC incubation end temperature (°C): modified mTEC incubation start date/time: modified mTEC incubation end date/time: Replicate number 1 | Analyst initials Sample Volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL £. co// No. colonies per filter E.coli per 100 mL Replicate number 2 Analyst initials Sample Volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL £. co// No. colonies per filter E.coli per 100mL Replicate number 3 Analyst initials Sample Volume filtered 100mL 10.0 mL 1.0 mL 0.1 mL £. co// No. colonies per filter E.coli per 100mL ------- Multiple-Tube Fermentation: Fecal streptococcus (SM 9230B) Laboratory: Sample Collection time: Sample Collection Date: Replicate Number 1 Azide Dextrose Broth (ADB) 24hrADBread 48 hr ADB read Analyst Initials 10 ml / 1.0 ml / 0.1 ml / 0.01 ml / 0.001 ml / Comments: Sampling point: BEA Plates 24 hr BEA read from 24 hr ADB 24 hr BEA read from 48 hr ADB Analyst Initials Final tube combination: 10 ml / 1.0 ml / 0.1 ml / 0.01 ml / 0.001 ml / Fecal streptococcus/1 00 ml_: Replicate Number 2 Azide Dextrose Broth (ADB) 24 hr ADB read 48 hr ADB read Analyst Initials 10 ml / 1.0 ml / 0.1 ml / 0.01 ml / 0.001 ml / Comments: BEA Plates 24 hr BEA read from 24 hr ADB 24 hr BEA read from 48 hr ADB Analyst Initials Final tube combination: 10 ml / 1.0 ml / 0.1 ml / 0.01 ml / 0.001 ml / Fecal streptococcus/1 00 ml_: Replicate Number 3 Azide Dextrose Broth (ADB) 24 hr ADB read 48 hr ADB read Analyst Initials 10 ml 1.0 ml / 0.1 ml / 0.01 ml / 0.001 ml / Comments: BEA Plates 24 hr BEA read from 24 hr ADB 24 hr BEA read from 48 hr ADB Analyst Initials Final tube combination: 10 ml / 1.0 ml / 0.1 ml / 0.01 ml / 0.001 ml / Fecal streptococcus/100 ml_: ADB incubation start date/time: Start temp: ADB incubation end date/time: End temp: BEA incubation start date/time: (From 24hrADB) Start temp: BEA incubation end date/time: (From 24hrADB) End temp: BEA incubation start date/time: (From 48 hr ADB) Start temp: BEA incubation end date/time: (From 48 hr ADB) End temp: ------- Membrane Filtration: Fecal Streptococcus (SM 9230C) Laboratory: Sample collection date: Sample collection time: Sampling point: mEnterococcus incubation start temperature (°C): mEnterococcus incubation end temperature (°C): mEnterococcus incubation start date/time: mEnterococcus incubation end date/time: Replicate number 1 | Analyst initials Sample volume filtered 100mL 10.0mL 1.0ml 0.1 ml Fecal streptococcus No. colonies per filter Fecal streptococcus per 100 ml Replicate number 2 | Analyst initials Sample volume filtered 100ml 10.0ml 1.0ml 0.1 ml Fecal streptococcus No. colonies per filter Fecal streptococcus per 100 ml Replicate number 3 | Analyst initials Sample volume filtered 100ml 10.0ml 1.0ml 0.1 ml Fecal streptococcus No. colonies per filter Fecal streptococcus per 100 ml ------- Membrane Filtration: Enterococcus (mE-EIA) (EPA1106.1,SM9230C) Laboratory: Sample collection date: Sample collection time: Sampling point: mE incubation start temperature (°C): mE incubation end temperature (°C): EIA incubation start temperature (°C): EIA incubation end temperature (°C): mE incubation start date/time: mE incubation end date/time: EIA incubation start date/time: EIA incubation end date/time: Replicate number 1 | Analyst initials Volume filtered 100mL 10.0mL 1.0ml 0.1 ml Enterococcus No. colonies per filter Enterococci per 100 ml Replicate number 2 Analyst initials Volume filtered 100ml 10.0ml 1.0ml 0.1 ml Enterococcus No. colonies per filter Enterococci per 100 ml Replicate number 3 Analyst initials Volume filtered 100ml 10.0ml 1.0ml 0.1 ml Enterococcus No. colonies per filter Enterococci per 100 ml ------- Laboratory: Membrane Filtration: Enterococcus (mEI) Sample collection time: Sample collection date: mEI incubation start temperature (°C): mEI incubation end temperature (°C): Sampling point: mEI incubation start date/time: mEI incubation end date/time: Replicate number 1 | Analyst initials Volume filtered 100mL 10.0mL 1.0ml 0.1 ml Enterococcus No. colonies per filter Enterococcus per 100ml Replicate number 2 | Analyst initials Volume filtered 100ml 10.0ml 1.0ml 0.1 ml Enterococcus No. colonies per filter Enterococcus per 100ml Replicate number 3 | Analyst initials Volume filtered 100ml 10.0ml 1.0ml 0.1 ml Enterococcus No. colonies per filter Enterococcus per 100ml ------- Batch-specific Cover Sheet: Method 1605 (Aeromonas) -ADA with Vancomycin Note: Please complete one sheet per week of analysis. Laboratory name: Section 1. Media Preparation Information 1 2 3 4 5 6 Kovac's expiration date Oxidase dry slides expiration date Date of nutrient agar plate preparation Date of nutrient agar slant preparation Date nutrient agar slants for positive controls and matrix spikes were inoculated Time nutrient agar slants for positive controls and matrix spikes were inoculated Section 2: Sample Processing Information 7 8 9 10 11 12 13 14 Dilution preparation date (for IDC, ODC, and MS/MSD samples) Dilution preparation time (for IDC, ODC, and MS/MSD samples) Analyst preparing dilutions for IDC, ODC, and MS/MSD samples) Sample spiking date Sample spiking time Analyst spiking samples Analyst performing filtration Funnel decontamination method Section 3: Sample Analysis Information 15 16 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Incubator temperature at start date Incubator temperature at read date ADA-V incubation start date / time ADA-V read date /time ADA-V read analyst Nutrient agar plate incubation start date / time Nutrient agar plate read date / time Nutrient agar plate read analyst Oxidase confirmation date / time Oxidase read analyst Trehalose incubation start date / time Trehalose read date / time Trehalose read analyst Tryptone (indole) incubation start date /time Tryptone (indole) read date / time Tryptone (indole) read analyst 0 C c ------- QC Checklist: Method 1605 (Aeromonas) - ADA with Vancomycin Note: Please complete one sheet per week of analysis. Please circle the appropriate response and provide supporting information as necessary. Laboratory name: Batch identification: 1 2 3 4 5 6 7 8 Did all method blanks (dilution/rinse water) exhibit the appropriate response? Yes No If no, please list contaminated method blank(s) and associated samples. Did ADA-V media sterility check exhibit the appropriate response? Yes No If no, please explain. Did nutrient agar plate media sterility check exhibit the appropriate response? Yes No If no, please explain. Did nutrient agar slant media sterility check exhibit the appropriate response? Yes No If no, please explain. Did trehalose media sterility check exhibit the appropriate response? Yes No If no, please explain. Did tryptone media sterility check exhibit the appropriate response? Yes No If no, please explain. Did the unspiked reagent water sample exhibit the appropriate response? Yes No If no, please explain. Did negative controls for oxidase, trehalose, and indoleexhibit the appropriate responses? Yes No If no, please explain. ------- Sample-specific Data Report Form: Method 1605 (Aeromonas) - ADA With Vancomycin Laboratory name: Section 1: Sample information 1 Sample number: 2 Utility: 3 Sampling point: QC Analysis or Matrix filtered sterility check, direct streak, streak with filter (please circle one): reage^ ^^ di|ution/rinse 5 6 7 8 Volume (ml_) of spike (QC samples only): Volume filtered (ml): Dilution bottle (for laboratory-prepared QC samples only, pli D If this report form is for a method blank, please indicate samples that are associated with this method blank: D2 Please note: It is important to record the number of colonies for each presumptively positive morphological type so that the final density otAeromonas per sample can be reported based on percent confirmation of each colony type. Section 2: Sample results 1 2 3 4 5 6 7 8 9 10 ADA-V colony descript (this section is option Color pale yellow dark yellow Morphology* 1358 1358 on al) size (mm) 2-4 mm 1-3 mm No. of presumptive positive colonies for this colony type 60 5 No. of presumptive colonies submitted to confirmation for this colony type 2 1 Nutrient agar colony description (this section is optional) Color off-white off-white Morphology* 1358 1358 size (mm) 2-4 mm 1-2 mm No. oxidase positive per colony type 2 of 2 1 of 1 No. trehalose positive per colony type 2 of 2 1 of 1 No. Indole positive per colony type 1 of 2 1 of 1 *Morphology choices (list all that apply): (1) Round, (2) oval, (3) symmetric, (4) asymmetric, (5) shiny, (6) dull, (7) translucent, (8) opaque, (9) grainy, (10) fuzzy, (11) other Section 3: Calculations (Use one row for each presumptive positive colony color and morphology. If more than five colony types, please attach another sheet.) A No. presumptive positive colonies for each colony type 60 5 B No. of each colony type submitted to confirmation 2 1 C How many submitted colonies per colony type confirmed (oxidase positive, ferments trehalose, and produces indole)? 1 1 A * (C/B) = D Calculated no. of confirmed Aeromonas per colony type D1= 30 D2= 5 D3= D4= D5= D1 + D2 + D3 + D4 + D5 Total confirmed Aeromonas per sample 30 + 5 = 35 Section 5: Comments ------- Sample-specific Data Report Form: Method 1605 (Aeromonas) - ADA With Vancomycin Laboratory name: Section 1: Sample information 1 Sample number: 2 Utility: 3 Sampling point: 4 QC Analysis or Matrix filtered sterility check, direct streak, streak with filter (please circle one): reagen( ^.^ di|ution/rinse 5 Volume (mL) of spike (QC samples only): 6 Volume filtered (mL): 7 Dilution bottle (for laboratory-prepared QC samples only, please circle one): D D2 If this report form is for a method blank, please 8 indicate samples that are associated with this method blank: I Please note: It is important to record the number of colonies for each presumptively positive morphological type so that the final density ofAeromonas per sample can be reported based on percent confirmation of each colony type. Section 2: Sample results 1 2 3 4 5 6 7 8 9 10 ADA-V colony description (this section is optional) Color Morphology* size (mm) No. of presumptive positive colonies for this colony type No. of presumptive colonies submitted to confirmation for this colony type Nutrient agar colony description (this section is optional) Color Morphology* size (mm) No. oxidase positive per colony type No. trehalose positive per colony type No. Indole positive per colony type "Morphology choices (list all that apply): (1) Round, (2) oval, (3) symmetric, (4) asymmetric, (5) shiny, (6) dull, (7) translucent, (8) opaque, (9) grainy, (10) fuzzy, (11) other Section 3: Calculations (Use one row for each presumptive positive colony color and morphology. If more than five colony types, please attach another sheet.) A No. presumptive positive colonies for each colony type B No. of each colony type submitted to confirmation C How many submitted colonies per colony type confirmed (oxidase positive, ferments trehalose, and produces indole)? A * (C/B) = D Calculated no. of confirmed Aeromonas per colony type D1 = D2= D3= D4= D5= D1 + D2 + D3 + D4 + D5 Total confirmed Aeromonas per sample Section 5: Comments ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Laboratory Name: Laboratory ID: Method 1622/23 Bench Sheet 1. Client sample number 2. Internal laboratory sample ID (if applicable) 3. Date and time of sample receipt 4. Received by 5. Temperature of sample and condition of sample upon arrival 6. Storage location and storage temperature 7. Sample turbidity, in NTU 8. Sample type (IPR, method blank, field sample, OPR, MS, PT sample) 9. Spiking suspension number (for IPR, OPR, MS, and PT samples only) 10. Estimated number of oocysts/cysts spiked (for IPR, OPR, MS, and PT samples only) 11. Spiking date and time 12. Sample volume spiked, in L 13. Sample filtration start date and time 14. Type of filter used (Envirochek, Envirochek HV, FiltaMax, CrypTest, other [specify]) and lot number: 15. Name of analyst performing filtration 16. Sample volume filtered, to nearest 1/4 L (do not include rinse volume) 17. Did filter clog? 18. Elution date and time (must be performed within 96 hours of sample collection/filtration) Crypto Giardia 19. Elution procedure: D wrist shaker D FiltaMax wash station stomacher D backflush/sonication 20. Name of analyst performing elution 21. Elution buffer: Elution buffer lot number and expiration date: 22. Concentration procedure (centrifugation, FiltaMax concentrator, other [specify]) 23. Name of analyst performing concentration 24. Pellet volume after concentration, in mL 25. (a) Total volume of resuspended concentrate; (b) volume transferred to IMS (in mL) 26. Number of subsamples processed independently through the remainder of the method 27. IMS system used (Dynal anti-Cryptosporidium, Dynal GC-Combo, other [specify]) and lot number 27. Name of analyst performing IMS procedure 28. Slide(s) used (Meridian, Dynal, other [specify]) and lot number 29. Date and time sample applied to slide(s) to dry (must be completed same working day as Row 18) 30. Detection kit used (Merifluor, AquaGlo, Crypt-a-Glo, Giardi-a-Glo, other [specify]) and lot number 31. Analyst performing staining procedure 32. Staining completion date and time (must be complete within 72 hours of Row 29) 33. Total number of oocysts and cysts counted in sample (sum of counts in subsamples, if applicable) (a) (b) Crypto Giardia Comments: Ftess steps must to eompfeted in cw working day HV=high volume NTU=nephelometric turbidity unit IPR=initial precision and recovery OPR=ongoing precision and recover MS=matrix spike PT=performance test ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Laboratory Name: Laboratory ID: Method 1622/1623 Cryptosporidium Report Form Client sample number: 10-mL subsample ID (if packed pellet > 0.5 ml): Analyst: Object located by FA No. 1 2 3 4 5 6 7 8 9 10 Shape (oval or round) Size LxW (Fm) DAPI- Light blue internal staining, no distinct nuclei, green rim (A) Internal laboratory sample ID (if applicable): Volume examined (in L) on this slide: Positive staining control acceptable YES NO Negative staining control acceptable YES NO DAPI + Intense blue internal staining (B) Total FA number from this slide: DAPI -: Total number (A): DAPI +: Total number (B): DAPI +: Total number (C): Total count DAPI + (C) that show structure by D.I.C. (F): Number of nuclei stained sky blue (C) D.I.C. Empty oocysts (D) Oocysts with amorphous structure (E) Oocysts with internal structure (F) Number of sporozoites Examination completion date: Examination completion time (must be complete within 7 days of staining): D.I.C. - Total number of empty oocysts (D): D.I.C. - Total number of oocysts with amorphous structure (E): D.I.C. - Total number of oocysts with internal structure (F): ------- EPA Microbiological Alternate Test Procedure (ATP) Protocol Laboratory Name: Laboratory ID (if applicable): Method 1623 Giardia Report Form Client sample number: 1 0-mL subsample ID (if packed pellet > 0.5 mL): Analyst: Object located by FA No. 1 2 3 4 5 6 7 8 9 10 Shape (oval or round) Size LxW (Fm) DAPI- Light blue internal staining, no distinct nuclei, green rim (A) Internal laboratory sample ID (if applicable): Volume examined (in L) on this slide: Pos. staining control acceptable YES NO Neg. staining control acceptable YES NO DAPI + Intense blue internal staining (B) Total FA number from this slide: DAPI-: Total number (A): DAPI+: Total number (B): DAPI+: Total number (C): Total number DAPI + (C) that show structure by D.I.C. (F): Number of nuclei stained sky blue (C) D.I.C. Empty cysts (D) Cysts with amorphous structure (E) Cysts with internal structure (F) Number of nuclei Median body Axonemes Examination completion date: Examination completion time (must be complete within 7 days of staining): D.I.C.: Total number of empty cysts (D): D.I.C.: Total number of cysts with amorphous structure (E): D.I.C.: Total number of cysts with one internal structure (F): D.I.C.: Total number of cysts with >one internal structure (F): ------- |