# EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking Water, Ambient Water, and Wastewater Monitoring Methods Guidance April 2004

## U.S. Environmental Protection Agency Office of Water Engineering and Analysis Division 1200 Pennsylvania Avenue, NW (4303T) Washington, DC 20460

EPA 821-B-03-004

July 2003

## A CKNOWLEDGMENTS This protocol was prepared under the direction of the U.S. Environmental Protection Agency’s (EPA’s) Office of Water. This document was prepared by the DynCorp Biology Studies Group.


## D ISCLAIMER The Engineering and Analy s is Division, of the Office of Science and Technology, has reviewed and approved this report for publication. The Office of Science and Technology directed, m a naged, and reviewed the work of DynCorp in preparing this re port. Neither the United States Governm e nt nor any of its employees, contractors, or their employees m a ke 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 com p any names, trade names, or com me rcial products in this protocol does not constitute endorsement or recom me ndation for use.
Questions concerning this report should be addressed to: Robin K. Oshiro Engineering and Analy s is Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsy l vania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov 202-566-1075 202-566-1053 (facsimile)

## F OREWORD This document describes a process for seeking EPA approval of m i crobiological alternate test procedures (ATPs) or new m e thods for use in monitoring drinki ng water, ambient water, and wastewater. This document serves as a supplement to the ATP guidelines at 40 CFR 136.4, 136.5, and 141.27. With this guidance document, EPA has revised a nd com b ined the three existing m i crobiological ATP protocols to m a ke the ATP process m o re transparent to applicants while m a intaining the same level of data quality

. The guidelines in Quantitative Membrane Filter Methods (Reference 10.16), Presence/Absence Liquid Culture Me thods for Finished Water (Reference 10.17), and Presence/Absence Me mbrane Filter M e thods for Finished W a ters (Reference 10.15) have been incorporated into this single protocol, which is applicable to a wi der range of analy t es and techniques. This revised ATP protocol describes a process for conducting side-by

- side m e thod com p arisons and for conducting quality control (QC) acceptance criteria- based m e thod studies for EPA-approved reference me thods with QC acceptance criteria. Additionally, in som e cases the revised protocol provides applicants an opportunity to demonstrate com p ar ability by m eeting QC acceptance criteria associated with the EPA-approved reference m e thods for differe nt com b inations of analy t e and determinative technique. Under EPA’s ATP program, any person m ay apply for approval of the use of an ATP or new m e thod to test for a regulated analy t e. 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 m et hods for com p liance monitoring under the National Pollution Discharge Elimination System (NPDES) permit program and national primary drinking water regulations (NPDWRs). This document is not a legal instrum e nt and does not establish or affect lega l obligations under Federal regulations. EPA reserves the right to cha nge this protocol without prior notice. Questions or com me nts regarding this document should be directed to: Robin K. Oshiro Engineering and Analy s is Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsy l vania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov 202-566-1075 202-566-1053 (facsimile)

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## T ABLE OF 

## C ONTENTS Section 1.0 I ntroduction

.......................................................... 1-1 1.1 B ackground and Objectives

.............................................. 1-1 1.2 Types of Applications

.................................................. 1-3 1.2.1 Limited Use

.................................................... 1-3 1.2.2 Nationwide Use

................................................. 1-3 1.3 Types of Studies

....................................................... 1-4 1.4 Scope of Alternate Test Procedures

........................................ 1-4 1.4.1 EPA-Approved Reference Methods

................................. 1-4 1.4.2 Modifications to Sample Preparation Techniques

...................... 1-5 Section 2.0 Application

........................................................... 2- 1 2.1 S ubmission Addresses and Approval Authority

.............................. 2-1 2.2 Application Information

................................................. 2-1 2.3 Reason for ATP

....................................................... 2-2 2.4 S tandard EPA Method Format

............................................ 2-3 2.5 M ethod Com p arison Table

.............................................. 2-3 2.6 M ethod Development Information

......................................... 2-3 2.7 Study Plan

........................................................... 2-3 2.8 Study Report

......................................................... 2-4 2.9 Proprietary Information in Applications

.................................... 2-4 Section 3.0 M ethod Format

........................................................ 3- 1 3.1 Scope and Application

.................................................. 3-1 3.2 Summary of Method

................................................... 3-1 3.3 M ethod Definitions

.................................................... 3-1 3.4 Interferences

.......................................................... 3-1 3.5 Safety

............................................................... 3-1 3.6 Equipment and Supplies

................................................ 3-2 3.7 Reagents and Standards

................................................. 3-2 3.8 S ample Collection, Preservation, and Storage

................................ 3-2 3.9 Q uality Control

....................................................... 3-2 3.10 Calibration and Standardization

........................................... 3-2 3.11 Procedure

............................................................ 3-2 3.12 Data Analysis and Calculations

........................................... 3-2 3.13 Method Performance

................................................... 3-3 3.14 Pollution Prevention

................................................... 3-3 3.15 Waste Management

.................................................... 3-3 3.16 References

........................................................... 3-3 3.17 Tables, Diagrams, Flowcharts, and Validation Data

........................... 3-3 Section 4.0 Study Plan

........................................................... 4- 1 4.1 B ackground

.......................................................... 4-1 4.2 Objectives

........................................................... 4-1 4.3 Study Design

......................................................... 4-1 4.4 Coordination

......................................................... 4-2 4.4.1 Study Management

.............................................. 4-2 4.4.2 Technical Approach

............................................. 4-2 4.5 Data Reporting

........................................................ 4-2

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol

Section 5.0 Q uality Assurance/Quality Control

........................................ 5-1 5.1 Q uality Assurance

..................................................... 5-1 5.2 Q uality Control

....................................................... 5-1 5.2.1 A nalyst Counting Variability

...................................... 5- 3 5.2.2 Autoclave Sterilization Verification

................................ 5-3 5.2.3 Dilution/Rinse Water Blanks

...................................... 5-3 5.2.4 Incubator/Waterbath Temperatures

.................................. 5-3 5.2.5 I nitial Demonstration of Capability

................................. 5-3 5.2.6 I nitial Precision and Recovery

..................................... 5-4 5.2.7 Matrix Spike and Matrix Spike Duplicate Samples

..................... 5-4 5.2.8 M edia Sterility Checks

........................................... 5-4 5.2.9 M ethod Blank

.................................................. 5-4 5.2.10 Ongoing Demonstration of Capability (ODC) Sam p les

.................. 5-5 5.2.11 Ongoing Precision and Recovery (OPR) Sam p les

...................... 5-5 5.2.12 Positive/Negative Controls

........................................ 5-5 5.2.13 Preparation Blanks (PB)

.......................................... 5-6 5.2.14 Refrigerator/Freezer Tem p eratures
.................................. 5-6 5.2.15 Sam p le Processing Equipment Sterility Checks

........................ 5-6 Section 6.0 Study Design

......................................................... 6- 1 6.1 Side-by-Side Comparison Studies

......................................... 6-1 6.1.1 Number of Laboratories

.......................................... 6-1 6.1.2 Number of Samples

.............................................. 6-1 6.1.3 Verification of Results

........................................... 6-3 6.2 Q C Acceptance Criteria-Based Com p arison Studies

........................... 6- 4 6.2.1 Number of laboratories

........................................... 6-4 6.2.2 Number of Matrices

............................................. 6-4 6.2.3 Number of Replicates per Matrix

................................... 6-4 Section 7.0 Sample Pr eparation and Analysis

......................................... 7-1 7.1 Collection of Samples for Analysis

........................................ 7-1 7.1.1 Source Water Characterization

..................................... 7-1 7.2 S ample Spiking and “Stressing” Procedures for Bacteriological Methods

.......... 7- 1 7.2.1 D rinking Water: Spiking and Chlorine-Stressing

....................... 7-2 7.2.2 Preparation of Enumerated Spiking Suspension

........................ 7-4 7.2.3 L og Phase Growth Curve

......................................... 7-5 7.2.4 Commercially Available Enumerated Spikes

.......................... 7-5 7.3 S piking Procedures for Virus Methods

..................................... 7- 6 7.3.1 C ell Monolayer Propagation

....................................... 7-6 7.3.2 Propagation of Virus Stock Suspension

.............................. 7-6 7.3.3 Titering of the Virus Stock Suspension

.............................. 7-6 7.4 S piking Procedures for Cryptosporidium and Giardia

......................... 7-6 7.5 Analysis of Samples

.................................................... 7-7 7.5.1 Side-by-Side Comparison Studies

.................................. 7-7 7.5.2 Q C Acceptance Criteria-Based Com p arison Studies

.................... 7-7 7.6 Verification of Results

.................................................. 7-7 7.6.1 V erification of Results from Bacteriological Methods

................... 7-8 7.6.2 V erification of Results from Virus Methods

........................... 7-8 7.6.3 V erification of Results from Cryptosporidium and Giardia Methods

....... 7-8

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol
Section 8.0 Review of Study Results

................................................ 8-1 8.1 A ssessment of Com p liance with Approved Study Plan

......................... 8-1 8.2 Data Review

.......................................................... 8-1 8.3 Data Validation

....................................................... 8-1 8.4 D evelopment of Descriptive Statistics

...................................... 8-1 8.4.1 Mean Recovery

................................................. 8-1 8.4.2 Precision

...................................................... 8-2 8.4.3 False Positive Rates, False Negative Rates, Sensitivity, and Specificity

..... 8-2 8.5 S tatistical Assessment of Method Com p arability

............................. 8-3 8.5.1 P resence / Absence Methods

...................................... 8-3 8.5.2 Q uantitative Methods

............................................ 8-5 8.5.3 Q C Acceptance Criteria-Based Com p arison Studies

................... 8-10 8.6 M ethod Recom me ndation and Approval

................................... 8-12 Section 9.0 Study R eport
......................................................... 9-1 9.1 B ackground

.......................................................... 9-1 9.2 Study Objectives and Design

............................................. 9-2 9.3 Study Implementation

.................................................. 9-2 9.4 Data Reporting and Validation

........................................... 9-2 9.5 Results

.............................................................. 9-2 9.6 Data Analysis and Discussion

............................................ 9-3 9.7 Conclusions

.......................................................... 9-3 9.8 Appendix A - Method

.................................................. 9-3 9.9 Appendix B - Study Plan

................................................ 9-3 9.10 Appendix C - Supporting Data

........................................... 9-3 9.10.1 Raw Data

...................................................... 9-3 9.10.2 Electronic Data Reporting

......................................... 9-4 9.10.3 Example Calculations

............................................ 9-4 9.11 Appendix D - Supporting References

...................................... 9-4 Section 10.0 R eferences

.......................................................... 10-1


## L IST OF 

## A PPENDICES Appendix A

.................................................................. Glo ssary Appendix B

....................................................... ATP Application For m Appendix C

................................................... Application I nventory Form Appendix D

....................................... Data Elements and Example B ench Sheets Appendix E

.................................... EPA Hea dquarters and Regional ATP Contacts

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 1.0 I NTRODUCTION 

### 1.1 B ackground 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 me thods) for data gathering and com p liance monitoring under National Pollution Discharge Elimination System (NPDES) permits and national primary drinki ng water regulations (NPDWRs). The approved test procedures can be found in the Code of Federal Re gulations (CFR) at 40 CFR Part 136 for wastewater and ambient water and 40 CFR Part 141 for drinking wa ter. 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 m e thod. Figure 1.1 below sum ma rizes the ATP or new m e thod review process within EPA. These regula tions are the basis for th e Agency ’s alternate test procedure (ATP) program for water m e thods that is adm i nistered by the Office of Water, Office of Science and Technology, Director of Analytical Methods. An ATP is a modification of an EPA-approved refe rence m e thod or a procedure that uses the sam e determinative technique (i.e., the physical and/or ch emical process used to de termine the identity and concentration of an analy t e) and measures the sam e analy t e(s) of interest as the EPA-approved reference me thod. The use of a different determinative techni que to measure the same analy t e(s) of interest as an EPA-approved reference m e thod is considered a new m e thod. Under the ATP program, an organization or individua l m ay apply for approval of an ATP or new m e thod to be used as an alternate to an EPA-approved refere nce m e thod. The applicant is generally responsible for characterizing m e thod performance of its proposed a lternate test procedure prior to submission to the ATP program

. EPA can provide assistance to applicants in the development of a study plan to demonstrate com p arability with the EPA-approved reference m e thod. Figure 1.1 sum ma rizes the ATP or new m e thod review process within EPA. The Agency reviews the ATP package, approves or disapproves the application, and, for nationwide applications, w ill generally propose to include successful ATPs in the CFR (unless the ATP is for limited use or constitutes a m i nor modification-- See Appendix A). Figure 1-1. Summary of the ATP or New Method Rev i ew ProcessLUW W &AW

ATP appl ic at io n Ap pl y thro ugh St ate D irector RA pr ov i des lett er of a pprov al or di sa pp ro va lNWDW, W W ,& AW R e gional re view Fo rwardForwardtoOWforreview

OW re view OW recom m e nds

app roval or di sa pp ro va l Ac ronym s: AW = Am bient W ater DW = Dr inking W ater LU = Li m ite d- use NW = Nationwi de-us e OW = O ffi ce of W a te r RA = Regional Ad m inistrator WW = Wa stewaterO W recommendationOptional EP A Ad m inistrator

ap prov es th rough ru lem aking

1-1

### EPA Microbiological Alternate Test Procedure (ATP) Protocol
To m a ke the ATP program mo re accessible to applican ts while m a intaining data quality, EPA has revised and com b ined the three m i crobiological ATP protocols into this com p rehensive protocol. The guidelines in Quantitative Membrane Filter Methods (Reference 10.16), Presence/Absence Liquid Culture Methods for Finished Water (Reference 10.17), and Presence/Absence Membrane F ilter Methods for Finished Waters (Reference 10.15) have been incorporated into a single protocol applicable to a wider range of analy t es and techniques. Additionally, the revised pr otocol illustrates how applicants can demonstrate com p arability by m eeting quality control (QC) acceptance criteria associated with EPA-approved reference m e thods for which tho se criteria have been developed. The ATP program provides laboratories and regulat ed facilities with an opportunity to enhance com p liance monitoring and encourages the use of i nnovative technologies. Approval for an ATP or new me thod m ay be sought when, for example, the altern ate procedure reduces analytical costs, overcomes ma trix interferences problems, improves laboratory productivity, or reduces the amount of hazardous ma terials used and/or produced in the laboratory

. Any person or organization m ay apply to gain a pproval for the use of an ATP or new m e thod for determination of a specific constituent that is regulated under the NPDES permit program or the NPDWRs. The ATP applicants generally may dem onstrate com p arability of its proposed ATP or new me thod with the EPA-approved reference m e thod using th e procedures described in this protocol. Other possible m e thod com p arison procedures include t hose 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 m ay depend on the intended use of the ATP. Methods intended for use in demonstrating com p liance with the NPDES pe rmit program (wastewater or ambient water ATPs) may be submitted for approval for limited-use (single la boratory ) or for nationwide-use (all laboratories). Because only the Adm i nistrator has the authority to a pprove an alternate analytical technique for SWDA purposes, EPA will generally consider proposed m et hods intended for use in demonstrating com p liance with NPDWRs (drinking water ATPs) that are submitted for approval for nationwide-use only

. 1.2.1 L imited Use The primary intent of the limited-use ATP is to allow use of an ATP or new m e thod by a single laboratory

. Limited-use ATPs can be applied to one or m o re m a trix types, excluding drinking water ma trices; limited-use applications generally will not apply to Office of Ground Water and Drinking Water (OGWDW) ATP applications (Reference 10.18). If a me thod developer intends to apply the m e thod to mo re than one m a trix type, m e thod studies should be conducted on each m a trix type. Generally, nine different wastewater types should be analyzed to demonstrate the ATP or new m e thod will be applicable to m o st other m a trix types. If m e thod modificati ons are within the specified flexibility of the EPAapproved reference m e thod and all QC acceptance crite ria are m e t, it generally will not be necessary to submit the modification to the ATP program

. 1.2.2 N ationwide Use The primary intent of a nationwide-use ATP is to allow use of an ATP or new m e thod by all regulated entities and laboratories for one or m o re m a trix ty p es including drinking water. Nationwide-use approval allows vendors to establish that new devices and reagents produce results that are acceptable for com p liance monitoring purposes, and allows environmen tal laboratories across the United States to apply new technologies or modified techniques throughout their chain of laboratories to one or m o re m a trix types. If a m e thod developer intends to apply the me thod to m o re than one m a trix type, m e thod studies should be conducted on each m a trix ty pe. Generally, nine different wastewater types should be analyzed
1-2

### EPA Microbiological Alternate Test Procedure (ATP) Protocol to demonstrate the ATP or new m e thod will be a pplicable to m o st other wastewaters. If m e thod m odifications are within the specified flexibility of the EPA-approved reference m e thod and all QC acceptance criteria are m e t, it generally will not be necessary to submit the modification to the ATP program . 

### 1.3 Typ es of Studies The type of study mo st useful in seeking approval of an ATP or new m e thod generally depends on whether or not the EPA-approved reference m e thods c ontain QC acceptance criteria. There are two basic types of studies described in this protocol:

• Side-by-Side Method Comparison Study. A side-by

- side m e thod com p arison study generally consists of parallel testing of an ATP or new m e thod along side an EPA-approved reference m e thod to determine whether the performance of the new or m odified m e thod is acceptable com p ared to the reference m e thod.

• QC Acceptance Criteria-Based Method Comparison Study. For EPA-approved reference me thods that contain (or are supplemented with) QC acceptance criteria for m o st com b inations of analy t e(s) and determinative technique(s), the goal of the study is for the applicant to demonstrate that its ATP or new m e thod is able to meet the QC acceptance criteria of the EPA-approved reference me thod (or other EPA-specified document) for the applicable com b ination of analy t e and determinative technique through a QC acceptance criteria-based com p arison study

. Specific guidelines for the studies can be found in Section 5.0: Quality Control, Section 6.0: Study Design, Section 7.0: Sam p le Preparation and Anal ys is, and Section 9.0: Review of Study Results. 

### 1.4 S cope of Alternate Test Procedures This protocol for demonstration of com p arability , submission, and approval of an ATP or new m e thod offers flexibility to modify EPA-approved refere nce m e thods. Generally, an applicant should demonstrate and document that the modified m et hod produces results better than or equal to those produced by an appropriate EPA-appr oved reference m e thod for the applicable com b ination of analy te and determinative technique. 1.4.1 EPA-Approved Reference Methods The ATP process is based on com p aring the performan ce of an ATP or new m e thod to an EPA-approved reference m e thod through a side-by

- side com p arison study or a QC acceptance criteria-based com p arison study

. Method com p arability is demonstrated when results produced by an ATP meet or exceed the performance criteria associated with the EPA-approved re ference m e thod. Table 1-1 below lists the EPAapproved reference m e thods for the analy t es covered by this protocol. This table will be updated as necessary as additional pathogens are added to the list or advances in technology me rit a change in the EPA-approved reference m e thod. When performing a study, the applicant should use the reference me thod that uses the same determinative technique (e.g., MF, MPN) as the ATP for the analy t e(s) of interest. If the applicant is validating a new m et hod, which generally will use a determinative technique that is not currently approved for use with the analy t e(s) of interest, then the applicant should consult EPA prior to com me ncing the study to determine which is the m o st appropriate reference m e thod.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol
Table 1-1. EPA-Approved Reference Methods Analy te M ethod Format 1 EPA-Approved Reference Method 2 40 CFR Citation

To tal coliforms MPN S M 9221B 136.3, 141.21, 141.74 MF SM 9222B Presence/Absence S M 9221D 141.21
Fecal coliforms MPN S M 9221E 136.3, 141.21, 141.74, 503.8(b) MF SM 9222D
E. coli MPN S M 9221F 136.3, 141.21, 141.74 MF SM 9222G, SM 9213D HPC P our Plate S M 9215B 141.74 Fecal streptococcus MPN S M 9230B 136.3 MF SM 9230C
Enterococcus MPN S M 9230B 136.3 MF SM 9230C Salmonella MPN K enner and Clark 136.3, 503.8(b) Enteric virus P laque Assay EPA Document 3 503.8(b) Helminth ova M icroscopy EPA Document 4 503.8(b) Aeromonas MF USEPA Method 1605 141.40 Coliphage Plaque Assay U SEPA Method 1601 141.403 Tw o-Step Enrichment USEPA Method 1602 Cryptosporidium Filtration/IMS/FA USEPA Method 1622/1623 136.3, 141.74 Giardia Filtration/IMS/FA USEPA Method 1623 1 MPN = most probable number, MF = membrane filtrati on, IMS/F A = immunomagnetic separation/fluorescent antibody 2 "SM” refers to Standard Methods for the Examinat ion of W a ter and W a stewater. Fo r the edition(s) approved for a specific method, consult the CFR se ctions referenced in the column headed “40 CFR Citation.” (References 10.2, 10.3, and 10.4) 3 See References 10.12 and 10.13 4 See References 10.12 and 10.19 1.4.2 M odifications 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 measurem e nt of the identity and concentration of an analy t e is m a de). Sam p le preparation techniques include the procedures, equipment, reagents, etc., that are u sed in the preparation and cleanup of a sample for analy s is. Laboratories generally may m odify sample preparation techniques, provided the modification is not explicitly prohibited in the EPA -approved reference m e thod that is being modified and provided the m odification can be demonstrated to produce results equal or superior to results produced by an EPAapproved reference m e thod for each com b inati on of analy t e and determinative technique.

1-4

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 2.0 A PPLICATION ATP applications should be submitted in triplicate to EPA to facilitate the review process. The application consists of a com p le ted ATP application form (a sample application form is provided in Appendix D) with any attachm e nts. Electronic subm issions are also generally acceptable and often m ay accelerate the review process. 

### 2.1 S ubmission Addresses and Approval Authority
A sum ma ry of ATP submission information and approval authorities is provided in Table 2-1. Table 2-1. Submission of Alternate Test Procedure Applications Lev e l of Use A pplicant S ubmit Application To 1 Approval Authority
EPA Regional laboratories EPA Regional Administrator (Regional ATP Coordinator) 2

Limited Use for W a stewater or Ambient W a ter States, commercial laboratories, individual dischargers, or permittees in States that do not have the authority to administer Clean W a ter Act and Safe Drinking Water Act monitoring programs States, commercial laboratories, individual dischargers, or permittees in States that have the authority to administer Clean W a ter Act and Safe Drinking Water Act monitoring programs EPA Regional Administrator (Regional ATP Coordinator) 2

Director of State Agency issuing the NPDES permit 2 EPA Regional Administrator

Nationwide Use for

Drinking Water, Wa stewater, Ambient Wate r All applicants Director, Analy t ical Methods, Attn: ATP Program Coordinator, EPA Headquarters EPA Administrator

1 See Appendix E for EPA addresses. 2 T he Regional Administrator or the Director of State Agency issuing the NPDES permit may choose to forward limited-use applications to the Directo r of Analy t ical 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 Directo r of Analy t ical Methods, Attn: ATP Program Coordinator. Generally, upon receipt, the application will be assigne d an identification number, and a confirm a tion letter referencing this identificati on number will be sent to the applicant. The applicant should use the identification number in all future co mm unications concerning the application. 

### 2.2 A pplication Information The following information should be provided on the ATP application form (Appendix B): • N ame, m a iling address, phone number, and email address of the applicant • D ate of submission of the application • M ethod number, title, and revision date of the ATP or new m e thod submitted for review • T he analy t e(s) included in the ATP or new m e thod submitted for review

2-1

### EPA Microbiological Alternate Test Procedure (ATP) Protocol
• T he m a trix or m a trices to which the ATP applies • EPA-approved reference m e thod used for demonstration of com p arability

• Typ e of application (i.e., wastewater, drinking wate r, ambient water, point source categories regulated at 40 CFR Parts 400-499) • T he level of use desired (i.e., limited use or nationwide use) • Typ e of study (side-by-side com p arison or QC acceptance criteria-based com p arison study )

• A pplicant’s NPDES permit number, the issuing agency, the type of permit and the discharge serial number (if applicable) The following item s should be submitted as attachm e nts to the initial application: • R eason for proposing the ATP or new m e thod • T he proposed ATP or new m e thod prepared in standardized format (Section 3.0)

• A me thod com p arison table that gives a side-by

- side com p arison of the steps of the proposed ATP or new m e thod and the EPA-approved reference m e thod (Section 2.5) • M ethod development information • S tudy plan for EPA review and com me nt (Section 4.0) A study plan is generally not needed with the application if an applicant is unsure whether or not a m odification is allowed within the m e thod-specified flexibility

. In such cases, the applicant m ay request that EPA determine the usefulness of a full ATP com p arability assessment based on the other information submitted with the application. From this in formation, EPA can determine whether a full ATP assessment will be helpful, whether the proposed modifi cation is considered to be a m i nor modification (i.e., employs the same chemistry and/or biological principles as the EPA-approved reference m e thod 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 w ithin the specified flexibility of the EPA-approved reference m e thod. The elements of a com p lete 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 A ppendix C. EPA will generally seek all application information and attachm e nts before the application is considered com p lete. Table 2-2. Application Information Application Information

• C ompleted application form • R eason for AT P • M ethod in EPA format • M ethod comparison table • M ethod development information • S tudy plan (to be approved by EPA before proceeding w i th study ) • S tudy report (final report generally c onsidered part of a complete application)
Note: Although the application process generally begins with the initial submission of ATP m a terials, the application is not usually considered to be com p le te until the final study report has been submitted, and all EPA questions on the report have been resolved. 

### 2.3 R eason 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: • T he ATP improves m e thod performance • T he ATP provides equivalent m e thod components for a lower cost • T he ATP enables laboratories to perform analyses m o re efficiently
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### EPA Microbiological Alternate Test Procedure (ATP) Protocol

• T he ATP successfully overcomes som e or all of the interferences associated with the EPA-approved reference m e thod • T he ATP significantly reduces the amount of hazardous wastes generated by the laboratory • T he ATP provides another means for measuring a contaminant (i.e., provides m o re choices) 

### 2.4 S tandard EPA Method Format In accordance with the standard EPA format advocated by EPA’ s Environmental Monitoring Managem e nt Council (EMMC), m e thods should contain 17 specific topical sections in a designated order. The 17 sections are listed in Section 3.0 of this document. Additional numbered sections m ay be inserted starting with Section 11.0, Procedure , as appropriate for a particular m e thod. For detailed information on the EPA format for proposed m e thods, see the Gu idelines and Format document (Reference 10.14). 

### 2.5 M ethod Comparison Table As part of the application, the applicant should pr ovide a two-colum n table com p aring the proposed ATP or new m e thod to the EPA-approved reference m et hod. The two-colum n me thod com p arison table should include the number and title of each m e thod, the latest revision date of the proposed ATP, and a detailed discussion of each of the 17 topics specified by the standard EPA m e thod format (as applicable). Each topic should be discussed on a separate row in the m e thod com p arison table. The applicant should highlight any differences between the proposed ATP and the EPA-approved reference m e thod. 

### 2.6 M ethod 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 ne w m e thod in the water m a trix for which the ATP or new m e thod is being applied. These data m ay have been generated during m e thod development by the vendor, or through independent tests by third-party la boratories. Examples include, but are not limited to, replicate spiked reagent water or re plicate spiked m a trix water tests. It is the responsibility of the applicant to provide sufficient data to demonstrate that the ATP or new m e thod performs sufficiently at a preliminary level in the m a trix of interest to m e rit evaluation of an ATP. If sufficient data is not available, EPA m ay request additional studies be conduc ted prior to the review of the ATP study plan. In addition to data, the following descriptive m e thod information will facilitate EPA’ s evaluation of the ATP application: • T he purpose and intended use of the m e thod

• T he analytical basis for the m e thod, noting any relationship of the m e thod to other existing analytical me thods and indicating whether the m e thod is associated with a sampling m e thod

• M ethod limitations and an indication of any m eans of recognizing cases where the m e thod m ay not be applicable to specific m a trix types (e.g., turbidity greater than 50 NTU) • T he basic steps involved in performing the test and data analy s is • O ptions to the m e thod, if applicable This information also will aid EPA in preparing th e 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 S tudy Plan Prior to conducting all studies, the applicant should submit a study design for EPA review and com me nt. A detailed procedure (Section 3.0) for the new m e thod or the modification should be included as an attachm e nt to the study plan. Generally, EPA will eval uate the study plan to ensure that the appropriate

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol data quality objectives identified in this protocol are defined and addressed. EPA com me nts will be incorporated into the study design and this pro cess will be repeated until EPA has approved the study design. Generally, the study plan should contain the elements listed below: • B ackground • O bjectives • S tudy Design • C oordination • D ata Reporting These elements are further described in Section 4.0. 

### 2.8 S tudy Report The applicant should conduct a study and provide a com p rehensive study report with the ATP or new me thod application. The study report should include the following elements: • B ackground • S tudy Objectives and Design • S tudy Implementation • D ata Reporting and Validation • R esults • D ata Analy s is and Discussion • C onclusions • A ppendix A - Method • A ppendix B - Study Plan • A ppendix C - Supporting Data • A ppendix D - Supporting References These elements are further described in Section 9.0. 

### 2.9 P roprietary Information in Applications All information provided to the Federal governm e nt is subject to the requirem e nts of the Freedom of Information Act. Therefore, any proprietary info rmation submitted with the proposed ATP application should be m a rked as confidential. However, EPA prefers that supporting documentation labeled as confidential business information not be submitted as part of the ATP application. If proprietary information is determined to be essential to the app lication, EPA staff will request the information and will handle such information according to the regul ations in subparts A and B of 40 CFR Part 2. Specifically, in accordance with 40 CFR §2.203, a busin ess that submits information to EPA m ay 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 m ay be submitted separately to facilitate identifica tion and handling by EPA. If the business desires confidential treatm e nt only until a certain date or until the occurrence of a certain event, the notice should so state. Please be advised, however, that any me thods proposed in the Federal Register cannot be claim e d as confidential business information. If a claim of business confidentiality is not m a de at the time of submission, EPA will m a ke such efforts as are adm i nistratively practicable to associate a late cl aim with copies of previously submitted information in EPA files. However, EPA cannot ensure that su ch efforts will be effective due to the nature of application review that m ay already be in progress.
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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 3.0 M ETHOD 

## F ORMAT Because alternate test procedures m ay be approved by EPA as com p arable to the reference m e thods, and may be implemented by mu ltiple laboratories, it is important that the written procedures include all of the information necessary to use the technique in the la boratory , including but not limited to: reagents and equipment, sample collection and preservation proce dures, quality control, and a detailed description of the procedure. The information described below s hould be provided in the m e thod. In addition, EPA recom me nds the Environmental Monitoring Methods C ouncil (EMMC) format described below be used. Sections 3.1 through 3.17 provide a list of EMMC m e thod sections and a general description of the type of information that should be included in each sec tion. The date and revision number of the m e thod should be included on the cover page. In addition, the date should be included as a footer on each page of the m e thod. A detailed description of m e thod format guidelines, as well as an example of a formatted me thod, is provided in Reference 10.14. The detailed information in Reference 10.14 is provided as guidance for the m e thod write-up and as such, specific suggestions for font size, m a rgins, etc. are optional. 

### 3.1 S cope and Application Include a list of target organisms (by com m on na me ), taxonomic group and their CAS registry numbers or other accepted numbering systems (if available), the m a trices to which the m e thod applies, a generic description of m e thod sensitivity (the m i nim um number of organisms the m e thod can detect per unit volume or mass, if known), and the data quality objectives that the m e thod is designed to meet or m onitoring programs for which the m e thod was designed to support. 

### 3.2 S ummary of Method Sum ma rize the m e thod in a few paragraphs. The purpose of the sum ma ry is to provide a succinct overview of the m e thod procedure to aid the review er or data user in understanding the m e thod and how the results are generated. Include a general descrip tion of the m e thod procedure, sample volume, type of me dia used, preparation steps, incubation time and tem p eratures, and the techniques used for qualitative or quantitative determinations. 

### 3.3 M ethod Definitions Provide definitions of terms that are necessary to unde rstand how the m e thod is used or what the results represent. This should include a definition of the targ et organism or group of organisms, relative to the determinative step of the m e thod. For extensive lists of definitions, this section m ay sim p ly refer to a glossary attached at the end of the m e thod document. 

### 3.4 I nterferences This section should discuss any known m e thod interfere nces such as toxic m a terials, particulates, nontarget organisms, etc. If known interferences in the reference m e thod are not interferences in the alternate me thod, this also should be clearly stated. 

### 3.5 S afety This section should discuss only those safety issues specific to the m e thod and beyond the scope of routine laboratory practices. Target analy t es or reag ents that pose specific health, toxicity, or safety issues should be addressed in this section.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 3.6 E quipment and Supplies For critical equipment that m ay affect the performance of the m e thod, cite the m a nufacturer, model nam e, and catalog or product number of the equipment that was used to develop or validate the m e thod; note that equivalent equipment can be used, if appli cable. Use generic language for standard laboratory glassware and disposables. 

### 3.7 R eagents and Standards Provide sufficient details on the concentration and pr eparation of reagents and standards to allow the work to be duplicated, but avoid lengthy discussions of com m on procedures. If only pre-prepared proprietary reagents can be used, specify this. In clude catalog and/or product numbers where appropriate. Indicate shelf life of packaged m a teri als and special storage specifications. 

### 3.8 S ample Collection, Pr eservation, and Storage Provide information on sample collection, preservati on, shipm e nt, storage conditions, and holding times. If effects of holding time were specifically eval uated, provide referen ce to relevant data. 

### 3.9 Q uality 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 measurem e nts using the m e thod, and that the measurem e nts are free from contamination. Sp ecific QC measures m ay include positive and negative controls, duplicate samples, m e thod blanks, and m e dia sterility checks. Indicate which QC measures are appropriate initially, before a laboratory uses the m e thod, and which are appropriate on an ongoing basis. Indicate frequencies for each QC measure and list mi nim um specifications or acceptance ranges (see Section 5.0). Indicate corrective actions that should be taken when QC measures are not m e t. Define all terms in m e thod definitions section. 

### 3.10 Calibration and Standardization Discuss initial calibration specifications for instru me nts used in the m e thod (e.g., water baths, refrigerators, therm ome ters, balances, pH m e ters, mi croscopes, etc.). Indicate frequency of such calibrations; refer to performance specifications; and indicate corrective actions that should be taken when performance specifications are not m e t. This section m ay also include procedures for calibration, verification, or continuing calibration, or these steps m ay be included in the procedure section. 

### 3.11 Procedure Provide a detailed description of the sample processing and analy s is steps. Avoid unnecessarily restrictive instructions, but provide sufficient detail for m a nual procedures so that analy s ts in other laboratories perform the m e thod consistently

. Ranges should be provided for tem p erature requirem e nts, time requirem e nts, etc. 

### 3.12 Data Analy s is and Calculations Identify qualitative and quantitative aspects of the m e thod. List criteria for the identification of target organism(s) and interpretation of results for all step s of the m e thod, including criteria for presum p tive and confirm e d results. Provide equations used to derive final sample results. Provide discussion of estimating detection limits, recoveries, specificity, fal se positive/false negative rates, etc., if appropriate.

3-2

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 3.13 Method Performance Provide detailed information on m e thod performance, including data on precision, bias (for quantitative me thods), specificity, detection limits (including the me thod by which they were determined and m a trices to which they apply ) , 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 m e thod, provide a sum ma ry of the side-b y- side com p arison of performance versus reference me thod specifications. 

### 3.14 Pollution Prevention Describe aspects of this m e thod that m i nim i ze or pr event pollution that m ay be attributable to the reference m e thod. 

### 3.15 Waste Management Cite how waste is m i nim i zed 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 m ay be presented at the end of the m e thod. Lengthy tables m ay be included here and referred to elsewhere in the text by number. Diagram s should only include new or unusual equipment or aspects of the m e thod.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 4.0 S TUDY 

## P LAN Applicants should submit a study design for EPA re view, com me nt, and approval prior to conducting the side-by

- side com p arison study or the QC acceptan ce criteria-based com p arison 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 m ay m odify the study design for a particular proposed m e thod. Data fro m studies conducted without EPA review and approval may not meet EPA’ s criteria, and m ay not adequately address the applicant’s study objectives. A detailed procedure (Section 3.0) for the ATP or new m e thod should be included as an attachm e nt 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 com me nts are incorporated into the study design. This review/revision process is re peated until EPA has approved the study design. Generally, the study design should include the info rmation described in Sections 4.1 through 4.5. 

### 4.1 B ackground This section of the study plan should include the following information: • A statement identify i ng the ATP as a new m e thod or a modification of a reference m e thod

• T he EPA program(s) to which the ATP or new m e thod applies (e.g., drinking water, wastewater, ambient water, point source categories regulated at 40 CFR Parts 400-499, etc.) • A short (one paragraph) sum ma ry of the ATP or new m e thod • T he organization and m e thod number of the reference m e thod 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

• T he m a trices (e.g., finished water, wastewater, am bient water, etc.), m a trix types (e.g., turbidity greater than 10 NTU, etc.), and/or m e dia to which the ATP or new m e thod is believed to be applicable

• A list of the analy t es measured by the ATP or new m e thod, including the corresponding CAS registry number (if available) or other identification number 

### 4.2 O bjectives Include a description of the new m e thod or modification, describe the goals of the study, and define data quality objectives. 

### 4.3 S tudy Design The following information should be included in the study design: • L aboratories that will participate in the study (Sections 5.0 and 6.0) • N umber and type of samples to be analyzed (Section 6.0) • D escription of the m a trices that will be used (Sections 6.0 and 7.0) • D escription of the spikes that will be used (Section 7.0) • D escription of the spiking procedure (Section 7.0) • P ositive and negative control organisms (Section 5.0) • Q uality control procedures that will be followed (Section 5.0)

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 4.4 C oordination Describe how the study will be coordinated, how the spikes will be shipped to the laboratories, and who will com p ile the data for submission. Data com p ila tion should not be performed by any of the analy s ts conducting the sample analyses. 4.4.1 S tudy Management This section of the study plan should include the following information: • T he organization responsible for m a naging the study • T he 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 analy s is, interpretation of sample results, com p letion of study, etc. 4.4.2 Technical Approach This section of the study plan should include the following: • A description of how sample m a trices and participating laboratories will be selected • A description of how samples will be collected and distributed • T he 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 D ata 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 attachm e nt to the Study Report (Section 9.0). Address the statistical analy s is 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 m e thod performance will generally be based on the statistical analyses described in Section 8.0.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 5.0 Q UALITY 

## A SSURANCE 

## /Q UALITY 

## C ONTROL For side-by

- side com p arison studies in which only one laboratory is performing analyses, the com p arability study should be conducted at an i ndependent laboratory and the laboratory should be certified to perform mi crobiological analyses under EPA’ s drinking water laboratory certification program . A laboratory with a vested interest in the me thod, instrum e ntation, apparatus, reagents, m e dia, or associated kits m ay not participate in the side-by

- side com p arison study

. For QC acceptance criteria-based com p arison studies, at least three independent laboratories should participate. A laboratory with a vested interest in the ATP also m ay participate in the study, but in such instances there should be at least three independent laboratories participate and the m a jority of the laboratories participating are independent laboratories. All laboratories should be certified to perform mi crobiological analyses under EPA’ s drinking water certification program, or a com p arable 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 m i crobiological analyses under EPA' s drinking water certification program

. If m o re than three laboratories participate, the m a jority of the laboratories should be certified for m i crobiological analyses under EPA' s drinking water certification program

. 

### 5.1 Q uality Assurance The laboratory should have a com p rehensive quality assurance (QA) program in place and operating at all times during the performance of the com p arability 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 m e thod(s). Laboratory QA/QC criteria for facilities, personne l, and laboratory equipment are included in Standard Me thods 9020-Quality Assurance (Reference 10.4) and the U.S. EPA Manual for the Certification of Laboratories Analyzing Drinking W a ter , Fourth Edition (March 1997) (Reference 10.13). The laboratory should adhere to st andard laboratory practices for cleanliness and environment, and to the me thods for glassware and apparatus, reagents, solv ents, 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 Contro l in Water and Wastewater Laboratories , EPA-600/4-79-019 (Reference 10.19). 

### 5.2 Q uality Control Laboratories participating in a com p arability study should perform all QC procedures specified in the me thods, except where explicitly stated in the approve d 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 m e thod. Other QC procedures m ay be necessary , based on the a pproved study plan. Laboratories participating in side-by

- side com p arison studies or QC acceptance criteria-based com p arison studies should perform all QC procedures specified in the m e thods, except where explicitly stated in the approved study plan. The laboratory should m a intain records to define the quality of data that are generated. The laboratory should ma intain a record of the date and results of all QC sample analyses. Laboratories should m a intain reagent and m a terial lot numbers along with samples analyzed using each of the lots. Laboratories should also ma intain m e dia preparation records. Table 5-1 lists quality control measures for each labor atory participating in side-by

- side com p arison studies and QC acceptance criteria-based com p arison st udies. Detailed descriptions of the QC measures are provided in Sections 5.2.1 to 5.2.15. If contamin ation is detected in any of the blanks or sterility checks described below, the source of contamin ation should be identified and corrected. The

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol blank/sterility check and all samples associated with that contaminated blank/sterility check should be reprepared and reanalyzed. Measures taken to eliminate contamination should be reported. Table 5-1. Quality control meas ures for each laboratory inv o lved in the study Sugge sted for Study Quality Co nt rol Measure Fr eq uency Side-by - Side Compa r ison Study QC acceptan ce criteria-based Compa r ison Study Analy s t counting variability (Section 5.2.1) 2 samples per study T T
Autoclave sterilization verification (Section 5.2.2) Wi thin one week prior to the start of the study T
Dilution/rinse w a ter blanks (Section 5.2.3) 1 per every 20 samples or 1 per day of study , wh ichever is greater T
Incubator/waterbat h temperatures (Section 5.2.4) 2 times per day w hen used in study T Initial demonstration of capability (IDC) (Section 5.2.5) or Initial precision and recovery (IPR) (Section 5.2.6) Each laboratory participating in the study should generate acceptable IDC/IPR data. T
Matrix spike/matrix spike duplicate (Section 5.2.7) Per approved study plan T
Media sterility checks (Section 5.2.8) 1 per each batch of media used in the study or per each test run, w h ichever is greater T
Method blank (Section 5.2. 9) Per approved study plan T Ongoing demonstration of capability (ODC) (Section 5.2.10) or Ongoing precision and recovery (OPR) (Section 5.2.11) Included as part of the study; not needed as separate QC. Optional

Positive and negative controls (Section 5.2.12) 1 positive control and 1 negative control for each media/stain used in the study. See 5.2.12 below for more details. T

Preparation blanks (Section 5.2.13) Fr equency depends on the type of method. See 5.2.13 below for more details. T Refrigerator/freezer temperatures (Section 5.2.14) Once per day w hen used in study T Sample processing equipment sterility checks (Section 5.2.15) Prior to the analy s is of samples. See 5.2.15 below for more details. T T

T

T

T

T

T

T

T

T

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.1 A naly s t Counting Variability If the laboratory has two or m o re analy s ts, each should count colonies, plaques, or positive wells on the same plate/tray from one positive field sample per month. Com p are each analy s t’s count of the colonies, plaques, or wells. Counts should fall within 10% be tween analy s ts. If counts fail to fall within 10% of each other, analy s ts should perform additional sets of counts, until the number of target colonies, plaques, or positive wells counted fall within 10% between analy s ts for at least three consecutive samples. If there is only one analy s t replicate counts should be done and be within 5% of original counts. 5.2.2 A utoclave 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 m i nutes. Place Bacillus stearothermophilus spore suspensions in try p ticase soy broth tubes and incubate at 55°C for 48 hours. Check for growth to ve rify 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 m i ni mum, autoclave sterilization is performed on a monthly basis. 5.2.3 D ilution/Rinse Water Blanks The laboratory should analyze dilution/rinse water bl anks 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 m o re frequent, and observed for contamination with agent of interest. 5.2.4 I ncubator/Waterbath Temperatures Incubator or waterbath tem p eratures should be measu red and recorded two times per day when in use. Tem p eratures should be taken at least 4 hours apart and should be within the range of the desired tem p erature as specified in each m e thod. Therm ome te rs used to measure “in-use” tem p eratures should be calibrated yearly against an NIST traceable therm ome ter. 5.2.5 I nitial Demonstration of Capability Laboratories participating in a QC acceptance crite ria-based com p arison study should have successfully performed an initial demonstration of capability (IDC) test for the new or modified m e thod 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 m e thod to demonstrate acceptable performance. The laboratory should com p lete any additional analyses as specified in the study plan. For the IDC test, the laboratory spikes and analyzes re ference m a trix (e.g., reagent water, buffered water, etc.) samples to demonstrate acceptable performance w ith the m e thod prior to the analy s is of field samples. The number of samples involved in the IDC varies by me thod. If the results of the IDC test m eets all IDC acceptance criteria (e.g., RSD, m i nim um 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 unacceptabl e. In this event, the laboratory should identify and correct the problem and repeat the test. IDC tests should be accom p anied by a m e thod blank (Section 5.2.9).

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.6 I nitial Precision and Recovery Laboratories participating in a QC acceptance crite ria-based com p arison study should have successfully performed an initial precision and recovery (IPR) test for the m e thod using the modified version of the me thod under evaluation in the study

. An IPR test is performed when QC acceptance criteria are available for the evaluation of both precision and r ecovery . The laboratory should perform an IPR as specified in the m e thod to demonstrate acceptable performance. The laboratory should com p lete any additional analyses as specified in the study plan. For the IPR test, the laboratory spikes and analyzes reference m a trix (e.g., reagent water, buffered water, etc.) samples to establish the laboratory ’ s ability to generate acceptable precision and recovery prior to the analy s is of field samples. Using results of the analyses, the laboratory calculates mean percent recovery and relative standard de viation (RSD) of the recoveries for the analy t e(s) and com p ares them with the corresponding limits for the IPR test criteria in the m e thod. If the RSD and the mean percent recovery m eet the acceptance criteria, system pe rformance is acceptable and analy s is of samples m ay begin. If the RSD or the mean percent recovery are unacceptable, system performance will generally be unacceptable. In this event, the laboratory should id entify and correct the problem and repeat the test. IPR tests should be accom p anied by me thod blank tests (Section 5.2.9). 5.2.7 M atrix Spike and Matrix Spike Duplicate Samples Matrix spike (MS) and m a trix spike duplicate (MSD ) samples are spiked matrix water samples analyzed by the laboratory to verify acceptable m e thod performan ce in the m a trix being monitored. During routine performance of the m e thod, MS/MSD samples are analy zed by the laboratory for the first sample of any new m a trix that will be monitored, and on the 21 st sample thereafter. During a QC acceptance criteriabased com p arison study for som e me thods (e.g., Cryptosporidium and Giardia me thods), the laboratory should analyze MS samples as part of routine laboratory QC, however these analyses m ay 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 M edia Sterility Checks Before using newly prepared m e dia, a representative portion of each m e dia batch needs to be checked for sterility

. The laboratory should test m e dia sterility by incubating one unit (tube or plate) from each batch of m e dium specified in the m e thod or per each test run, whichever is m o re frequent, at the appropriate tem p erature for the length of the m e thod-specified incubation time and observing for growth. 5.2.9 M ethod Blank Method blanks are reagent water blanks or other bl anks including but not limite d to buffered water, tap water, etc., depending on the m e thod analyzed to demonstrate freedom from contamination. Method blanks should be analyzed at the frequency specifi ed in the approved study plan. For QC acceptance criteria-based com p arison studies, the laboratory need s to analyze a m e thod blank with the IPR and IDC tests. During routine performance of the m e thod, the laboratory should analyze at least one m e thod blank per every 20 test samples or per every week. During a QC acceptance criteria-based com p arison study for som e me thods (e.g., Cryptosporidium and Giardia me thods), the laboratory should analyze m e thod blanks as part of routine laboratory QC, but m e thod blanks also should be shipped to the laboratory as double-blind samples by the study coordinator.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.10 Ongoing Demonstration of Capability (ODC) Samples Ongoing demonstration of capability samples are spiked reference m a trix (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 refe rence m a trix (e.g., reagent water, buffered water, etc.) samples that are analyzed by the laboratory to verify that m e thod performance criteria are being m e t. During a performance based com p arison study for som e me thods (e.g., Cryptosporidium and Giardia me thods), the laboratory should analyze OPR samples as part of routine laboratory QC because generally OPR samples using blinded spiking suspensions distribut ed 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 confirm a tion test results are appropriate. 5.2.12.1 P ositive/Negativ e Culture Controls (Culture-Based Methods) Positive and negative culture controls refer to cultu res that, when analyzed exactly like field samples, produce a known positive or a known negative result, respectively , for a given type of m e dia. One positive culture control and one negative culture control should be prepared and analyzed for every me dia (including confirm a tion m e dia) used in the m e thod when ever a new batch of m e dium or reagents is used, every day of the study, or every 20 samples, or as specified in the m e thod, whichever is m o re frequent. Each control should be carried through the entire procedure and should exhibit the expected positive or negative result. 5.2.12.2 P ositive/Negativ e Staining Controls ( Cryptosporidium and Giardia Methods) A positive staining control for Cryptosporidium and Giardia me thods is a slide containing positive antigen or intact Cryptosporidium oocy s ts 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 sam e procedure as used for field samples or test samples. The laboratory should prepare and examine positive a nd 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 P ositive/Negativ e Staining Controls (Other) At a m i nim um, the laboratory should prepare and examine a positive and negative control using the sam e procedure as used for field or test samples whenever a new batch of m e dia or reagents is used every day of the study or every 20 samples, whichever is m o re frequent. Each control should be carried through the entire procedure and should exhibit the expected positive or negative result.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 5.2.13 Preparation Blanks (PB) 5.2.13.1 M embrane Filter Pr eparation Blank (PB-MF) If m emb rane filtration is used, at the beginning and the end of each filtration series, a PB-MF is performed by filtering 20-30 m L of dilution water thr ough the m emb rane 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 m i nutes or m o re elapse between sample filtrations. 5.2.13.2 M ultiple-Tube Fermentation Test Preparation Blank (PB-MTF) If a m u ltiple-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 m o re frequent. The preparation blank s hould 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 m u ltiple-tube fermentation m e dia should be included. 5.2.13.3 O ther 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 fi eld sample should be analyzed for every day of the study or every 20 samples, whichever is m o re frequent

. The preparation blank should be incubated with the sample batch and observed for growth of the targ et 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 tem p eratures should be m easured and recorded once per day when in use. Refrigerator tem p erature should be m a intained at 1° C to 4°C. Freezer tem p eratures should be m a intained at -15°C to -20°C. Special freezers capable of long-te rm storage of cultures or virus should be m a intained at -70°C to -80°C. Therm ome ters used to measu re “in-use” tem p eratures should be calibrated yearly against an NIST traceable therm ome ter. 5.2.15 Sample Processing Equipment Sterility Checks A representative portion of non-disposable item s 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 approxim a tely 500 m L (or appropriate volume based on the size of the equipment being used) of a sterile non-selective broth (e.g., try p tic soy , try p ticase soy , or try p 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 m e thod, the length of the inc ubation time for the sample processing equipment sterility check m ay be increased.

5-6

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 6.0 S TUDY 

## D ESIGN This section provides a description of the study design that is important for assessing com p arability using either side-by

- side m e thod com p arison studies (Section 6.1) or QC acceptance criteria-based studies (Section 6.2). The sections below address the numbe r of laboratories, number of m a trices, and the number of samples that should be analyzed in the studies. 

### 6.1 S ide-by-Side Comparison Studies Methods are com p ared using the following param e ters:

• Recovery. Does the new m e thod have similar, better or worse recoveries of the target organism as the reference m e thod?

• Precision. Are the recoveries by the new m e thod significantly less or m o re variable than the reference m e thod?

• False positive rate/specificity. Is the new m e thod significantly mo re likely or less likely to detect non-target organisms or other sample constituents th at would be reported as the target organism by the analy s t when com p ared to the reference m e thod?

• False negative rate/sensitivity. Is the new m e thod significantly mo re likely or less likely to exhibit non-detects for samples with the target organism or to exhibit results that are biased low when com p ared to the reference m e thod? To generate these param e ters, samples are analyzed by a single laboratory (6.1.1). The number of samples (and m a trix types) used in the study are dete rmined using a historical EPA standard (6.1.2.1) 6.1.1 N umber of Laboratories A single laboratory should be used for a side-by

- side com p arability study

. Since the study should be conducted in a single independent la boratory with no conflict of interest; the laboratory selected cannot be the m e thod developer’s laboratory and cannot be affiliated with the m e thod developer. 6.1.2 N umber of Samples The following standards generally provide the m i ni mum number of samples that should be analyzed. Additional data are generally acceptable and m ay be very helpful when reviewing an ATP or new me thod.
6.1.2.1 Method Com p arison Study Design Sum ma ry Table 6.1 provides a sum ma ry of the m e thod com p arability requirem e nts for nationwide m i crobiological ATPs and new m e thods.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol

6-2

### EPA Microbiological Alternate Test Procedure (ATP) Protocol Number of M a trices Ten different water/wastewater m a trices from geogr aphically diverse locations should be included to obtain, as m u ch as is practical, a good presentation of the wide range of water types with an even wider range of target organisms to which the m e thod shoul d appropriately respond. Each water or wastewater sample should be collected in sufficient volume to co mp lete all replicate analyses of sample or dilution volumes by both the ATP or new m e thod and the EPA -approved reference m e thod. For ambient water studies, the turbidity of at least one m a trix should be greater than 10 NTU. Generally, for m a trices other than finished drinking water or ambient water, m a trix composition will be addressed on a study-specific basis. Number of Samples and Replicates Twenty replicate analyses should be performed by each m e thod for each of the 10 m a trices for a total of 200 replicate analyses per m e thod. The replicate an alyses should be performed on the same day for both the proposed and reference m e thods. Verification of Results For quantitative reference m e thods, 10 typical colonies m u st be verified from 4 randomly chosen replicates (of the 20 replicates) of each of the 10 sam ples 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 verificati on 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 F alse Positiv es To assess whether the false positive rates are significan tly different between m e thods, replicates known to contain non-target organisms that could be falsely id entified as the target organism should be analyzed by both the ATP or new m e thod. The determination that the samples do not contain the target organism should be based on a third independent standard m e thod (see Section 7.6) rather than by the EPAapproved reference m e thod being used in the com p arison. This is because it should not be assum e d that the accepted m e thod has a false positive or negative rate of zero. In side-by

- side com p arison studies using the EPA standard for determining the number of m a trices and the number of samples, at least 200 positive results should be verified for the ATP or new m e thod in order to adequately com p are false positive rates. 6.1.3.2 F alse Negatives To assess whether the false negative rates are signifi cantly different between m e thods, replicates known to contain target organisms should be analyzed by both the ATP or new m e thod. The determination that the samples do not contain the target organism shoul d be based on a third independent standard m e thod (see Section 7.6) rather than by the EPA-approved re ference m e thod being used in the com p arison. This is because it should not be assum e d that the accepted m e thod has a false positive or negative rate of zero. In side-by

- side com p arison studies using the EPA st andard for determining the number of m a trices and the number of samples, at least 200 negative results should be verified for the ATP or new m e thod in order to adequately com p are false negative rates.

6-3

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 6.2 Q C Acceptance Criteria-Based Comparison Studies QC acceptance criteria-based com p arison studies are ge nerally conducted to demonstrate that the ATP or new m e thod is able to meet the QC acceptance crite ria of the EPA-approved reference m e thod. In som e instances, the quality control (QC) acceptance criteri a specified in a m e thod m ay not be sufficient to demonstrate com p arability between the ATP and th e EPA-approved reference m e thod and a side-by

- side com p arison study should be conducted. Generally, EPA will m a ke this decision based on review of the application m a terials. 6.2.1 N umber of laboratories A m i nim um of three laboratories should be used for a QC acceptance criteria-based com p arison study

. All three laboratories should meet all QC acceptance criteria in the EPA-approved reference m e thod. If mo re than three laboratories participate, at least 75% of the participating laboratories should meet all QC acceptance criteria in the EPA-approved reference m e thod. 6.2.2 N umber of Matrices For all QC acceptance criteria-based com p arison studies, there should be one m a trix per laboratory

. For ambient water studies, the turbidity of at least one m a trix should be greater than 10 NTU. Generally, for ma trices other than finished drinking water or ambi ent water, m a trix composition should be addressed on a study-specific basis. 6.2.3 N umber of Replicates per Matrix The number of replicates per m a trix and laboratory s hould be specific to the QC criteria to which the results will be com p ared. Generally, the number of reagent results should be 4 and the number of source water results per m a trix should be 2.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 7.0 S AMPLE 

## P REPARATION AND 

## A NALYSIS 

### 7.1 C ollection of Samples for Analy s is Each sample should be collected in sufficient volum e to com p lete all replicate analyses by both the ATP or new m e thod and the EPA-approved reference m e thod. Sam p les should be spiked (if necessary ) and analyzed as soon as possible after collection. 7.1.1 S ource Water Characterization Source water characterization information should be collected at the time of sample collection. This information will be useful in characterizing the m a trix to identify potential interferences and generally includes, but is not limited to, the following: • S ample collection location • S ource of water (e.g., ground water, stream, river, lake, etc.) • P lant treatm e nt processes (if sample is collected from a water treatm e nt facility ) • T emperature • pH • T urbidity • T otal organic carbon (TOC) • F ree and total disinfectant residual at time of sample collection

• H eterotrophic plate count (HPC) unless heterotrophic bacteria are the target analy t es, in which case total coliform s should be measured In addition to this information, data on the con centration of potential interferences (e.g., com p eting bacteria, interfering chemicals, etc.) in the water collected for analy s is m ay be necessary

. Known interferences should be discussed in the m e thod and addr essed in the study plan. The final results, as well as bench sheets, log sheets, instrum e nt printouts, and any associated quality control analyses should be submitted as part of Appendix C of the study report (Section 9.10). 

### 7.2 S ample Spiking and “Stressing” Pr ocedures for Bacteriological Methods Depending on the m a trix and the analy t e of interest, it m ay not alway s be necessary to spike samples prior to analy s is. Rangefinding analyses should be perfo rmed to assess the ambient concentration of target organism(s) in the m a trix of interest to determin e whether sample spiking will be useful. Sam p les are collected and dilutions are prepared in order to e numerate the number of organisms in the sample without qualifiers (i.e. less than, greater than, or too numerous to count). In addition, rangefinding 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 m ay exhibit different recovery and precision characteristics than natural flora. NELAC ( http://www.epa.gov/ttn/nelac/standard/5qs-15-0-jhrev-corr.pdf , Reference 10.9) and ATCC ( http://www.atcc.org/SearchCatalogs/faqBacteriology.cfm#Q15 , Reference 10.6) recom me nd that bacterial cultures be transferred monthly and passed no m o re 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.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol 7.2.1 D rinking Water: Spiking and Chlorine-Stressing Drinking water samples should be spiked and stressed w ith chlorine as described in Sections 7.2.1.1 and 7.2.1.2 below. 7.2.1.1 D rinking Water Spiking Because finished drinking water does not typically cont ain the target analy t e(s) of interest, finished drinking water should be spiked. For the evaluation of drinking water samples, a single finished drinking water m a trix is spiked with target organism(s) from other m a trices, 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 typica lly have the advantage of providing organisms m o re variable in quality

. Depending on the study design (Section 6.0), the source of spiking suspensions (i.e., wastewaters) should com e from geographically dispersed sites. To prepare spiked drinking water samples: (1) C ollect at least five liters of each non-chlori nated secondary sewage effluents or polluted surface water to be used as spiking suspensions. (2) P erform rangefinding 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. (3) F or each spiking suspension, sp ike a sufficient volume of drinking water with a sufficient volum e of spiking suspension (based on rangefinding) to obtain 10 3 -10 5 target organisms/100 m L. Please note : A single drinking water, with negligible concentrations of oxidants and reductants (e.g., chlorine and sodium thiosulfate, respectively ) s hould be used for the entire study

. Refrigerate all spiked drinking waters at 1°C - 4°C for use in preliminary chlorination study and com p arability study

. High oxidant or reductant levels in th e drinking water could interfere with organism stressing. 7.2.1.2 P reliminary 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 tem p eratures under conditions similar to those in drinking water treatm e nt facilities. The goal of chlorinating the spiked samples is to sim u late drinking water treatm e nt by reducing the number of organisms in the spiked drinking water samples from 10 3 -10 5 target organisms/100 m L to 1-10 chlorine-stressed target organisms/100 m L fo r m o st probable number m e thods or to 20 - 100 chlorine-stressed target organisms/100 m L for m emb ra ne filtration m e thods (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 disinf ected (e.g., spiked with sewage effluent or polluted source water), the initial concentration of the target organism(s), the chlorine dem a nd of the spiked sample, the type and concentration of chlorinating agent, the exposure time, the sample m i xing , pH, and tem p erature. As a result, a preliminary chlorinati on study should be performed to establish the exposure time necessary to reduce the number organisms in the spiked drinking water samples from 10 3 -10 5 target organisms/100 m L to 1-10 chlorine-stressed target organisms/100mL (or a 2-4 log removal). During this preliminary chlorination exposure time study, the physi cal and biological param e ters in Section 7.1.1 should be carefully m onitored and recorded for each sample.

7-2

### EPA Microbiological Alternate Test Procedure (ATP) Protocol The exposure time is directly dependent upon the initial concentration of the target organism present, the ma trices’ chlorine dem a nd, and the form of chlorine present. For testing, the spiked sample is generally exposed to 2.0-2.5 m g total chlorine/L for over a range of times such as 10, 20, and 30 m i nutes to reduce the density of target organisms from 10 3 -10 5 CFU/100mL to 1-10 CFU/100 m L sample. However, the period of exposure of a sample with a low chlori ne dem a nd m ay be significantly shorter than 20-30 mi nutes. Suggested Preliminary Chlorination Study (1) D eterm i ne and record the total residual and free residual chlorine concentrations using an EPAapproved N, N diethyl-p-phenylenediamine (DPD ) colorim e tric m e thod (e.g., Standard Method 4500-Cl - G) initially, at m i dpoint, and at the end of the exposure time just prior to dechlorination. (2) F or each exposure time, place 2 L of each spiked drinking water sample (from Section 7.2.1.1) in a glass container. (3) A dd 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 achie ve the desired level of chlorinating agent and stir the sample continuously during exposure to chlorination. If the spiked sample has an appreciable chlorine dem a nd (e.g., spiked with a primary effluent or a sewage sample), add dilute sodium hypochlorite solution until a total residua l chlorine level between 2.0 and 2.5 m g /L is ma intained in the absence of free chlorine. If a sample has a low chlorine dem a nd, avoid overstressing or killing the organisms by prolonged exposure to free residual chlorine. The free residual chlorine concentration should not exceed 0.5-1.0 m g /L. (4) S top the chlorine oxidation (dechlorinate) at the end of the exposure period by adding 0.8 m L of a 10% (w/v) sodium thiosulfate solution/L sample. (5) E numerate the target organism density in an aliquot of the spiked, chlorine-stressed, dechlorinated drinking water sample using the appropriate EPA-approved reference m e thod from Table 1-1. (6) F or each exposure time, repeat Steps 3 - 5, above. 7.2.1.3 S uggested Procedure for Chlorination a nd Dilution of Samples for Comparability Study (1) D eterm i ne and record the total residual and free residual chlorine concentrations initially, at mi dpoint, and at the end of the exposure time just prior to dechlorination using an EPAapproved N, N diethyl-p-phenylenediamine (DPD) colorim e tric m e thod (e.g., Standard Method 4500-Cl - G). (2) P lace a sufficient volume of each spiked drinking water in a glass container to perform sufficient repeat analyses at m u ltiple dilutions. (3) I mme diately prior to chlorination, perform e numeration 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.

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol (4) A dd reagent-grade sodium hypochlorite to achieve the same concentration, as in the preliminary chlorination exposure study

. (5) T o reduce the density of target organisms from 10 3 -10 5 CFU/100mL to 1-10 CFU/100 m L, chlorinate each spiked drinking water sampl e for the appropriate time, based upon the preliminary chlorination exposure study

. Stir the sample continuously during the chlorination. (6) S top the chlorine oxidation (dechlorinate) at the end of the exposure period by adding 0.8 m L of a 10% (w/v) sodium thiosulfate solution/L sample. (7) E numerate target organism density in an aliquot of the spiked, chlorine-stressed, dechlorinated drinking water sample using th e appropriate EPA-approved reference m e thod from Table 1-1. (8) R efrigerate the spiked, chlorine-stressed, dechlorinated drinking water samples at 1°C - 4°C for use in com p arability testing. (9) R ead the plates to determine the approxim a te density of the target organisms. Use these results to estimate the appropriate dilution n ecessary to reach the target organism density of 1-10 CFU/100 m L. (10) Evaluate three dilutions of each spiked dr inking water sample. Sam p les 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 m L for m o st probable number me thods or 20-100 target organisms/100 m L fo r m emb rane filtration m e thods. 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. Im me diately conduct the com p arability analyses with each sample using these three dilutions. Please note: For the evaluation of presence/absence m e thods, the data used for com p arison should be from the dilution(s) which produces results closest to an equal number of positive and negative results for the reference m e thod. A 25% to 75% split in res ponses (in either direction) should be sought. For com p arability, the evaluated results for the ATP or new m e thod should be from the same dilution as the reference m e thod. If one of the dilutions does not produce an acceptable split in positive and negative results for the reference m e thod, the applicant should return to the original, spiked sample. 7.2.2 P reparation of Enumerated Spiking Suspension This dilution scheme is adapted from Standard M e thods for the Examination of W a ter and W a stewater , 19th Edition , Section 9020 B (Reference 10.3). This entire process should be performed quickly to avoid loss of viable organisms. There should be approxim a tely 10 10 organisms per slant. Therefore, dilution bottles “A” through “E,” below contain approxim a tely 10 10 , 10 8 , 10 6 , 10 4 , and 10 3 organisms per dilution bottle, respectively

. Depending on the growing cond itions, these numbers m ay vary . As a result, until experience has been gained, m o re dilutions should be filtered to determine the appropriate dilution. Inoculate bacterial culture onto the entire surface of several nutrient agar slants with a slope approxim a tely 6.3 cm long in a 125 × 16 m m screw-cap tube. Incubate for 24 ± 2 hours at 35°C ± 0.5°C.

7-4

### EPA Microbiological Alternate Test Procedure (ATP) Protocol From the slant that has the best growth, prepare ser ial dilutions using four dilution bottles with 99 m L of sterile buffered dilution water (bottles A, B, C, and D) and one dilution bottle containing 90-m L of sterile buffered dilution water (bottle E). Pipette 1 m L 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 pipe tte, being careful not to tear the agar. Pipette the suspension back into dilution bottle “A.” Repeat this procedure a second time to remove any rem a ining growth on the agar slant, without disturbing the agar. Make serial dilutions as follows: (1) S hake bottle “A” vigorously and pipette 1 m L to bottle “B” containing 99 m L buffer (2) S hake bottle “B” vigorously and pipette 1 m L to bottle “C” containing 99 m L buffer (3) S hake bottle “C” vigorously and pipette 1 m L to bottle “D” containing 99 m L buffer (4) S hake bottle “D” vigorously and pipette 10 m L to bottle “E” containing 90 m L buffer; this should result in a final dilution of approxim a tely 10 organisms / m L. If it is more convenient for your laboratory, an acceptable alternative to the dilu tion 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- to 5-m L portions in triplicate from bottles “D” and “E" according to standard m emb rane filtration m e thods to determine the number of CFU in the dilutions. The recom me nded target dilution and spike volume depends on the m e thod and the target anal yt e(s). Typically, dilutions should be stored at 1°C to 5°C and m ay 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 m L of broth m e dia with a single isolated colony and incubate organisms at optimum tem p erature. If possible, shake at 200 RPM during incubation. Take optical density (OD) measurem e nts at 550-600 nm at 30 m i nute intervals for the first 8 hours and record readings. In addition to OD readings at 30 m i nute intervals, using aseptic technique, remove a 0.1-m L portion of the culture and m a ke a series of 1:10 dilutions in sterile buffer. Initially plate 0.1 m L of the 1.0, 10 -1 , and 10 -2 dilutions in duplicate and incubate overnight at optimum tem p erature. Count colonies and record data. As the optical density increases, evaluate serial dilutions to accom m odate the increased numbers of bacteria (i.e., when the optical density exceeds 1.0 then plate 0.1 m L from dilutions 10 -1 , 10 -2 , and 10 -3 ). Based on OD and the CFU/m L results, the OD of fresh cultures (in log-phase growth) can be used to determine the concentration of bacteria in the tub es by a sim p le graphic representation of the com b ined OD and CFU/m L results. Com me rcially available McFarland standards m ay be used to determine the bacterial density instead of actually doing a growth curve within the lab. In or der to determine bacterial densities using McFarland standards, the OD of the standards are co mp ared to the OD of the log phase culture. 7.2.4 C ommercially Available Enumerated Spikes One time use, com me rcially available spiking susp ensions m ay be obtained from a variety of vendors. Prepare spiking suspensions according to m a nufacturer’ s instructions. It should be noted that a different spiking volume than that recom me nded by the m a nufacturer m ay be necessary to achieve the target density

.

7-5

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 7.3 S piking Procedures for Virus Methods 7.3.1 C ell Monolayer Propagation In order to propagate virus stock suspensions cell m onolayers should be propagated. The type of assay cytopathic effect (CPE) or plaque assay (PA), bei ng performed determines the size of the flask and the number of day s the monolayer incubates prior to use for the assay

. Inoculate T75 or T25 flasks with 1 x10 5 cells containing cell specific growth m e dium

. Fl asks for CPE should incubate for 5-7 day s at 37°C ± 0.5°C with 95% relative humidity (and 5% CO 2 concentration, if necessary ). One CPE flask should be stained with cry s tal violet and m i croscopically check ed to ensure a 95% confluent cell monolayer prior to use in the assay

. Flasks used for PA incubate fo r 7-10 day s 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 P ropagation of Virus Stock Suspension For (CPE) analy s is, utilizing the appropriate cell line (e.g., Buffalo green monkey kidney cells; BGMK), inoculate the cell monolayer with virus and incuba te culture flasks at 37°C ± 0.5°C until the entire m onolayer has been destroy e d by virus replication (approxim a tely 72 - 96 hours). Freeze (to approxim a tely -80°C) and thaw the flasks three times, then pool contents of the flasks and spin at 10,000 x g for 30 m i nutes. Filter supernatant using a 0.2μm pore nylon filter to remove any rem a ining cell debris. The filtrate is the virus stock suspension. 7.3.3 T itering of the Virus Stock Suspension Titer the virus stock suspension (from Section 7.3.2) by performing a plaque assay

. Inoculate monolayers with 0.2 m L of serial diluted viral stock suspension. After rocking and rinsing the monolayers, add the agar overlay to the monolayer. Incubate the cultu re flasks for seven day s while reading the number of plaques each day for the entire seven day s. After th e viral stock suspension has been titered, appropriate volumes of the suspension can be used to spike test ma trices to obtain plaque forming units (PFU)/m L. 

### 7.4 S piking Procedures for Cryptosporidium and Giardia Enumerated spiking suspensions are needed fo r initial and ongoing precision and recovery (IPR and OPR) samples (often referred to as positive cont rols) and m a trix spike (MS) samples. Flow cytom e ter–sorted organisms are n ecessary for these spiking suspensions, rather than m a nual techniques. Flow cytom e ter–sorted spikes generally are character ized by lower variability than m a nually enumerated spikes. Spiking suspensions should be prepared using unstained organisms that have not been irradiated, heatfixed or formalin-fixed. Im me diately before sor ting spiking suspensions, initial calibration of the flow cytom e ter should be performed by conducting a series of sequential sorts directly onto m emb ranes or well slides. These initial sorts should be stained and count ed m i croscopically to verify the accuracy of the system
. When sorting the spiking suspensions, ongoi ng 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-cy t ometer-sorted spiking suspensi ons should be used by the expiration date noted on the suspension. Flow-cy t ometer-sorted spiking suspensions containing live organisms should be used within two weeks of the preparation date. Gene ral procedures for preparing flow-cy t ometer-counted spikes for Cryptosporidium and Giardia can be found in EPA Method 1622 and 1623 (April 2001).

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### EPA Microbiological Alternate Test Procedure (ATP) Protocol A potential com me rcial source of flow-sorted Cryptosporidium and Giardia spiking suspensions is: Wisconsin State Laboratory of Hygiene Flow Cytom e try 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 oocy s ts and Giardia intestinalis cysts that have not been treated to reduce viability

. 

### 7.5 A nalysis of Samples
Sam p les 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 r eceive approval for the modification to the study plan. Deviations from the approved study plan should be docum ented in the study report (Section 9.0). Example analy s is schemes are provided in Sections 7.5.1 and 7.5.2 below. Generally, the analy s is schemes will differ depending on the type of organism a nd the type of m e thod. The study plan (Section 4.0) should detail the order in which samples will be analyzed. 7.5.1 S ide-by-Side Comparison Studies Method Blank 1 Sam p le 1 up to Sam p le 20 Method Blank 2 Positive Control Negative Control Method Blank 3 Media Sterility Checks 7.5.2 Q C 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 Sam p le 

### 7.6 V erification of Results The number of positive and negative results to be veri fied in a side-by

- side com p arison study is discussed in Section 6.1.3. Sections 7.6.1 to 7.6.3 below discuss the types of independent standards that m ay be used for the verification of results from bacteriological m e thods, virus m e thods, and Cryptosporidium and Giardia me thods, respectively

.

7-7

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 7.6.1 V erification of Results from Bacteriological Methods 7.6.1.1 B iochemical Tests that May be Used for Verifications Oxidase Citrate Catalase Lysine decarboxylase ONPG Methy l Red and Voges Proskauer (MRVP) Indole T riple sugar iron (TSI) Coagulase Lysine iron agar (LIA) Esculin Urease Sugars (e.g., Trehalose, lactose, m a nnitol, and sorbitol) It is recom me nded that m u ltiple biochem i cal tests be utilized to verify colony identification. The above list of biochem i cal tests is not exhaustive. The choi ce of which biochem i cal tests to use is based on type of organism (i.e., gram stain results) being identifie d/verified. Biochem i cal 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 biochem i cal identification. A description of the bi ochem i cal tests listed above, as well as additional tests is provided in Standard M e thods for the Examination of W a ter and W a stewater, Method 9225. 7.6.1.2 C ommercially Available Biochemical Testing Products Com me rcial biochem i cal test systems incorporate m u ltip le biochem i cal tests to allow for identification to the genus and/or species level, which m ay be difficu lt when using individual biochem i cal tests prepared in house. Com me rcial biochem i cal test systems are available in two formats: systems that depend on the analy s t to ma nually interpret the results (e.g., API strips, BBL cry s tal, and enterotube) and systems that autom a te the interpretation of results (e.g., Vitek and Biolog). 7.6.2 V erification of Results from Virus Methods Viral protocol testing confirm a tions tend to be m e thod-specific, therefore a detailed description of the confirm a tion/verification procedure s hould be included in the study plan and reviewed on a study-specific basis. 7.6.3 V erification of Results from Cryptosporidium and Giardia Methods Verification of results m ay not be necessary for Cryptosporidium and Giardia IFA m e thods beyond the analy s t’s m i croscopic examination of the organism

. If verification is necessary , a detailed description of the confirm a tion/verification procedure should be included in the study plan and reviewed on a study

specific basis.

7-8

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 8.0 R EVIEW OF 

## S TUDY 

## R ESULTS 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 com p liance with the approved study plan 2. Data review 3. Data validation 4. Development of descriptive statistics 5. Statistical assessment of m e thod com p arability Methods that are deemed acceptable will genera lly be recom me nded for approval (Section 8.6). 

### 8.1 A ssessment of Compliance w i th 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. Devia tions from the approved study plan that occur without prior approval from EPA m ay result in the rejection of som e or all study data. 

### 8.2 D ata Review Upon receipt of the applicant’s data, EPA will generally verify that all raw data described in Section 9.10 are present and com p lete. Generally, all calculations used in the m e thod will be verified. This m ay include calculations used for spiking enumeration, preliminary or presum p tive stages of the m e thod, and the determination of the final result. 

### 8.3 D ata Validation After verifying data com p leteness and reviewing of all calculations, EPA will generally verify that all m easurem e nts were performed in acco rdance with the m e thod. This m ay include, but is not limited to, the following: • T emperature logs for incubator/waterbath/refrigerator • M edia preparation records • S ample incubation times • A ssociated QC samples (as described in Section 5.0) • M ethod blanks • P reparation blanks • S terility checks • P ositive and negative controls 

### 8.4 D evelopment of Descriptive Statistics 8.4.1 M ean Recovery To determine if m a trix characteristics effect m et hod performance, mean recoveries should be calculated separately for each m a trix and m e thod. Mean recoveries should also be calculated for each m e thod over all m a trices.

8-1

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 8.4.2 P recision 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 refe rred to as coefficient of variation) is calculated as the standard deviation divided by the mean, expressed as a percent. For the purpose of sum ma rizing the data, both standard deviations and RSDs should be calculated, and the one which is m o st appropriate for assessing com p arability will be used in analyses. Generally, RSDs are m o st appropriate for sum ma rizing precision when variability increases as concentration increases. To give an indication of the effect of m u ltiple ma trices on precision, standard deviations should be calculated separately for each m a trix and m e thod. Standard deviations also should be calculated for each me thod over all m a trices. 8.4.3 False Positive Rates, False Negative Rates, Sensitivity, and Specificity False positive (FP) and false negative (FN) rates of approved and reference m et hods should be evaluated when assessing com p arability

. An independent standa rd (described in Section 7.6) m ay som e times be necessary to confirm positives and negatives of both the ATP or new m e thod and the EPA reference me thod. The confirm a tion m e thods used should be discussed in the validation study plan. Generally, performance of the ATP or new m e thod a nd EPA-approved reference m e thods will be defined in terms of false positive rates and false negative ra tes. 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 sam ples correctly identified as negative, and sensitivity is defined as the percent of positive samples correctly id entified as positive (see equations on the next page). In order to calculate estimates of false positives, false negatives, sensitivity, and specificity for each me thod, 2-by-2 tables for each m a trix, and over all ma trices, should be set up as follows in Table 8-1. Table 8-1. Standard Format for 2-by-2 Tables Independent Standard

+ - Tota l
AT P or New

Method + TP 1 FP 1 TP 1 + FP 1

- FN 1 TN 1 FN 1 + T N 1 Tota l TP 1 + FN 1 FP 1 + T N 1 TP 1 + FP 1 + T N 1 + FN 1

Independent Standard

+ - Tota l EPA A pprov ed Reference

Method + TP 2 FP 2 TP 2 + FP 2

- FN 2 TN 2 FN 2 + T N 2 Tota l TP 2 + FN 2 FP 2 + T N 2 TP 2 + FP 2 + T N 2 + FN 2

8-2

### EPA Microbiological Alternate Test Procedure (ATP) Protocol Estimates of sensitivity, specificity, false positive rates, and false negative rates as percentages for the two me thods should be calculated as follows:

TP i Sensitivity i = * 100 % TP i + FN i
TN i * 100 % TN i + FP i Specificity i =

FP i TN i * 100 % = ( 1 - ) * 100 % = 1 - Specificity i TN i + FP i TN i + FP i False positive rate i =

FN i TP i * 100 % = ( 1 - ) * 100 % = 1 - Sensitivity i TP i + FN i TP i + FN i False negative rate i = Where F = False, N = Negative, T = True and “ i” refers to the specified method (i=1 for new method, i=2 for reference method) 

### 8.5 S tatistical Assessment of Method Comparability 8.5.1 P resence / Absence Methods For presence/absence m e thods, the chi-square test a nd Breslow-Day test will generally be used to com p are the percent of false positives and false ne gatives in the proposed m e thod analyses with the percent of false positives and false negatives in the reference m e thod analyses. Generally, tests for significant differences in false positive and false negative rates between m e thods will be run using the results sum ma rized in Table 8-1 in S ection 8.4. To perform the chi-square and BreslowDay tests, it m ay help to first rearrange the data as follows in Tables 8-2 and 8-3:

8-3

### EPA Microbiological Alternate Test Procedure (ATP) Protocol
Table 8-2. False Negativ e Rate Comparison Method New R eference Tot al

Result True + TP 1 TP 2 TP 1 + T P 2 Fa lse - FN 1 FN 2 FN 1 + FN 2 Tota l TP 1 +FN 1 TP 2 + FN 2 TP 1 + T P 2 + FN 1 + FN 2

Table 8-3. False Positiv e Rate Comparison Method New R eference Tot al

Result Fa lse + FP 1 FP 2 FP 1 + FP 2 True - TN 1 TN 2 TN 1 + T N 2 Tota l FP 1 + T N 1 FP 2 + T N 2 FP 1 + FP 2 + T N 1 + T N 2 Before running the chi-square tests, positives and negatives should be confir me d using the independent standard m e thod. The chi-square test will be used to determine whether the false positive rate and false negative rate have a statistically significant diffe rence between the ATP or new m e thod and the EPAapproved reference m e thod. 8.5.1.1 Assessing Method Differences (Chi-Square Test) In order to assess whether the false positive and false negative rates differ between m e thods, chi-square tests should be run over all m a trices; additional tests in each m a trix also m ay be necessary depending on the presence of m a trix interactions (see Section 8.5.1.2)

. For false negative rates, the chi-square test(s) indicate whether the proportions of negative samples correctly identified as negative by the two m e thods are significantly different, and for false positive rat es, the chi-square test(s) indicate whether the proportions of true positive results correctly iden tified as positive for the two m e thods are significantly different. 8.5.1.2 Assessing Method/Matrix Inte ractions (Breslow-Day Test) In order to assess whether there are false positive and false negative rate differences between m e thods, it is also necessary to establish if there is a m a trix effect on these param e ters. The effect of m a trices on false positive and false negative rate differences between m e thods is assessed using the Breslow-Day test. (Reference 10.7). The Breslow-Day test is used to test whether there is an interaction between m a trix and me thod 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 signi ficant interaction is found between m e thod and m a trix, then the chi-square tests for a difference between m e thods for that attribute should be done separately for each m a trix. Otherwise, separate chi-square tests for each m a trix are not necessary , and a single chisquare test can be run using the data from all m a trices. 8.5.1.3 Method Comparability Conclusions A decision on whether the ATP or new m e thod is co mp arable to the EPA-approved reference m e thod will generally be m a de based on the results of the false positive and false negative rate com p arisons.

8-4

### EPA Microbiological Alternate Test Procedure (ATP) Protocol Generally, the decision on acceptability of the new m e thod should be m a de based on the chi-square test using data from all m a trices. However, if the results of the Breslow-Day test indicate that there is a significant interaction between m e thod and m a trix fo r false positive and/or false negative rates, then further review should be m a de for those m a trices which y i elded a higher false positive and/or false negative rate for the proposed m e thod. If the results of the chi-square tests indicate that the false positive and false negative rates of the ATP or new m e thod are not significantly different from th e EPA reference m e thod, this generally will be interpreted as not having enough evidence to conclude that the performance of the ATP or new m e thod is worse than the EPA-approved reference m e thod. However, if the results of the chi-square test indicate that the false positive and/or false negative rates of the new m e thod are significantly less than that of the EPA-approved reference m e thod, this will generally be interpreted as worse performance and would lead to rejection of the ATP or new m e thod. 8.5.2 Q uantitative Methods 8.5.2.1 T esting for Normality Many of the statistical analyses used to assess di fferences in recovery and precision between m e thods require certain assum p tions about the data be m e t. Because the validity of these assum p tions affect which statistical tests will be used to assess recovery a nd precision differences, testing these assum p tions is a prerequisite as a first step. There are two assum p tions that should be evaluated prior to com p aring the two m e thods statistically

. One assum p tion is that th e variability of the replicates is constant for each ma trix and m e thod. This assum p tion is discussed in Section 8.5.2.2. The other m a in assum p tion that should be m e t is that the data follow a norm a l distribution. An assessment of norm a lity can be done either graphically or with statistical tests, or both. A norm al distribution looks like a bell curve (i.e., sy mme tric around the mean). A graphical assessment can be done using a histogram, stem-and-leaf or Normal probab ility plot. Appropriate statistical tests include the Shapiro-Wilk test, D’Agostino test, and Kolmogorov-Smirnov test. These tests are computation intensive, and it m ay be necessary to use software (e .g., SAS) to run them

. The Shapiro-Wilk test is generally inappropriate for testing the norm a lity assum p tion of a data set with greater than 50 results. For environmental data (especially field data, t hough som e times spiked data as well), the distribution of results will often be positively skewed (i.e., with a few unusually high results). A com m on corrective action of this that can be utilized is the logarithm ic transform a tion, base e (natural log). If the data are positively skewed, this transform a tion should be attem p ted, assum i ng that none of the results are negative or equal to zero. When zero-valued results are presen t in a skewed data set, a com m on approach is to add a small constant to every result prior to transform a ti on. However, the arbitrary choice of this constant will have a large effect on the transform e d data (the log of 1 and the log of 0.1 are very different, for example), and this approach is not recom me nded. Th e use of non-param e tric tests, discussed later in this guidance, are considered to be the appropriate alternatives. If no zero-valued results are present in a positively skew ed data set, then the log-transform e d data should be tested for norm a lity

. If the log-transform e d data do follow a Normal distribution, then all analyses should be done using the log-transform e d results. 8.5.2.2 Eva luating Precision The decision of what statistical test to use to eval uate precision will generally be m a de on the results of the norm a lity test described in Section 8.5.2.1. If th e assum p tion of norm a lity is m e t, then the F-test for differences in variance should be used. If the assum p tion of norm a lity is not m e t, a non-param e tric test

8-5

### EPA Microbiological Alternate Test Procedure (ATP) Protocol such as the Conover Squared-Rank test should be used. The F-test is based on com p arisons of variances (i.e., the squared standard deviations). The C onover Squared-Rank test is based on ranking absolute deviations from the mean. Figure 8-1 provides a sum ma ry of the procedure for evaluating precision. Prior to testing whether there is a precision difference between m e thods, the precision of the different ma trices should be com p ared for each m e thod. If the results of the F-test indicate that variances diffe r significantly by ma trix, or if the results of the Conover Squared-Rank test indicate that absolute deviations from the mean differ significantly by ma trix, then com p arisons of precision between m e thods should be done separately for each m a trix. Otherwise, a single com p arison of m e thod precision can be done using the data from all m a trices. If the results of the F-test indicate that there is not enough evidence to conclude that the variances differ significantly by ma trix, then the pooled within-matrix variance (i.e., the average of the m a trix variances for the given me thod) should be calculated for each m e thod. The F-test should then be used to com p are the pooled within-matrix variance for the two m e thods. If the Conover Squared-Rank test was used and there was not enough evidence to conclude that the mean absolu te deviation differed significantly between m a trices, then the Conover Squared-Rank test should be used to com p are the mean absolute deviations from the overall m e thod mean for the two m e thods.

8-6

### EPA Microbiological Alternate Test Procedure (ATP) Protocol Figure 8-1. Evaluating Precision

Co mpare absolut e devi ati ons fr om met hod mean f or tw o m et hod s us ing Co nover square d rank test. R un t est for

no rmality

Data foll ow Norm al di stri bu ti on Dat a do not fo llow Norm al di stri bu ti on

Comp ar evar iances betw een mat rices for each met h od usi ng F- test.

Are al l mat ric es equal ? Yes No

Pool var i ances over mat ric es for ea ch met hod. Co mpar e pool ed var i ances for tw

o methods us in g t he F- test. Co mpar e va ri an ces b etween met hod s se parately f or each m atrix us ing t he F- test.

Is t he new met hod at l eas t as prec is e f or at le as t 80% of

mat ric es ?

Evaluat e Re covery Yes

Fai l ur e No Is t he new met h od sign ific an tl y less pr ec is e?

No Yes

Evaluat e Re covery Fai l ur e

Is t he new

method si gni fic ant ly less prec ise?

No

Fai l ur e Yes

Evaluat e Rec overy

8-7

### EPA Microbiological Alternate Test Procedure (ATP) Protocol
8.5.2.3 Eva luating Recov e ry Com p arisons of mean recovery of the different m e thods should be done using either an analy s is of variance (ANOVA) model or non-param e tric test such as the Wilcoxon-Mann-Whitney (WMW) test. If the assum p tion of norm a lity is not m e t, or if the precision differs between m e thods or m a trices based on the precision evaluation described in the previous section, the WMW test should be used. If all assum p tions are m e t, the ANOVA model should be used. Figure 8-2 provides a sum ma ry of the procedure for evaluating recovery

. If an ANOVA model is used, m e thod-by-matrix interactions should be tested for significance first using the corresponding ANOVA F-test. If the m e thod-by-matrix interaction is significant, it would mean that the difference between m e thods is not the same for each ma trix. In this case, no conclusion regarding an overall difference between the recovery of m e thods can be m a de. If the interaction is not significant, then the test for a significant difference between m e thods can be run in the ANOVA model, using the F-test for a significant m e thod m a in effect. If the WMW test is used, between-matrix and between-laboratory variability cannot be separated from replicate variability

. Therefore, significant interac tions between m e thod and laboratory or m a trix cannot be tested when the WMW test is used. Instead the WMW model only tests for an overall difference between m e thods. The model can be run separately for each m a trix, but the dim i nished power will limit the value of this approach. It is therefore recom me nded that a single WMW test be run com p aring me thod recoveries, without stratify i ng by or controlling for m a trix type.

8-8

### EPA Microbiological Alternate Test Procedure (ATP) Protocol Figure 8.2 Eva luating Recov e ry

Ev aluate f alse po

sitive an d fals e negat ive ra

tes. Is

recovery si gni fic ant ly lower for t he n ew met hod? Run te st fo r nor mality

Da ta do not foll ow Norm al di stri bu ti on

Fa il ur e Run AN OVA m odel te sting fo r me th od di ffere nc e, controlling fo r matr ix

. No

inte ract io n In teraction

No Yes R un s i ngl e WMW test to com pare m et hod recovery, di sr egarding ma

trices.

Is

recovery signif ic ant ly lo wer fo r th e new met hod ?

Fai l ur e No Ye s

Ev

aluate f alse po

sitive an d fals e negat ive ra

tes. Eval uate f alse pos itive and fals e nega ti ve rate s.

Fai l ur e No Ye s Run AN OVA m odel te sting fo r me th od di ff erence separatel y for

each m atrix. C ount number of mat rices w

here the new method is

equal to or si gn ificantl y bet ter.

Is

reco ve ry signif ic ant ly lower for t he ne w met hod f or at l eas t 20% of al l matricies? Da

ta fo llow Normal

di stri bu ti on

Usi ng ana lysis of va riance (ANOVA), te st fo r met hod /m at rix inte raction.

8-9

### EPA Microbiological Alternate Test Procedure (ATP) Protocol
8.5.2.4 F alse Positiv e Rates, False Negativ e Rates, Sensitivity , and Specificity Com p arisons of false positive rates, false negative ra tes, sensitivity, and specificity should be conducted according to the m e thods described in Sections 8.5.1.1 and 8.5.1.2. 8.5.2.5 M ethod Comparability Conclusions If there is not enough evidence to conclude that th ere is a significant interaction between m e thod and ma trix for recovery, or if there is not enough evidence to conclude that there is a statistically significant difference in precision between m a trices for either me thod, then it can be concluded that any such differences of the m e thod will be consistent. Theref ore, lack of sufficient evidence to conclude a significant difference between m e thods will generally be interpreted as equal or better performance, and mi ght lead to acceptance of the ATP or new m e thod, 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 m e thod, and would generally lead to rejection of the new m e thod. In cases where significant interactions between m a trix and m e thod are found when assessing recovery, or in cases where differences in precision between m a trices for at least one m e thod (and therefore a com p arison of m e thod precision could not be done ove r all m a trices), som e judgment will be necessary in deciding whether the proposed m e thod should be deem ed acceptable. The decision should be based on the attribute (i.e., recovery or precision) for each ma trix. As a general rule, if there was not enough evidence to conclude that the new m e thod was similar or better than the EPA-approved reference m e thod for that attribute for at least 80% of the m a trices u sed in the study, then the new m e thod can generally be recom me nded for approval, pending review of false positive and false negative rates. If there is not enough evidence to conclude that the new m e thod is worse than the EPA-approved reference m e thod for at least 80% of the m a trices fo r both precision and recovery, the false negative rate and false positive rate should next be com p ared, based on the m e thods described in sections 8.5.1.1 and 8.5.1.2. If the results of the chi-square test indicat e that there is not enough evidence to conclude that the false negative rate or false positive rate of the A TP or new m e thod is worse than that of the EPAapproved reference m e thod, this w ill be interpreted as equal or better performance and m i ght lead to acceptance of the new m e thods. However, if the results of the chi-square test indicate that the false negative rate or false positive rate of the new m et hod is significantly greater than that of the EPAapproved reference m e thod, this will generally be interpreted as worse performance for 100% of the ma trices used in the study, and would lead to rejection of the ATP or new m e thod. For example, suppose side-by

- side testing is done in one laboratory for ten different m a trices. For recovery, there is not enough evidence to conclude th at the new m e thod is significantly worse than the EPA-approved reference m e thod for eight of the ten m a trices. For precision, there is not enough evidence to conclude a significant m a trix effect, and ther e is not enough evidence to conclude a significant difference between m e thods. There is not enough eviden ce to conclude the false negative rate or false positive rate of the ATP or new m e thod is worse than the EPA-approved reference m e thod. Under these circumstances, the ATP or new m e thod m ay be reco mme nded for approval. However, if there is not enough evidence to conclude that the new m e thod is not worse than the approved m e thod for only seven of ten m a trices for recovery, then the ATP or new m e thod will not, generally, be recom me nded for approval. 8.5.3 Q C Acceptance Criteria-Based Comparison Studies For m e thods with QC acceptance criteria, necessary calculations and QC criteria are typically provided in the m e thod. Generally, QC criteria will include both recovery and precision specifications. Recovery criteria take the form of a recovery interval for eith er a single result or mean of m u ltiple results. Precision

8-10

### EPA Microbiological Alternate Test Procedure (ATP) Protocol criteria will generally be either a m a xim um standard de viation, RSD, or RPD. Details on calculations that may be necessary for the com p arisons are described below. 8.5.3.1 Recov e ry Recovery results will generally be necessary for data sp iked 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 = * 100 % Spike where Result is the amount recovered from the sample after spiking, and Spike is the estimated amount spiked into the sample. For source water, percent recovery should be calculated as follows: Result - Background %Recovery = * 100 % Spike where Result is the amount recovered fr om the sample after spiking, Spike is the estimated amount spiked into the sample, and Background is the estimated background amount me asured in the sample prior to spiking. For som e QC criteria, it m ay be necessary to calculate the mean of the sample recoveries prior to com p aring the results to the criteria. Generally , this will be specified with that given m e thod. 8.5.3.2 Precision Calculation of an RSD, RPD, or standard devia tion 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 com p ared to the total amount recovered. For r eagent water-spiked data, where there the background count is zero, the calculation can be m a de either using the recovered amount or the percent recovery

. Where the precision criteria is to be based on two results, the precision criterion will generally be an RPD. The equation for an RPD is given below: * Amount 1 - Amount 2 * RPD = * 100 % ( Amount 1 + Amount 2 ) / 2
Where Amount 1 and Amount 2 are the two recovered amounts.

8-11

### EPA Microbiological Alternate Test Procedure (ATP) Protocol Where the precision criterion is to be based on m o re 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 % Mea n Where SD is the standard deviation of all recovered amounts, and Mean is the mean of all recovered amounts. 8.5.3.3 P resence/Absence Criteria QC criteria for presence/absence m e thods m ay be defined as false positive and/or false negative rates, sensitivity and/or specificity, or as a specified proporti on of positive and/or negative results out of a given number of samples. All calculations should be specific to the given situati on and should be defined explicitly therein. 

### 8.6 M ethod Recommendation and Approval Generally, after comp letion 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 recom me ndation for approval/disapproval of the ATP or new m e thod and notify the applicant of the recom me ndation. If the data evaluation demonstrates that the app licant’s m e thod performs at least as well as the EPAapproved reference m e thod, the Director of Analy tical Methods, Attn: ATP Program Coordinator will generally recom me nd 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 com me nt on the proposed m e thod, and (depending on public com me nt) a final rule published in the Federal Register that approves the m e thod. Generally, the regulation development process m ay take one year or m o re. For limited-use ambient water or wastewater applica tions the Regional Adm i nistrator (see Table 2-1 and Appendix E) will generally prepare a recom me ndation of approval/disapproval of the ATP or new m e thod and notify the applicant of the recom me ndation after co mp letion of the technical and statistical reviews.

8-12

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 9.0 S TUDY 

## R EPORT Laboratories or other organizations responsible fo r developing ATPs or ne w m e thods should document the results of the side-by

- side com p arison study or QC acceptance criteria-based com p arison 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 m e thod and m a ke a decision on whether it is com p arable to the reference m e thod. The applicant is responsible for ensuring that all m e thod-specified criteria are m et by the laboratory ( ies) involved in the study and that the study report contains all data from the laboratory ( ies). A copy of all com p arison study data should be m a intained at the participant laboratory ( ies) or other organization responsible for developing the ATP or new m e thod. 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 anal ys is 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 m a de 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: • B ackground • S tudy Objectives and Design • S tudy Implementation • D ata Reporting and Validation • R esults • D ata Analy s is and Discussion • C onclusions • A ppendix A - Method • A ppendix B - Study Plan • A ppendix C - Supporting Data • A ppendix D - Supporting References Details on the information that should be include d in each of these sections are provided below, in Sections 9.1 through 9.11. 

### 9.1 B ackground This section of the study report should describe the ATP or new m e thod that was tested, and identify the organization responsible for deve loping the ATP or new m e thod. The background section of the study report should include the following information: • A me thod sum ma ry • T he organization, m e thod number, and title for the ATP or new m e thod

• T he m e thod number or title and publication numbe r (given in 40 CFR parts 136, 141, and 405 - 503) for the EPA-approved reference m e thod that is be ing used for demonstrating m e thod com p arability (i.e., the reference m e thod) • A description of the nature of the ATP (e.g., altern ate m e dia, alternate concentration technique, etc.)

9-1

### EPA Microbiological Alternate Test Procedure (ATP) Protocol

• T he m a trices, m a trix types, and/or m e dia to which the ATP or new m e thod is believed to be applicable

• T he analy t e(s) measured by the ATP or new m e thod, including corresponding CAS Registry or other identification numbers, when available 

### 9.2 S tudy Objectives and Design This section of the study report should identify th e 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 S tudy Implementation This section of the study report should describe the me thodology and approach undertaken in the study

. This section should include the following information: • T he organization that was responsible for m a naging the study

• T he laboratories, facilities, and other organizati ons that participated in the study; describe how participating laboratories were selected; and explai n the role of each organization involved in the study • T he type of study performed (i.e., side-by

- side or QC acceptance criteria-based com p arison study ) • T he study schedule that was followed

• A brief description of how sample m a trices were chosen, including, for QC acceptance criteria-based com p arison studies, a statement of com p liance w ith recom me ndations for m a trix type selection

• A description of any preliminary testing conducte d prior to the side-by

- side or QC acceptance criteria-based com p arison study (e.g., m e thod validation, physical and chemical assessment of the ma trices, preliminary range-finding analyses) • T he numbers and types of analyses perfo rmed by the participating laboratories • A description of how samples were collected, distributed, and stored • T he source and strain of the organism used for sample spiking

• If spikes were quantified, a description of how es timated true spike values were determined and provide all supporting data

• T he type of water used in the preparation of sample dilutions if not specified by the m e thod (e.g., reagent water, phosphate buffered wate r, phosphate buffered saline, etc.)

• A ny problems encountered with samples, spiking organisms, equipment, etc. and their subsequent resolution

• A ny com m unications with EPA relevant to the study, such as clarification of the study design or approved changes to the study plan • A ny deviations from the study plan and their impact on study performance and/or results 

### 9.4 D ata Reporting and Validation This section of the study report should describe the pr ocedures 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 R esults This section of the study report presents the study r esults. Results m ay be presented in a sum ma ry table that lists the recovery or concentration of each sample, by test m e thod, laboratory , and m a trix.

9-2

### EPA Microbiological Alternate Test Procedure (ATP) Protocol For QC acceptance criteria-based com p arison studies, r esults should indicate the QC test associated with each sample (e.g. IPR, m e thod blank, MS/MSD, unspiked m a trix sample). Raw data and example calculations should be submitted, a nd should be included in an appendix to the study report (see Section 9.10.1). 

### 9.6 D ata Analy s is and Discussion This section of the study report provides a statistical analy s is and discussion of the study results. Recovery, precision, false positive rates, false negativ e rates, specificity, and sensitivity, as appropriate, should be calculated by test m e thod, laboratory , a nd m a trix, and sum ma rized in a tabular format that includes the mean, standard deviation, and relative standard deviation. The discussion should address any discrepancies between the results and com p arability guidelines, or, for QC acceptance criteria-based com p arison studies, any discrepancies between the r esults and the QC acceptance criteria of the EPAapproved reference m e thod. 

### 9.7 C onclusions This section of the study report should describe the conclusions drawn from the study based on the data analy s is discussion. The section should contain a statement(s) regarding achievem e nt of the study objective(s). 

### 9.8 A ppendix A - Method
The ATP or new m e thod, prepared in accordance w ith EPA' s Guidelines and Format document (Section 3.0 and Reference 10.14), should be appended to the study report. 

### 9.9 A ppendix 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 accom p anied by raw data, quality control information, and example calculations that support the sum ma ry results presented in the report. 9.10.1 R aw Data Appendix C of the study report should include sufficien t 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 m et hod to the final result reported. The raw data are, generally, m e thod-specific and m ay include but are not limited to the following: • S ample numbers or other identifiers used by the laboratory • S ample collection dates and times

• V erification that m e thod-specified QC procedures were m e t for test samples and all associated QC samples • A nalysis dates and times for all steps in the m e thod • S ample volum e • A ny m easurem e nts of ancillary param e ters (i.e., tem p erature, pH, turbidity, percent solids, etc.) • R esults for all intermediate steps in the m e thod • P reliminary data steps to determine the final result • F inal result
9-3

### EPA Microbiological Alternate Test Procedure (ATP) Protocol

• If appropriate, quantitation reports, data system outputs, and other data to link the raw data to the results reported

• L aboratory bench sheets and copies of all per tinent logbook pages for all sample preparation and cleanup steps, and for all other parts of the determination • T emperature logs for waterbaths, incubators, refrigerators, etc. • M edia preparation information • If appropriate, direct instrum e nt 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, m a trix spikes and duplicates, and other QC analyses specified in the EPA-approved reference m e thod. Data should be organized so that a m i crobiologist can clearly understand how the analyses were performed. The names, titles, addr esses, and telephone numbers of the analy s ts who performed the analyses and of the quality assurance offi cer who verified the analyses should be provided. For instrum e nts involving data systems, raw data on ma gnetic tape or disk s hould be m a de available on request. 9.10.2 E lectronic 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 to create a database of study results. EPA anticipates that this database will facilitate autom a ted review and statisti cal analy s is of study results. The information included in electronic format m ay include: laboratory , analy s t, m e thod, sample type, sample number, date and time of analy s is, volume analyzed, replicate numbe r, raw data, and calculated results. The applicant should discuss an appropriate electronic form at with EPA prior to data submission. 9.10.3 E xample Calculations Generally, the study report should provide example cal culations 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 analy t es and undetected analy t es. If the laboratory or the m e thod employs a standardized reporting level for undetected analy t es, this should be ma de clear in the example, as should adjustm e nts for sample volume, etc. 

### 9.11 Appendix D - Supporting References Hard copies of all references and supporting doc umentation for the ATP or new m e thod should be attached to the study report as an appendix. The lis t of references m ay contain links to web sites, or documents available on-line. However, a hard c opy should be submitted with the final study report.

9-4

### EPA Microbiological Alternate Test Procedure (ATP) Protocol 

## S ECTION 

## 10.0 R EFERENCES
10.1 AOAC. 1999. Qualitative and Quantitative Microbi ology Guidelines for Methods Validation, Journal of AOAC International , Vol. 82, No. 2.
10.2 APHA. 1998. Standard M e thods for the Examination of W a ter and W a stewater . 20 th Edition. American Public Health Association. 1015 15 th Street, NW, Washington, DC 20005.
10.3 APHA. 1995. Standard M e thods for the Examination of W a ter and W a stewater . 19 th Edition. American Public Health Association. 1015 15 th Street, NW, Washington, DC 20005.
10.4 APHA. 1992. Standard M e thods for the Examination of W a ter and W a stewater . 18 th Edition. American Public Health Association. 1015 15 th Street, NW, Washington, DC 20005.
10.5 ASTM. 1999. D4855-91: Standard Practice for Co mp aring Test Methods, ASTM Standards on Precision and Bias for Various Applications. 1999 Annual Book of ASTM Standards: Water and Environmental Technology, Volum e 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, 2 nd ed., John Wiley & Sons, New York, NY.
10.8 ISO. 2001. CD17994. Water Quality - Criteria fo r the Establishment of Equivalence Between Microbiological Methods, Fina l Version, June 15, 2001.
10.9 National Environmental Laboratory Accreditation Conference. 2001. National Environmental Laboratory Accreditation Conference: Quality Sy stems. Approved May 25, 2001, effective July 1, 2003 unless otherwise noted. Appendix D - Essential Quality Control Requirem e nts, section D.3, pp D-15 to D-16.
10.10 Title 40, Code of Federal Regulati ons, Sections 136.4, 136.5, and 141.27.
10.11 USEPA. 2001. EPA Requirem e nts for Quality Managem e nt 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. 1997. Manual for the Certification of La boratories Analy z ing Drinking Water: Criteria and Procedures Quality Assurance, Fourth Edition. USEPA Office of Ground Water and Drinking Water. EPA-815-B-97-001.
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.

10-1

### EPA Microbiological Alternate Test Procedure (ATP) Protocol
10.15 USEPA. 1995a. Presence/Absence Mem b rane Filte r Methods for Finished Waters, USEPA Protocol for Alternate Test Procedures for Colif orm Bacteria in Com p liance with Drinking Water Regulations, Version 1.2, December 1995. U SEPA Office of Research and Development, Cincinnati, OH.
10.16 USEPA. 1995b. Quantitative Mem b rane Filter Met hods, USEPA Protocol for Alternate Test Procedures for Coliform Bacteria in Com p lian ce 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 Colif orm Bacteria in Com p liance with Drinking Water Regulations, Version 1.2, December 1995. U SEPA Office of Research and Development. Cincinnati, OH.
10.18 USEPA 1989. Mem o randum: Analytical Methods for Com p liance and Limited Alternate Test Procedures Approvals. December 27, 1989.
10.19 USEPA. 1979. Handbook for Analytical Quality Contro l in Water and Wastewater Laboratories

. EPA-600/4-79-019. Environmental Monitoring and Support Laboratory , Cincinnati, OH. March 1979.

10-2

## A PPENDIX 

## A G LOSSARY

## A PPENDIX 

## A: G LOSSARY
40 CFR part 136—Title 40, part 136 of the Code of Federal Regulations . This part specifies approved test procedures for the analy s is 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 National Primary Drinking Water Regulati ons pursuant to the Safe Drinking Water Act; Subpart C of 40 CFR part 141 lists analytical m e thods required for monitoring under the Act. 95% confidence interval— A statistical level indicating a 95% probability that the param e ter variable is enclosed within the given data interval. 

## A Acceptable version— An acceptable version is a m e thod that is either identical to the approved



## B

C me thod or exercises the flexibility explicitly allowed in the m e thod. 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 analy s is. Aliquot— A representative portion of a sample. Ambient water— Ambient water refers to any fresh, m a rine, or estuarine surface water used for recreation; propagation of fish, shellfish, or w ildlife; 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 decompos ition of the variance of the response variable. Analyte— The target organism or class of organisms that are measured by the m e thod. Analyte of concern— An analy t e designated by EPA to adversely affect or have the potential to adversely affect human health, the environment, aesthetics, or the senses. Analy t es of concern are listed in approved m e thods. Approved method— A testing procedure (analytical m e thod) 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 measurem e nt process that deprives the result of representativeness; i.e., the expected sample measu rem e nt 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.

Calibration— The process of establishing the relationship between the concentration or amount of m a terial introduced into an instrum e nt or measurem e nt process and the output signal. Calibration verification— Means of establishing that the instrum e nt performance rem a ins within pre-established limits.

## D Code of Federal Regulations — A codification of the general and permanent rules published in the Federal Register by the Executive departm e nts and agencies of the Federal Governm e nt. Comparability test— See side-by

- side com p arison. Confidence interval— The numerical interval constructed around a point estimated of a population param e ter, com b ined with a probability statement (the confidence coefficient) linking it to the population’s true param e ter value. If th e same confidence interval construction technique and assum p tions are used to calculate future intervals, they will include the unknown population param e ter with the same specified probability. (EMMC) Confirmed counts— Organism counts that have been verified to ensure proper identification. Conover Squared-Rank test— A nonparam e tric test for equality of variability, based on the joint squared ranks of deviations from the means. (SPRENT) Contract laboratory— Private, academic, or com me rcial 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 norm al or log-norm a l 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-m a ker is willing to accept in results or decisions derived from environmental data. Data quality objectives provi de the statistical fram e work for planning and ma naging environmental data ope rations consistent with the data user' s needs. (EMMC) Determinative technique— The physical and/or chemical process by which measurem e nt of the identity and concentration of an analy t e is m a de. Differential medium— A solid culture m e dium that m a kes 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 becom e contaminated by the introduction of a fo reign m i croorganism through poor technique. Discharge —Generally, any spilling, leaking, pumping, pouring, emitting, empty i ng or dumping (40 CFR 109.2; 110.1; 116.3); also, see "discharge of a pollutant" (40 CFR 122.2); the m e dium that is spilled, leaked, pumped, poured, emitted, emptied, or dumped. Discharge of pollutant— Any addition of any pollutant or com b ination 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) Duplicate— A second sample collected from the same sampling point at the same time the original sample is collected and analyzed exac tly like the original sample. Duplicate samples can be used as a measure of sample variability

. Effluent— A m e dium that flows out of a point source, e.g., the discharge from a sewage treatm e nt plant. 

## E

## F

G

H

I Enrichment— Using a culture m e dium to enhance growth of the target organism prior to isolation of that organism

. Explicit flexibility— Modifications that are explicitly allowed in an approved m e thod.
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 Analy s is of Variance, a test for the equality of factor level means (such as for different m e thods) or for the presence of an inte raction between two factors (such as m e thod and ma trix). (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 m u ltiple water supplies, discharges, waste streams, or other environmental m e dia that are subject to com p liance 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 st ream, all of which are subject to com p liance m onitoring. False negative— A target organism incorrectly identified as a non-target organism or not identified at all using the m e thod of interest. False positive— A non-target organism incorrectly identified as the target organism using the me thod of interest. False negative error rate— The proportion of target organisms incorrectly identified as a nontarget organism or not identified at all using the me thod of interest, equal to (1 - Sensitivity). In statistical testing, the rate at which one fal sely accepts a statistical hypothesis (such as that a difference between m e thods does not exist) based on a statistical test, when the hypothesis is actually false. Abbreviated as $ , 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 m e thod 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 me thods does not exist) based on a statistical test, when the hypothesis is actually true. Abbreviated as " , 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 docum ents. Docum e nts published in the Federal Register ma ke changes to the CFR to k eep the CFR current. (OFR) Guidelines and Format— The document titled Guidelines and Format for M e thods to be Proposed at 40 CFR Parts 136 and 141 ; available from the National Technical Information Service (NTIS), U.S. Departm e nt of Com me rce, Springfield, Virginia, 22161 (703-487-4600) as NTIS publication PB96-210448. Histogram — A bar diagram of the distribution of a set of analytical results. The range of values is categorized into sets of subintervals, or bi ns, and the number of results within each bin are display e d 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.

## J K
L

M Initial precision and recovery— The analy s is of a m i nim um of four spiked reagent water samples under the same conditions as will be used for analy s is of environmental samples. The IPR is used to demonstrate that a laboratory is able to produce reliable results with the m e thod prior to analy s is of environmental samples. Interaction— The situation where the effect of one variable (such as m e thod type) on a dependent variable (such as recovery) is affected by the value of a third variable (such as m a trix). Interference— A positive or negative effect on a measurem e nt caused by a substance other than the one being investigated. (QAD) Interlaboratory— Occurring in m u ltiple laboratories. Intralaboratory— Occurring within a single laboratory

.
Kolmogorov-Smirnov test— A statistical test for determining whether a given set of results follow a norm a l distribution, or any other specified distribution. (GILBERT)
Limited use— Use of a m e thod by a single regulated entity or laboratory for analy s is of one or mo re m a trix types. Log-normal— A distribution of a random variable X such that the natural logarithm of X is norm a lly distributed. Log-phase— Bacterial growth phase in which the l ogarithm of the bacterial biomass increases linearly with time.
Main effect— Situation where a variable (such as m e thod type) has a consistent effect on a dependent variable (such as recovery). Matrix— The component or substrate that contains the analy t es of interest. Matrix effect— Variability in the analytical performance of a m e thod that can be attributed to the type of sample analyzed. Matrix spike— A sample prepared by adding a known mass of target analy t e to a specified amount of a sample m a trix for which an independent estimate of target analy t e concentration is available. A m a trix spike is used, for example, to determine the effect of the m a trix on a me thod’s recovery efficiency. (QAMS) Matrix spike duplicate— A replicate of the m a trix spike to test precision. The MS/MSD are used in com b ination to test the precision of an analy s is. (QAD) Matrix type— A sample m e dium with com m on characteristics across a given industrial category or subcategory

. For example, C-stage efflue nts from chlorine bleach mills, effluent from the continuous casting subcategory of the iron and st eel industrial category , POTW sludge, and inprocess streams in the Atlantic and Gulf Coast Hand-shucked Oyster Processing subcategory are each a m a trix type. For the purposes of this initiative all drinking waters constitute a single ma trix type. May— This action, activity, or procedural st ep is neither required nor prohibited. May not— This action, activity, or procedural step is prohibited.

## N Measurement quality— Critical level which, if exceeded, is considered to append objective additional, and possibly unacceptable, measure me nt uncertainty to the corresponding data. Method— A body of procedures and techniques for performing a task (e.g. sampling, characterization, quantitation) systematically presented in the order in which they are to be executed. (QAD) Method blank— An aliquot of reagent water or designated m a trix that is treated exactly as a sample, including exposure to all glassware, equi pment, solvents, and procedures that are used with samples. The m e thod blank is used to de termine if analy t es or interferences are present in the laboratory environment, the reagents, or the apparatus. Method-defined analyte— An analy t e without a specific, known composition where the analytical result depends totally on the measurem e nt procedure. Method modification— A change m a de to an approved m e thod. Method validation— A process by which a laboratory or vendor establishes the performance of a new m e thod or substantiates the performance of a m e thod modification. Methods and Criteria— The document titled: Analysis of Pollutants in Municipal W a ter and Industrial Wastewater: Test Procedures and Quality Control Acceptance Criteria ; available from the National Technical Information Service (NTIS), U.S. Departm e nt of Com me rce, Springfield, Virginia, 22161 (703-487-4600) as NTIS publi cation PB96-210463, 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 m e thod that has been reviewed by EPA and has been determined to be technically equivalent to a me thod approved for use in com p liance monitoring. A m i nor modification employs the same chemistry and/or biological principles as the approved me thod to determine the presence/absence or to quan tify the amount of the target organism in a sample. Supporting data m ay be necessary to demonstrate that a m i nor modification will y i eld results equivalent to those obtained using the approved m e thod but does not require approval as an alternate test procedure through proposal and promulgation in the Federal Register. Modified method— An approved m e thod that has been modified to change a front-end technique. EPA will judge a modified m et hod to be: 1) an acceptable version or m i nor m odification of a previously promulgated m et hod, which does not require approval as an ATP or 2) a significantly different m e thod which re quires an application for an ATP approval.

Nationwide use— Use of a m e thod by all regulated entities and laboratories for analy s is of one or m o re m a trix 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 confirm a tion test results are appropriate.

## O

P New method— A m e thod that employs a determinative technique for an analy t e of concern that differs from determinative techniques employed fo r that analy t e in m e thods previously approved at 40 CFR part 136 or 141. Nonparametric— A type of statistical analy s is for which no assum p tions about the underly i ng distribution of the data are necessary

. Non-selective media— An enrichm e nt m e dia that allows m o st bacteria to grow. Normal probability plot— A graphical depiction of the distribution of a set of analytical results. A norm a l probability plot is a scatterplot depicti ng the observed results com p ared to the expected results based on a norm a l distribution. If the observed data follow a norm a l 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 analy s is of ODC samples (positive control/positive control duplicate). Ongoing precision and recovery— A reagent water sample m e thod blank spiked with known quantities of analy t es. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory rem a in w ithin the limits specified within the m e thod for precision and recovery

. Other approved methods— Promulgated m e thods that are not designated as a reference m e thod, but continue to carry the same regulatory status. Physical phase— The physical phase of a sample m a trix (e.g., air, water, soil). Positive control— A target organism that is analyzed to ensure that the laboratory is performing the m e thod acceptably and that the m e dia is providing appropriate results. Power— The probability that a statistical test will conclude that a difference (for example, between m e thods) exists, when a difference truly does exist. Equal to 1 - $

. Precision— The degree to which a set of observations or measurem e nts of the same property , usually obtained under similar conditions, confor m to themselves; a data quality indicator. Precision is usually expressed as standard deviati on, variance, or range, in either absolute or relative terms. The precision obtainable from an environmental measurem e nt m e thod m ay be estimated from replicate analyses of subsamples taken from the same (homogenous) sample. Generally speaking, the m o re carefully one executes the various steps of a m e thod and controls the variables affecting the m e thod’s capability, the m o re precise will be the results. The use of a nonhomogeneous sample will compound the precision estimate with the sample variability

. Preparation— Processing performed on a sample prior to analy s is, e.g. extraction, concentration, cleanup, etc. Presumptive counts— Num b ers of organisms based on results that have not been confirm e d or verified. Procedures— A set of systematic instructions for performing an activity. (QAD)

## Q

R Promulgated m e thod— A m e thod 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 me thod). 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 day s out of the year. Such term includes (1) any collection, treatm e nt, 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 pretreatm e nt storage facilities not under such control which are u sed primarily in connection with such system

. A public water system is either a “com m unity water system” or a “noncom m unity water system.” Quality assurance — An integrated system of activities involving planning, quality control, quality assessment, reporting, and quality improvem e nt 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 , ad equate, dependable, and economical. (QAD) QC acceptance criteria— Performance specifications develope d from validation data and used to control the limits within which an analytical m e thod is operated. QC acceptance criteria-based comparison study— A study performed to evaluate the performance of a modified m e thod against the quality control acceptance criteria of a reference me thod. Range finding— Preliminary analyses conducted to assess the ambient concentration of the target organism in a m a trix to be used in a study or preliminary analyses of spiked samples involving few, if any , replicates, to assess m et hod performance to identify the spike dose to be used in a study

. Raw data— Data that have not been processed. Reagent water— Water conform i ng to Specification D 1193, Annual Book of ASTM Standards, or specifications in Standard Methods 9020B.4.d. Recovery— The total amount of the analy t e found in the sample divided by the amount of the analy t e added into the sample as a spike. Reference method— A m e thod that serves as a standard against which m e thod modifications can be com p ared. Regulated entity— Permittees, PWSs, POTWs, and other entities responsible for com p liance with provisions of the CWA or SDWA.

## S Relative percent difference (RPD)— An estimate of the variability of two numbers expressed in relative terms. Calculated as the absolute valu e of the difference of the two numbers, divided by their mean: A B

− RP D = 1 * 100% 2 *( A B ) + Equal to the relative standard deviation of the two numbers m u ltiplied by the square root of 2. Relative standard deviation (RSD)— The standard deviation expressed as a percentage of the m ean (100 F /X); i.e., the coefficient of variation. Replicate— Multiple samples collected from the sam e 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 analy s is. Sam p le preparation techniques do not include conditions and/or procedures for the collection, preservation, shipm e nt, and storage of the sample. Sensitivity— In presence/absence testing, sensitivity is th e proportion of target organisms in the sample that were correctly detected by the m e thod of interest. Shapiro-Wilk test— A statistical test for determining whether a given set of results follow a norm a l or log-norm a l distribution. Best used for datasets with at m o st 50 results. (GILBERT) Side-by-side comparison— Parallel testing of a new or modified m e thod and a reference m e thod to determine whether the performance of the ne w or modified m e thod is acceptable com p ared to the reference m e thod. 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 m e thod of interest. Spike— The process of adding a known amount of target analy t e to a sample; used to determine the recovery efficiency of the m e thod. (QAD) Spiking suspension— Diluted stock suspension containing the organism(s) of interest at a concentration appropriate for spiking samples. Standard deviation ( F )— 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 arithm e tic mean of the set, divided by one less than the number of values in the set. For a total of n numbers:


## SD = n X ean i i n



## − −

= 

# ∑ 1 1 2 1 

## ( M )

Statistical power— See power.

## T U V Stem-and-leaf plot— A graphical depiction of a set of analytical results, that convey s information about the shape of the distribution while retaining the numerical information. The stem-and-leaf plot separates the digits of the va lues as leaves (the last digit of the values) and stems (the rem a ining 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, com me nt, and approval prior to conducting the side-by

- side or QC acceptance criteria-based m e thod com p arability study

. This process protects the applicant by providing written approval of the study design before resources are spent to conduct the study

. Data from st udies conducted without EPA review and approval may not adequately address the applicant’s study objectives. A detailed procedure for the ATP or new m e thod should be included as an attachm e nt 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 com me nts are incorporated in to 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 m e thods documenting the results of the side-by

- side or QC acceptance criteria-based m e thod com p arability 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 me thod and m a ke a decision on whether it is com p ar able or superior to the reference m e thod. The approved study report should also identify and discuss any deviations from the study plan that were m a de in implementing the study, a nd 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 m e thod is designed to detect.

Validate— Reliably assess the performance (bias and precision) of a m e thod in a reference ma trix (such as reagent water) and the m a trix in which the validated m e thod will be used (such as drinking water, surface water, or municipal wastewater effluent). Validation, single-laboratory— Assessment of m e thod performance (see “validate”) in one laboratory

. Validation, interlaboratory— Assessment of m e thod performance (see “validate”) at m u ltiple 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 arithm e tic 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 nonparam e tric analy s is used to determine whether the m e dians from two levels of a given factor (such as m e thod) differ from each other, based on the ranks of all results. (SPRENT) 

## W

X Y Z

The above definitions are referen ced to the following organizations: ASTM ASTM D 4855-91. Standard Practice for Com p aring Test Methods. BERRY Berry, D. A. and B. Lindgren. Statistics Theory and Methods. Wardsworth, Belmonst, CA, 1990. EMMC Environmental Monitoring Managem e nt Council GILBERT Gilbert, R. O. Statistical Methods for Environmental Pollution Monitoring. Van Nostrand Reinhold, New York, 1987. NELAC QS National Environmental Laboratory Accreditation Conference, Quality System s 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 Analy s is, Second Edition. Wadsworth, Belmont, CA, 1995. SPRENT Sprent, P. Applied Nonparam e tric Sta tistical Methods, Second Edition. Chapm an & Hall, London, 1993.

## A PPENDIX 

## B ATP A PPLICATION 

## F ORMEPA Office of Water Alternate Test Procedure or New Method Preliminary Application Form for Microbiological Analy t es Applicant Name and Address: EPA Use Only ATP Case Number:

Date Application Submitted: Type of Application: Circle appropriate application type Alternate Test Procedure New Method
Method Number Title of Method Rev i sion Date EPA-Approved Reference Method: Analy t e(s): Applicable Matrices: Circle all that apply Ambient Water Drinking Water Wastewater
Lev e l of Use: Limited Use N ationwide Study Design: Circle appropriate study design 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 G Justification for ATP G Alternate Test Procedure or New Method (in standard EPA format) G Method Comparison Table G Study Plan G Other: Submit Application and Attachments in Triplicate BiosolidsEPA Office of Water Alternate Test Procedure or New Method Final Application Form for Microbiological Analy t es
Applicant Name and Address: EPA Use Only ATP Case Number:
Date Application Submitted: Type of Application: Circle appropriate application type Alternate Test Procedure
Method Number Title of Method Rev i sion Date EPA-Approved Reference Method: Analy t e(s): Applicable Matrices: Circle all that apply Ambient Water Drinking Water Wastewater
Lev e l of Use: Limited Use N ationwide Study Design: Circle appropriate study design 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 G Justification for ATP New Method

Biosolids

G Alternate Test Procedure or New Method (in standard EPA format) G Method Comparison Table G Study Plan G Study Report G Other: Submit Application and Attachments in Triplicate

## A PPENDIX 

## C A PPLICATION 

## I NVENTORY 

## F ORM1. Completed applic at ion form. Includes the name and address of the applicant; the date of submissi on of the application; the method number, title, and revision date; the EPA-approved reference method; the analy t e(s) for w h ich the ATP or new method is proposed; the type of application; applicable matrices; study design; level of use; NP DES permit information, if applicable; and the attachments submitted w i th the application. Section 2.2 Appendix C 

## G

2. Just ific at ion for A T P or ne w method. Brief justification for w hy the ATP or new method is being proposed. Section 2.3 

## G
3. Method in EPA format. Scope and application Section 3.1 

## G Summary of method Section 3.2 

## G
Definitions of method Section 3.3 

## G
Interferences Section 3.4 

## G Safety Section 3.5 

## G Equipment and supplies S ection 3.6 

## G Reagents and standards Section 3.7 

## G Sample collection, preserva tion, and storage Section 3.8 

## G Quality control Section 3.9 

## G
Calibration and standardization Section 3.10 

## G
Procedure Section 3.11 

## G Data analy s is and calculations Section 3.12 

## G Method performance Section 3.13 

## G Pollution prevention Section 3.14 

## G
Wa ste management Section 3.15 

## G
References Section 3.16 

## G
T ables, diagrams, flowcharts , and validation data S ection 3.17 

## G
4. Method c o mpa r ison table
. A two-column table comparing the propos ed ATP or new method w i th the EPA-approved reference method. T h is table should include the number and title of each method, the latest revision date of the ATP or new me thod, and a detailed discussion of each of the method sections listed in Sect ion 3.0. Each topic should be discussed in a separate row of the table and the applicant s hould highlight any differences between the ATP or new method and the EPA-approved reference method. Section 2.5 

## G5. Study pla n. Background Section 4.1 

## G Objectives Section 4.2 

## G Study design Section 4.3 

## G
Coordination Section 4.4 

## G Data reporting Section 4.5 

## G
6. Study report. Background Section 9.1 

## G Study objectives and design S ection 9.2 

## G Study implementation Section 9.3 

## G Data reporting and validation S ection 9.4 

## G
Results Section 9.5 

## G Data analy s is and discussion Section 9.6 

## G
Conclusions Section 9.7 

## G Appendix A: Method Section 9.8 

## G Appendix B: Approved st udy plan Section 9.9 

## G Appendix C: Supporting data S ection 9.10 

## G Appendix D: Supporting re ferences Section 9.11 

## G

## A PPENDIX 

## D D ATA 

## E LEMENTS AND 

## E XAMPLE 

## B ENCH 

## S HEETS

### Data Elements The data elements listed below should be reporte d on the bench sheets or in the lab notebook for each me thod, as applicable. EPA will review the informati on during the data validation process to ensure the me thod-specific QC measures are m e t, as agreed to in the approved study plan. • L aboratory nam e • M ethod number • M edia • P rocedure • M atr ix • S ample collection date/tim e • D ates and times for all m e thod steps associated with holding times or incubation times • A nalyst initials for each processing step in the m e thod • P resum p tive results for all applicable m e dia • C onfirm e d/com p leted results for all applicable m e dia • A ll measured volumes • D ilution information • F inal result per units of measurem e nt 

### Example Benchsheets Example bench sheets for the following EPA-approved re ference m e thods are included in this appendix: • Aeromonas (USEPA Method 1605) • Cryptosporidium (USEPA Methods 1622 and 1623) • E. coli (SM 9221F, SM 9222G) • E nterococci (SM 9230C) • F ecal coliform s (SM 9221E, 9222D) • F ecal streptococcus (SM 9230B, SM 9230C) • Giardia (USEPA Method 1623) • T otal coliform s (SM 9221B, SM 9222B)

Note: Additional example bench sheets or electronic copi es of the attached bench sheets are available upon request.Multiple-Tube Fermentation: Total Coliform, Fecal Coliform,E. coli(SM 9221B, SM 9221E, SM 9221F)Laboratory:Sample Collection Time:Sample Collection Date:Sampling Point:AnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mLAnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mLAnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mL24 hr fecal read(From 24 hr LTB)24 hr fecal read(From 48 hr LTB)24 hrE. coliread(From 24 hr LTB)LTB start24 hrE. coliread(From 48 hr LTB)LTB 24 hr readFinal tube combination:Total coliforms/100 mL:Fecal tube combination:Fecal/100 mL:E. colitube combination:E. coli/100 mL:AnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mLAnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mLBGB 48 hr read (from 48 hr LTB)24 hr fecal read(From 24 hr LTB)24 hr fecal read(From 48 hr LTB)24 hrE. coliread(From 24 hr LTB)AnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mL24 hrE. coliread(From 48 hr LTB)Final tube combination:Total coliforms/100 mL:Fecal tube combination:Fecal/100 mL:E. colitube combination:E. coli/100 mL:AnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mLAnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mLAnalystInitials Read Temp10mL1.0mL0.1mL0.01mL0.001mL24 hr fecal read(From 24 hr LTB)24 hr fecal read(From 48 hr LTB)24 hrE. coliread(From 24 hr LTB)24 hrE. coliread(From 48 hr LTB)Final tube combination:Total coliforms/100 mL:Fecal tube combination:Fecal/100 mL:E. colitube combination:E. coli/100 mL:Comments:24 hr / 48 hrBGB read(From 48 hr LTB)LTB: Replicate 3BGB: Replicate 3LTB: Replicate 224 hr / 48 hrLTB readBGB: Replicate 224 hr / 48 hrBGB read(From 24 hr LTB)24 hr / 48 hrBGB read(From 24 hr LTB)LTB: Replicate 1BGB: Replicate 1EC-MUG: Replicate 124 hr / 48 hrLTB read24 hr / 48 hrBGB read(From 48 hr LTB)Please enter date and time for the following:LTB 48 hr read,BGB 24 hr read (from 24 hr LTB),ECMUG 24 hr read (from 24 hr LTB)EC-MUG: Replicate 324 hr / 48 hrBGB read(From 24 hr LTB)EC-MUG: Replicate 2BGB 48 hr read (from 24 hr LTB),BGB 24 hr read (from 48 hr LTB),ECMUG 24 hr read (from 48 hr LTB)24 hr / 48 hrBGB read(From 48 hr LTB)24 hr / 48 hrLTB readMembrane Filtration: mEndo/NA-MUG (SM 9222B/SM 9222G) Laboratory: Sample collection time: Sample collection date: Sampling point:

mEndo incubation start temperature ( o C): mEndo incubation start date/time: mEndo incubation end temperature ( o C): mEndo incubation end date/time:

NA-MUG incubation start temperature ( o C): NA-MUG incubation start date/time: NA-MUG incubation end temperature ( o C): NA-MUG incubation end date/time:
Replicate number 1

Analyst initials Sample volume filtered No. colonies per filter Total Coliforms per 100 mL E. coli per 100 mL

100 mL 10.0 mL 1.0 mL 0.1 mL

Replicate number 2

Analyst initials Sample volume filtered No. colonies per filter Total Coliforms per 100 mL E. coli per 100 mL

100 mL 10.0 mL 1.0 mL 0.1 mL

Replicate number 3

Analyst initials Sample volume filtered No. colonies per filter Total Coliforms per 100 mL E. coli per 100 mL

100 mL 10.0 mL 1.0 mL 0.1 mL E. coli Total coliforms E. coli
No. colonies

per filter Total coliforms

E. coli No. colonies

per filter

No. colonies

per filter Total coliformsMembrane Filtration: mFC/NA-MUG (SM 9222D/SM 9222G) Laboratory: Sample collection time: Sample collection date: Sampling point:

mFC incubation start temperature ( o C): mFC incubation start date/time: mFC incubation end temperature ( o C): mFC incubation end date/time:
NA-MUG incubation start temperature ( o C): NA-MUG incubation start date/time: NA-MUG incubation end temperature ( o C): NA-MUG incubation end date/time:
Replicate number 1

Analyst initials Sample volume filtered No. colonies per filter Fecal coliforms per 100 mL E. coli per 100 mL

100 mL 10.0 mL 1.0 mL 0.1 mL

Replicate number 2

Analyst initials Sample volume filtered No. colonies per filter Fecal coliforms per 100 mL E. coli per 100 mL

100 mL 10.0 mL 1.0 mL 0.1 mL

Replicate number 3

Analyst initials Sample volume filtered No. colonies per filter Fecal coliforms per 100 mL E. coli per 100 mL

100 mL 10.0 mL 1.0 mL 0.1 mL E. coli No. colonies

per filter

No. colonies

per filter Fecal coliforms E. coli
No. colonies

per filter Fecal coliforms

E. coli Fecal coliformsE. coli Membrane Filtration: mTEC (EPA 1103.1, SM 9213D) Laboratory: Sample collection time: Sample collection date: Sampling point:

mTEC incubation start temperature ( o C): mTEC incubation start date/time: mTEC incubation end temperature ( o C): mTEC incubation end date/time:

Urease substrate incubation start date/time: Urease substrate incubation end date/time:
Replicate number 1

Analyst initials

Replicate number 2

Analyst initials

Replicate number 3

Analyst initials E. coli

E. coli Sample volume filtered

100 mL 10.0 mL 1.0 mL

10.0 mL 1.0 mL 100 mL 10.0 mL 1.0 mL 0.1 mL

E. coli per 100 mL Sample volume filtered No. colonies per filter E. coli per 100 mL E. coli

Sample volume filtered No. colonies per filter E. coli per 100 mL

100 mL

0.1 mL 0.1 mL No. colonies per filterE. coli Membrane Filtration: Modified mTEC (EPA 1603)
Laboratory: Sample collection time: Sample collection date: Sampling point:

modified mTEC incubation start temperature ( o C): modified mTEC incubation start date/time: modified mTEC incubation end temperature ( o C): modified mTEC incubation end date/time:

Replicate number 1

Analyst initials

Replicate number 2

Analyst initials

Replicate number 3

Analyst initials

100 mL E. coli

E. coli 0.1 mL 100 mL 10.0 mL 1.0 mL 10.0 mL 1.0 mL

E.coli per 100 mL

E.coli per 100 mL No. colonies

per filter

1.0 mL 0.1 mL 10.0 mL Sample Volume filtered No. colonies per filter E.coli per 100 mL E. coli

100 mL

No. colonies per filter 0.1 mL

Sample Volume filtered Sample Volume filteredMultiple-Tube Fermentation: Fecal streptococcus(SM 9230B)Laboratory:Sample Collection time:Sample Collection Date:Sampling point:10mL1.0mL0.1mL0.01mL0.001mL10mL1.0mL0.1mL0.01mL0.001mL24 hr ADB read24 hr BEA readfrom 24 hr ADB48 hr ADB read24 hr BEA readfrom 48 hr ADBFinal tube combination:Fecal streptococcus/100 mL:10mL1.0mL0.1mL0.01mL0.001mL10mL1.0mL0.1mL0.01mL0.001mL24 hr ADB read24 hr BEA readfrom 24 hr ADB48 hr ADB read24 hr BEA readfrom 48 hr ADBFinal tube combination:Fecal streptococcus/100 mL:Replicate Number 2BEA PlatesAnalystInitialsReplicate Number 1Comments:Azide Dextrose Broth (ADB)AnalystInitialsAnalystInitialsComments:Azide Dextrose Broth (ADB)AnalystInitialsBEA Plates10mL1.0mL0.1mL0.01mL0.001mL10mL1.0mL0.1mL0.01mL0.001mL24 hr ADB read24 hr BEA readfrom 24 hr ADB48 hr ADB read24 hr BEA readfrom 48 hr ADBFinal tube combination:Fecal streptococcus/100 mL:BEA PlatesComments:Azide Dextrose Broth (ADB)AnalystInitialsAnalystInitialsReplicate Number 3ADB incubation start date/time:Start temp:ADB incubation end date/time:Start temp:End temp:Start temp:End temp:BEA incubation end date/time:(From 48 hr ADB)End temp:BEA incubation start date/time:(From 24 hr ADB)BEA incubation end date/time:(From 24 hr ADB)BEA incubation start date/time:(From 48 hr ADB)Membrane Filtration: Fecal Streptococcus (SM 9230C) Laboratory: Sample collection time: Sample collection date: Sampling point:
mEnterococcus incubation start temperature ( o C): mEnterococcus incubation start date/time: mEnterococcus incubation end temperature ( o C): mEnterococcus incubation end date/time:
Replicate number 1

Analyst initials

Replicate number 2

Analyst initials

Replicate number 3

Analyst initials Fecal streptococcus Fecal streptococcus

Fecal streptococcus

1.0 mL 0.1 mL Sample volume filtered

100 mL 10.0 mL

100 mL 10.0 mL 1.0 mL 0.1 mL Sample volume filtered 0.1 mL

Sample volume filtered

100 mL 10.0 mL 1.0 mL

No. colonies per filter Fecal streptococcus per 100 mL Fecal streptococcus per 100 mL

Fecal streptococcus per 100 mL No. colonies per filter

No. colonies per filterMembrane Filtration: Enterococcus (mE-EIA) (EPA 1106.1, SM 9230C) Laboratory: Sample collection time: Sample collection date: Sampling point:

mE incubation start temperature ( o C): mE incubation start date/time: mE incubation end temperature ( o C): mE incubation end date/time:
EIA incubation start temperature ( o C): EIA incubation start date/time: EIA incubation end temperature ( o C): EIA incubation end date/time: Replicate number 1

Analyst initials

Replicate number 2

Analyst initials

Replicate number 3

Analyst initials Enterococcus Enterococcus

Enterococcus

0.1 mL 0.1 mL

Volume filtered

100 mL 10.0 mL 0.1 mL

Volume filtered

1.0 mL Enterococci per 100 mL 100 mL 10.0 mL 1.0 mL

No. colonies per filter Enterococci per 100 mL

Enterococci per 100 mL No. colonies

per filter Volume filtered

100 mL

No. colonies per filter 10.0 mL 1.0 mL

### Membrane Filtration: Enterococcus (mEI) (EPA 1600) Laboratory: Sample collection time: Sample collection date: Sampling point:

mEI incubation start temperature ( o C): mEI incubation start date/time: mEI incubation end temperature ( o C): mEI incubation end date/time:

Replicate number 1
Analyst initials

Replicate number 2
Analyst initials

Replicate number 3
Analyst initials Enterococcus 0.1 mL 100 mL 10.0 mL 1.0 mL 0.1 mL

Enterococcus

Volume filtered No. colonies per filter Enterococcus per 100 mL
100 mL 10.0 mL Volume filtered

100 mL 10.0 mL 1.0 mL No. colonies per filter Enterococcus per 100 mL Enterococcus per 100 mL No. colonies

per filter Enterococcus Volume filtered

1.0 mL 0.1 mLBatch-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 Kovac's expiration date
2 Oxidase dry slides expiration date
3 Date of nutrient agar plate preparation
4 Date of nutrient agar slant preparation

5 Date nutrient agar slants for positive

controls and matrix spikes were inoculated

6 Time nutrient agar slants for positive

controls and matrix spikes were inoculated Section 2: Sample Processing Information

7 Dilution preparation date (for IDC, ODC, and MS/MSD samples)
8 Dilution preparation time (for IDC, ODC, and MS/MSD samples)
9 Analyst preparing dilutions for IDC, ODC, and MS/MSD samples)
10 Sample spiking date
11 Sample spiking time
12 Analyst spiking samples
13 Analyst performing filtration
14 Funnel decontamination method Section 3: Sample Analysis Information
15 Incubator temperature at start date o C 16 Incubator temperature at read date o C 16 ADA-V incubation start date / time
17 ADA-V read date / time 18 ADA-V read analyst
19 Nutrient agar plate incubation start date / time
20 Nutrient agar plate read date / time
21 Nutrient agar plate read analyst
22 Oxidase confirmation date / time 23 Oxidase read analyst
24 Trehalose incubation start date / time
25 Trehalose read date / time 26 Trehalose read analyst
27 Tryptone (indole) incubation start date / time
28 Tryptone (indole) read date / time 29 Tryptone (indole) read analyst

### QC Checklist: Method 1605 (Aeromonas) - ADA with Vanomycin
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 Yes No Did all method blanks (dilution/rinse water) exhibit the appropriate response? If no, please list contaminated method blank(s) and associated samples.

2 Yes No Did ADA-V media sterility check exhibit the appropriate response? If no, please explain.

3 Did nutrient agar plate media sterility check exhibit the appropriate response? Yes No If no, please explain.

4 Did nutrient agar slant media sterility check exhibit the appropriate response? Yes No If no, please explain.

5 Did trehalose media sterility check exhibit the appropriate response? Yes No If no, please explain.

6 No Did tryptone media sterility check exhibit the appropriate response? Yes If no, please explain.

7 Did the unspiked reagent water sample exhibit the appropriate response? Yes No If no, please explain.

8 Did negative controls for oxidase, trehalose, and indoleexhibit the appropriate responses? Yes No If no, please explain.12345678910Sample-specific Data Report Form:Method 1605 (Aeromonas) - ADA With VanomycinLaboratory name:Section 1: Sample information1 Sample number:5 Volume (mL) of spike (QC samples only):2 Utility:6 Volume filtered (mL):3 Sampling point:7 Dilution bottle(for laboratory-prepared QC samples only, plea DD2sterility check, direct streak, streak with filterreagent, finished, dilution/rinse4QC Analysis or Matrix filtered(please circle one):8If this report form is for a method blank,please indicate samples that areassociated with this method blank:Please note:It is important to record the number of colonies for each presumptively positive morphological type so that the final density ofAeromonasper sample can be reported based on percent confirmation of each colony type.Section 2: Sample resultsADA-V colony description(this section is optional)No. of presumptivepositive colonies forthis colony typeNo. of presumptive coloniessubmitted to confirmationfor this colony typeNutrient agar colony description(this section is optional)No. oxidasepositiveper colony typeNo. trehalosepositiveper colony typeNo. Indole positiveper colony typeColorMorphology*size (mm)ColorMorphology*size (mm)pale yellow13582-4 mm602off-white13582-4 mm2 of 22 of 21 of 2dark yellow13581-3 mm51off-white13581-2 mm1 of 11 of 11 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) otherSection 3: Calculations (Use one row for each presumptive positive colony color and morphology. If more than five colony types, please attach another sheet.)ANo. presumptive positive coloniesfor each colony typeBNo. of each colony typesubmitted toconfirmationCHow many submitted colonies percolony type confirmed (oxidasepositive, ferments trehalose, andproduces indole)?A * (C/B) = DCalculated no. ofconfirmedAeromonasper colony typeD1 + D2 + D3 + D4 + D5Total confirmed Aeromonasper sample6021D1=3030 + 5 = 35511D2=5D3=D4=D5=Section 5: Comments12345678910Sample-specific Data Report Form:Method 1605 (Aeromonas) - ADA With VanomycinLaboratory name:Section 1: Sample information1 Sample number:5 Volume (mL) of spike (QC samples only):2 Utility:6 Volume filtered (mL):3 Sampling point:7 Dilution bottle (for laboratory-prepared QC samples only, please circle one):DD2sterility check, direct streak, streak with filterreagent, finished, dilution/rinse4QC Analysis or Matrix filtered(please circle one):8If this report form is for a method blank, pleaseindicate samples that are associated with thismethod blank:Please note:It is important to record the number of colonies for each presumptively positive morphological type so that the final density ofAeromonasper sample can be reported based on percent confirmation of each colony type.Section 2: Sample resultsADA-V colony description(this section is optional)No. of presumptivepositive colonies forthis colony typeNo. of presumptive coloniessubmitted to confirmationfor this colony typeNutrient agar colony description(this section is optional)No. oxidasepositiveper colony typeNo. trehalosepositiveper colony typeNo. Indole positiveper colony typeColorMorphology*size (mm)ColorMorphology*size (mm)*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) otherSection 3: Calculations (Use one row for each presumptive positive colony color and morphology. If more than five colony types, please attach another sheet.)ANo. presumptive positive coloniesfor each colony typeBNo. of each colony typesubmitted toconfirmationCHow many submitted colonies percolony type confirmed (oxidasepositive, ferments trehalose, andproduces indole)?A * (C/B) = DCalculated no. ofconfirmedAeromonasper colony typeD1 + D2 + D3 + D4 + D5Total confirmed Aeromonasper sampleD1=D2=D3=D4=D5=Section 5: CommentsMethod 1622/23 Bench Sheet Laboratory name: Laboratory ID:

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 tur b idity , in NT U 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 oocy s ts/cy s ts spik ed (for IPR, OPR, MS, and PT samples only ) Cr ypto Giardi a 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, Cry p Te st, other [specify ] ) and lot number: 15. Name of analy s t performing filtration 16. Sample volume filtered, to nearest 1⁄4 L ( do not include rinse volum e ) 17. Did filter clog? 18. Elution date and time (must be performed within 96 hours of sam p le collection/filtration)These steps must be completed in one working day 19. Elution procedure: 9 wr ist shaker 9 FiltaMax w a sh station 9 stomacher 9 backflush/sonication 20. Name of analy s t performing elution 21. Elution buffer:_________________________ Elution buffer lot number and expiration date: 22. Concentration procedure (centrifugation, FiltaMax concentrator, other [specify ]) 23. Name of analy s t performing concentration 24. Pellet volume after concentration, in mL 25. (a) Total volume of resuspended concentrate; (b) volume transferred to IMS (in mL) ( a) (b) 26. Number of subsamples processed i ndependently through the remainder of the method 27. IMS system used (Dynal anti- Cryptosporidium , Dynal GC-Combo, other [specify ] ) and lot number 27. Name of analy s t 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 com p leted sam e working day as Row 18) 30. Detection kit used (Merifluor, AquaGlo, Cry p t- a-Glo, Giardi-a-Glo, other [specify ] ) and lot number 31. Analy s t performing staining procedure 32. Staining completion date and time (must be com p lete within 72 hours of Row 29) 33. Total number of oocy s ts and cysts counted in samp le (sum of counts in subsamples, if applicable) Cr ypto Giardi a Comments:

HV=high volume IPR=initial precis ion and recovery June 15, 2001, Revision - Draft N TU=nephelometric turbidit yunit O PR=ongoing precision and recover P T=performance test MS=matrix spike12345678910Method 1622/1623CryptosporidiumReport FormLaboratoryname:Laboratory ID:Client sample number:Internal laboratory sample ID (if applicable):10-mL subsample ID (if packed pellet > 0.5 mL):Volume examined (in L) on this slide:Analyst:Positive staining control acceptable9YES9NONegative staining control acceptable9YES9NOObjectlocatedby FANo.Shape(ovalorround)SizeL x W(Fm)DAPI -DAPI +D.I.C.Light blue internalstaining, nodistinct nuclei,green rim(A)Intense blueinternal staining(B)Number ofnuclei stainedsky blue(C)Empty oocysts(D)Oocysts withamorphousstructure(E)Oocysts with internal structure (F)Number of sporozoitesTotal FA number from this slide:Examination completion date:Examination completion time (must be complete within 7 days of staining):DAPI -: Total number (A):D.I.C. - Total number of empty oocysts (D):DAPI +: Total number (B):D.I.C. - Total number of oocysts with amorphous structure (E):DAPI +: Total number (C):D.I.C. - Total number of oocysts with internal structure (F):Total count DAPI + (C) that show structure by D.I.C. (F):June 15, 2001, Revision - Draft12345678910Method 1623GiardiaReport FormLaboratory name:Laboratory ID (if applicable):Client sample number:Internal laboratory sample ID (if applicable):10-mL subsample ID (if packed pellet > 0.5mL):Volume examined (in L) on this slide:Analyst:Pos. staining control acceptable9YES9NONeg. staining control acceptable9YES9NOObjectlocatedby FANo.Shape(ovalorround)SizeL x W(Fm)DAPI -DAPI +D.I.C.Light blueinternal staining,no distinctnuclei, green rim(A)Intense blueinternalstaining(B)Number ofnuclei stainedsky blue(C)Empty cysts(D)Cysts withamorphousstructure(E)Cysts with internal structure (F)Number ofnucleiMedianbodyAxonemesTotal FA number from this slide:Examination completion date:Examination completion time (must be complete within 7 days of staining):DAPI-: Total number (A):D.I.C.: Total number of empty cysts (D):DAPI+: Total number (B):D.I.C.: Total number of cysts with amorphous structure (E):DAPI+: Total number (C):D.I.C.: Total number of cysts with one internal structure (F):Total number DAPI + (C) that show structure by D.I.C. (F):D.I.C.: Total number of cysts with >one internal structure (F):June 15, 2001, Revision - Draft

## A PPENDIX 

## E EPA H EADQUARTERS AND 

## R EGIONAL 

## ATP C ONTACTSHeadquarters William Telliard Director, Analytical Methods Attn: ATP Program Coordinator Mail Code 4303T U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsy l vania Avenue, NW Washington, DC 20460 Region 1 Arthur Clark QA Chemist USEPA Region 1 EQA 60 Westview Street Lexington, MA 02173 Region 2 Carol Lynes ATP Program Coordinator USEPA Region 2 Division of Science and Monitoring 2890 Woodbridge Avenue (MS-220) Building 10 Edison, NJ 08837-3679 Region 3 Charles Jones Regional QA Officer USEPA Region 3 Environmental Assessment and Protection Division 1650 Arch Street, 3ES-10 Philadelphia, PA 19103-2029 Region 4 Way n e Turnbull Chemist/ATP Program Coordinator USEPA Region 4 Room: SESD 960 College Station Road Athens, GA 30605-2720 Region 5 Kenneth Gunter ATP Program Coordinator USEPA Region 5 77 W. Jackson Blvd., WT-15J Chicago, IL 60604 Region 6 David Stockton USEPA Region 6 Laboratory Houston Branch 10625 Fallstone Road (6MD-HI) Houston, TX 77099 Region 7 Doug Brune ATP Program Coordinator USEPA Region 7 726 Minnesota Avenue, ENSV/QA Kansas City, KS 66101 Region 8 Rick Edmonds Regional Quality Assurance Officer USEPA Region 8 999 18th Street - Suite 500 (8TMS-L) Denver, CO 80202-2466 Region 9 Roseanne Sakam o to ATP Program Coordinator USEPA Region 9 75 Hawthorne Street, PMD-3 San Francisco, CA 94105 Region 10 Bruce Woods QAO USEPA Region 10 200 Sixth Avenue, OEA-095 Seattle, WA 98101