United States Office of Water EPA-821-R-03-016
Environmental Protection 4303
Agency
Results of the Intel-laboratory Validation of
EPA Method 1602 for Enumeration of Male-
specific (F+) and Somatic Coliphage in Water
by Single Agar Layer (SAL)
July 2003
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Acknowledgments
The EPA technical lead for this report was Paul Berger, of the Standards and Risk Management Division
within the Office of Water. This document was prepared under an EPA, Engineering and Analysis
Division Contract No. 68-C-98-139 by DynCorp Information & Enterprise Technology, Inc.
The contributions of the following persons and organizations to the development of this method are
gratefully acknowledged:
Sobsey, Mark, Ming Jing Wu, and Greg Lovelace, University of North Carolina, Department of
Environmental Sciences and Engineering, CB#7400, MC/G Hall, Chapel Hill, NC 27599
Hsu, Fu-Chih, and Jim Larkin, Environmental Health Laboratories, 110 South Hill Street, South Bend, IN
46617
Chambers, Yildiz, City of San Diego Marine Microbiology Laboratory, 5530 Kiowa Drive, La Mesa, CA
91942
Cliver, Dean, Tadesse Mariam, and Mulugeta Tamene, University of California Davis, Department of
Health and Reproduction, School of Veterinary Medicine, Davis, CA 95616-8743
Danielson, Richard, BioVir Laboratory, 685 Stone Road Unit # 6, Benicia, CA 94510
Fujioka, Roger and Geeta Rijal, University of Hawaii, Water Resources Center, Holmes Hall 283, 2540
Dole Street, Honolulu, HI 96822
Karim, Mohammad and Dale Young, American Water Works System Research Laboratory, 1115 South
Illinois Street, Belleville, IL 62220-3731
Margolin, Aaron and Nicola Ballester, University of New Hampshire, Department of Microbiology,
Biological Sciences Building, Rudman Hall Room 285, Durham, NH 03824
Pillai, Suresh and Elisa Camacho, Texas A&M University, Department of Poultry Science, Kleberg
Center Room 418D, College Station, TX 77843
Pope, Misty, Kevin Connell, Ken Miller, Jason Kempton, and Jessica Pulz, DynCorp Information and
Enterprise Technologies, 6101 Stevenson Avenue, Alexandria, VA 22304
Williams, Fred and Ron Stetler, U.S. Environmental Protection Agency, 26 West Martin Luther King
Drive, Cincinnati, OH, 45268
Yates, Marylynn, Omid Bakhtar, and Andre Salazar, University of California Riverside, Department of
Environmental Sciences, 2217 Geology, Riverside, CA 92521-0424
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Disclaimer
Mention of company names, trade names, or commercial products in this report does not constitute
endorsement or recommendation for use.
Questions concerning this report should be addressed to:
U.S. EPA Office of Water
Analytical Methods Staff
1200 Pennsylvania NW
Mail Code 4303-T
Washington, DC 20460
Requests for additional copies of this publication should be directed to:
Water Resource Center
Mail Code RC-4100
401 M Street, SW
Washington, DC 20460
(202) 260-7786 or (202) 260-2814
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Table of Contents
Executive Summary vi
Section 1 Background 1
1.1 History of Method Development 1
1.2 Summary of Method 1
Section 2 Study Design and Objectives 2
2.1 Identification of Laboratories 2
2.2 UNC-prepared sewage filtrate spiking suspensions 2
2.3 Participant preparation of sewage filtrate spiking suspensions 3
2.4 Analysis of Water Samples 3
2.5 Quality Control Analyses 4
Section 3 Study Implementation 5
3.1 Study Management 5
3.2 Laboratory Participants 5
3.3 Minimum Validation Study Requirements 6
3.4 Schedule 6
3.5 Reagents and Materials Provided by EPA 7
3.6 Materials Provided by the Referee Laboratory 7
Section 4 Data Reporting and Validation 8
Section 5 Results 9
Section 6 Development of QC Acceptance Criteria 11
6.1 Outlier Analyses 11
6.2 Calculations for Development of Initial Precision and Recovery (IPR)
and Ongoing Precision and Recovery (OPR) Criteria 11
6.3 Calculations for Development of Matrix Spike/Matrix Spike Duplicate Criteria 14
Section 7 Data Analysis and Discussion 16
Section 8 Conclusions 17
Section 9 References 18
Section 10 Flowchart 19
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List of Tables
Table 1. Summary of Valid 100-mL Single Agar Layer Results vi
Table 2. Laboratories Participating in the Interlaboratory Validation of Method 1602 5
Table 3. Comparison of PBMS Tier 2 Requirements, ASTM Recommendations, and the Study . 6
Table 4. Sequence of Events for the Method 1602 Interlaboratory Validation Study 6
TableS. Male-Specific Coliphage Results by Laboratory for 100-mL Single Agar Layer Tests . 9
Table 6. Somatic Coliphage Results by Laboratory for 100-mL Single Agar Layer Tests 10
Table 7. Method 1602 QC Acceptance Criteria 14
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Executive Summary
This report presents the results of the U.S. Environmental Protection Agency's (EPA's) interlaboratory
validation study (the "Study") of EPA Method 1602: Male-specific (F+) and Somatic Coliphage in Water
by Single Agar Layer Procedure (the "SAL Method"). The purpose of the Study was to determine the
precision and recovery for male-specific and somatic coliphage in reagent water and ground water
matrices in multiple laboratories using the Method.
Coliphage presence in ground water is an indication of fecal contamination. Method 1602 is a
performance-based method for the enumeration of male-specific and somatic coliphage in ground water
and other waters. Laboratories are permitted to modify or omit any steps or procedure, with the exception
of the coliphage stock enumeration procedure, provided that all performance requirements set forth in the
validated method are met.
The SAL Method requires the addition of host bacteria, magnesium chloride, and double-strength molten
agar medium to the sample, followed by pouring the total volume of the mixture into plates. All plates
from a single sample are examined for plaque formation (zones of bacterial host lawn clearing). The
quantity of coliphage in a sample is expressed as plaque forming units (PFU) / 100 mL.
One referee laboratory and 10 participating laboratories were involved in the Study. The Study was
conducted during the week of August 9, 1999. The referee laboratory provided sewage filtrate spiking
suspensions, log-phase host bacteria, and male-specific and somatic positive controls.
Each laboratory in the study conducted a double agar layer (DAL) enumeration of the spiking suspension
to determine spike level, and analyzed four, 100-mL, spiked reagent water samples; two, 100-mL, spiked
ground water samples; and one, 100-mL unspiked ground water sample for each coliphage type according
to the July 1999 version of the Method, as amended by technical clarifications. Concurrent with these
analyses, each laboratory also analyzed a positive control, a method blank, and an unspiked ground water
sample for each coliphage type.
Sample results submitted by laboratories were validated using a standardized data review process to
verify that results were generated in accordance with the SAL Method and the Study specifications. A
summary of the overall precision and recovery for the Study is provided in Table 1. This summary
includes all valid results.
Table 1. Summary of Valid 100-mL Single Agar Layer Results
Coliphage
type
Male-specific
Somatic
Matrix
Reagent water
Ground water
Reagent water
Ground water
Mean spike
(PFU/sample)a
72
84
Mean spike
RSD
48%
20%
Mean recovery based on
individual lab spike dose
75%
61%
137%
178%
Mean
RSD/RPDb
for recovery
20%
14%
11%
8%
Spike level is based on the mean of the double agar layer (DAL) enumeration of the spiking suspension
performed by each laboratory on the day that the 100-mL test samples were spiked.
Mean of relative standard deviation (RSD) of the four reagent water replicates for each phage type in each
laboratory or the relative percent difference (RPD) of the two ground water samples for each phage type in each
laboratory.
VI
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As indicated in Table 1, the SAL procedure was characterized by 61% to 75% recoveries of male-specific
coliphages from ground water and reagent water, based on spike values determined using the double agar
layer (DAL) procedure. The number of somatic coliphages detected by the SAL procedure, however, was
considerably greater, on average, than the somatic coliphage spike values determined using the DAL
procedure. As indicated in Table 1, the SAL procedure recovered 137% and 178% of the somatic
coliphage recovered by the DAL procedure, which was used throughout the Study to estimate the number
of coliphages spiked into the test samples. Although the SAL somatic recoveries were unusually high
relative to the DAL results, the overall results for both coliphage types were consistent with the test
results generated previously by the method developer's laboratory. (Please see discussion in Section 7).
Study results from laboratory-prepared spikes were used in the development of quality control (QC)
acceptance criteria for Method 1602 and, as a result, these criteria reflect method performance. In
development of Quality Control (QC) acceptance criteria, laboratories and individual results disparate
from the average results produced by all laboratories were eliminated from consideration through outlier
analysis according to American Society for Testing and Materials (ASTM) procedures D2777-98 (Section
9.1). The QC acceptance criteria for Method 1602 are provided in Table 7.
Based on the Study results, the DAL procedure appears to be more accurate than SAL in enumerating
male-specific coliphage, but is less accurate than SAL in enumerating somatic coliphage. Because the
DAL is limited to analysis of small volumes (a maximum of 20 mL, which would require at least twice as
many plates as those required for the SAL procedure), the DAL procedure was not considered as an
option for monitoring ground water. Somatic results for both reagent water and ground water samples
were significantly higher using the SAL procedure than using the DAL. This could be the result of
increased adsorption by the host bacteria because of the addition of magnesium ions in the SAL
procedure.
Based on the Study results, Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single
Agar Layer (SAL) Procedure (EPA 821-R-01-029) is valid for use in enumeration of male-specific and
somatic coliphage in ground water.
vn
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 1 BACKGROUND
The Method 1602 single agar layer (SAL) method was optimized for use in the Environmental Protection
Agency's (EPA's) data gathering and monitoring programs under the Safe Drinking Water Act and the
Clean Water Act. EPA submitted the Method to interlaboratory validation, in anticipation of Ground
Water Rule proposal (May 10, 2000, 63 FR 10274), which proposed coliphage monitoring. The goal of
the Ground Water Rule is to reduce public health risk associated with the consumption of waterborne
pathogens from fecal contamination.
1.1 History of Method Development
Method 1602 was optimized by the University of North Carolina-Chapel Hill (UNC). Method 1602 is a
performance-based quantitative method for enumerating male-specific (F+) and somatic coliphage in
ground water and other waters. The Method 1602 SAL procedure requires the addition of host bacteria,
magnesium chloride, and double-strength molten agar medium to the sample, followed by pouring the
total volume of the mixture into plates. All plates from a single sample are examined for plaque formation
(zones of bacterial host lawn clearing). The quantity of coliphage in a sample is expressed as plaque
forming units (PFU) / 100 mL. EPA's Office of Water developed a draft method for coliphage in February
1999. The February 1999 draft method was significantly revised based on the February 16 and 17, 1999
Ground Water Rule Indicator Evaluation Workshop to provide the July 1999 version of the method which
was submitted to interlaboratory evaluation. The method was revised in April 2000, based on comments
from peer reviews and the interlaboratory validation study laboratories. Method 1602 was updated in
April 2001 to include performance criteria and comments from peer reviewers.
The July 1999 draft of the SAL Method was submitted to interlaboratory validation during August 1999
at 10 laboratories. This report describes the design, results, and conclusions of this study.
1.2 Summary of Method
Method 1602 describes the single agar layer (SAL) procedure. A 100-mL ground water sample is assayed
by adding MgCl2 (magnesium chloride), log-phase host bacteria (E. coll Famp for male-specific coliphage
and E. coll CN-13 for somatic coliphage), and 100-mL of double-strength molten tryptic soy agar to the
sample. The sample is thoroughly mixed and the total volume is poured into 5-10 plates (dependant on
plate size). After an overnight incubation, circular lysis zones (plaques) are counted and summed for all
plates from a single sample. The quantity of coliphage in a sample is expressed as plaque forming units
(PFU) /100 mL. For quality control purposes, a coliphage-positive sample and an unspiked sample are
analyzed for each coliphage type and matrix (reagent water and ground water).
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 2 STUDY DESIGN AND OBJECTIVES
The following objectives were established for the interlaboratory validation study of the precision and
recovery of the SAL Method:
• Meet the method validation requirements in EPA's Guide to Method Flexibility and Approval of EPA
Water Methods performance-based measurement system (PBMS) Tier 2 requirements
• Meet the method validation recommendations in ASTM D2777-98
• Determine the performance capabilities of the Method
• Establish QC acceptance criteria for performance tests in the Method
• Ensure that all samples and data produced during the Study were generated according to the
analytical and QA/QC procedures in the current version of the SAL Method
A summary flow chart of the Method 1602 validation study is provided in Section 10.
2.1 Identification of Laboratories
Because of limited funding, EPA sought participation from qualified laboratories on a volunteer basis.
2.1.1 Referee Laboratory
Referee laboratory activities were performed by the University of North Carolina (UNC), Department of
Environmental Sciences and Engineering. The referee laboratory prepared and distributed sewage filtrate
spiking suspensions, log-phase host bacteria, and pure-culture positive control spiking suspensions (male-
specific: MS2 ATCC#15597-B1 and somatic: phi-X 174 ATCC#13706-B1).
2.1.2 Participant Laboratories
Volunteer participant laboratories were chosen primarily based on experience propagating host bacteria
and performing coliphage assays. Each week, participant laboratories enumerated the sewage filtrate
spiking suspension using the double agar layer (DAL) technique described in the Method, spiked and
analyzed samples, and provided EPA with data on Method performance. The DAL technique was selected
to enumerate the sewage filtrate spiking suspensions because this technique was expected yield the most
accurate estimate of the coliphage levels in the concentrated sewage filtrate spiking suspensions.
(However, it should be noted that the DAL procedure appears to be more accurate than SAL in
enumerating male-specific coliphage, but is less accurate than SAL in enumerating somatic coliphage.
Please see Sections 7 and 8 below for a detailed discussion.)
2.2 UNC-prepared sewage filtrate spiking suspensions
On Sunday of each week during which analyses were conducted, a UNC microbiologist initiated
enumeration of a sewage filtrate spiking suspension for each coliphage type (male-specific and somatic)
using the DAL procedure described in the Method. After enumeration was completed on Monday, the
suspensions were shipped to participant laboratories via Federal Express Priority Overnight service for
analyses on Tuesday.
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
2.3 Participant preparation of sewage filtrate spiking suspensions
Participant laboratories enumerated the sewage filtrate twice per week using the DAL procedure
described in the Method 1602 (1st and 2nd DAL enumeration of sewage filtrate). The first enumeration was
conducted to determine the appropriate volume of sewage filtrate spiking suspension to use during each
week of analyses. The second enumeration was conducted to evaluate the true coliphage spiking
concentration of sewage filtrate on the day that samples were spiked.
2.3.1 1st DAL enumeration of sewage filtrate. Upon receipt of the UNC-prepared sewage filtrate
spiking suspensions on Tuesday, each participant laboratory enumerated the spiking suspensions
for both coliphage types using the DAL procedure described in the Method (four dilutions
evaluated in duplicate). DAL results were read and reported to DynCorp on Wednesday. The
laboratories prepared and spiked samples based on the mean number of coliphage observed across
all laboratories except University of Hawaii. Because Hawaii's results were not available until the
afternoon that samples were spiked, Hawaii's results were not included in determination of the
spiking volume. However, Hawaii did spike with the same volume of sewage filtrate as the other
participants. For each matrix (reagent water and ground water) the laboratories analyzed ten,
sewage-filtrate spiked samples per coliphage type and sample volume. Results of these analyses
were used in the development of Method 1602 QC acceptance criteria (see Section 6).
2.3.2 2nd DAL enumeration of sewage filtrate. On the day that samples were spiked, each laboratory
re-evaluated the sewage filtrate spiking suspension coliphage concentrations by the DAL
procedure described in the Method (four dilutions evaluated in duplicate) to determine the true
coliphage spike concentrations at each laboratory.
2.4 Analysis of Water Samples
Participant laboratories spiked reagent water and ground water samples for each coliphage type. To help
ensure that samples within each laboratory were homogenous, samples were spiked in-bulk (rather than
individually) and aliquots dispensed. The samples were analyzed according to the July 1999 version of
the Method, as amended by technical clarifications. These clarifications have been incorporated in the
April 2001 version of the Method.
Reagent water was analyzed to provide a means for assessing the performance of the Method on a matrix
that could be duplicated in each laboratory in the Study and in the future. Ground water samples were
analyzed to provide a means for assessing the performance of the Method on waters comparable to the
ground waters that would be analyzed during Ground Water Rule monitoring. Details on how reagent
water and ground water (matrix) samples were used in the Study are provided in Sections 2.4.1 and 2.4.2.
2.4.1 Reagent Water Sample Analysis
Each participant laboratory was required to analyze four, 100-mL reagent water samples per coliphage
type (male-specific and somatic). One, 100-mL unspiked reagent water sample per coliphage type was
also analyzed. Analysis of these reagent water samples was designed to provide EPA with the following:
• Sufficient data to assess method precision and recovery through analysis of samples by multiple
laboratories in the absence of interfering materials
• Sufficient data to develop initial and ongoing quality control (QC) acceptance criteria for reagent
water samples
• Identification of laboratory contamination through analysis of the unspiked reagent water samples
• An indication of intralaboratory performance through analysis of four replicate samples
• An indication of interlaboratory performance through analysis of samples by multiple laboratories
3 July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
2.4.2 Ground Water Sample Analysis
With the exception of San Diego Marine Microbiology Laboratory, each laboratory collected their own
ground water samples. The referee laboratory provided San Diego Marine Microbiology with ground
water for use during the study. All ground water samples used in the Study were spiked within 48 hours
of collection.
Each participant laboratory was required to analyze two, sewage filtrate spiked, 100-mL ground water
samples per coliphage type (male-specific and somatic). One, 100-mL unspiked ground water sample per
coliphage type was also analyzed. Analysis of these ground water samples by the participant laboratories
was designed to provide EPA with sufficient data to develop matrix spike (MS) QC acceptance criteria
for Method 1602.
2.5 Quality Control Analyses
Each participant laboratory also performed the following Quality Control (QC) analyses during each
week of the Study (quality control violations are discussed in Section 4):
• Reagent water method blank. At a minimum, each laboratory analyzed one method blank (a reagent
water sample containing no coliphage) for each coliphage type. Each participant laboratory identified
the samples associated with each method blank on the method blank report form, enabling
contamination problems to be tracked to associated samples. This ensured that the reagent water was
not contaminated.
• Unspiked ground water. At a minimum, each laboratory analyzed one unspiked ground water
sample for each coliphage type for assessment of background coliphage. None of the laboratories
observed coliphage in the unspiked ground water samples.
• Positive control. Each laboratory analyzed positive controls from pure stock coliphage suspensions
(MS2, ATCC#15597-B1 and phi-X 174, ATCC#13706-B1) to ensure that the host bacteria and
media were performing properly. Each laboratory evaluated one positive control per coliphage type.
Positive controls were analyzed on the same day as validation study samples.
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 3 STUDY IMPLEMENTATION
3.1 Study Management
This Study was designed under the direction of the Office of Science and Technology, Engineering and
Analysis Division within the U.S. Environmental Protection Agency's (EPA's) Office of Water (OW).
The EPA technical lead was Paul Berger, of the Office of Ground Water and Drinking Water, Standards
and Risk Management Division within the Office of Water. Coordination of activities for the Study were
performed by DynCorp I&ET. Referee laboratory activities were performed by the University of North
Carolina (UNC), Department of Environmental Sciences and Engineering.
3.2 Laboratory Participants
The participating laboratories involved in the interlaboratory validation of Method 1602 are listed in
Table 2.
Table 2. Laboratories Participating in the Interlaboratory Validation of Method 16023
American Water Works System Research Laboratory
Mohammad Karim and Dale Young
1115 South Illinois Street
Belleville, IL 62220-3731
U.S. Environmental Protection Agency
Fred Williams and Ron Stetler
26 West Martin Luther King Drive
Cincinnati, OH 45268
BioVir Laboratory
Richard Danielson
685 Stone Road Unit #6
Benicia, CA 94510
University of California Davis
Department of Health and Reproduction
School of Veterinary Medicine
Dean Cliver, Tadesse Mariam, and Mulugeta Tamene
Davis, CA 95616-8743
City of San Diego Marine Microbiology Laboratory
Yildiz Chambers
5530 Kiowa Drive
La Mesa, CA 91942
University of California Riverside
Department of Environmental Sciences
Marylynn Yates, Omid Bakhtar, and Andre Salazar
2217 Geology
Riverside, CA 92521-0424
Environmental Health Laboratories
Fu-Chih Hsu and Jim Larkin
110 South Hill Street
South Bend, IN 46617
University of Hawaii
Water Resources Center
Roger Fujioka and Geeta Rijal
Holmes Hall 283, 2540 Dole Street
Honolulu, HI 96822
Texas A&M University
Department of Poultry Science
Suresh Pillai and Elisa Camacho
Kleberg Center Room 418D
College Station, TX 77843
University of New Hampshire
Department of Microbiology
Aaron Margolin and Nicola Ballester
Biological Sciences Bldg. Rudham Hall Rm285
Durham, NH 03824
Referee laboratory:
University of North Carolina
Department of Environmental Sciences and Engineering
Mark Sobsey, Ming Jing Wu, and Greg Lovelace
CB #7400 MC/G Hall
Chapel Hill, NC 27599
No endorsement of these laboratories is implied, nor should any be inferred. Participant laboratories have been
randomly assigned numbers for purposes of presenting data in this report.
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
3.3 Minimum Validation Study Requirements
The Study exceeds the EPA performance-based measurement system (PBMS) Tier 2 method validation
requirements for development of quality control (QC) acceptance criteria for initial and ongoing
demonstration of laboratory capability, and matrix spike/matrix spike duplicate (MS/MSD) tests, as set
forth in Table 4-2 of EPA's Guide to Method Flexibility and Approval of EPA Water Methods (Table 3).
The Study also exceeds the ASTM D2777-98 method validation recommendations (Table 3) with every
respect except number of concentrations evaluated and matrix spike samples evaluated.
Table 3. Comparison of PBMS Tier 2 Requirements, ASTM Recommendations, and the Study
PBMS Tier 2 Requirements a
3 participant laboratories
1 matrix type plus reagent water
Samples from 3 public water
systems
1 concentration
12 IPR c samples (4 replicate
sample analyses in 3 laboratories)
6 MS/MSD d samples (1 MS and 1
MSD sample in 3 laboratories)
Minimum ASTM
Recommendations
6 participant laboratories
1 matrix type plus reference matrix
(typically reagent water)
1 matrix type plus reference matrix
(typically reagent water)
~3 concentrations (3 sets of
Youden pairs)
36 spiked reagent water samples (6
samples at 6 laboratory)
36 spiked matrix samples (6
samples at 6 laboratories)
1602 Study
10 participant laboratories
1 matrix type (ground water) plus
reagent water
Samples from 1 1 public water
systems
1 concentration
40 IPR samples (4 replicate
analyses in 10 laboratories)
20 MS samples (2 replicate MS
analyses in 10 laboratories)
USEPA. EPA Guide to Method Flexibility and Approval of EPA Water Methods, EPA 821-D-96-004, December
1996.
ASTM. Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D-19
on Water (ASTM D2777-98), October 1998.
IPR: Initial precision and recovery
MS/MSD: Matrix spike/matrix spike duplicate
3.4 Schedule
The SAL Study schedule is provided in Table 4.
Table 4. Sequence of Events for the Method 1602 Interlaboratory Validation Study
Date
May 25 -June 21, 1999
August 9, 1999
August 10 -12, 1999
Event
Laboratories were invited to participate in the validation of the SAL Method
Referee laboratory shipped sewage filtrate spiking suspensions, positive
controls, and host bacteria for 100-mL sample SAL analyses
Participant laboratories collected ground water samples; received sewage
filtrate spiking suspensions, positive controls, and host bacteria for sample
analyses; and conducted 100-mL analyses
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
3.5 Reagents and Materials Provided by EPA
EPA procured and distributed the following reagents and materials to each of the participant laboratories
through DynCorp I&ET: tryptic soy broth, nalidixic acid, ampicillin sodium salt, agar, sterile dilution
tubes, sterile serological pipets, sterile disposable petri dishes, and 1-L sample bottles.
3.6 Materials Provided by the Referee Laboratory
The referee laboratory provided the following materials:
3.6.1 Sewage filtrate spiking suspensions. The referee laboratory prepared and enumerated sewage
filtrate spiking suspensions according to the Method and provided spiking suspensions to the
participants for each week of analyses.
3.6.2 Host bacteria. The referee laboratory provided log-phase host bacteria (E. coll famp,
ATCC#700891 and E. coll CN-13, ATCC#700609) to the participants for each week of analyses.
3.6.3 Positive controls. The referee laboratory provided pure coliphage stock (MS2, ATCC#15597-B1
and phi-X 174, ATCC#13706-B1) for use as positive controls to the participants for each week of
analyses.
3.6.4 Trip controls. Sewage filtrate trip control spiking suspensions were shipped to each laboratory
and DynCorp for each week of analyses. The ice packs were replaced, and the trip controls from
every laboratory returned to the referee laboratory via FedEx Priority Overnight Service. The
referee laboratory received and evaluated the trip controls from every laboratory on the same day
that laboratories spiked and analyzed their samples each week. These trip controls were analyzed
by the referee laboratory approximately 48 hours after the suspensions were prepared using the
DAL procedure as described in the Method. The purpose of the trip control analyses was to
determine the effects of shipping on the suspensions.
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 4 DATA REPORTING AND VALIDATION
Each laboratory was required to submit data on standardized bench sheets and report forms designed for
use with Method 1602. Participant laboratories submitted the following data to DynCorp I&ET for review
and validation:
• Completed sample traffic reports
• Completed DAL report form for enumeration of sewage filtrate spiking suspensions for each
coliphage type
• Completed report form for 100-mL samples with QC sample results, reagent water sample results,
and ground water sample results
• Documentation of any additional information that would assist in evaluating the data
All 10 laboratories completed the Study and submitted data packages.
DynCorp reviewed each data package for completeness and determined if the sample result met the
requirements of the Study and Method 1602. Items reviewed for each sample included confirmation that:
• Original forms were submitted
• Incubation times were met
• All method blanks tested negative for each coliphage type
• Positive controls were performed and exhibited the appropriate response
• Calculations were correct
Based on the DynCorp data review, the following data were considered invalid and unacceptable for
inclusion in subsequent data analysis:
• Male-specific and somatic results for reagent and ground water tests from Laboratories 8 and 9 were
considered invalid because several of the sample/bacterial host mixtures remained in the waterbath(s)
for an extended period of time. As a result, coliphage replication occurred with recoveries of up to
>3800%. In addition to the extended time that the sample/host mixture spent in the waterbath(s), the
male-specific reagent water method blanks for Laboratory 9 tested positive for the presence of male-
specific coliphage.
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 5 RESULTS
Individual laboratory results for spike level (based on individual laboratory's 2nd DAL enumeration) and
100-mL sample analyses are summarized in Tables 5 and 6. Concurrent with these analyses, each
laboratory also analyzed a positive control, a method blank, and an unspiked ground water sample for
each coliphage type.
Table 5. Male-Specific Coliphage Results by Laboratory for 100-mL Single Agar Layer Tests
(invalid results are shaded
Lab
1
2
3
4
5
6
7
gb,C
9b
10
Trip Control
(PFU/mL)
Based on DAL
Enumeration by the
Referee
200
243
243
190
143
247
207
260
123
160
Spike level
(PFU/sample)a
based on 2nd
DAL
69
61
87
148
36
46
55
na
nad
75
Recovery based on individual laboratory spike enumeration
Reagent Water
Sample
1
43%
79%
67%
35%
113%
190%
79%
3199%
njj?
77%
Sample
2
30%
72%
41%
29%
124%
203%
77%
2892%
•»*
81%
Sample
3
35%
85%
53%
39%
83%
136%
139%
3844%
nae •'
81%
Sample
4
42%
97%
85%
36%
105%
226%
48%
3082%
™£ '
74%
Ground Water
Sample
1
26%
49%
58%
32%
110%
175%
72%
42%
TNTC
50%
Sample
2
16%
49%
65%
31%
85%
211%
92%
18%
/
JNTC;
42%
Spike level is based on the result of the double agar layer (DAL) enumeration of the spiking suspension
performed by each laboratory concurrent with the 100-mL test sample analyses.
Some of the laboratory's SAL sample/bacterial host mixtures were left in the waterbath(s) for an extended period
of time.
Laboratory did not perform spiking enumeration tests (2nd DAL analyses). As a result, the laboratory's percent
recoveries are based on mean DAL enumeration of spiking suspensions performed by all laboratories on the day
that the 100-mL samples were spiked.
Laboratory's male-specific DAL enumeration had bacterial host that grew in clumps.
Laboratory's reagent water male-specific method blank for the SAL analyses were positive.
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
Table 6. Somatic Coliphage Results by Laboratory for 100-mL Single Agar Layer Tests (invalid
results are shaded)
Lab
1
2
3
4
5
6
7
gb,C
9b
10
Trip Control
(PFU/mL)
Based on DAL
Enumeration by the
Referee
1053
933
970
1257
1013
1040
1017
1033
810
417
Spike level
(PFU/sample)a
based on 2nd
DAL
79
92
70
67
97
115
78
na
598
71
Recovery based on individual laboratory spike enumeration
Reagent Water
Sample
1
126%
463%
123%
82%
114%
117%
112%
81%
TNTC
171%
Sample
2
155%
412%
153%
100%
146%
145%
111%
,,8S%
TNTC
169%
Sample
3
127%
720%
107%
82%
162%
137%
126%
1375%-'
TNTC
153%
Sample
4
160%
591%
119%
132%
135%
129%
126%
TNTC
, /
160%
Ground Water
Sample
1
142%
383%
161%
139%
207%
183%
128%
TNTC
TNTC
265%
Sample
2
132%
813%
146%
103%
216%
155%
138%
/TNTC '
TNTC:
261%
Spike level is based on the result of the double agar layer (DAL) enumeration of the spiking suspension
performed by each laboratory concurrent with the 100-mL test sample analyses.
Some of the laboratory's SAL sample/bacterial host mixtures were left in the waterbath(s) for an extended period
of time.
Laboratory did not perform spiking enumeration tests (2nd DAL analyses). As a result, the laboratory's recoveries
are based on mean DAL enumeration of spiking suspensions performed by all laboratories on the day that the
100-mL samples were spiked.
July 2003
10
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 6 DEVELOPMENT OF QC ACCEPTANCE CRITERIA
6.1 Outlier Analyses
After evaluating laboratory results against method and study requirements, valid results were screened for
outliers using two approaches.
6.1.1 Evaluation of spike concentrations based on 2nd DAL enumeration. The first approach was
designed to ensure that the round-robin participants' spiked sample results were comparable, and
that no laboratory had spiked their samples with significantly higher or lower coliphage levels
than the other laboratories, thereby biasing the recoveries high or low. This was accomplished by
performing Grubb's test for individual outliers at 99% and 95% confidence levels each
laboratory's enumeration of the spiking suspension (based on 2nd DAL enumeration performed
the day that samples were spiked) and trip control results.
No outliers were identified at the 99% confidence interval. The somatic trip control result for
Laboratory 10 was identified as an outlier at the 95% confidence interval, but the nature of the
outlier was not supported by the enumeration of the spiking suspension or the spiked sample
results. As a result, the spiking results from Laboratory 10 were considered to be valid. Therefore,
all valid laboratory results were considered comparable and suitable for screening according to
the second approach: Youden's test for outlying laboratories.
6.1.2 Evaluation of SAL results. The valid SAL results were tested for the presence of an outlying
laboratory using Youden's laboratory ranking test. This test is designed to identify Study
participants with significantly higher or lower SAL results than the other laboratories. The test
was run separately for each coliphage type, and data for both matrices (reagent water and ground
water) were combined for each laboratory. Sample results for each laboratory were ordered based
on sample number. Based on the test, one laboratory was identified as biased high in the dataset
for each coliphage type: Lab 6 for male-specific and Lab 2 for Somatic.
6.2 Calculations for Development of Initial Precision and Recovery (IPR) and Ongoing
Precision and Recovery (OPR) Criteria
Laboratories demonstrate the ability to generate acceptable performance with this Method by performing
an IPR test before analyzing any field samples. On an ongoing basis, the laboratory demonstrates that the
analytical system is in control through analysis of OPR samples. IPR and OPR acceptance criteria were
developed based on the reagent water data from the validation study, as these QC tests will be performed
with reagent water by laboratories using the method during monitoring.
Estimates of each variance component (between laboratory and within laboratory) were calculated with
PROC MIXED from SAS 6.12 using the maximum likelihood method of estimation. Details on the
maximum likelihood estimation can be found in SAS/STAT User's Guide, Volume 2, published by the
SAS Institute.
11 July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
Estimates of between-laboratory variance and within-laboratory variance were labeled s2L and s2w,
respectively.
The combined standard deviation for IPR (isc) is:
Where:
L = the number of labs (7),
«; = the number of combined reagent water sample results for laboratory i, and
nT = the total number of reagent water results from all laboratories.
Upper and lower limits for IPR samples were then calculated as: (equation fixed to remove log-trans.)
-X-Mean - ^(0.975; idf) lSc
Where idf is calculated using Satterthwaite 's estimate as given below:
iv 4
idf =
' ( L \
I"?
1 4. i=l
n2
nT
\ )
*s2
2
_L
V4 nT) w
2
L-l
nT- L
The combined standard deviation (osc) for OPR is:
Where:
n ; = the number of reagent water sample results for lab i,
L = the number of labs, and
nT = the total number of reagent water results from all laboratories.
July 2003
12
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
Upper and lower limits for OPR samples were then calculated as: (equation fixed to remove log-
transformation)
Y + f
^ Mean ~ l (0.975;odf)
'OS
Where odfis calculated using Satterthwaite 's estimate as given below:
ns4
odf =
Y L \
n2T
\ )
L-l
c
*s2
™c
2
fi 0 * 2
V nT) w
nT- L
o
The precision criterion for IPR samples was calculated as a maximum relative standard deviation (RSD),
with the pooled within-laboratory standard deviation sw calculated by PROC MIXED from SAS 6.12
using the maximum likelihood method of estimation. The pooled RSD was first calculated as:
The maximum RSD was then calculated as:
Max ~ J
= \F,
(0.95;3,nT-L)
••RSD
Pool
Where:
nT = the total number of reagent water results from all laboratories, and
L = the number of laboratories.
The IPR and OPR QC acceptance criteria are provided in Table 7.
13
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
Table 7. Method 1602 QC Acceptance Criteria
Performance test
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
Matrix spike (MS)
MS percent recovery
Matrix spike, matrix spike duplicate (MS/MSD)
Mean percent recovery for MS/MSD
Precision (as maximum relative percent difference of MS/MSD)
Male-specific
acceptance
criteria
9% -130%
46%
4% -135%
Detect -120%
Detect -120%
57%
Somatic
acceptance
criteria
86% -177%
23%
79% -183%
48% -291%
48% -291%
28%
6.3
Calculations for Development of Matrix Spike/Matrix Spike Duplicate Criteria
QC acceptance criteria for matrix spikes (MS) and matrix spike duplicates (MSB) were developed based
on ground water data from the Study, as these QC tests will be performed with ground water samples by
laboratories using the method during monitoring.
Estimates of each variance component (between laboratory and within laboratory) were calculated using
PROC MIXED from SAS 6.12 using the maximum likelihood method of estimation. Details on the
maximum likelihood estimation can be found in SAS/STAT User's Guide, Volume 2, published by the
SAS Institute.
Estimates of between laboratory variance and within laboratory variance were labeled s2L and s2w,
respectively.
The combined standard deviation for MS/MSD (sc) is:
1
(!+•
L
)
XI
i '
SL ~*
Wy
1 ?
/I , \ 02
•^1+ )s
nT
Where:
L = the number of labs (7),
nt = the number of source water sample results for laboratory i, and
nT = the total number of source water results from all laboratories.
July 2003
14
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
Upper and lower limits for MS/MSD samples were then calculated as:
Y + t * v
^ Mean ~ '(0.975; df) ^c
Where dfis calculated using Satterthwaite 's estimate as given below:
Sc
' ( L
l^\ni
1 ~*~ 2
nT
V
L-
>
* r.2
)
2
r ~" °
i
/ ^ \
\+—\ *s2
( nT) w_
z.
1 nT - L
The precision criterion for MS/MSD samples was calculated as a maximum relative percent difference
(RPD), with the pooled within-laboratory standard deviation sw calculated by PROC MIXED from SAS
6.12 using the maximum likelihood method of estimation. The pooled RSD was first calculated as:
Mean
The maximum RPD was then calculated as:
RSD
pool
Where:
nT = the total number oflPR and OPR results from all laboratories, and
L = the number of laboratories.
The MS/MSD QC acceptance criteria are provided in Table 7, above.
15
July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 7 DATA ANALYSIS AND DISCUSSION
The male-specific summary results presented in Table 1 indicate that Study participants were able to
achieve mean recoveries of 75% for reagent water and 61% for ground water. As indicated in Table 1, on
average the SAL procedure recovered 137% and 178% (reagent water and ground water, respectively) of
the somatic coliphage recovered by the DAL procedure, which was used throughout the Study to estimate
the number of coliphages spiked into the test samples. Although the SAL somatic recoveries were
unusually high relative to the DAL results, the overall results for both coliphage types were consistent
with the test results generated previously by the method developer's laboratory.
The Study results were analyzed to determine whether there was a statistically significant difference
between the DAL and SAL results. The spike concentration (PFU / sample, based on 2nd DAL
enumeration of the spiking suspensions) and the mean SAL enumeration (PFU / sample) for each
coliphage type and matrix were log transformed (due to concerns over the normality of the data) and
subjected to a Paired-T test at 95% and 99 % confidence levels to determine whether there were
significant differences between the two enumeration methods. The data were also evaluated without log
transformation using a non-parametric, one-sample sign test at 95% confidence (data could not be
evaluated at 99% confidence because of the low number of samples). Based on the statistical analyses,
male-specific ground water sample enumerations were significantly less at 95% confidence level (for both
tests) when analyzed by the SAL procedure (compared to the DAL); reagent water sample results were
not significantly different. Somatic results for both reagent water and ground water samples were
significantly higher (99% confidence for Paired-T and 95% confidence for one-sample sign test) using the
SAL procedure than using the DAL. It is possible that the differences in recoveries between the DAL and
SAL procedures could be related to the differences in the analytical volumes. However, because this
difference in volume is the same for both phage types, this would not explain the interaction between
procedure and phage type on recovery (i.e., that male-specific recoveries were greater for the DAL
procedure, but somatic recoveries were greater for the SAL procedure). It is possible that the greater
somatic recoveries using the SAL procedure were the result of increased adsorption by the host bacteria
because of the addition of magnesium ions in the SAL media.
July 2003 16
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 8 CONCLUSIONS
Based on the Study results, the DAL procedure appears to be more accurate than SAL in enumerating
male-specific coliphage, but is less accurate than SAL in enumerating somatic coliphage. Because the
DAL is limited to analysis of small volumes (a maximum of 20 mL, which would require at least twice as
many plates as those required for the SAL procedure), the DAL procedure was not considered as an
option for monitoring ground water. The April 2001 version of Method 1602 includes the QC acceptance
criteria and comments and recommendations provided by laboratories participating in the Study. Based on
the results of this Study, EPA Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single
Agar Layer Procedure (821-R-01-029) is considered valid for the enumeration of male-specific and
somatic coliphage in ground water.
17 July 2003
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EPA Method 1602 Validation Study Results for Coliphage by Single Agar Layer (SAL)
SECTION 9 REFERENCES
9.1 Annual Book of ASTMStandards, Vol. 11.01. American Society for Testing and Materials.
Philadelphia, PA 19103.
9.2 EPA Guide to Method Flexibility and Approval of EPA Water Methods, EPA 821-D-96-004,
December 1996.
9.3 EPA Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer
(SAL) Procedure, EPA-821-R-01-029, April 2001.
July 2003 18
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SECTION 10 FLOWCHART
Method 1601/1602 Interlaboratory Validation Study Sample and Data Flow
Referee Laboratory
1. Collected/enumerated sewage filtrate spikes
2. Prepared log-phase host bacteria
3. Prepared and enumerated positive controls
4. Enumerated trip control samples
1. Sewage filtrate spiking suspensions
2. Log-phase host bacteria
3. Trip controls
4. Positive controls
Results for sewage filtrate, trip
control, and positive control
enumeration
Trip controls
Trip controls
Trip controls
Results of sewage filtrate
spiking suspension enumeration
Participant Laboratories
1. Enumerated sewage filtrate spikes upon receipt using DAL
2. Spiked reagent water and ground water samples with sewage filtrate/
3. Analyze spiked samples, positive controls, and blanks
DynCorp Biology Studies Group
Coordinated all sample shipments
Trouble-shot analyses at labs
Received all analytical data
Reviewed/validated data
Analyzed and summarized data
6. Drafted report summarizing method performance
Summary of method performance
data and study report
Results of sewage filtrate
spiking suspension enumeration
Provided spiking instructions based on results/
of sewage filtrate enumeration at all labs
Results of spiked sample, postive control,
and blank sample analyses
EPA Office of Water
1. Reviewed results
2. Evaluated method performance
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