United States Office of Water EPA-821-R-03-015
Environmental Protection 4303
Agency
Results of the Intel-laboratory Validation of EPA
Method 1601 for Presence/Absence of Male-
specific (F+) and Somatic Coliphage in Water by
Two-Step Enrichment
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 Technology, 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 Avenue 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 2
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 Schedule 6
3.4 Reagents and Materials Provided by EPA 6
3.5 Materials Provided by the Referee Laboratory 6
Section 4 Data Reporting and Validation 7
Section 5 Results 8
5.1 Individual Laboratory Results for 100-mL Sample Tests 8
5.2 Individual Laboratory Results for 1-L Sample Tests 10
Section 6 Data Analysis and Development of QC Acceptance Criteria 12
6.1 Evaluation of spike concentrations based on 2nd DAL enumeration and
trip control results 12
6.2 Two-Step Enrichment Sample Outlier Analyses 12
6.3 Criteria calculations for initial demonstration of capability (IDC) and
Expanded matrix spike (expanded MS) tests 12
6.4 Calculations for matrix spike (MS) and on-going demonstration of
capability (ODC) tests 15
Section 7 Discussion and Conclusion 16
Section 8 References 17
Section 9 Flow Chart 18
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List of Tables
Table 1. Summary of Valid, 100-mL Two-Step Enrichment Results vi
Table 2. Summary of Valid, 1-L Two-Step Enrichment Results vii
Table 3. Laboratories Participating in the Interlaboratory Validation of Method 1601 5
Table 4. Sequence of Events for the Method 1601 Interlaboratory Validation Study 6
Table 5. Summary of 100-mL, Male-specific Results (invalid results are shaded) 8
Table 6. Summary of 100-mL, Somatic Results (invalid results are shaded) 9
Table 7. Summary of 1-L, Male-specific Results (invalid results are shaded) 10
Table 8. Summary of 1-L, Somatic Results (invalid results are shaded) 11
Table 9. QC acceptance criteria for initial demonstration of laboratory capability (IDC) 14
Table 10. Expanded MS acceptance criteria for evaluation of method modification performance 14
Table 11. MS and ODC sample spiking requirements for ongoing evaluation of method
performance 15
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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 1601: Male-specific (F+) and Somatic Coliphage in Water
by Two-Step Enrichment (the "Method"). The purpose of the Study was to determine Method
performance for detection of very low levels of male-specific and somatic coliphage in reagent water and
ground water matrices in multiple laboratories when analyzing 100-mL and 1-L samples.
One referee laboratory and 10 participant laboratories were involved in the Study. During each week of
analysis, the referee laboratory provided sewage filtrate spiking suspensions, log-phase host bacteria, and
male-specific and somatic positive controls.
100-mL sample analyses. During the week of July 19, 1999 each laboratory in the study conducted a
double agar layer (DAL) enumeration of the spiking suspension to determine spike level. Each laboratory
analyzed ten, 100-mL, spiked reagent water samples; ten, 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. Concurrent with these analyses, each laboratory also analyzed a positive control, a method
blank, and an unspiked ground water sample for each coliphage type.
1-L sample analyses. During the week of July 26, 1999 each laboratory in the study conducted a double
agar layer (DAL) enumeration of the spiking suspension to determine spike level. Each laboratory
analyzed ten, 1-L, spiked reagent water samples; ten, 1-L, spiked ground water samples; and one, 1-L
unspiked ground water sample for each coliphage type according to the July 1999 version of the Method.
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 the laboratories were validated using a standardized data review process to
verify that results were generated in accordance with Method and Study specifications. A summary of the
100-mL and 1-L test results are provided in Tables 1 and 2. These tables summarize all valid results.
Table 1.
Summary of Valid, 100-mL Two-Step Enrichment Results
Coliphage
type
Male-specific
Somatic
Matrix
Reagent water
Ground water
Reagent water
Ground water
Mean spike level
(PFU per sample)3
0.9
1.6
Mean
Spike
RSD
27%
41%
Mean no. of positive
samples
across laboratories
(out of 10)
8.5
7.8
8.6
9.3
RSD for
no. of
positive
samples
14%
19%
12%
11%
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.
VI
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Table 2. Summary of Valid, 1-L Two-Step Enrichment Results
Coliphage
type
Male-specific
Somatic
Matrix
Reagent water
Ground water
Reagent water
Ground water
Mean spike level
(PFU per sample)3
1.1
1.6
Spike
RSD
39%
18%
Mean no. of positive
samples
across laboratories
(out of 10)
6.7
6.4
8.9
8.7
RSD for
no. of
positive
samples
30%
54%
14%
21%
Spike level is based on the mean of the DAL enumeration of the spiking suspension performed by each
laboratory on the day that the 1-L test samples were spiked.
The summary results presented in Tables 1 and 2 indicate that Study participants were able to achieve
positive rates of 60% to 90% for 100-mL and 1-L reagent water and ground water samples spiked with
approximately one plaque forming unit (PFU) per sample. When comparing 100-mL sample results to 1-
L sample results, it appears that positive rates for male-specific coliphages are higher, on average, in the
lower sample volume. However, it should be noted that because each 100-mL or 1-L sample was spiked
with approximately 1 PFU, each 100-mL sample received 10 times more coliphage per unit volume than
each 1-L sample.
The QC acceptance criteria for Method 1601 are provided in Tables 9,10, and 11. These criteria were
developed using Study data and reflect method performance. Based on results of the Study, Method 1601:
Male-specific (F+) and Somatic Coliphage in Water by Two-step Enrichment Procedure (EPA 821-R-01-
030) is valid for use in the determination of male-specific and somatic coliphage in ground water.
vn
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
SECTION 1 BACKGROUND
Method 1601 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 the 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 1601 was optimized by the University of North Carolina-Chapel Hill (UNC). Method 1601 is a
performance-based qualitative method for detecting the presence of male-specific (F+) and somatic
coliphage in ground water and other waters. The two-step enrichment procedure requires enrichment of
coliphage in tryptic soy broth with host bacteria followed by spotting onto a lawn of host bacteria and
assessing lysis zone formation in the lawn. 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
Method 1601 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 1601
was updated in April 2001 to include performance criteria and comments from peer reviewers.
The July 1999 draft of Method 1601 was submitted to interlaboratory validation during July 1999 at 10
laboratories. This report describes the design, results, and conclusions of the Study.
1.2 Summary of Method
Method 1601 provides a qualitative (presence/absence) two step enrichment procedure for coliphage. A
100-mL or 1-L ground water sample is supplemented with MgCl2 (magnesium chloride), log-phase host
bacteria (E. coll Fampfor male-specific coliphage and E. coll CN-13 for somatic coliphage), and tryptic soy
broth (TSB) as an enrichment step for coliphage. After an overnight incubation, enriched samples are
"spotted" onto a lawn of host bacteria specific for each type of coliphage, incubated, and examined for
circular lysis zones, which indicate the presence of coliphages.
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
SECTION 2 STUDY DESIGN AND OBJECTIVES
The following objectives were established for the interlaboratory validation study of Method 1601:
• 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 Method
A summary flow chart of the Method 1601 validation study is provided in Section 9.
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 to yield the
most accurate estimate of the coliphage levels in the concentrated sewage filtrate spiking suspensions.
(However, it should be noted that, based on the Method 1602 Single Agar Layer (SAL) study results, the
DAL procedure appears to be more accurate than SAL in enumerating male-specific coliphage, but less
accurate than SAL in enumerating somatic coliphage. Please see Sections 7 and 8 of the Method 1602
validation study report (EPA-821-R-03-016) 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.
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 (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.
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
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 1601 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 Method 1601 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 Method 1601 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 ten, 100-mL and ten 1-L reagent water samples per
coliphage type (male-specific and somatic). A 100-mL and a 1-L unspiked reagent water sample per
coliphage type was also analyzed. Analysis of these reagent water samples by at least ten participant
laboratories was designed to provide EPA with:
• Sufficient data to assess method performance through analysis of identical 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
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.
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
Each participant laboratory was required to analyze ten, 100-mL ground water samples and ten 1-L
ground water samples spiked with each coliphage type (male-specific and somatic). An unspiked 100-mL
and an unspiked 1-L 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 1601.
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 1601 Validation Study Results for Coliphage by Two-Step Enrichment
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 laboratories involved in the interlaboratory validation of Method 1601 are listed in Table 3.
Table 3. Laboratories Participating in the Interlaboratory Validation of Method 1601
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 6822
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 - Chapel Hill
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 1601 Validation Study Results for Coliphage by Two-Step Enrichment
3.3 Schedule
The Study schedule is provided in Table 4.
Table 4. Sequence of Events for the Method 1601 Intel-laboratory Validation Study
Date
May 25 -June 21, 1999
July 19, 1999
July 20 -23, 1999
July 26, 1999
July 27 -30, 1999
Event
Laboratories were invited to participate in the validation of Method 1601
Referee laboratory shipped sewage filtrate spiking suspensions, positive
controls, and host bacteria for 100-mL sample analyses
Participant laboratories collected ground water samples; received sewage
filtrate spiking suspensions, positive controls, and host bacteria for 100-mL
sample analyses; and conducted 100-mL analyses
Referee laboratory shipped sewage filtrate spiking suspensions, positive
controls, and host bacteria for 1-L sample analyses
Participant laboratories collected ground water samples; received sewage
filtrate spiking suspensions, positive controls, and host bacteria for 1-L sample
analyses; and conducted 1-L analyses
3.4 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.
3.5 Materials Provided by the Referee Laboratory
The referee laboratory provided the following materials:
3.5.1 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.
3.5.2 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.
3.5.3 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.
3.5.4 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 1601 Validation Study Results for Coliphage by Two-Step Enrichment
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 1601. 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 sample spiking information, QC sample results,
reagent water sample results, and ground water sample results
• Completed report form for 1-L samples with sample spiking information, 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 1601. 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 coliphage results for 100-mL reagent water tests from Laboratories 5, 6, and 9 were
considered invalid because the reagent water positive controls were negative.
• Male-specific coliphage results for 100-mL ground water tests from Laboratories 6 and 9 were invalid
because the ground water positive controls were negative.
• Male-specific coliphage results for 100-mL ground water tests from Laboratory 3 were invalid
because the laboratory did not analyze a ground water positive control sample.
• Male-specific and somatic coliphage results for 100-mL reagent water tests from Laboratory 9 were
invalid because spots from several samples ran together on spot plate.
• The preliminary report for the Study indicated that Laboratory 8's 100-mL sample results were
removed as an outlier. However, after further discussion with the laboratory regarding data quality
concerns, the Laboratory 8, 100-mL results were considered invalid.
• Male-specific and somatic coliphage results for 1-L reagent water tests from Laboratory 3 were
considered invalid because of a positive response in their method blank.
• Male-specific and somatic coliphage DAL sewage filtrate enumeration results for Laboratory 8 were
considered invalid because no plaques were observed on the DAL plates and the DAL positive
control results were negative.
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
SECTION 5 RESULTS
5.1 Individual Laboratory Results for 100-mL Sample Tests
Each laboratory in the study conducted a 2nd DAL enumeration of the sewage filtrate spiking suspension
to determine spike level, and analyzed ten, 100-mL reagent water samples and ten, 100-mL ground water
samples for each coliphage type. Individual laboratory results for DAL enumeration and 100-mL 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. Summary of 100-mL, Male-specific Results (invalid results are shaded)
Lab
1
2
3
4
5
6
7
8
9
10
Trip Control
(PFU/mL) Based on DAL
Enumeration by the Referee
385
355
512
445
321
279
321
337
297
130
Spike Level (PFU/sample)a
Based on the 2nd DAL
Enumeration
1.36
0.67
0.92
1.37
0.70
0.82
0.87
9f°
0.84
0.91
No. of Positive Samples (Out of 10)
Reagent Water
9
7
7
10
/1tf
Sc
9
2
nafi?
9
Ground Water
7
6
^
10
8
X
9
2
X
7
Spike level is based on the result of the double agar layer (DAL) enumeration of the sewage filtrate spiking
suspension performed by each laboratory on the day that the 100-mL test samples were spiked.
Laboratory did not analyze positive control sample.
Laboratory's reagent water positive control was negative.
Laboratory's ground water positive control was negative.
Laboratory did not perform spiking enumeration tests (2nd DAL) analyses.
Spots from several samples ran together on spot plate (no data).
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
Table 6. Summary of 100-mL, Somatic Results (invalid results are shaded)
Lab
1
2
3
4
5
6
7
8
9
10
Trip Control
(PFU/mL) Based on DAL
Enumeration by the Referee
806
718
706
730
484
636
621
653
852
667
Spike Level (PFU/sample)a
Based on the 2nd DAL
Enumeration
1.13
1.20
1.40
3.06b
1.25
2.11
1.02
X
1.47
1.53
No. of Positive Samples (Out of 10)
Reagent Water
9
8
8
7
8
10
9
4
W1
10
Ground Water
10
10
8
10
10
10
8
z
,/
8
10
Spike level is based on the result of the double agar layer (DAL) enumeration of the sewage filtrate spiking
suspension performed by each laboratory on the day that the 100-mL test samples were spiked.
Although the calculated mean spike level noted in Table 2 included this value, this result was identified as an
outlier (see discussion below). If results from this laboratory were eliminated from the mean spike level
calculation noted in Table 2, the mean level would be 1.4 PFU, rather than 1.6 PFU, per sample.
Laboratory did not perform spiking enumeration tests (2nd DAL) analyses.
Spots from several samples ran together on spot plate (no data).
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
5.2 Individual Laboratory Results for 1-L Sample Tests
Each laboratory in the study conducted a 2nd DAL enumeration of the sewage filtrate spiking suspension
to determine spike level, and analyzed ten, 1-L reagent water samples and ten, 1-L ground water samples
for each coliphage type. Individual laboratory results for DAL enumeration and 1-L sample analyses are
summarized in Tables 7 and 8. 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 7. Summary of 1-L, Male-s
)ecific Results (invalid results are shaded)
Lab
1
2
3
4
5
6
7
8
9
10
Trip Control
(PFU/mL) Based on DAL
Enumeration by the Referee
1067
1433
1067
733
1133
967
1067
B|? /
1067
306
Spike Level (PFU/sample)a
Based on the 2nd DAL
Enumeration
1.59
1.23
0.47
1.85
1.11
0.94
1.10
*f
1.18
0.59
No. of Positive Samples (Out of 10)
Reagent Water
7
8
>#
9
6
8
9
5
4
4
Ground Water
5
9
10
9
4
10
9
4
4
Od
Spike level is based on the result of the double agar layer (DAL) enumeration of the spiking suspension
performed by each laboratory on the day that the 1-L test samples were spiked
Laboratory's reagent water method blank was positive
Laboratory had no DAL plaques and the DAL positive controls were negative
Laboratory performed these analyses in duplicate and had no positives in either. However, the laboratory did
have six positives after respotting and reincubating. Zero positive is the result used in data analysis.
Laboratory did not return trip controls to the referee laboratory for analysis.
July 2003
10
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
Table 8. Summary of 1-L, Somatic Results (invalid results are shaded)
Lab
1
2
3
4
5
6
7
8
9
10
Trip Control
(PFU/mL) Based on DAL
Enumeration by the Referee
2067
2000
2033
2333
2167
2300
2233
BS?
2133
1227
Spike Level (PFU/sample)a
Based on the 2nd DAL
Enumeration
1.99
1.74
1.64
1.05
1.51
1.80
1.50
Bf^
1.55
1.29
No. of Positive Samples (Out of 10)
Reagent Water
10
9b
9G
10
7
10
10
7
8
9
Ground Water
10
10
9
9
10
10
8
5
6
10
Spike level is based on the result of the double agar layer (DAL) enumeration of the sewage filtrate spiking
suspension performed by each laboratory on the day that the 1-L test samples were spiked.
One sample outcome could not be determined, 9 samples were positive.
Laboratory's reagent water method blank was positive.
Laboratory had no DAL plaques and the DAL positive controls were negative.
Laboratory did not return trip controls to the referee laboratory for analysis.
11
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
SECTION 6 DATA ANALYSIS AND DEVELOPMENT OF QC ACCEPTANCE
CRITERIA
6.1 Evaluation of spike concentrations based on 2nd DAL enumeration and trip control
results
After evaluating laboratory results against method and study requirements, data from each laboratory with
valid results were screened for outliers to ensure that the Study 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 number of positive samples high or low. This was
accomplished by performing the Grubb's test for individual outliers on each laboratory's calculated spike
level (based on 2nd DAL enumeration performed the day that samples were spiked) and on the trip control
results. Grubb's test was run at both 99% and 95% confidence levels.
6.1.1 100-mL samples. No outliers were identified at the 99% confidence level. One outlier was
identified at the 95% confidence level (Laboratory 4's calculated somatic coliphage spike level
based on the 2nd DAL was biased high), but the nature of the outlier was not supported by spiked
sample results (see table 6). As a result, we consider all valid laboratory 100-mL results
comparable.
6.1.2 1-L samples. No outliers were found using Grubb's test at either the 95% or 99% confidence
level. As a result, we consider all valid laboratory 1-L results comparable.
6.2 Two-Step Enrichment Sample Outlier Analyses
The two-step enrichment results could not be evaluated using Youden's test for outlying laboratories
because a balanced dataset (all labs have same number of results) is necessary. The dataset is unbalanced
because some laboratories produced invalid data points that could not be used for data analysis, while
others did not. It should be noted that the preliminary report for the Study indicated that Laboratory 8's
100-mL sample results were removed as an outlier. However, after further discussion with the laboratory
regarding data quality concerns, the Laboratory 8, 100-mL results were considered invalid (see Section
4).
6.3 Criteria calculations for initial demonstration of capability (IDC) and Expanded
matrix spike (expanded MS) tests
An IDC test is performed by the laboratory to demonstrate the ability to generate acceptable performance
with this method before analyzing any field samples. An expanded MS test is used to demonstrate the
performance of the modified method in at least one real-world matrix before analyzing field samples
using the modified method. These QC acceptance criteria calculations were based on the lower limit of a
prediction interval for a value following the binomial distribution. General QC specification calculations
were not used because of the discrete nature of the data. The prediction interval was designed based on
two sources of variability included in prediction limits: the variability of the pooled estimate obtained
from the study data (in this case the pooled proportion positive), and the variability of a proportion of 10
replicates positive from the same population as the collected study data.
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
6.3.1 The pooled proportion positive, p-hat, for each volume, coliphage type and matrix was calculated
as:
Ill
I",
7=1
Where: m = the number of laboratories after the outlier test had been applied,
Xi = the number of positive replicates for lab I for a given matrix, volume and
coliphage type, and
Nt = the number of total replicates for lab i for a given matrix volume and coliphage
type (always 10).
6.3.2 The first part of the variability in the prediction limit is the variance of p-hat, calculated as:
Var(p) = —
I",
7=1
6.3.3 The second part of variability in the prediction limit is the variance of pNew, the proportion
positive out of 10 to be collected and compared to the limit. However, the variability of pNew
depends on what pNew will actually be. Therefore, this variance was calculated using the pNew that
yields the highest variance. This would occur when pNew is equal to 0.5 (5 positive out of 10).
Therefore,
New
Where NNew is the number of reps to be compared to the limit in the future, set as 10.
6.3.4 The upper and lower limits are calculated as:
, »*(!-»)
= p-l.96* ( J; )+ 0.025,
7 = 1
= £+1.96* (^——)+ 0.025
7=1
The multiplier 1.96 is used because under the standard normal distribution, 2*(1-P(Z<=1.65)) = 0.05.
~3 July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
6.3.5 The lower limit for positive replicates out of 10 is calculated as:
LLX= 10*LLp, and rounded down to the nearest whole number.
6.3.6 The upper limit for positive replicates out of 10 is calculated as:
ULX= 10*ULp, and rounded up to the nearest whole number.
6.3.7 The IDC test is performed to demonstrate acceptable performance with the method as written
prior to analysis of field samples or to evaluate acceptable performance of a method modification.
The resulting IDC acceptance criteria are provided in Table 9.
Table 9. QC acceptance criteria for initial demonstration of laboratory capability
(IDC)
Coliphage
type
Male-specific
Male-specific
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
"Bulk"
volume to be
spiked
1000mL
10L
1000mL
10L
Bulk spike
concentration
(PFU per bulk volume)
13
12
15
14
Minimum number
of positive
samples out of 10
5
3
5
5
6.3.8 Method modification validation/equivalency demonstration requirements. Although not
required, the laboratory also should perform an Expanded Matrix Spike (MS) test to demonstrate
the performance of the modified method in at least one real-world matrix before analyzing field
samples using the modified method. The resulting Expanded MS acceptance criteria are provided
in Table 10.
Table 10. Ex
)anded MS acceptance criteria for evaluation of method modification performance
Coliphage
type
Male-specific
Male-specific
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
"Bulk"
volume to be
spiked
1000-mL
10-L
1000-mL
10-L
Target bulk spike
(PFU per bulk
volume)
13
12
15
14
Minimum number
of positive
samples out of 10
4
2
5
5
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
6.4 Calculations for matrix spike (MS) and on-going demonstration of capability
(ODC) tests
An ODC test is performed by laboratories to demonstrate that the analytical system is in control on an
ongoing basis. The laboratory also spikes and analyzes field samples from each ground water source to
assess method performance in each matrix. In order to estimate the number of replicates needed to
confirm detection of a positive sample at 95% confidence, the number of replicates that would result in
less than a 5% chance of yielding no positive samples was calculated. In order to do this, it must be
assumed that the probability of getting a positive will be the same as observed during the Study. It must
also be assumed that the probability of a false positive equals zero. Therefore, the estimated number of
replicates would only apply in situations where the amount of coliphage spiked and volume analyzed
would be equal to those evaluated in this Study. The calculated number of replicates will be based on the
pooled proportion positive, p-hat, for the corresponding coliphage type, matrix and sample volume.
The number of replicates identified as positive follows a Binomial distribution. This is because each
replicate can only have two possible outcomes (positive or negative), and the Binomial distribution
describes the number of possible positives (or negatives) out of a given maximum number of trials (in this
case each replicate is considered a trial). Using the probability function of the Binomial distribution, the
number of replicates must be solved for using the equation below:
n
0.05 < P[0 positives out of n replicates] = | p\\ -
= (1-PT
and therefore,
n>
ln(0.05)
The analysis of ODC test samples demonstrates that the analytical system is in control on an ongoing
basis. The analysis of MS test samples from each ground water source assesses matrix-specific method
performance The resulting MS and ODC acceptance criteria are provided in Table 11.
Table 11. MS and ODC sample spiking requirements for ongoing evaluation of method
performance
Coliphage
type
Male-specific
Male-specific
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
"Bulk"
volume
to be spiked
300-mL
3-L
300-mL
3-L
Target bulk spike
(PFU per bulk
volume)
3.9
3.6
4.5
4.2
Number of samples
that must be spiked
(i1 must be positive)
3
3
3
3
15
July 2003
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
SECTION 7 DISCUSSION AND CONCLUSION
Study results indicate that on average Study participants were able to achieve positive rates of 60% to
90% for 100-mL and 1-L reagent water and ground water samples spiked with approximately one plaque
forming unit (PFU) per sample. Results of the Study demonstrate that EPA Method 1601 is valid for use
in determining the presence or absence of male-specific and somatic coliphage in ground water. Results
of the Study enabled assessment of the Method's performance in reagent water and ground water, and
enabled development of quality control (QC) acceptance criteria that will be used to confirm acceptable
laboratory and Method performance on an ongoing basis in ground water monitoring surveys and other
studies.
The April 2001 version of Method 1601 includes the QC acceptance criteria and comments and
recommendations provided by laboratories participating in the Study. Based on Study results and
revisions to the Method, EPA Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-
step Enrichment Procedure (EPA 821-R-01-030) is valid for the determination of male-specific and
somatic coliphage in ground water.
July 200 3 16
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EPA Method 1601 Validation Study Results for Coliphage by Two-Step Enrichment
SECTION 8 REFERENCES
8.1 EPA Method 1601 '.Male-specific (F*) and Somatic Coliphage in Water by Two-step Enrichment
Procedure, EPA 821-R-01-030, April 2001.
17 July 2003
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SECTION 9 FLOW CHART
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
2. Trouble-shot analyses at labs
3. Received all analytical data
Reviewed/validated data
5. 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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