EPA-821 -R-04-009
vvEPA
United States
Environmental Protection
Agency
Results of the I nterlaboratory Validation of
EPA Method 1603 (modified mTEC) for
E. coli in Wastewater Effluent
February 2004
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-04-009
U.S. Environmental Protection
Region 5, Library (PL-12J)
77 West Jackson Boulevard, 12th FSoc*
Chicago. 11 60604-3590
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Acknowledgments
This report was prepared by the DynCorp/CSC Biology Studies Group under the direction of Robin K. Oshiro, of the
Office of Science and Technology's Engineering and Analysis Division (BAD) within the U.S. Environmental
Protection Agency (EPA's) Office of Water.
The contributions of the following persons and organizations to this study are gratefully acknowledged:
Volunteer Research Laboratory
• EPA Office of Research and Development, National Risk Management Research Lab: Mark C. Meckes
Volunteer Participant Laboratories
• City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda,
and Zora Bahariance
• County Sanitation Districts of Los Angeles County, JWPCP: Kathy Walker, Michele Padilla, and Albert Soof
• County Sanitation Districts of Los Angeles County, SJC: Shawn Thompson and Julie Millenbach
• Environmental Associates (EA): Susan Boutros and John Chandler
• Hampton Roads Sanitation District (HRSD): Anna Rule, Paula Hogg, and Bob Maunz
• Hoosier Microbiological Laboratories (HML): Don Hendrickson, Katy Bilger, and Lindsey Shelton
• Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofhsteyn
• North Shore Sanitation District (NSSD): Robert Flood
Texas A&M University: Suresh Pillai and Reema Singh
• University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord
Wisconsin State Laboratory of Hygiene (WSLH): Jon Standridge, Sharon Kluender, Linda Peterson, and Jeremy
Olstadt
• Utah Department of Health: Sanwat Chaudhuri and Devon Cole
Volunteer Verification Laboratory
• City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda,
and Zora Bahariance
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Disclaimer
This document has been reviewed and approved by the EPA/EAD. Mention of company names, trade names, or
commercial products does not constitute endorsement or recommendation for use.
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Questions concerning this report should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov
202.566.1075
202.566.1053 (facsimile)
Requests for additional copies of this publication should be directed to:
Water Resource Center
Mail Code RC-4100
1200 Pennsylvania Avenue, NW
Washington, DC 20460
202.566.1729 or 202.566.1730
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Table of Contents
Section 1.0 Background 1
1.1 Summary of the Method 1
Section 2.0 Study Objectives and Study Design 2
2.1 Study Objectives 2
2.2 Phase 1 Technical Approach: Identification of Laboratories 3
2.2.1 Research Laboratory 3
2.2.3 Verification Laboratory 3
2.3 Phase 2 Technical Approach: BioBall™ Spikes 3
2.4 Phase 3 Technical Approach: Lab-Prepared Spiking Suspensions 4
2.5 Phase 4 Technical Approach: Sample Analysis 4
2.5.1 Range-finding Analyses 5
2.5.2 Assessment of Method Sensitivity and Specificity 5
2.5.3 Assessment of Method Accuracy (Precision and Recovery) 6
2.5.4 Development of Quantitative QC Criteria for Initial (IPR) and Ongoing (OPR)
Method/Laboratory Performance Assessments 6
2.5.5 Development of Quantitative QC Criteria for Matrix Spikes (MS) 7
2.5.6 Quality Control (QC) Analyses 7
2.5.7 Minimum Validation Study Requirements 7
Section 3.0 Study Implementation 8
3.1 Study Management 8
3.2 Schedule 8
3.3 Research, Participant, and Verification Laboratories 9
Section 4.0 Data Reporting and Validation 10
4.1 Data Reporting 10
4.2 Data Validation 10
4.3 Censored Data 11
Section 5.0 Results 12
5.1 Unspiked Sample Results 12
5.2 Spiked Disinfected Sample Results 14
5.3 Spiked PBS Results 16
5.4 Spiking Suspension Results 18
Section 6.0 Development of QC Acceptance Criteria 21
6.1 Outlier Analyses 21
6.1.1 Youden's Laboratory Ranking Test 21
6.1.2 Grubbs Test for Individual Outlying Sample Results 21
6.2 Initial Precision and Recovery (IPR) and Ongoing Precision and Recovery (OPR) 22
6.3 Matrix Spike (MS) and Matrix Spike Duplicate (MSD) Recovery and Precision 24
Section 7.0 Assessment of Method Performance: Discussion and Conclusions 27
7.1 Spiking Suspension (BioBalls™ and Laboratory-Prepared) Evaluation 27
7.2 Method Performance 28
7.3 Conclusion 29
Section 8.0 References 30
Section 9.0 Acronyms 31
IV
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List of Tables
Table 1.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
Table 7.
Table 8.
Table 9.
Table 10.
Table 11.
Table 12.
Table 13.
Table 14.
Table 15.
Method 1603 Validation Study Analyses Performed by Each Laboratory 5
Comparison of ASTM Recommendations and the Method 1603 Study 7
Laboratories Participating in the Interlaboratory Validation of Method 1603 9
Summary of E. coli Results from Unspiked Disinfected Wastewater Samples 12
Summary of False Positive and False Negative Rates Associated with Unspiked Disinfected
and Unspiked Secondary Wastewater Effluents 13
Laboratory-Specific False Positive and False Negative Rates Associated with Unspiked
Wastewater Effluents 13
Summary of E. coli Results from Disinfected Samples Spiked with BioBalls™ 14
Summary E. coli Results from Disinfected Samples Spiked with Laboratory-Prepared
Spiking Suspensions 15
Summary of E. coli Results from PBS Samples Spiked with BioBalls™ 16
Summary off. coli Results from PBS Samples Spiked with Laboratory-Prepared
Spiking Suspensions
17
BioBall™ Spike Enumeration by Participant Laboratories 18
Laboratory-Prepared Spiking Suspension Enumeration 19
BioBall™ Stability Evaluation Results (Counts from Nutrient Agar Spread Plates) 20
Calculated Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria 24
Calculated Matrix Spike Precision and Recovery Acceptance Criteria 26
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List of Appendices
Appendix A: Method 1603 Spiking Protocol A-l
Appendix B: Wastewater Laboratory Capabilities Checklist B-1
VI
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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 a membrane filtration procedure for the analysis of Escherichia coll (E. coif) in
wastewater: EPA Method 1603 which uses modified membrane-thermotolerant Escherichia coli agar medium
(modified mTEC). The September 2002 version of the Method (EPA-821-R-02-023) was followed during the
Study. The purposes of the Study were to characterize method performance (sensitivity, specificity, precision,
and recovery) across multiple laboratories and disinfected wastewater matrices and to develop quantitative quality
control (QC) acceptance criteria.
Eight volunteer participant laboratories, an E. coli verification laboratory, and a research laboratory participated in
the Study which was conducted during the week of November 10, 2003. During the Study, each laboratory
spiked samples with BioBall™ E. coli spikes and laboratory-prepared E. coli spikes. Samples were spiked in
accordance with the Method 1603 spiking protocol (the "Spiking Protocol," Appendix A). Results from samples
spiked with laboratory-prepared spikes were used to assess method performance and for the development of (QC)
acceptance criteria to support future assessments of method and laboratory performance for disinfected
wastewater matrices. Results from samples spiked with BioBalls™ were not used to develop QC acceptance
criteria or assess method performance because the enumeration of BioBalls™ on TSA at the participant
laboratories indicated that the BioBall™ spikes were highly variable (see Section 7.1 for detailed discussion of
this issue).
Results submitted by laboratories were validated using a standardized data review process to confirm that results
were generated in accordance with study-specific instructions and the September 2002 version of EPA Method
1603. Method 1603 recovery of E. coli was acceptable, with mean laboratory-specific recoveries of E. coli from
disinfected wastewater samples spiked with laboratory-prepared spikes ranging from 47.8% to 106%, with an
overall mean recovery of 80.7%. Laboratory-specific relative standard deviations (RSDs) from disinfected
wastewater samples spiked with laboratory-prepared spikes ranged from 6.1% to 51.4%, with an overall pooled,
within-laboratory RSD of 25.9%.
False positive rates were also acceptable, with laboratory-specific false positive rates for unspiked
disinfected/secondary results combined, ranging from 0% - 22.2%. For secondary wastewater (excluding
disinfected results), only one of 41 typical colonies submitted to verification were non-E. coli, resulting in a false
positive rate of 2.4% for secondary wastewater. For disinfected wastewater (excluding secondary results), only
four of 70 typical colonies submitted to verification were non-E. coli, resulting in a false positive rate of 5.7% for
disinfected wastewater.
Laboratory-specific false negative rates for unspiked disinfected/secondary results combined, ranged from 0% -
6.7%. For secondary wastewater (excluding disinfected results), two of 33 atypical colonies submitted to
verification were identified as E. coli, resulting in a false negative rate of 6.1% for secondary wastewater. For
disinfected wastewater (excluding secondary results), three of 75 atypical colonies submitted to verification were
identified as E. coli, resulting in a false negative rate of 4.0% for disinfected wastewater.
Based on the results of this Study, Method 1603 is considered valid for use in determining the concentration of E.
coli in disinfected wastewater.
VII
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EPA Method 1603 Validation Study Results
SECTION 1.0 BACKGROUND
The evaluation of E. coli is recommended as a measure of recreational water quality. Epiderniological studies
have led to the development of criteria which can be used to promulgate recreational water standards based on
established relationships between health effects and water quality. Method 1603 was recently approved for
monitoring ambient waters for E. coli (68 FR 43272, July 21,2003). Prior to this study, none of the approved E.
coli methods had been validated for wastewater analyses. National Pollutant Discharge Elimination System
(NPDES) permit holders and others have requested that EPA validate one or more E. coli methods for evaluation
of wastewater effluents.
The initial Study analyses were conducted in September 2003, but due to problems with materials used during the
analyses none of the data generated were considered valid or useable. Upon identification and resolution of these
problems by the research laboratory, repeat Study analyses were conducted during the week of November 10,
2003. This report describes the design, results, and conclusions of the repeat analyses.
1.1 Summary of the Method
Method 1603 is a membrane filtration procedure for the detection of E. coli in water samples. In Method 1603, a
water sample is filtered through a membrane (0.45 urn pore-size), which retains the bacteria. After filtration, the
membrane containing the bacteria is placed on a selective medium, modified mTEC agar, incubated at 35.0°C ±
0.5°C for 2 hours, and then incubated at 44.5°C ± 0.2°C for 22 to 24 hours. All red and magenta colonies are
considered E. coli.
February 2004
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EPA Method 1603 Validation Study Results
SECTION 2.0 STUDY OBJECTIVES AND STUDY DESIGN
2.1 Study Objectives
The following objectives were established for the Study:
• Generate at least six sets of useable, valid data to characterize method performance
• Characterize Method 1603 sensitivity and specificity across multiple laboratories and disinfected wastewater
matrices through the assessment of false positive and negative rates
• Characterize Method 1603 accuracy (recovery and precision) across multiple laboratories and disinfected
wastewater effluents
Establish Method 1603 quantitative Quality Control (QC) acceptance criteria for initial and ongoing
laboratory and method performance assessments
Establish Method 1603 quantitative QC acceptance criteria for matrix spikes
To accomplish these objectives, this Study was conducted in four phases (the technical approach for each phase is
described below):
• Phase 1 involved identification of qualified laboratories to participate in the Study, including a research
laboratory to provide additional technical and laboratory support as necessary; participant laboratories to
analyze both BioBall™ spikes and laboratory-prepared spiking suspensions; and a verification laboratory to
speciate colonies from the participant laboratories for the assessment of false positive and negative rates.
• Phase 2 involved enumeration and use of BioBall™ (BTF Pty Ltd, Sydney, Australia) spikes of E. coli
ATCC #11775 (Manassas, VA) at each of the participant laboratories. BioBalls™ are pre-packaged, water-
soluble balls containing a precise number of bacteria. BioBall™ products are freeze-dried and have a shelf
life of 6 months when stored at -20°C. BioBalls™ were selected for this study because they minimize the
burden on the participant laboratories and because BioBalls™ are typically very precise spikes.
• Phase 3 involved enumeration and preparation of a laboratory-prepared spiking suspension of E. coli ATCC
#11775 at each of the participant laboratories.
• Phase 4 involved the analysis of unspiked/spiked wastewater samples and unspiked/spiked sterile phosphate
buffered saline (PBS) samples at the participant laboratories.
The following data quality objective was established for this Study:
• Data produced under this Study were required to be generated according to the analytical and quality
assurance (QA)/QC procedures in the September 2002 version of Method 1603 (EPA-821-R-02-023) or
approved changes to these procedures as provided to participant laboratories during the course of the Study.
This data quality objective was c /eloped to ensure that data were of known and reliable quality, thereby
allowing EPA to use the results 01 the Study to identify the need for further revision of the method.
February 2004
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EPA Method 1603 Validation Study Results
2.2 Phase 1 Technical Approach: Identification of Laboratories
The Study required three types of laboratories: a research laboratory, participant laboratories, and a centralized
verification (identification) laboratory.
2.2.1 Research Laboratory
EPA's National Risk Management Research Laboratory (NRMRL) in Cincinnati, OH, served as the research
laboratory for this Study. Prior to sample analysis at the participant laboratories, the research laboratory
evaluated three strains of E. coli (ATCC #8739, ATCC #11775, and ATCC #35421) and two buffers [buffered
water and phosphate buffered saline (PBS)] following Method 1603 and the Spiking Protocol (Appendix A).
The research laboratory confirmed that E. coli ATCC #11775 and the PBS were appropriate to use during the
Study. ATCC #11775 was considered preferable to ATCC #8739 and ATCC #35421 because it was available in
BioBall™ format
2.2.2 Participant Laboratories
Participant laboratories analyzed samples to provide EPA with the data necessary to assess method performance
and develop QC acceptance criteria. The participant laboratories also provided typical and atypical colonies to
the verification laboratory for identification. Participant laboratories (Section 33) were representative of the
general user community, with experience analyzing wastewater or ambient water samples for E. coli using
membrane filtration techniques. Laboratory availability was also considered. A detailed Wastewater Laboratory
Capabilities Checklist (Appendix B) was used to collect information from laboratories and screen potential
participants to ensure that laboratories were qualified. Participants also needed to have access to representative
disinfected and secondary treated wastewater effluents from the same facility.
Qualified volunteer laboratories were recruited in an effort to reduce costs. To reduce the burden on participant
laboratories, EPA provided the media, reagents, and supplies necessary for the Study.
2.2.3 Verification Laboratory
The City of Los Angeles Bureau of Sanitation Microbiology Laboratory in Playa del Rey, CA served as the
centralized verification laboratory. To assess false positive and negative rates, the verification laboratory
speciated all typical and atypical colonies submitted by the participant laboratories. Colonies were identified
using the Vitek® automated identification system. A detailed Wastewater Laboratory Capabilities Checklist
(Appendix B) was used to collect information from laboratories and screen potential verification laboratories to
ensure the centralized verification laboratory was qualified.
The verification laboratory was also recruited as a volunteer in an effort to reduce costs. To reduce the burden on
the verification laboratory, EPA provided all necessary verification media and supplies.
2.3 Phase 2 Technical Approach: BioBall™ Spikes
Phase 2 involved enumeration and preparation of BioBall™ spikes ofE. coli ATCC #11775 according to the
Spiking Protocol (Appendix A). The "lot mean value" provided by the manufacturer was used as the "true spike
concentration." In addition, participant laboratories enumerated BioBalls™ on the day of analysis in triplicate,
using the spread plate technique and tryptic soy agar (TSA) as described in the Spiking Protocol (Appendix A),
to confirm stability of the BioBalls™.
February 2004
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EPA Method 1603 Validation Study Results
2.4 Phase 3 Technical Approach: Lab-Prepared Spiking Suspensions
Phase 3 involved each laboratory preparing and enumerating spiking suspensions of E. coli ATCC #11775
according to the Spiking Protocol (Appendix A). Results from samples spiked with laboratory-prepared spikes
were used to assess inter- and intra-laboratory precision and recovery (method performance) and to develop QC
acceptance criteria.
Spiking suspensions were enumerated in triplicate, using the spread plate technique and TSA as described in the
Spiking Protocol (Appendix A). To estimate the "true spike concentration," the participant laboratories
enumerated the laboratory-prepared spiking suspensions on the same day that the validation study samples were
spiked and analyzed.
2.5 Phase 4 Technical Approach: Sample Analysis
Phase 4 entailed the use of Method 1603 at multiple laboratories to analyze ir spiked/spiked wastewater samples
and PBS samples for E. coli.
The following objectives were established for Phase 4:
• Generate false positive and negative rate data for Method 1603 in disinfected wastewater effluents for the
assessment of sensitivity and specificity. It should be noted that while the objective of the Study was to
assess sensitivity and specificity for Method 1603 in disinfected wastewater, this was not possible because of
the low numbers of colonies from disinfected wastewater samples. As a result, colonies from secondarily
treated wastewater were also used to assess sensitivity and specificity of the Method. It was not possible to
assess sensitivity/specificity solely in the matrix of interest (disinfected wastewater) during this study.
• Generate precision and recovery data for Method 1603 in disinfected wastewater effluents
• Develop quantitative QC acceptance criteria for Method 1603 in sterile PBS to support future assessments of
laboratory performance
• Develop quantitative QC acceptance criteria for Method 1603 in the matrix of interest (disinfected
wastewater) to support future assessments of method performance
• Generate a minimum of six sets of useable (as recommended by ASTM for method validation), valid data
from the interlaboratory validation study to characterize method performance
Table 1 summarizes the number of samples that were evaluated to meet the objectives listed above. A detailed
discussion is included in Sections 2.5.1 through 2.5.5 below.
February 2004
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EPA Method 1603 Validation Study Results
Table 1. Method 1603 Validation Study Analyses Performed by Each Laboratory
Matrix
Disinfected
wastewater
Secondary
Disinfected
wastewater
Secondary *
Disinfected
wastewater
Disinfected
wastewater
Sterile PBS
Sterile PBS
Spiking
Description
Unspiked
Unspiked
Unspiked
Unspiked
BioBalls™
Lab-prepared
BioBalls™
Lab-prepared
Sample
Number
N/A"
N/A
1-4
5-6
7-8
9-10
11-14
15-18
Verification
N/A
N/A
5 typical and
5 atypical
5 typical and
5 atypical
N/A
N/A
N/A
N/A
Purpose of Analysis
Range-finding (Section 2.5.1)
Range-finding (Section 2.5.1)
Evaluation of ambient E. coli
concentrations (Section 2.5.2)
False positive and negative rates
(Section 2.5.2)
False positive and negative rates
(Section 2.5.2)
Assessment of method accuracy
(Section 2.5.3)
Assessment of matrix spike
QC criteria (Section 2.5.5)
Assessment of method accuracy
(Section 2.5.3)
Assessment of matrix spike QC
criteria (Section 2.5.5)
Develop quantitative QC criteria for
IPR c and OPR d (Section 2.5.4)
Develop quantitative QC criteria for
IPR and OPR (Section 2.5.4)
Results
Provided in
the Following
Tables
N/A
N/A
4
5 and 6
5 and 6
7
8
9
10
* Colonies from these samples were only submitted to verification when a sufficient number of colonies from disinfected
samples were not available
" N/A: Not applicable
c IPR: Initial Method/Laboratory Performance Assessment
d OPR: Ongoing Method/Laboratory Performance Assessment
2.5.1 Range-finding Analyses
Range-finding analyses were conducted on the secondary and disinfected wastewater effluents during the week of
validation study analyses. These analyses were conducted one day prior to the analysis of validation study
samples to determine the filtration volume(s) necessary to obtain plates within the optimum counting range for the
method.
2.5.2 Assessment of Method Sensitivity and Specificity
Sensitivity and specificity of Method 1603 was assessed through the evaluation of false positive and false
negative rates. Each of the 8 participant laboratories evaluated four unspiked disinfected wastewater samples for
false positive/negative results by submitting five typical and five atypical colonies from each of the four
disinfected wastewater samples to verification through biochemical evaluation. Because very few colonies were
February 2004
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EPA Method 1603 Validation Study Results
expected to be available for verification from the unspiked disinfected samples, two unspiked secondary effluent
samples were also filtered to ensure that a sufficient number of colonies were available for verification during the
Study. Colonies from the secondary samples were only submitted to verification when a sufficient number of
colonies from disinfected samples were not available. As a result, the study was designed to verify a total of 160
typical and 160 atypical colonies (including colonies from disinfected and secondary samples).
For each colony submitted to verification, the laboratory streaked the colony onto an modified mTEC agar plate
for isolation, inverted the plate, incubated at 35.0°C ± 0.5°C for 2 hours, and then incubated at 44.5°C ± 0.2°C for
22 to 24 hours. Plates were labeled with sample identification information and colony type. To prepare plates for
shipping, the laboratory wrapped the edges of the plates with parafilm, wrapped the stack of plates associated with
each sample with bubble wrap, placed the plates into a cooler lined with a trash bag, and surrounded the plates
with ice packs. Plates were shipped to the verification laboratory via Federal Express Priority Overnight Service.
Upon receipt at the verification laboratory, a single isolated colony was picked from each modified mTEC plate
and streaked for growth onto a tryptic soy agar (TSA) plate with 5% sheep blood (one TSA plate for every
modified mTEC plate) and incubated at 35°C ± 0.5°C for 18 to 24 hours. A suspension was prepared by placing
growth from the blood agar plates in 3 mL of physiological saline. The suspensions were then evaluated using a
Vitek® (bioMerieux, Hazelwood, Missouri), which is an automated biochemical identification system that utilizes
test cards with either 30 or 45 microwells containing identification substrates and antimicrobials. For
identification of E. coll during the Study, gram negative identification + (GNI+) cards were filled with the
suspension using the Vitek®'s automated card filler and placed into the card reader/incubator. Readings were
taken by the Vitek® every 15 minutes until identification (speciation) was complete.
2.5.3 Assessment of Method Accuracy (Precision and Recovery)
To collect data necessary to evaluate Method bias each laboratory analyzed two disinfected wastewater samples
spiked with BioBalls™ and two disinfected wastewater samples spiked with laboratory-prepared E. coll spiking
suspensions. Recovery was assessed by comparing E. coli concentrations in the spiked samples (minus the
ambient concentrations assessed from the analysis of the unspiked disinfected samples from Section 2.5.2) to the
BioBall™ lot mean value or the laboratory enumeration of the laboratory-prepared spiking suspension. Precision
was assessed based on the relative standard deviation of the two replicate recoveries.
2.5.4 Development of Quantitative QC Criteria for Initial (IPR) and Ongoing (OPR)
Method/Laboratory Performance Assessments
To collect the data necessary to develop quantitative QC recovery and precision criteria for use in initial and
ongoing method and laboratory performance, each participant laboratory analyzed four sterile 100-mL PBS
samples spiked with BioBalls™ and four sterile 100-mL PBS samples spiked with laboratory-prepared spiking
suspensions, usin? ATCC #11775 in both cases. Samples spiked with BioBalls™ were spiked with
approximately 29.2 CFU per sample and samples spiked with laboratory-prepared spiking suspensions were
spiked with approximately 32.5 CFU per sample.
February 2004
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EPA Method 1603 Validation Study Results
2.5.5 Development of Quantitative QC Criteria for Matrix Spikes (MS)
Quantitative QC criteria for matrix spikes were developed for use in assessing matrix interferences, should the
methods be implemented for use in disinfected wastewater by EPA. To collect the data necessary to develop
these criteria, each participant laboratory analyzed two disinfected wastewater samples spiked with BioBalls™
and two disinfected wastewater samples spiked with laboratory-prepared spiking suspensions using ATCC
#11775 in both cases. Samples spiked with BioBalls™ were spiked with approximately 29.2 CPU per aliquot
filtered and samples spiked with laboratory-prepared spiking suspensions were spiked with approximately 68.5
CPU per aliquot filtered. It should be noted that several laboratories were instructed to spike less than 100 mL of
disinfected wastewater because of high E. coli concentrations in the samples (see Section 4.2). It should be noted
that the same disinfected wastewater samples spiked with either BioBall™ or laboratory-prepared spikes were
used to assess method accuracy (Section 2.5.3) and to develop quantitative QC criteria for matrix spikes.)
2.5.6 Quality Control (QC) Analyses
Participating laboratories completed the following QC requirements: media sterility checks, dilution water
sterility checks, method blanks (sterile unspiked PBS), filtration blanks, positive controls, and negative controls.
E. coli (ATCC #11775) served as the positive control and E.faecalis (ATCC #19433) as the negative control.
2.5.7 Minimum Validation Study Requirements
The Study met or exceeded the ASTM D2777-98 (Reference 8.2) method validation recommendations in every
respect except number of concentrations and matrix spike samples. Only one spike concentration (instead of
three) was evaluated because the Study was designed to evaluate samples spiked at levels similar to what was
expected to be observed in disinfected wastewater samples at most laboratories. A limited number of matrix spike
samples were evaluated because only eight laboratories were able to obtain disinfected wastewater during the time
of year that the study was repeated. Table 2 presents a comparison of the Study with ASTM D2777-98 validation
study requirements.
Table 2. Comparison of ASTM Recommendations and the Method 1603 Study
Minimum ASTM Recommendations '
6 participant laboratories
1 matrix type plus reference matrix (typically reagent
water)
1 matrix type plus reference matrix (typically reagent
water)
-3 concentrations
36 spiked reagent water samples (6 samples at 6
laboratories)
36 spiked matrix samples (6 samples at 6
laboratories)
Method 1603 Study
8 participant laboratories
1 matrix type plus reference matrix (PBS)
Samples from a total of 8 facilities plus reference matrix (PBS)
1 concentration
64 (PR samples (for each of 8 participants, 4 replicate PBS
samples spiked with BioBalls™ and 4 replicate samples spiked
with lab-prepared spikes)
32 MS samples (for each of 8 participants, 2 duplicate MS
samples spiked with BioBalls™ and 2 duplicate MS samples
spiked with lab-prepared spikes)
ASTM. Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D-19 on Water
(ASTM D2777-98), October 1998.
February 2004
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EPA Method 1603 Validation Study Results
SECTION 3.0 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 Robin K. Oshiro. Coordination of activities for the Study were performed by DynCorp/CSC Biology
Studies Group. Evaluation of three strains of E. coli (ATCC #8739, ATCC #11775, and ATCC #35421) and two
buffers [buffered water and phosphate buffered saline (PBS)] following Method 1603 and the Spiking Protocol
(Appendix A) was performed by the EPA's Office of Research and Development (ORD), National Risk
Management Laboratory (NRMRL).
3.2 Schedule
The Study schedule was as follows: practice analyses occurred the week of August 25, 2003 and the initial
validation study analyses occurred the week of September 15,2003. Repeat validation study analyses were
conducted the week of November 10,2003. Range-finding analyses were conducted on Monday, November
10th. Isolates were received at the verification laboratory on November 19"1 and 20*. Verifications were started
on November 20,2003 and completed on December 24,2003.
February 2004
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EPA Method 1603 Validation Study Results
3.3 Research, Participant, and Verification Laboratories
The 12 participating laboratories, verification laboratory, and research laboratory involved in the Study are shown
in Table 3.
Table 3. Laboratories Participating in the Intel-laboratory Validation of Method 1603 *
City of Los Angeles Bureau of Sanitation b
Farhana Mohamad, Ann Dalkey, loannice Lee, Genevieve
Espineda, and Zora Bahariance
Hyperion Treatment Rant
12000 Vista del Ma* Playa del Rey, CA 90293
County Sanitation Districts of LA. County (JWPCP) "
Kathy Walker, Michele Padilla, and Albert Soof
24501 South Figueroa Street, Carson. CA 90745
County Sanitation Districts of LA. County (SJC) "
Shawn Thompson and Julie Millenbach
1965 South Workman Mill Road, Whlttier, CA 90601
Environmental Associates Ltd."
Susan Boutros and John Chandler
24 Oakbrook Drive, Ithaca, NY 14850
Hampton Roads Sanitation District
Anna Rule, Paula Hogg, and Bob Maunz
1 432 Air Rail Avenue, Virginia Beach, VA 23471
Hoosier Microbiological Laboratories
Don Hendrickson, Katy Bilger and Lindsey Shetton
912 West McGalliard, Muncie, IN 47303
Massachusetts Water Resources Authority
Steve Rhode and Mariya Gofhsteyn
1 90 Tafts Avenue, Winthrop, MA 021 52
North Shore Sanitation District
Robert Rood
William Koespel Drive, Guemee, IL 60031
Texas A&M University b
Suresh Pillai and Reema Singh
41 8D Kleberg Center, 2472 TAMUS, College Station, TX
77843
University of Iowa, Hygienic Laboratory b
Nancy Hall and Cathy Lord
Oakdale Campus # H101 OH, Iowa City, IA 52242
Wisconsin State Laboratory of Hygiene "
Jon Standridge, Sharon Kluender, Linda Peterson, and
Jeremy Olstadt
2601 Agriculture Drive, Madison, Wl 53718
Utah Department of Health
Sanwat Chaudhuri and Devon Cole
46 North Medical Drive, Salt Lake City, UT 841 13
Verification laboratory: City of Los Angeles Bureau of Sanitation, Microbiology Laboratory
Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve Espineda, and Zora Bahariance
Hyperion Treatment Plant, 12000 Vista del Mar, Playa del Rey, CA 90293
Research laboratory: EPA Office of Research and Development, National Risk Management Research Lab
Mark C. Meckes
26 West Martin Luther King Dr., Cincinnati, OH 45268-1320
No endorsement of these laboratories is implied, nor should any be inferred. Participant laboratories have been randomly
assigned numbers for purposes of presenting data in this report.
b Laboratories participated during both the initial Study analyses and the repeat Study analyses
February 2004
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EPA Method 1603 Validation Study Results
SECTION 4.0 DATA REPORTING AND VALIDATION
4.1 Data Reporting
Laboratories submitted the following data to DynCorp/CSC Biology Studies Group for review and validation:
• Completed cover sheet with sample collection and QC information
• Completed sample-specific reporting forms
• Documentation of any additional information that would assist in evaluating the data
4.2 Data Validation
DynCorp/CSC Biology Studies Group used data review checklists to ensure that each data package was complete
and to ensure that each sample result met the study-specific and method-specific requirements. Items reviewed
for each sample included the following:
• Confirmation that original forms were submitted
• Confirmation that incubation times were met
• Confirmation that incubation temperatures were met
• Confirmation that pre-filtration blank and phosphate buffer blank tested negative for E. coli
• Confirmation that media sterility checks were performed and acceptable
• Confirmation that positive and negative controls were performed and exhibited the appropriate response
• Confirmation mat samples were spiked with the appropriate dilution
• Confirmation that calculations were correct
This process was performed independently by two data reviewers, each of whom entered the results into separate
spreadsheets designed for data review and validation for this study. The results were compared to verify
consistency and identify potential data entry errors.
Based on data review, the data from Laboratory 2 were considered invalid and unacceptable for inclusion in
subsequent data analysis because the laboratory did not meet the method specific incubation requirements for 14
of the 18 samples analyzed during the validation study.
Although the QC checks for Laboratory 6 were acceptable, the laboratory expressed concern regarding their
analysis of the samples. Based on data review, the recoveries from Laboratory 6 were considerably lower than
recoveries from the other participating labs. As a result of the laboratory's concern and the very low recoveries
observed, results from Laboratory 6 were considered invalid and were not included in subsequent data analysis.
Laboratory-prepared spike analyses were not conducted by Laboratory 4 because the laboratory-prepared
spiking suspension did not grow (as indicated by the lack of turbidity) in the 1% LTB following the 18-24 hour
incubation period. The laboratory attempted to enumerate and spike with the 24 hour 1% LTB, however, the
spike enumeration resulted in no growth.
Laboratory 10 observed confluent growth on two of the three TSA plates used for BioBall™ enumeration and on
two of the three TSA plates used for laboratory-prepared spiking suspension enumeration. As a result, spike
enumeration data from this laboratory were not included in subsequent data analyses.
It should be noted that three laboratories were instructed to spike less than 100 mL of disinfected wastewater
because of high E. coli concentrations in the samples (based on range-finding analyses). Spiking and filtering a
smaller volume of sample helped ensure that plates within the optimum counting range were obtained.
Adjustments were made as follows:
February 2004 10
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EPA Method 1603 Validation Study Results
• Laboratory 11: Due to high E. coli concentrations, Laboratory 11 was instructed to spike 50 mL of
disinfected wastewater per sample.
• Laboratories 9 and 12: Due to high E. coli concentrations, Laboratories 9 and 12 were instructed to spike 10
mL of disinfected wastewater per sample.
4.3 Censored Data
During the evaluation of validation study samples, results below the analytical range of the method (less-than
results, also referred to as left-censored) were observed for some of the unspiked disinfected effluent samples.
Left-censored results were observed with the following frequency: <1 E. coli per 100 mL, seven results and <10
E. coli per 100 mL, one result. The censor limit was replaced with one half of the "less than" value for
subsequent data analyses for these samples.
11 February 2004
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EPA Method 1603 Validation Study Results
SECTION 5.0 RESULTS
All of the results included in this section were considered valid. Please see Section 4 for detailed data
invalidation information. Results of unspiked wastewater samples and false positive/negative rates (Section 5.1),
spiked disinfected wastewater samples (Section 5.2), spiked PBS samples (Section 5.3), and spiking suspension
evaluations (Section 5.4) are included in this section. Please see Section 6 for the development of QC acceptance
criteria and Section 7 for a discussion of method performance.
5.1 Unspiked Sample Results
Results from unspiked disinfected wastewater sample analyses are provided in Table 4. These data were used to
estimate the background concentration of E. coli in disinfected wastewater samples.
Results of the verification analysis (assessment of false positive and negative rates based on unspiked disinfected
and secondary confirmations) were used to assess method performance (see discussion in Section 7.2). Valid
verification results from unspiked disinfected and secondary wastewater effluent samples are summarized in
Table 5. Valid, laboratory-specific verification results are summarized in Table 6. Any typical colony that was
identified as non-E. coli by the Vitek® was considered a false positive result. Any atypical colony that was
identified as an E. coli by the Vitek® was considered a false negative result. It should be noted that some of the
isolates submitted to verification did not exhibit growth on modified mTEC. Colonies that did not grow after
streaking for isolation on modified mTEC agar plates, were considered to be non-E. coli and included in data
analyses as non-£. coli colonies. The decision to include the colonies that exhibited no growth on the modified
mTEC as non-£. coli was based on the selective nature of the medium, as the modified mTEC was considered the
first step in the verification process.
Table 4. Summary of E. coli Results from Unspiked Disinfected Wastewater Samples (see Table 1 for
cross-reference to sample number)
Lab
2
3
4
6
9
10
11
12
Sample number (CFU/100 mL) '
1
•
1
<1
'ft.',;, ';
3
30
72
10
2
^ |*M^i
<1
<1
: 4s-*
10
50
54
20
3
'^/^
<1
<1
v «* ^*
4
40
48
10
4
-;- - «"
-------
EPA Method 1603 Validation Study Results
Table 5. Summary of False Positive and False Negative Rates Associated with Unspiked Disinfected
and Unspiked Secondary Wastewater Effluents
Matrix
Disinfected
Secondary
Disinfected/Secondary
False Positive Assessment
Typical
colonies
submitted
70
41
111
Number of false
positives
4
1
5
False Positive
Rate (%)
5.71
2.44
4.50
False Negative Assessment
Atypical
colonies
submitted
75
33
108
Number of false
negatives
3
2
5
False Negative
Rate (•/.)
4.00
6.06
4.63
Table 6. Laboratory-Specific False Positive and False Negative Rates Associated with Unspiked
Wastewater Effluents (Disinfected and Secondary Results Combined, Samples 1-6, see cross-
reference in Table 1)
Lab
2
3
4
6
9
10
11
12
False Positive Assessment
Typical
colonies
submitted
19
19
Number of false
positives
False Positive
Rate (%)
False Negative Assessment
Atypical
colonies
submitted
Number of false
negatives
False Negative
Rate(%)
<-.---• . £$&"'- --'9.
0
0
0.00
0.00
... v,,t|||*i
18
19
19
17
4b
0
0
1
22.22
0.00
0.00
5.88
19
20
18
20
15
16
1
1
0
1
1
1
5.26
5.00
0.00
5.00
6.67
6.25
Data validation discussed in Section 4.2
" Three of the four false positive results were due to no growth while streaking for isolation onto modified mTEC (discussed
in Section 7.2)
13
February 2004
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EPA Method 1603 Validation Study Results
5.2 Spiked Disinfected Sample Results
Results from disinfected wastewater samples spiked with laboratory-prepared spikes (Table 8) were used to
assess method performance (see discussion in Section 7.2) and develop QC acceptance criteria for matrix spikes
for use in assessing matrix interferences (see discussion in Section 6).
Table 7. Summary of E. coll Results from Disinfected Samples Spiked with BioBalls™ (see Table
1 for cross-reference to sample number)
Lab
2
3
4
6
9
10
11
12
Spike level
(CFU/100 mL)"*
Percent Recovery by sample
7
29.2
29.2
292
292
58.4
292
59.93
77.05
8
Mean Percent
Recovery
SDe
IF
73.63
87.33
'- '^iSI
56.51
107.02
47.09
71.06
46.23
79.62
26.54
71.06
Overall (n = 12)
66.35
82.19
51.37
93.32
36.82
71.06
66.92
9.69
726
726
19.37
14.53
0.00
11.44'
RSDd(%)
14.60
8.84
14.14
20.76
39.47
0.00
20.32*
Colony forming units
" Spike level is based on the lot mean value provided by the manufacturer and the volume of sample that was spiked (it was
necessary to spike <100 mL at some laboratories because of high background concentrations of E. coli)
c Standard deviation
" Relative standard deviation
* Data review and validation discussed in Section 4.2
' Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances
9 Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab
RSDs
February 2004
14
-------
EPA Method 1603 Validation Study Results
Table 8. Summary £. coli Results from Disinfected Samples Spiked with Laboratory-Prepared
Spiking Suspensions (see Table 1 for cross-reference to sample number)
Lab
2
3
4
6
9
10
11
12
Spike level
(CFU/IOOmL)*"
Percent Recovery by sample
9
10
* i "• -: • : y?4fSlfe^
32.9
101.82
110.94
Mean Percent
Recovery
SDC
RSD"(%)
' >>.:• ft •:> •;•• ,;j?l| < ;•' ;• ^ -
106.00
6.45
6.08
;^: •%/: #XV
30.3
357
53.4
367
34.65
87.54
55.24
94.69
74.26
101.54
40.26
105.59
Overall (n = 10)
rtr-1^^:*^^
54.46
94.54
47.75
100.14
80.65
^f4fS¥W"^-- '%•
3 ^^V^^K&'A
28.00
9.90
10.59
7.71
14.80*
A:r "•'.;; A- "'
51.43
10.48
22.18
7.70
25.86"
Colony forming units
" Spike level is based on laboratory enumeration of spiking suspension and the volume of sample that was spiked (it was
necessary to spike <100 mL at some laboratories because of high background concentrations of £. coli)
0 Standard deviation
" Relative standard deviation
8 Data validation discussed in Section 4.2
f Laboratory-prepared spiking suspension did not grow in the 1% LTB , please see discussion in Section 4.2
9 Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances
h Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab
RSDs
15
February 2004
-------
EPA Method 1603 Validation Study Results
5.3 Spiked PBS Results
Results from PBS samples spiked with laboratory-prepared spiking suspensions (Table 10) were used to develop
QC acceptance criteria for use in assessing initial and on-going method/laboratory performance (see discussion in
Section 6).
Table 9. Summary of E. coli Results from PBS Samples Spiked with BioBalls™ (see Table 1 for
cross-reference to sample number)
Lab
2
3
4
6
9
10
11
12
Spike level
(CFU/IOOmL)"*
Percent Recovery by sample
11
•;;f Jci-.'f'- "f^X^l
29.20
29.20
^
29.20
29.20
29.20
29.20
3.42
54.79
;'
54.79
78.77
95.89
82.19
12
3.42
30.82
13
3.42
37.67
14
Mean Percent
Recovery
SDC
'
10.27
54.79
^filE'"* '*''"' ' ' g ^'^rC^*^^'' " ' "s ^r^; ' '• "
92.47
78.77
44.52
85.62
89.04
85.62
58.22
92.47
85.62
102.74
78.77
102.74
Overall (n=24)
5.14
44.52
80.48
86.47
69.35
90.75
62.79
3.42
12.19
RSDd(%)
66.67
27.38
17.35
11.32
22.61
9.06
14.04'
21.56
13.09
32.60
9.98
34.14*
Colony forming units
Spike level is based on the lot mean value provided by the manufacturer
Standard deviation
Relative standard deviation
Data validation discussed in Section 4.2
Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances
Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab
RSDs
February 2004
16
-------
EPA Method 1603 Validation Study Results
Table 10. Summary of EL coli Results from PBS Samples Spiked with Laboratory-P
Spiking Suspensions (see Table 1 for cross-reference to sample number
Lab
2
3
4
6
9
10
11
12
Spike level
(CFU/IOOmL)"*
Percent Recovery by sample
15
16
17
18
Mean Percent
Recovery
repared
SDC
RSDd(%)
Y Jt?, . - - : £*(, ^:m^S-"'M. --i^i-''-'s^Ssi Jflvafef *; J^'-v&fi^'S:, «i|»:^>' '*f.U-*i^*«'
' ' , '^ ' ' '^ Vv ''* ~^ sC&£&&^Jj$ij- *' '•• '/''JffiPfy rf'^i?^* £», , ^"» ' =^i *»J»WWt ^ 3, \ .« C-^T '' '*$%, ^'^&^^^^^^^^'^4^/^"'',f"'~i, V* * ?i" Y ?•'-, %%£}",
32.9
•':, >;'X >' •-
- ' ^ • .
• :-,.
30.3
35.7
26.7
36.7
99.01
67.23
89.89
103.54
132.01
78.43
86.14
73.57
118.81
95.24
74.91
70.84
89.11
44.82
86.14
65.40
Overall (n-20)
109.74
71.43
84.27
78.34
82.89
19.31
21.15
6.49
17.14
15.86"
17.60
29.61
7.70
21.88
19.63"
Colony forming units
" Spike level is based on laboratory enumeration of spiking suspension on tryptic soy agar (ISA) plates
0 Standard deviation
d Relative standard deviation
" Data validation discussed in Section 4.2
' Laboratory-prepared spiking suspension did not grow in 1% LTB, please see discussion in Section 4.2
9 Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances
Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab
RSDs
17
February 2004
-------
EPA Method 1603 Validation Study Results
5.4 Spiking Suspension Results
Results from BioBall™ enumerations conducted at the participant laboratories using TSA spread plates are
provided in Table 11 and results of laboratory-prepared spiking suspension enumerations is provided in Table 12.
Results from BTF's assessment of BioBall™ shipping effects are provided in Table 13. Results from this section
were used to evaluate the BioBall™ and laboratory-prepared spiking suspensions (see discussion in Section 7.1)
Table 11.
BioBall™ Spike Enumeration by Participant Laboratories
Lab
Counts from TSA Plates
Plate 1
Plate 2
PlateS
Mean
SD'
RSD
(%)
Var
16
25
17
19.33
4.93
25.51
24.33
29
15
20
21.33
7.09
33.26
50.33
27
25
17
23.00
5.29
23.01
28.00
10
21
23
29
20.33
10.26
50.47
105.33
12
26
29
31
28.67
2.52
8.78
6.33
Overall (n = 16)
22.53
6.55"
31.34'
42.87
' Standard deviation
b Relative standard deviation
c Data review and validation discussed in Section 4.2
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances
* Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab
RSDs
February 2004
18
-------
EPA Method 1603 Validation Study Results
Table 12.
Laboratory-Prepared Spiking Suspension Enumeration
Lab
2
3
4"
6
9
10
11
12
Counts from TSA Plates
Plate 1
Plate 2
> «'r;N, „ )'/^(/' 4
117
107
Plate 3
Mean
f >toi*>tA£ r&
-•*&,/ ™* *£* ^ *! **.# .'./
105
109.67
SD'
' ^
6.43
RSD»(%)
't'V, ffi
5.86
Var
41.33
-~ > T > ,/' ^',i~k>>',>? ^*km*i*A^ /?y , V*? ,:"- * :
t * v * ' * ^ *s x <( *f %/YM* ^f^. v ^ * "** ' "- /rfi * %; ',* * ** ** °< «** ^° ^ ^
». #»<%» -- .
*** "'''' ' '*
109
119
87
127
"*' : •'' ^f^ff^'^M
* «X fc^^vv, £/«,
102
96
115
92
84
125
Overall (n = 13)
0 '
101.00
Wif&f^^m
89.00
122.33
105.50
j. x'*' A *" *t /
i - ,,-^p x
8.54
8.46
6.24
6.43
6.98'
7.02
5.26
6.76'
l\- ,.
' 4fyff* * ,
73.00
39.00
41.33
48.67
Standard deviation
" Relative standard deviation
c Data review and validation discussed in Section 4.2
d Laboratory-prepared spiking suspension did not grow in the 1% LIB , please see discussion in Section 4.2
8 Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab variances
f Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the squared lab
RSDs
19
February 2004
-------
EPA Method 1603 Validation Study Results
Table 1 3. BioBall™ Stability Evaluation Results (Counts from Nutrient Agar S
Replicate
1
2
3
4
5
6
7
8
9
Mean
SDa
RSD " (%)
1. Container filled
with dry ice
29
31
34
31
31
31
31
31
29
30.89
1.45
4.70
2. Container without ice
31
28
26
30
29
33
26
27
26
28.44
2.51
8.81
3. New super Insulated
container with blue ice
30
27
31
30
30
27
32
32
29
29.78
1.86
6.23
pread Plates)
4. Validation study
simulation
24
29
27
28
28
30
25
30
24
27.22
2.39
8.77
Standard deviation
b Relative standard deviation
February 2004
20
-------
EPA Method 1603 Validation Study Results
SECTION 6.0 DEVELOPMENT OF QC ACCEPTANCE CRITERIA
All data analyses and QC acceptance criteria calculations described below were performed using the results of
disinfected wastewater and PBS samples spiked with laboratory-prepared spiking suspensions. Results from
samples spiked with BioBalls™ were not used to develop QC acceptance criteria or assess method performance
because the enumeration of BioBalls™ on ISA at the participant laboratories indicated that the BioBall™ spikes
were highly variable. BioBall™ variability is discussed in greater detail in Section 7.1.
Although samples spiked with BioBalls™ were not used in the development of QC criteria for Method 1603,
laboratories may choose to spike QC samples with BioBalls™, instead of laboratory-prepared spiking
suspensions, and compare the results with the QC criteria developed based on samples spiked with laboratory-
prepared spiking suspensions (Tables 14 and 15). The use of BioBalls™, instead of laboratory-prepared spiking
suspensions, may reduce the time spent conducting QC analyses. However, based on the results of this study, it
should be noted that laboratories may have difficulty meeting demonstrating acceptable performance if samples
are spiked with BioBalls™ .
6.1 Outlier Analyses
Valid results from samples spiked with laboratory-prepared spiking suspensions were screened for outliers in
accordance with the procedures described in American Society for Testing and Materials (ASTM) guidance
D2777-98 (Reference 8.2). Outlying data were identified and removed in two steps: identification of outlying
laboratories, followed by identification of individual sample results. First, outlying laboratories were identified
using Youden's laboratory ranking test (Reference 8.2). For this test, laboratories were ranked and screened to
identify laboratories with significantly higher or lower results than the other laboratories. The second test for
identification of outlying data is the Grubbs test (Reference 8.2), which identifies individual sample results for
outlying observations. While the D2777-98 guidance states that outlier tests should not be run unless valid data
are available from a minimum of six laboratories, these tests were still run to ensure that the dataset used to
calculate QC acceptance criteria represented the population of qualified laboratories. It should be noted that no
outlying laboratories (Section 6.1.1, below) or outlying individual results (Section 6.1.2, below) were identified.
It should also be noted that outlier analyses were only performed for development of QC acceptance criteria
(Section 6). Outlier analyses were not conducted for the assessment of method performance (Section 7.2), as all
valid data were included in the assessment of method performance.
6.1.1 Youden's Laboratory Ranking Test
The valid data were first tested for the presence of an outlying laboratory using Youden's laboratory ranking test.
For this test, lab-prepared spike recoveries from both disinfected wastewater and PBS samples were used.
Sample results for each laboratory were sorted based on sample number. Based on the Youden's test, no
laboratories were identified as being outliers, and therefore no laboratories were removed for the QC criteria
calculations.
6.1.2 Grubbs Test for Individual Outlying Sample Results
After performing Youden's lab ranking test, the remaining data were then tested for the presence of individual
outlying results using Grubbs test. Grubbs test was run separately for each matrix (disinfected wastewater and
PBS) for the lab-prepared spike recoveries. Grubbs test was run without performing any data transformations.
This was because statistical and graphical evaluations did not reveal any major departures from the assumption of
the data following a Normal distribution. Application of Grubbs test did not result in the identification of any
21 February 2004
-------
EPA Method 1603 Validation Study Results
outliers for either wastewater or PBS samples. Therefore, no results were removed for the QC criteria
calculations.
6.2 Initial Precision and Recovery (IPR) and Ongoing Precision and Recovery (OPR)
QC acceptance criteria for initial precision and recovery (IPR) and ongoing precision and recovery (OPR) were
developed based on the results from PBS (reference matrix) samples spiked with laboratory-prepared spiking
suspensions during the Study, as these QC tests will be performed using PBS as the reference matrix by
laboratories using the method during monitoring.
The IPR and OPR recovery criteria were calculated based on within and between laboratory variance components.
These variance components were calculated with PROC MIXED from the SAS version 8 program using the
maximum likelihood method of estimation on the recovery results. Details on the maximum likeuhood estimation
can be found in the user's guide for this program (Reference 8.3).
Estimates of between laboratory variance and within laboratory variance were labeled s2L and s2w, respectively.
The combined standard deviation for IPR (isc) is:
z«
4 nT
Where:
L = number of labs
nt = number of PBS sample results for laboratory i
nT = total number of PBS results from all laboratories
Upper and lower limits for IPR samples were then calculated as:
Mean * '(0.975; W)
Where idfis calculated using Satterthwaite 's estimate as given below:
idf =
is.
'( L \
I"'
1 1 "'
4
\ )
**;
2
4--
'&-}"•
\4 nT)
L-l
nr-L
February 2004
22
-------
EPA Method 1603 Validation Study Results
The combined standard deviation (osc) for OPR is:
Where:
L = number of labs
n, - number of PBS sample results for laboratory i
nT = total number of PBS results from all laboratories
Upper and lower limits for OPR samples were then calculated as:
y + f
•^ Mean — l(0.97S;o »T) J
L-l
nT - L
The precision criterion for IPR samples was calculated as a maximum relative standard deviation (RSD). The
RSD for each laboratory and each spiking procedure was calculated by dividing the standard deviation of the
recoveries by the mean of the recoveries for that laboratory and procedure. The RSDs were pooled directly,
rather than calculating a pooled RSD by pooling standard deviations and dividing by an overall mean, because the
pooled RSD calculated using the latter approach was unduly affected by laboratories which exhibited high bias
and low variability.
The pooled RSD was then calculated as:
RSD_ =
(n,-l)*RSD?
Where:
L = number of laboratories
n, = number of PBS sample results for laboratory i
nT ~ total number of PBS sample results for all laboratories
RSD, = RSD calculated using the recoveries for laboratory i
23
February 2004
-------
EPA Method 1603 Validation Study Results
The maximum RSD was then calculated as:
RSD
p°°'
Where:
nT = total number oflPR and OPR results from all laboratories
L — number of laboratories
The calculated IPR and OPR QC acceptance criteria are provided in Table 14.
Table 14. Calculated Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria
Performance test
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
Lab-prepared spike acceptance criteria
47% -119%
36%
39% -127%
6.3 Matrix Spike (MS) and Matrix Spike Duplicate (MSD) Recovery and Precision
QC acceptance criteria for matrix spikes (MS) and matrix spike duplicates (MSD) were developed based on
lab-prepared spike data from the spiked disinfected wastewater matrices used in the validation study.
Recovery criteria were based on estimates of each variance component (between laboratory and within laboratory)
and were calculated using PROC MIXED from SAS version 8 using the maximum likelihood method of
estimation on the recovery results. Details on the maximum likeHhood estimation can be found in the user's guide
for this program (Reference 8.3). Between matrix variability could not be separated from between laboratory
variability because each laboratory analyzed a different disinfected wastewater sample, and therefore the estimate
of between laboratory variance also includes matrix variability.
Estimates of between laboratory variance and within laboratory variance were labeled s\and s2w, respectively.
The combined standard deviation for MS/MSD (s,.) is:
I«,2
Where:
L = number of labs
nt = number of disinfected sample results for laboratory i
nr = total number disinfected sample results from all laboratories
February 2004
24
-------
EPA Method 1603 Validation Study Results
Upper and lower limits for MS/MSD samples were then calculated as:
y + / * «•
•^ Mean — l\0.975;df) ^
Where:
Xmam ~ tne >"eaH recovery of all disinfected wastewater samples
dfis calculated using Satterthwaite 's estimate as given below:
'{ L \
I"?
] x M
1 M2
nT
V /
*c2
•»£
2
[t'^H
L-l
nr- L
The precision criterion for MS/MSD samples was calculated as a maximum relative percent difference (RPD).
The RSD for each laboratory and matrix was calculated by dividing the standard deviation of the two recoveries
by the mean of those recoveries for that lab and matrix. The RSDs were pooled directly, rather than calculating a
pooled RSD by pooling standard deviations and dividing by an overall mean, because the pooled RSD calculated
using the latter approach was unduly affected by laboratories/matrices which exhibited high bias and low
variability.
The pooled RSD was then calculated as:
i=l
Where:
L = number of laboratories
RSD: = RSD calculated using the recoveries for laboratory i
The maximum RPD was then calculated as:
RSD
pool
Where:
L =
2 =
the total number of laboratories
a constant used to convert an RSD to an RPD (when calculated using 2 values, the RSD and RPD
differ by a factor of V2 ).
25
February 2004
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EPA Method 1603 Validation Study Results
The calculated MS/MSD QC acceptance criteria are listed in Table 15.
Table 15. Calculated Matrix Spike Precision and Recovery Acceptance Criteria
Performance test
Mean percent recovery for MS or MS/MSD
Precision (as maximum relative percent difference of MS/MSD)
Lab-prepared acceptance criteria
13% -149%
94%
February 2004
26
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EPA Method 1603 Validation Study Results
SECTION 7.0 ASSESSMENT OF METHOD PERFORMANCE: DISCUSSION AND
CONCLUSIONS
Results of this Study enabled assessment of the Method's performance in PBS and disinfected wastewater and
the development of quality control (QC) acceptance criteria that can be used to confirm acceptable laboratory and
method performance on an ongoing basis in disinfected wastewater monitoring surveys and other studies.
Method performance was evaluated through the assessment of false positive and negative rates in unspiked
disinfected and unspiked secondary wastewater samples and the evaluation of precision and recovery in
disinfected wastewater samples spiked with laboratory-prepared spiking suspensions. It should be noted that
outlier analyses were not conducted for the assessment of method performance, as all valid data were included in
the assessment of method performance.
7.1 Spiking Suspension (BioBalls™ and Laboratory-Prepared) Evaluation
Results from samples spiked with BioBalls™ were not used to develop QC acceptance criteria or assess method
performance because the enumeration of BioBalls™ on TSA at the participant laboratories indicated that the
BioBall™ spikes were highly variable. The pooled within-lab RSD of the participant laboratory TSA
enumerations was 28.2% and 6.8% for BioBalls™ and laboratory-prepared spiking suspensions, respectively.
Based on these participant laboratory TSA enumerations, BioBall™ variability was significantly greater than
lab-prepared spiking suspension variability, based on an F-test analysis of the log-transformed spike results. The
high level of BioBall™ variability was unexpected based on previous studies using this product.
In an effort to determine the cause of the high BioBall™ spike variability, the manufacturer of this product, BTF,
enumerated 50 BioBalls™ that remained from the batch used during the Study (B149) using nutrient agar spread
plates. The RSD of these enumerations was 7.0%, compared to 28.2% observed by the Study participants,
indicating that either the BioBalls™ were damaged during shipment or that the laboratories were not able to
accurately enumerate the BioBalls™. During the Study, the BioBall™ shipment from BTF (Sydney, Australia) to
DynCorp/CSC (Alexandria, VA) was delayed and resulted in an increased shipping temperature. Upon receipt at
DynCorp/CSC, very little dry ice remained in the shipping container, and while the BioBalls™ were cool, they
were not cold. In an effort to assess the effect of shipping conditions on BioBall™ organism viability and
variability, BTF conducted a study simulating four shipping conditions:
1. shipping container filled with dry ice (approximately 5 kg)
2. shipping container without ice
3. new super insulated shipping container with blue ice
4. validation study simulation using shipping container with 0.5 kg of dry ice
For each shipping condition, 9 vials, each containing one BioBall™, were placed in a shipping container and
packed on a Friday afternoon as stated above. After the containers were packed, they were placed in the trunk of
a car. Containers for the first three shipping conditions were held in the trunk over the weekend and enumerations
conducted on Monday morning using nutrient agar spread plates. For the fourth shipping condition, which was
designed to simulate shipping during the validation study, the container was removed from the trunk Saturday
evening and placed in a -20°C freezer overnight, returned to the trunk Sunday morning, removed from the trunk
Monday morning, packed with blue ice, and returned to the trunk overnight Enumerations were conducted on
Tuesday morning. This simulated the shipping conditions from the manufacturer to DynCorp/CSC and from
DynCorp/CSC to the participating laboratories. A comparison of the mean counts using an ANOVA indicated a
significant difference among shipping conditions and the Tukey's test indicated that mean count from the
validation study shipping simulation (27.2 mean count) was significantly lower than the mean count from
BioBalls™ stored in containers filled with dry ice (30.9 mean count). Based on the Hartley's test (Reference 8.5)
27 February 2004
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EPA Method 1603 Validation Study Results
for non-constant variance, the variability between the four shipping conditions was not significantly different. So,
while the Tukey's test indicated that E. coll counts were slightly lower for the validation study shipping
simulation, based on the results of the Hartley's test, it does not appear that the delayed shipment contributed to
the BioBall™ variability issue, though it is possible that BTF simply was unable to simulate the same conditions.
Another potential cause of the BioBall™ variability problem may be inadequate BioBall™ dissolution.
BioBalls™ must be completely dissolved in order to obtain an accurate spread plate count. During previous
validation studies using BioBalls™, some laboratories observed confluent growth on the BioBall™ TSA spread
plates. As a result, BTF reduced the recommended amount of sterile phosphate buffered saline (PBS) solution
used to dissolve BioBalls™ on the spread plates from 200 uL to 100 uL. A few laboratories indicated that with
the reduced amount PBS, the BioBalls™ were more difficult to dissolve and were somewhat fibrous. The
BioBall™ moisture content may cause the BioBalls™ to adhere to the spreader when enumerating the BioBalls
on the TSA spread plates and could also interfere with BioBall™ dissolution.
7.2 Method Performance
All laboratories participating in the Study reported pinpoint-size E. coll colonies that were extremely difficult to
read without the use of a microscope. Seven of the eight participant laboratories used modified mTEC media
from lot #3007935, and the eighth laboratory used media from lot #3112205. During preliminary analyses
conducted at EPA's NRMRL laboratory (using modified mTEC lot #3231326), E. coli colony size was generally
2 to 3 mm. The modified mTEC media utilized by the participant laboratories required a pH adjustment to
achieve the required pH of 7.3 ± 0.2, with most laboratories reporting a pH of 6.8 prior to adjustment. The
modified mTEC media used at NRMRL's laboratory had an initial pH of 7.1, which did not require a pH
adjustment. The different pH observations at participant vs. the NRMRL lab indicates that there may be an issue
with lot-to-lot media variability. Variability between lots of medium may impact colony size and possibly method
performance.
Method 1603 recovery of E. coli was acceptable, with mean laboratory-specific recoveries of E. coli from
disinfected wastewater samples spiked with laboratory-prepared spikes ranging from 47.8% to 106%, with an
overall mean recovery of 80.7%. Laboratory-specific RSDs from disinfected wastewater samples spiked with
laboratory-prepared spikes ranged from 6.1% to 51.4%, with a pooled, within-laboratory RSD of 25.9%.
False positive rates were also acceptable, with laboratory-specific false positive rates for unspiked
disinfected/secondary results combined, ranging from 0% at four labs, to 5.8% at one lab, and 22.2% at one lab.
The laboratory that observed the 22.2% false positive rate submitted 18 typical colonies to verification, of which
four did not confirm as E. coli. It should be noted that three of the four isolates were considered false positives
because they did not produce growth when streaked for isolation onto modified mTEC plates. The lack of growth
may be have been caused by failing to allow the loop to cool prior to streaking or possibly by using an insufficient
amount of growth to inoculate the plates. For secondary wastewater (excluding disinfected results), only one of
41 typical colonies submitted to verification were non-f. coli, resulting in a false positive rate of 2.4% for
secondary wastewater. For disinfected wastewater (excluding secondary results), only four of 70 typical colonies
submitted to verification were non-£. coli (either identified as Enterobacter aerogenes or Klebsiella pneumoniae
or had no growth), resulting in a false positive rate of 5.7% for disinfected wastewater.
False negative rates were also acceptable, with laboratory-specific false negative rates for unspiked
disinfected/secondary results combined, ranging from 0% - 6.7%. For secondary wastewater (excluding
disinfected results), only two of 33 atypical colonies submitted to verification were identified as E. coli, resulting
in a false negative rate of 6.1% for secondary wastewater. For disinfected wastewater (excluding secondary
results), three of 75 atypical colonies submitted to verification were identified as E. coli, resulting in a false
negative rate of 4.0% for disinfected wastewater.
February 2004 28
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EPA Method 1603 Validation Study Results
7.3 Conclusion
Based on the results of this Study, Method 1603 is considered valid for use in determining the concentration of E.
coll in disinfected wastewater.
29 February 2004
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EPA Method 1603 Validation Study Results
SECTION 8.0 REFERENCES
8.1 American Public Health Association, American Water Works Association, and Water Environment
Federation. 1995. Standard Methods for Water and Wastewater. 20* Edition. Sections: 9020, 9221,
9222.
8.2 American Society for Testing and Materials. 1998. Annual Book of ASTM Standards, Vol. 11.01.
Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee
D-19 on Water, ASTM D2777-98, October 1998.
8.3 SAS Institute Inc. 1994. SAS/STAT User's Guide, Volume 2, GLM-VARCOMP. Version 6,4*
Edition, June 1994.
8.4 USEPA. 2002. EPA Method 1603: Escherichia coli (E. coli) Water by Membrane Filtration Using
Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC), EPA-821-R-02-023,
September 2002.
8.5 Neter, John, W. Wasserman, and M. H. Kutner. Applied Linear Statistical Models. 3ri Edition. Richard
D. Irwin, Inc. Burr Ridge, IL, 1990. Pages 619-620.
February 2004 30
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EPA Method 1603 Validation Study Results
SECTION 9.0 ACRONYMS
CPU Colony forming unit
IPR Initial precision and recovery
MS Matrix spike
MSD Matrix spike duplicate
OPR Ongoing precision and recovery
QA Quality assurance
QC Quality control
RPD Relative percent difference
RSD Relative standard deviation
SAS Statistical analysis software
SD Standard deviation
31
February 2004
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Appendix A:
Method 1603 Spiking Protocol
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EPA Method 1603 Validation Study Results
E. co/i Spiking Protocol
Intel-laboratory Wastewater Validation Study of Method 1603
(August 20, 2003)
The purpose of this protocol is to provide laboratories with E. coli spiking procedures for the interlaboratory
wastewater validation study of Method 1603. During this study, laboratories will spike samples using laboratory-
prepared spiking solutions and with BioBalls™ (a commercially available product from BioTechnology Frontiers,
Sydney, Australia). The following sections are included in this protocol:
Laboratory-Prepared Spiking Solutions
Section 1: Preparation of Laboratory-Prepared Spiking Suspensions
Section 2: Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration
Section 3: Calculation of Laboratory-Prepared Spike Percent Recovery
BioBalls™
Section 4: BioBall™ Sample Spiking and BioBall™ Enumeration
Section 5: Calculation of BioBall™ Spike Percent Recovery
1.0 Preparation of Laboratory-Prepared Spiking Suspensions
1.1 Stock Culture. Prepare a stock culture by inoculating a trypticase soy agar (TSA) slant (or other non-
selective media) with Escherichia coli ATCC #11775 and incubating at 35°C ± 3°C for 20 ± 4 hours. This
stock culture may be stored in the dark at room temperature for up to 30 days.
1.2 1% Lauryl Tryptose Broth (LTB). Prepare a 1% solution of LTB by combining 99 mL of sterile
phosphate buffered saline (Method 1603, Section 7.4) and 1 mL of sterile single strength lauryl tryptose
broth in a sterile screw cap bottle or re-sealable dilution water container. Shake to mix.
1.3 Spiking Suspension (Undiluted). From the stock culture of E. coli ATCC #11775 in Section 1.1, transfer
a small loopful of growth to the 1 % LTB solution and vigorously shake a minimum of 25 times. Incubate
at 35°C ± 3°C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0 * 107 to 1.0
* 10s E. coli colony forming units (CFU) per mL. This is referred to as the "undiluted spiking
suspension." Note: During the Method 1603 validation study, growth of spiking suspensions will begin on
Monday, so the spiking suspensions are ready to be spiked into the samples on Tuesday.
1.4 Proceed to Section 2.0 for sample spiking and enumeration of spiking suspension.
2.0 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration
Since the objective of spiking the sample is to establish percent recovery, it is necessary to determine the number
ofE. coli in the undiluted spiking suspension prepared in Section 1.3. This section provides instructions for
sample spiking (Section 2.1) and spiking suspension enumeration (2.2).
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EPA Method 1603 Validation Study Results
2.1 Sample spiking
2.1.1 Dilute spiking suspension
2.1.1.1 Mix the spiking suspension by vigorously shaking the bottle a minimum of 25 times.
Use a sterile pipette to transfer 1.0 mL of the undiluted spiking suspension (from
Section 1.3 above) to 99 mL of sterile phosphate buffered saline (Method 1603,
Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "A". A 1.0-mL volume of dilution "A" is 10"2 mL
of the original undiluted spiking suspension.
2.1.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspension ^lution "A" (from
Section 2.1.1.1 above) to 99 mL of sterile phosphate bufferec iline (Method 1603,
Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "B". A 1.0-mL volume of dilution "B" is 10"4 mL
of the original undiluted spiking suspension.
2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" (from
Section 2.1.1.2 above) to 99 mL of sterile phosphate buffered saline (Method 1603,
Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "C". A 1.0-mL volume of dilution "C" is 10"5 mL
of the original undiluted spiking suspension.
2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" (from
Section 2.1.1.3 above) to 99 mL of sterile phosphate buffered saline (Method 1603,
Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "D". A 1.0-mL volume of dilution "D" is 10"6 mL
of the original undiluted spiking suspension.
2.1.2 Spike sample(s)
2.1.2.1 To spike sample, add 0.3 mL of spiking suspension dilution "D" (from Section 2.1.1.4
above) to 100-mL of unspiked sample and mix by vigorously shaking the bottle a
minimum of 25 times. This is the "spiked" sample. The volume (mL) of undiluted
spiking suspension added to each 100 mL of sample is 3.0 x 10"7 mL per 100 mL [(0.3
mL x 10"6 mL) per 100 mL of sample], which is referred to as V5piked p^ 100 mL samplem
Section 3.2 below. This is the "spiked" sample. Analyze the spiked sample according to
the instructions provided in Method 1603, Section 11.0.
2.2 Enumeration of undiluted spiking suspension (prepared in Section 1.3)
2.2.1 Prepare trypticase soy agar (TSA) according to manufacturer's directions, add 10 - 15 mL of TSA
per 100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note: Agar
plates must be dry and free from condensation prior to use. To ensure that the agar surface is
dry, plates should be made several days in advance and stored inverted at room temperature or
dried using a laminar-flow hood.
2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread
plates:
• Pipet 0.1 mL of dilution "B" (Section 2.1.1.2) onto surface of pre-dried TSA plate [ 10'5 mL
(0.00001) of the original spiking suspension].
• Pipet 0.1 mL of dilution "C" (Section 2.1.1.3) onto surface of pre-dried TSA plate [10"* mL
(0.00000l)of the original spiking suspension].
• Pipet 0.1 mL of dilution "D" (Section 2.1.1.4) onto surface of pre-dried TSA plate [ 1O'7 mL
(0.000000 l)of the original spiking suspension].
February 2004 A-2
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EPA Method 1603 Validation Study Results
2.2.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum over the
surface of medium by rotating the dish by hand or on a turntable. Note: Please ensure that the
inoculum is evenly distributed over the entire surface of the plate.
2.2.4 Allow inoculum to absorb into the medium completely.
2.2.5 Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours.
2.2.6 Count and record number of colonies per plate. Refer to Section 3.0 for calculation of spiking
suspension concentration.
3.0 Calculation of Spiked E. coli Percent Recovery
Spiked E. coli percent recovery will be calculated in three steps as indicated in Sections 3.1 through 3.3 below.
Note: The example calculated numbers provided in the tables below have been rounded at the end of each step. If
your laboratory recalculates the examples using a spreadsheet and rounds only after the final calculation (Step
3), the percent recoveries may be slightly different.
3.1 Step 1 : Calculate Concentration of E. coli (CPU I ml_) in Undiluted Spiking Suspension
3.1 .1 The number of E. coli (CPU / mL) in the undiluted spiking suspension (prepared in Section 1 .3
above) will be calculated using all TSA plates from Section 2.2 yielding counts within the ideal
range of 30 to 300 CPU per plate.
3.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if the colonies are not discrete,
results should be recorded as "too numerous to count" (TNTC).
3.1 .3 Calculate the concentration of E. coli (CPU / mL) in the undiluted spiking suspension according
to the following equation. (Example calculations are provided in Table 1 below.)
E. coli undilutedsf,ke = (CPU, + CFU2 + ... + CFUn) / (V, + V2 + ... + Vn)
Where,
E. coli
CPU
E. coli (CPU / mL) in undiluted spiking suspension
Number of colony forming units from TSA plates yielding
counts within the ideal range of 30 to 300 CPU per plate
Volume of undiluted sample on each TSA plate yielding
counts within the ideal range of 30 to 300 CPU per plate
Number of plates with counts within the ideal range
A-3
February 2004
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EPA Method 1603 Validation Study Results
TABLE 1.
EXAMPLE CALCULATIONS OF E. COLI SPIKING SUSPENSION CONCENTRATION
Examples
Example
1
Example
2
CPU / plate (triplicate analyses) from
TSA plates in Section 2.2.5
10*mL plates
TNTC, TNTC,
TNTC
269, 289, 304
10* mL plates
94, 106, 89
24, 30, 28
10'7mL plates
10,0,4
0,2,0
£. coli CFU / mL in undiluted
spiking suspension
(^C undiluted »pika)
(94+106+89) / (lO^+IO^+IO"6) =
289 / (3.0 x 10"6) = 96,333,333 =
9.6x107CFU/mL
(269+289+30) / (lO^+IO^+IQ-6) =
588 / (2.1 x 10-5) =28,000,000 =
2.8x107CFU/mL
*EC
undiluted spike
is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate
3.2 Step 2: Calculate "True" Spiked E. coli (CFU /100 mL)
3.2.1 Calculate true concentration of spiked E. coli (CFU /100 mL) according to the following
equation. Example calculations are provided in Table 2 below.
'Spiked B. coli= (E- CO" undiluted spike) X ( ^ spiked per 100 mL sample)
Where,
1 Spiked £ coli
spiked per 100 mL sample
Number of spiked E. coli (CFU / 100 mL)
E. coli (CFU / mL) in undiluted spiking suspension
(calculated in Section 3.1.3)
mL of undiluted spiking suspension per 100 mL sample
(Section 2.1.2.1)
Table 2. Example Calculations of Spiked E. coli
EC undiluted spike
(Table 1 above)
9.6x107CFU/mL
2.8x107CFU/mL
w
* spMnd par ISO mL umpte
(Section 2.1.2.1 above)
3.0X10-7mLper100ml_
of sample
3.0X10-7mLper100mL
of sample
' Spik»d E. CO//
(9.6 x 107 CFU / mL) x (3.0 x 10'7 mL / 100 mL) =
28.8 CFU / 100 mL
(2.8 x 107 CFU / mL) x (3.0 x 10'7 mL / 100 mL) =
8.4CFU/100mL
February 2004
A-4
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EPA Method 1603 Validation Study Results
3.3 Step 3: Calculate Percent Recovery
3.3.1 Calculate percent recovery (R) using the following equation.
R=100x
(NS-NU)
Where,
R
N,
K,
= Percent recovery
E. coli (CFU /100 mL) in the spiked sample (Method 1603, Section 12)
E. coli (CPU /100 mL) in the unspiked sample (Method 1603, Section
12)
= True spiked E. coli (CFU / 100 mL) in spiked sample (Section 3.2,
above)
Note: During the validation study, Nu (unspiked sample) is the mean E. coli (CFU /100 mL) of the 4
unspiked disinfected wastewater samples.
3.3.2 Example percent recovery calculations are provided in Table 3.
Table 3. Example Percent Recovery Calculations
N. (CFU / 100 mL)
42
34
16
10
Nu(CFU/100mL)
<1
. 10
<1
<1
TsoiKedE.cc// (CFU/ 100 mL)
28.8
28.8
8.4
8.4
Percent recovery (R)
100 x (42 -1)/ 28.8
= 142%
100 x (34-10)728.8
= 83%
100x(16-1)/8.4
= 179%
100 x (10-1) 78.4
= 107%
4.0 BioBall™ Sample Spiking and Enumeration
During the validation study, each laboratory will enumerate E. coli in the BioBalls™ so that percent recovery can
be evaluated. This section provides instructions for sample spiking (Section 4.1) and spiking suspension
enumeration (4.2).
After receipt at your laboratory, the BioBalls™ should be stored at -20°C. Preparation of the BioBalls™ prior to
spiking is not necessary, as they can be spiked directly into the sample once the vial is opened.
A-5
February 2004
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EPA Method 1603 Validation Study Results
4.1 Sample spiking
4.1.1 Open BioBall™ vial by removing the c up and cap. To spike a sample, aseptically add 1
BioBall™ to 100 mL of unspiked sample and mix by vigorously shaking the bottle a minimum of
25 times. This is the "spiked" sample. Analyze the spiked sample according to the instructions
provided in Method 1603, Section 11.0.
4.2 Enumeration of BioBall™ (used in Section 4.1 above)
4.2.1 Prepare trypticase soy agar (TSA) according to manufacturer's instructions, add 10 -15 mL of
TSA per 100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note:
Agar plates must be dry and free from condensation prior to use. To ensure that the agar surface
is dry, plates should be made several days in advance and stored inverted at room temperature or
dried using a laminar-flow hood.
4.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of three spread
plates:
• Open BioBall™ vial by removing the crimp and cap. Aseptically place one BioBall™ onto
the center of each pre-dried TSA plate by tipping the vial over the medium.
Immediately pipette 100 \il of sterile phosphate buffered saline solution (Method 1603,
Section 7.4) directly onto the BioBall™.
Allow the BioBall™ to dissolve.
4.2.3 For each spread plate, using a sterile bent glass rod or spreader, distribute the BioBall™ inoculum
over surface of medium by rotating the dish by hand or on a turntable. Note: Please ensure that
the inoculum is evenly distributed over the entire surface of the plate.
4.2.4 Allow inoculum to absorb into the medium completely.
4.2.5 Invert plates and incubate at 35°C ± 3°C for 20 ± 4 hours.
4.2.6 Count and record number of colonies per plate. Refer to Section 5.0 for calculation of the
concentration of E. coli in the BioBall™.
5.0 Calculation of BioBall™ Spike Percent Recovery
Spiked BioBall™ percent recovery will be calculated following the steps indicated below. Note: The example
calculated numbers provided in the tables below have been rounded at the end of each step. If your laboratory
recalculates the examples using a spreadsheet and rounds only after the final calculation (Step 3), the percent
recoveries may be slightly different.
5.1 Step 1: Calculate Mean E. coli per BioBall and "True" Spiked E. coli (CPU /100 mL)
The mean concentration of E. coli (CPU) in the BioBalls™ will be calculated using all three TSA plates
from Section 4.2. Since one BioBall is spiked per 100 mL sample, use the mean number of E. coli per
BioBall as the "true" spiked E. coli per 100 mL sample. For example,
Tspiked £. co//
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EPA Method 1603 Validation Study Results
5.2 Step 2: Calculate Percent Recovery
Calculate percent recovery (R) using the following equation.
R= 100 x
(N.-NJ
Where,
R
N,
Nu
^Spiked£ coli
Percent recovery
E. coli (CPU /100 mL) in the spiked sample (Method 1603, Section 12)
E. coli (CPU /100 mL) in the unspiked sample (Method 1603, Section 12)
True spiked E. coli (CPU /100 mL) in spiked sample (Section 5.2, above)
Note: During the validation study, Nu (unspiked sample) is the mean E. coli (CPU /100 mL) of the 4
unspiked disinfected wastewater samples.
Example percent recovery calculations are provided in Table 4.
Table 4.
Example Percent Recovery Calculations
Ns (CPU / 100 mL)
24
36
Nu(CFU/100mL)
<1
10
T(CFU/100mL)
32
32
Percent recovery (R)
100 x (24 -1)/ 32 = 72%
100 x (36 - 10) / 32 = 81%
A-7
February 2004
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Appendix B:
Wastewater Laboratory Capabilities Checklist
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EPA Method 1603 Validation Study Results
Wastewater Laboratory Capabilities Checklist
(June 17, 2003)
EPA plans to invite 11 laboratories (10 participants and 1 referee) to participate in a study to validate the modified
mTEC (E. coif) and mEI (enterococci) methods in wastewater effluent. EPA will provide all media and disposable
materials for the study and will also cover all shipping costs. Volunteers will be acknowledged in both the method
and validation study reports.
The schedule will include two weeks (range-finding and validation study) of analyses per method with an
additional week of practice analyses prior to the study. The study is tentatively scheduled to begin in September,
with some potential analyses being conducted by the referee laboratory in August.
If your laboratory is interested in participating in the validation study as either a participant laboratory or the
referee, please provide the requested information below and fax the signed, completed checklist to Mike Chicoine
at 703.461.8056 by Thursday July 3. In addition, please send the form electronically to Mike Chicoine at
mike.chicoine(a).dvncorp.com.. Mike will confirm receipt of the checklist. If you have any questions pertaining to
the information requested below or the validation study, please do not hesitate to contact Yildiz Chambers at
703.461.2165 or vildiz.chambers@,dvncorp.com.
Section 1. Laboratory Capabilities and Experience
a. Please complete the requested capabilities and experience information below. The information requested in
Table 1 pertains to experience with a given method, regardless of matrix (i.e., surface water, wastewater)
analyzed.
Table 1. Analyst Experience
Analyst
Years of experience or estimated number of samples analyzed
Methods to be validated
modified
mTEC
mEI
Other membrane filter methods
mEndo or
LES Endo
mFC
NA-MUG
mTEC
mE/EIA
b. Primary analyst's name:
c. Primary analyst's years of experience performing wastewater analyses:
d. What certifications does your laboratory have for microbial analyses?
e. Additional comments:
Section 2. Background Information
B-l
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EPA Method 1603 Validation Study Results
a. Does your laboratory have access to wastewater samples? Yes No
If yes, please indicate in Table 2 or Table 3, as appropriate, the types ofwastewater to which you have
access.
b. If your laboratory has experience analyzing wastewater samples for E. coli and/or enterococci, in Table 2,
place a check " /" next to the wastewater type(s) to which you have access and indicate the method(s) used
for analysis and typical concentrations of each analyte. If your laboratory does not have experience analyzing
wastewater samples for E. coli and/or enterococci, please complete Table 3.
Table 2. E. coli and Enterococci
Access?
Example
/
Wastewater type
Primary treated
Raw
Primary treated
Secondary treated
Tertiary treated
Chemically
disinfected
Monitoring
frequency
1 per month
E. coli
Methods
SM 9221 B/F
Typical range
30x1^
Enterococci
Methods
SM 9230B
Typical range
12 x 103
c. If your laboratory has experience analyzing wastewater samples for total coliforms, fecal coliforms, fecal
streptococci, or other indicator organisms, in Table 3, place a check " /" next to the wastewater rype(s) to
which you have access and indicate the method(s) used for analysis and typical ranges.
Table 3. Other Indicator Organisms
Access
?
Wastewater type
Raw
Primary treated
Secondary treated
Tertiary treated
Chemically disinfected
Monitoring
frequency
Other indicator organisms
Organism(s)
Methods
Typical range
February 2004
B-2
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EPA Method 1603 Validation Study Results
d. How many membrane filtration funnels will be available for use during the study?
e. How many funnels may be used at one time (i.e., the size of the manifold that will be used to analyze samples
3, 6, etc.)?
f. In Table 3, below, please indicate the medium used for isolation prior to inoculation of each type of
verification procedure used in your laboratory.
Table 3. Verification Procedures
Verification procedure
API20E®
VITEK®
BIOLOG
BBL Crystal™
Other (please describe below)*
Isolation medium
*If other, please describe:
g. Is your laboratory potentially interested in verifying isolates from other laboratories? Yes No
Section 3. Referee Laboratory
If your laboratory is interested in participating in this study as the referee laboratory, please respond to the
following questions.
a. Has your laboratory served as a referee laboratory for other studies? If yes, please briefly describe the study
or studies.
b. Does your laboratory have experience preparing and enumerating spiking suspensions? (Please provide a
brief description including organism(s) and associated study.)
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EPA Method 1603 Validation Study Results
b. Does your laboratory have experience isolating and propagating environmental (wild type) strains from
environmental samples? (Please provide a brief description including organism(s) and associated sample(s).
c. Is your laboratory's shipping and receiving department familiar with the shipment of dangerous goods?
(Dangerous goods shipping containers and associated shipping documentation will be provided.)
d. If preparation of spiking suspensions is necessary, does your laboratory have sufficient personnel, supplies
(e.g., flasks, etc.), and equipment (e.g., incubator space) to propagate spiking suspensions
(E. coli and enterococci) and ship the suspensions to the participant laboratories?
e. If the participant laboratories spike samples with wastewater from their own facility, does your laboratory
have sufficient personnel, supplies and equipment to enumerate spikes in triplicate for each participant?
f. Additional comments:
I certify that the information provided above is accurate and complete:
Primary Analyst or Lab Manager:
Laboratory name:
Signature:
Date:
February 2004 B-4
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