EPA-821 -R-04-008
vxEPA
United States
Environmental Protection
Agency
Results of the Interlaboratory Validation of
EPA Method 1600 (mEI) for Enterococci
in Wastewater Effluent
February 2004
-------
U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-04-008
U.S. Environmental Protection
Region 5, Library (PL-12J)
77 West Jackson Boulevard, 12th Floor
Chicago, IL 60604-3590
-------
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 (HAD) 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
-------
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.
-------
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
-------
Table of Contents
Section 1 .0 Background
1.1 Summary of the Method
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
2.2.2 Participant 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 [[[ 9
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 .................................. . ............................ ... 1 1
Section 5.0 Results ......... . [[[ 12
5.1 Unspiked Sample Results [[[ 12
5.2 Spiked Disinfected Sample Results .................................................. 15
5.3 Spiked PBS Results [[[ 17
Section 6.0 Development of QC Acceptance Criteria .............................................. 19
-------
List of Tables
Table 1. Method 1600 Validation Study Analyses Performed by Each Laboratory 5
Table 2. Comparison of ASTM Recommendations and the Method 1600 Study 7
Table 3. Laboratories Participating in the Interlaboratory Validation of Method 1600 9
Table 4. Summary of Enterococci Results from Unspiked Disinfected Wastewater Samples 13
Table 5. Summary of False Positive and False Negative Rates Associated with Unspiked Disinfected
and Unspiked Secondary Wastewater Effluents 13
Table 6. Laboratory-Specific False Positive and False Negative Rates Associated with Unspiked
Wastewater Effluents 14
Table 7. Summary of Enterococci Results from Disinfected Samples Spiked with BioBalls™ 15
Table 8. Summary Enterococci Results from Disinfected Samples Spiked with Laboratory-Prepared
Spiking Suspensions 16
Table 9. Summary of Enterococci Results from PBS Samples Spiked with BioBalls™ 17
Table 10. Summary of Enterococci Results from PBS Samples Spiked with Laboratory-Prepared
Spiking Suspensions 18
Table 11. Calculated Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria 22
Table 12. Calculated Matrix Spike Precision and Recovery Acceptance Criteria 24
-------
List of Appendices
Appendix A: Method 1600 Spiking Protocol A-1
Appendix B: Wastewater Laboratory Capabilities Checklist B-l
VI
-------
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 enterococcus in wastewater: EPA
Method 1600 which uses membrane-enterococcus indoxyl-|3-D-glucoside agar medium (mEI). The September
2002 version of the Method (EPA-821-R-02-022) 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.
Twelve volunteer participant laboratories, an enterococci verification laboratory, and a research laboratory
participated in the Study which was conducted during the week of September 22,2003. Each laboratory spiked
samples with BioBall™ Enterococcus faecalis (E.faecalis) spikes and laboratory-prepared E.faecalis spikes.
Samples were spiked in accordance with the Method 1600 spiking protocol (the "Spiking Protocol," Appendix
A). Results from samples spiked with BioBall™ 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. QC criteria were also developed based on results of the laboratory-prepared
spikes (in addition to criteria developed based on BioBalls™) to ensure that QC criteria will be available for these
methods, if BioBalls™ become unavailable.
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
1600. Method 1600 recovery of enterococci was acceptable, with mean laboratory-specific recoveries of
enterococci from disinfected wastewater samples spiked with BioBalls™ ranging from 77.1% to 114.9%, with an
overall mean recovery of 90.8%. Laboratory-specific relative standard deviations (RSDs) ranged from 0% to
69.5%, with an overall pooled, within-laboratory RSD of 22.6%.
False positive rates were also acceptable, with laboratory-specific false positive rates for unspiked
disinfected/secondary results combined, ranging from 0% - 27.8%. For secondary wastewater (excluding
disinfected results), only 11 of 132 typical colonies submitted to verification were non-enterococci, resulting in a
false positive rate of 8.3% for secondary wastewater. For disinfected wastewater (excluding secondary results),
only three of 69 typical colonies submitted to verification were non-enterococci, resulting in a false positive rate
of 4.3% for disinfected wastewater.
In contrast, laboratory-specific false negative rates for unspiked disinfected/secondary results combined, ranged
from 13.3% - 100.0%. For secondary wastewater (excluding disinfected results), 62 of 118 atypical colonies
submitted to verification were identified as enterococci, resulting in a false negative rate of 52.5% for secondary
wastewater. For disinfected wastewater (excluding secondary results), eight of 12 atypical colonies submitted to
verification were identified as enterococci, resulting in a false negative rate of 66.7% for disinfected wastewater.
Results of this study indicate that Method 1600 precision, recovery, and false positive rates are acceptable for the
determination of enterococci in disinfected wastewater. However, false negative rates observed during this study
were high and should be taken into consideration when using results from this method. When evaluating
wastewater using Method 1600, it is recommended that the false negative rate for each matrix be evaluated
through biochemical confirmation and results adjusted accordingly, especially if large numbers of atypical
colonies are observed in a particular matrix. If very few atypical colonies are observed in samples for a particular
matrix, the high false negative rates observed during this study may be less of a concern.
VII
-------
EPA Method 1600 Validation Study Results
SECTION 1.0 BACKGROUND
The enterococci test is recommended as a measure of recreational water quality. Epidemiological 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 1600 was recently approved for monitoring
ambient waters for enterococci (68 FR 43272, July 21,2003). Prior to this study, none of the approved
enterococci 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 enterococci methods
for evaluation of wastewater effluents.
1.1 Summary of the Method
Method 1600 is a membrane filtration procedure for the detection of enterococci in water samples. In Method
1600, a water sample is filtered through a membrane (0.45 um pore-size), which retains the bacteria. After
filtration, the membrane containing the bacteria is placed on a selective medium, mEI agar, and incubated at 41°C
± 0.5°C for 24 hours. All colonies that produce a blue halo (regardless of color) are considered enterococci.
February 2004
-------
EPA Method 1600 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 1600 sensitivity and specificity across multiple laboratories and disinfected wastewater
matrices through the assessment of false positive and negative rates
• Characterize Method 1600 accuracy (recovery and precision) across multiple laboratories and disinfected
wastewater effluents
• Establish Method 1600 quantitative Quality Control (QC) acceptance criteria for initial and ongoing
laboratory and method performance assessments
Establish Method 1600 quantitative QC acceptance criteria for matrix spikes
To accomplish these objectives, this Study was conducted in five 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 confirm that the spiking approach for the Study was acceptable; 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 ofE.faecalis
ATCC #19433 (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 ofE.faecalis
ATCC #19433 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 1600 (EPA-821-R-02-022) or
approved changes to these procedures as provided to participant laboratories during the course of the Study.
This data quality objective was developed to ensure that data were of known and reliable quality, thereby
allowing EPA to use the results of the Study to identify the need for further revision of the method.
February 2004
-------
EPA Method 1600 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 the procedure for preparation of the E.faecalis spiking suspensions that would be used as the
laboratory-prepared spiking suspensions during the Study. The research laboratory (Section 3.1) confirmed that
appropriate spike levels could be obtained by growing E.faecalis in 1% azide dextrose broth as specified in the
Method 1600 Spiking Protocol (Appendix A).
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 3.3) were representative of the
general user community, with experience analyzing wastewater or ambient water samples for enterococci 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 of E.faecalis ATCC #19433 according to the
Method 1600 Spiking Protocol (Appendix A). Results from samples spiked with BioBalls™ were used to assess
inter- and intra-laboratory precision and recovery (method performance) and to develop QC acceptance criteria.
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
3 February 2004
-------
EPA Method 1600 Validation Study Results
technique and tryptic soy agar (TSA) as described in the Method 1600 Spiking Protocol (Appendix A), to
confirm stability of the BioBalls™.
2.4 Phase 3 Technical Approach: Lab-Prepared Spiking Suspensions
Phase 3 involved each laboratory preparing and enumerating spiking suspensions ofE.faecalis ATCC #19433
according to the Method 1600 Spiking Protocol (Appendix A). To ensure that QC criteria are available for this
method if BioBalls™ become unavailable, QC criteria were also developed based on results of the laboratory-
prepared spikes. Results from samples spiked with laboratory-prepared spiking suspensions were not used to
assess method performance.
Spiking suspensions were enumerated in triplicate, using the spread plate technique and TSA as described in the
Method 1600 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 1600 at multiple laboratories to analyze unspiked/spiked wastewater samples
and PBS samples farE.faecalis.
The following objectives were established for Phase 4:
• Generate false positive and negative rate data for Method 1600 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 1600 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 1600 in disinfected wastewater effluents
• Develop quantitative QC acceptance criteria for Method 1600 in sterile PBS to support future assessments of
laboratory performance
• Develop quantitative QC acceptance criteria for Method 1600 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
-------
EPA Method 1600 Validation Study Results
Table 1. Method 1600 Validation Study Analyses Performed by Each Laboratory
Matrix
Disinfected
wastewater
Secondary
Disinfected
wastewater
Secondary a
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 enterococci
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 matrix spike
QC criteria (Section 2.5.5)
Develop quantitative QC criteria for
IPR c and OPR " (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
1 Colonies from these samples were only submitted to verification when a sufficient number of colonies from disinfected
samples were not available
b N/A: Not applicable
c IPR: Initial Method/Laboratory Performance Assessment
" 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 1600 was assessed through the evaluation of false positive and false
negative rates. Each of the 12 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
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
February 2004
-------
EPA Method 1600 Validation Study Results
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 240
typical and 240 atypical colonies (including colonies from disinfected and secondary samples).
For each colony submitted to verification, the laboratory streaked the colony onto an mEI agar plate for isolation,
inverted the plate, and incubated at 41°C ± 0.5°C for 24 ±2 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 mEI plate and streaked
for growth onto a tryptic soy agar (TSA) plate with 5% sheep blood (one TSA plate for every mEI 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 enterococci
during the Study, gram positive identification (GPI) 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)
Method bias was evaluated through the analysis of two disinfected wastewater samples spiked with BioBalls™ at
each of the participant laboratories. Each sample was spiked with a single BioBall™ containing a lot mean value
(provided by the manufacturer) of 32.1 E.faecalis ATCC #19433 colony forming units (CFU). Recovery was
assessed by comparing E.faecalis 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. 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 using ATCC #19433 in both cases. Samples spiked with BioBalls™ were spiked with approximately
32.1 CFU per sample and samples spiked with laboratory-prepared spiking suspensions were spiked with
approximately 68.5 CFU per sample. QC criteria were developed based on results of the laboratory-prepared
spikes, in addition to developing criteria based on BioBalls™, to ensure that QC criteria are available for Method
1600, if BioBalls™ become unavailable.
February 2004
-------
EPA Method 1600 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
#19433 in both cases. Samples spiked with BioBalls™ were spiked with approximately 32.1 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 either turbidity or high enterococci concentrations in the samples (see Section
4.1). It should be noted that the same two disinfected wastewater samples spiked with BioBalls™ 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.faecalis (ATCC #19433) served as the positive control and E. coll (ATCC #11775) as the negative control.
2.5.7 Minimum Validation Study Requirements
The Study met or exceeded the ASTM D2777-98 (Reference 10.2) method validation recommendations with
every respect except number of concentrations. 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. Table 2 presents a comparison of the Study with ASTM
D2777-98 validation study requirements.
Table 2. Comparison of ASTM Recommendations and the Method 1600 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 1600 Study
12 participant laboratories
1 matrix type plus reference matrix (PBS)
Samples from a total of 12 facilities plus reference matrix (PBS)
1 concentration
96 IPR samples (for each of 12 participants, 4 replicate PBS
samples spiked with BioBalls™ and 4 replicate samples spiked
with lab-prepared spikes)
48 MS samples (for each of 12 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 02777-98), October 1998.
February 2004
-------
EPA Method 1600 Validation Stvdy Results
SECTION 3.0 STUDY IMPLEMENTATION
3.1 Study Management
This study was designed under the direction of the Office of Science ana 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 the 1% azide dextrose broth and Enterococcus faecalis (ATCC #19433) following
the Method 1600 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 actual
validation study analyses occurred the week of September 22,2003. Range-finding analyses were conducted on
Monday, September 22nd. Isolates were received at the verification laboratory on September 26, 2003.
Verifications were started on October 2,2003 and completed on October 30,2003.
February 2004
-------
EPA Method 1600 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 1600 *
City of Los Angeles Bureau of Sanitation
Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve
Espineda, and Zora Bahariance
Hyperion Treatment Plant
12000 Vista del Mar, Playa del Rey, CA 90293
County Sanitation Districts of L.A. County (JWPCP)
Kathy Walker, Michele Padilla, and Albert Soof
24501 South Figueroa Street, Carson, CA 90745
County Sanitation Districts of L.A. County (SJC)
Shawn Thompson and Julie Millenbach
1965 South Workman Mill Road, Whittier, 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
1432 Air Rail Avenue, Virginia Beach, VA 23471
Hoosier Microbiological Laboratories
Don Hendrickson, Katy Bilger and Lindsey Shelton
912 West McGalliard, Muncie, IN 47303
Massachusetts Water Resources Authority
Steve Rhode and Mariya Gofhsteyn
190 Tafts Avenue, Winthrop, MA 02152
North Shore Sanitation District
Robert Flood
William Koespel Drive, Guemee, IL 60031
Texas A&M University
Suresh Pillai and Reema Singh
4180 Kleberg Center, 2472 TAMUS, College Station, TX
77843
University of Iowa, Hygienic Laboratory
Nancy Hall and Cathy Lord
Oakdate 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.
February 2004
-------
EPA Method 1600 Validation Study Results
SECTION 4.0 DATA REPORTING AND VALIDATION
4.1 Data Reporting
Laboratories submitted the following data to DynCorp 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 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 enterococci
• Confirmation that media sterility checks were performed and acceptable
• Confirmation that positive and negative controls were performed and exhibited the appropriate response
• Confirmation that 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 perform all of the required quality control checks (pre-
filtration blank and negative control) during the validation study.
It should be noted that several laboratories were instructed to spike less than 100 mL of disinfected wastewater
because of either turbidity or high enterococci concentrations in me 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:
• Laboratory 1: Due to high enterococci concentrations, Laboratory 1 was instructed to spike 1 mL of
disinfected wastewater per sample.
Laboratory 9: Laboratory 9 spiked 100 mL of disinfected effluent and filtered three volumes (i.e., 50, 30,
and 10 mL) to obtain countable plates.
• Laboratory 10: Laboratory 10 could not filter volumes greater than 25 mL due to the turbidity of the
disinfected effluent samples. As a result, Laboratory 10 was instructed to spike 25 mL of disinfected
wastewater per sample. Note/Results from Laboratory 10, Sample 8 (disinfected wastewater spiked with a
BioBall™) were not available because of confluent growth.
• Laboratories 11 and 12: Due to high enterococci concentrations, Laboratories 11 and 12 were instructed to
spike 10 mL of disinfected wastewater per sample.
Results from Laboratory 10, Sample 8 (disinfected wastewater spiked with a BioBall™) were not available
February 2004 10
-------
EPA Method 1600 Validation Study Results
because of confluent growth.
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 enterococci per 100 mL, 18 results; <4
enterococci per 100 mL, four results; <10 enterococci per 100 mL, one result; and <100 enterococci 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. It should be noted that at first glance, replacing the < 100 enterococci per 100 mL observed in
Laboratory 1's unspiked disinfected wastewater with 50 enterococci per 100 mL seems unreasonable. However,
that laboratory's other results for unspiked disinfected wastewater were 700, 300, and 300 enterococci per 100
mL. Since the other results were much higher than the < 100 value, simply removing the < 100 from the data set
for estimating the concentration of background enterococci in the disinfected sample was deemed inappropriate
and half the censor limit was used instead.
11 February 2004
-------
EPA Method 1600 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 are provided in
Section 5.1, results of spiked disinfected wastewater samples are provided in Section 5.2, and results of spiked
PBS samples are provided in Section 5.3. 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 enterococci 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). Valid
verification results from unspiked disinfected and secondary wastewater effluent samples are summarized in
Table 5. Valid, laboratory-specific verification results are summarized in Tabk 6. Any typical colony that was
identified as non-enterococci by the Vitek® was considered a false positive result. Any atypical colony that was
identified as an enterococci 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 one of the two verification streak plates (mEI or
blood agar). Isolates that did not grow on blood agar at the verification laboratory were treated as if they had not
been submitted to verification and eliminated from subsequent data analyses. Colonies that did not grow after
streaking for isolation on mEI agar plates, were considered to be non-enterococci and included in data analyses as
non-enterococci colonies. The decision to include the colonies that exhibited no growth on the mEI as non-
enterococci was based on the selective nature of the medium, as the mEI was considered the first step in the
verification process.
February 2004 12
-------
EPA Method 1600 Validation Study Results
Table 4. Summary of Enterococci Results from Unspiked Disinfected Wastewater Samples (see Table 1
for cross-reference to sample number)
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Sample number (CPU a/100 mL)
1
<100
; ^*A^
<1
<1
<1
<1
4
<1
84
<4
30
10
2
700
> "'", '3&K
'»"** :' ',&?:"
0.88
0.50
0.63
0.50
6.00
0.50
76.25
2.00
47.50
8.75
43.73
SDb
268.87
I'V- ' ' " ^
0.75
0.00
0.25
0.00
2.83
0.00
5.44
0.00
22.17
2.50
81.4*
Colony forming units
" Standard deviation
c 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
Table 5. Summary of False Positive and False Negative Rates Associated with Unspiked Disinfected
and Unspiked Secondary Wastewater Effluents
Matrix (Sample No.)
Unspiked Disinfected
(Samples 1 - 4)
Unspiked Secondary
(Samples 5, 6)
Unspiked
Disinfected/Secondary
(Samples 1-6)
False Positive Assessment
Typical
colonies
submitted
69
132
201
Number of false
positives
3
11
14
False positive
rate (%)
4.3
8.3
7.0
False Negative Assessment
Atypical
colonies
submitted
12
118
130
Number of false
negatives
8
62
70
False negative
rate (%)
66.7
52.5
53.9
13
February 2004
-------
EPA Method 1600 Validation Study Results
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
1
2
3
4
5
6
7
8
9
10
11
12
False Positive Assessment
Typical
colonies
submitted
20
14
17
18
20
17
18
20
19
20
18
Number of false
positives
0
0
0
0
2
0
5
0
4
3
0
False positive
rate(%)
0.0
0.0
0.0
0.0
10.0
0.0
27.8
0.0
21.1
15.0
0.0
False Negative Assessment
Atypical
colonies
submitted
15
Number of false
negatives
14
3
6
17
3
6
17
False negative
rate (%)
93.3
;^/'^%?^
100.0
100.0
100.0
' -
11
15
18
14
18
13
4
2
8
4
7
5
36.4
13.3
44.4
28.6
38.9
38.5
Data validation discussed in Section 4.2
b Atypical colonies were only observed on filters with colonies that were too numerous to count (TNTC)
February 2004
14
-------
EPA Method 1600 Validation Study Results
5.2 Spiked Disinfected Sample Results
Results from disinfected wastewater samples spiked with BioBalls™ (Table 7) were used to assess method
performance (see discussion in Section 7) and develop QC acceptance criteria for matrix spikes for use in
assessing matrix interferences (see discussion in Section 6). Results from disinfected wastewater samples spiked
with laboratory-prepared spiking suspensions (Table 8) were used to develop QC acceptance criteria (Section 6,
these data were not used to assess method performance).
Table 7. Summary of Enterococci Results from Disinfected Samples Spiked with BioBalls™ (see Table
1 for cross-reference to sample number)
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Spike Level
(CFU/100mL)''b
3210
Percent Recovery by Sample
7
95.4
8
95.4
.iL^iL^v^* ^ ,<:> * "w
32.1
32.1
32.1
32.1
32.1
32.1
32.1
128.4
321
321
87.6
76.3
82.2
95.1
96.6
101.2
58.4
82.6
81.8
93.8
87.6
91.9
91.5
73.2
90.3
85.7
171.3
72.4
97.0
Overall (n = 21)
Mean Percent Recovery
95.4
SDe
0.0
«^;:,^;w , ^-''<^*T«
87.6
84.1
86.8
84.1
93.5
93.5
114.9
82.6
77.1
95.4
90.8
0.0
11.0
6.6
15.4
4.4
11.0
79.9
"' " " NtM flS!
* , ^^*™!
6.6
2.2
25.2'
RSD'(%)
0.0
<;<~'^r^
0.0
13.1
7.6
18.3
4.7
11.8
69.5
sessed :
8.6
2.3
22.6<>
Colony forming units
b Spike level is based on the tot 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 either high turbidity or high background concentrations of
enterococci)
c Standard deviation
d 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
8 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 1600 Validation Study Results
Table 8. Summary Enterococci Results from Disinfected Samples Spiked with Laboratory-Prepared
Soikinq Suspensions (see Table 1 for cross-reference to sample number)
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Spike Level
(CFU/100 mL)"*
3440
Percent Recovery by Sample
9
26.5
55.2
64.2
44
193
60.7
62.52
32
114
1080
710
87.2
75.5
105.4
43.8
42.8
90.4
84.6
107.0
74.3
28.3
10
25.1
103.5
81.8
78.1
46.4
41.2
71.2
58.6
86.0
81.7
28.3
Overall (n = 22)
Mean Percent Recovery
25.8
95.3
78.7
91.8
45.1
42.0
80.8
71.6
96.5
78.0
28.3
66.7
SDe
1.0
11.5
4.4
19.3
1.8
1.2
13.6
18.4
14.9
5.2
0.0
10.9'
RSD"(%)
4.0
«4:ftv«'i-';i^,-
VJ, ... , ,„* .
12.1
5.6
21.0
4.1
2.8
16.8
25.7
15.4
6.7
0.0
13.1"
Colony forming units
b 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 either high turbidity or high background concentrations of
enterococti)
c 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
8 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 1600 Validation Study Results
5.3 Spiked PBS Results
Results from PBS samples spiked with BioBalls™ (Table 9) and 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 Enterococci Results from PBS Samples Spiked with BioBalls™ (see Table 1 for
cross-reference to sample number)
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Spike Level
(CFU/IOOmL)"*
32.1
32.1
32.1
32.1
32.1
32.1
32.1
32.1
32.1
32.1
32.1
Percent Recovery by Sample
11
93.5
99.7
102.8
102.8
90.3
87.2
87.2
99.7
93.5
90.3
96.6
12
99.7
13
96.6
14
102.8
Mean Percent
Recovery
98.1
e •' ^Slii^:^^ • ''.:• '"' *:>$!$*$:'' •'-.':
96.6
96.6
102.8
87.2
99.7
87.2
93.5
93.5
87.2
99.7
109.0
90.3
99.7
93.5
71.7
96.6
105.9
96.6
99.7
90.3
93.5
84.1
115.3
96.6
93.5
84.1
77.9
99.7
109.0
102.8
Overall (n = 44)
99.7
93.5
105.1
91.9
88.0
88.8
94.2
95.8
96.6
97.4
95.4
SDe
4.0
t >': •>;;?;• =
6.7
8.0
6.9
4.0
12.0
5.4
12.0
3.0
9.9
5.3
7.6'
«-•«
4.1
4
6.8
8.6
6.6
4.4
13.7
6.1
12.8
3.1
10.2
5.5
8.1-
Colony forming units
" Spike level is based on the tot mean value provided by the manufacturer
c Standard deviation
" Relative standard deviation
8 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
9 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 1600 Validation Study Results
Table 10. Summary of Enterococci Results from PBS Samples Spiked with Laboratory-Prepared
Spiking Suspensions (see Table 1 for cross-reference to sample number)
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Spike Level
(CFU/IOOmL)1*
34.4
•*?•', ' 4
Ife-'Jfllfe^SC
55.2
64.2
44
193
60.7
62.5
32
28.5
108
71
Percent Recovery by Sample
15
49.4
90.6
76.3
81.8
46.1
37.9
76.8
93.8
80.7
86.1
42.3
16
26.2
123.2
77.9
86.4
51.8
44.5
120.0
93.8
77.2
80.6
26.8
17
34.9
83.3
84.1
84.1
51.3
34.6
96.0
125.0
98.2
82.4
23.9
18
20.3
Mean Percent
Recovery
32.7
65.2
82.6
118.2
43.0
32.9
88.0
81.3
77.2
82.4
36.6
Overall (n = 44)
90.6
80.2
92.6
48.1
37.5
95.2
98.4
83.3
82.9
32.4
70.4
SDe
12.6
RSD-(%)
38.7
24.2
3.7
17.1
4.2
5.1
18.3
18.7
10.1
2.3
8.5
13.4'
26.7
4.6
18.5
8.8
13.6
19.2
19.0
12.1
2.8
26.3
20.0"
Colony forming units
b Spike level is based on laboratory enumeration of spiking suspension on tryptic soy agar (ISA) plates
c Standard deviation
d Relative standard deviation
' Data validation discussed in Section 4.2
' Pooled withln-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
18
-------
EPA Method 1600 Validation Study Results
SECTION 6.0 DEVELOPMENT OF QC ACCEPTANCE CRITERIA
All data analyses described below were performed using the results of disinfected wastewater and PBS samples
spiked with either BioBalls™ or laboratory-prepared spiking suspensions. Separate QC acceptance criteria were
calculated for BioBall™ and laboratory-prepared spike results, to ensure that QC criteria are available for this
method if BioBalls™ become unavailable.
6.1 Outlier Analyses
Valid results from samples spiked with BioBalls™ and 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.
It should 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), 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, results were stratified by spike type (BioBalls™ vs laboratory-prepared), using recoveries from both
disinfected wastewater and PBS samples. Youden's test was conducted separately by spike type, because separate
criteria were to be calculated for BioBall™ and laboratory-enumerated spiked samples. Sample results for each
laboratory were sorted based on sample number. Prior to conducting the Youden's laboratory ranking test, one
missing result was replaced by the mean of the non-missing results for the given spike type, matrix, and
laboratory. This replacement value was only used for the Youden test and was not used in any other data
analyses. Based on the Youden's test, two laboratories were removed for laboratory-prepared sample analyses.
These two laboratories (Laboratories 1 and 12) were both biased low compared to the remaining laboratories,
and were not used in the development of QC acceptance criteria. (The low recoveries from the laboratory-
prepared spiking suspensions may be related to spiking suspensions not being sufficiently homogenized prior to
spiking samples or enumerating the spiking suspensions.) No laboratories were removed for BioBall™ spiked
sample analyses.
6.1 .2 Grubbs Test for Individual Outlying Sample Results
After removing outlying laboratories identified using 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) and spike type. 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 resulted in the removal of
one BioBall™ disinfected wastewater sample result analyzed by Laboratory 9 with a recovery of 171%. This
result was not used in the development of QC acceptance criteria.
February 2004
-------
EPA Method 1600 Validation Study Results
6.2 Initial Precision and Recovery (IPR) and Ongoing Precision and Recovery (OPR)
QC acceptance criteria for initial precision and recovery (DPR) and ongoing precision and recovery (OPR) were
developed based on the results from PBS (reference matrix) samples spiked with BioBalls™ and 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. Again, separate QC acceptance criteria were
calculated for BioBall™ and laboratory-prepared spike results
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 likelihood 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, respe. vely.
The combined standard deviation for IPR (isj is:
Where:
L = number of labs for the given spiking procedure
n, = number of PBS sample results for laboratory if or the given spiking procedure
nT = total number of PBS results from all laboratories for the given spiking procedure
Upper and lower limits for IPR samples were then calculated as:
y +1 *iv
•* Mem - '(0.975; Uf) "c
Where idfis calculated using Satterthwaite 's estimate as given below:
IY L \
1"'
1 i '•'
i i r"
V /
1
*«2
SL
2
\(l 0* *1
\A nj " _
L-\ nT-L
February 2004
20
-------
EPA Method 1600 Validation Study Results
The combined standard deviation (osc) for OPR is:
Where:
L — number of labs for the given spiking procedure
n, = number of PBS sample results for laboratory if or the given spiking procedure
nT = total number of PBS results from all laboratories for the given spiking procedure
Upper and lower limits for OPR samples were then calculated as:
y + t * s*e
•"- Mean — l(0.975;oay) UAc
Where odfis calculated using Satterthwaite 's estimate as given below:
odf = -
OS,
'( I >
Z-?
1 1 '•'
n\
V. )
*s2
•*i
2
\-
"M'-1-"
V nT)
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:
*SD^- =
(n,-l)*RSD?
Where:
L = number of laboratories for the given procedure
n, = number of PBS sample results for laboratory ifor the given spiking procedure
nT = total number of PBS sample results for all laboratories for the given spiking procedure
RSD, - RSD calculated using the recoveries for laboratory ifor the given spiking procedure
21
February 2004
-------
EPA Method 1600 Validation Study Results
The maximum RSD was then calculated as:
-Ł)
pool
Where:
nT - total number oflPR and OPR results from all laboratories
L = number of laboratories
The calculated DPR and OPR QC acceptance criteria are provided in Table 11.
Table 11. 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
BioBall™
acceptance criteria
85% - 106%
14%
78% -113%
Lab-prepared spike
acceptance criteria
31% - 127%
28%
27% -131%
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 data
from the spiked disinfected wastewater matrices used in the validation study. Separate QC acceptance criteria
were calculated for BioBall™ and laboratory-prepared spike results
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 likelihood 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.
February 2004
22
-------
EPA Method 1600 Validation Study Results
Estimates of between laboratory variance and within laboratory variance were labeled s\and s2w, respectively.
The combined standard deviation for MS/MSD (sc) is:
1
Where:
L = number of labs for the given spiking procedure
n, = number of disinfected sample results for laboratory ifor the given spiking procedure
nT = total number disinfected sample results from all laboratories for the given spiking procedure
Upper and lower limits for MS/MSD samples were then calculated as:
4- / *
Mean — ' (0.975;df)
Where:
Km*™ = tne wea/i recovery of all disinfected wastewater samples, and for the given spiking procedure
dfis calculated using Satterthwaite 's estimate as given below:
*/
T v 2!
i . i=l
1+ j
nT
\ 1
•*s2
SL
2
— j_ -
rr i
1+ — *s2
\ nT)
L-l
nT- 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.
23
February 2004
-------
EPA Method 1600 Validation Study Results
The pooled RSD was then calculated as:
lv
= ,->. RSD?
1=1
Where:
L = number of laboratories for the given spiking procedure, and
RSD, = RSD calculated using the recoveries for laboratory if or the given spiking procedure
The maximum RPD was then calculated as:
RSD
pool
Where:
L -
2 =
the total number of 'laboratories for the given spiking procedure, and
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 ).
The calculated MS/MSD QC acceptance criteria are listed in Table 12.
Table 12. 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)
BioBall™
acceptance criteria
63% -110%
28%
Lab-prepared
acceptance
criteria
29% - 122%
47%
February 2004
24
-------
EPA Method 1600 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
enabled 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 through the evaluation of precision and recovery in
disinfected wastewater samples spiked with BioBalls™. (Results from samples spiked with laboratory-prepared
spiking suspensions were not used to assess method performance.) 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.
Results from the laboratory enumerations of the BioBalls™ indicate that BioBall™ spikes ofE.faecalis (ATCC
#19433) were stable and precise, as laboratory enumeration of the BioBalls™ estimated a mean of 29.9 CPU per
BioBall™ and a pooled within-laboratory RSD of 10.5%, compared to the manufacturer's lot mean value of 32.1
CPU per BioBall™ and RSD of 7.8%. In contrast, the laboratory-prepared spiking suspensions (used for
development of QC acceptance criteria) were significantly less precise (based on an F-test comparing the pooled
within-laboratory RSDs at a=0.05), with a pooled within-laboratory RSD of 24.0%.
Method 1600 recovery of enterococci was acceptable, with mean laboratory-specific recoveries of enterococci
from disinfected wastewater samples spiked with BioBalls™ ranging from 77.1% to 114.9%, with an overall
mean recovery of 90.8%. Laboratory-specific RSDs ranged from 0% to 69.5%, with a pooled, within-laboratory
RSD of 22.6%.
False positive rates were also acceptable, with laboratory-specific false positive rates for unspiked
disinfected/secondary results combined, ranging from 0% - 27.8%. For secondary wastewater (excluding
disinfected results), only 11 of 132 typical colonies submitted to verification were non-enterococci, resulting in a
false positive rate of 8.3% for secondary wastewater. For disinfected wastewater (excluding secondary results),
only three of 69 typical colonies submitted to verification were non-enterococci, resulting in a false positive rate
of 4.3% for disinfected wastewater.
In contrast, laboratory-specific false negative rates for unspiked disinfected/secondary results combined, ranged
from 13.3% - 100.0%. For secondary wastewater (excluding disinfected results), only 62 of 118 atypical colonies
submitted to verification were identified as enterococci, resulting in a false negative rate of 52.5% for secondary
wastewater. For disinfected wastewater (excluding secondary results), eight of 12 atypical colonies submitted to
verification were identified as enterococci, resulting in a false negative rate of 66.7% for disinfected wastewater.
Results of this study indicate that Method 1600 precision, recovery, and false positive rates are acceptable for the
determination of enterococci in disinfected wastewater. However, false negative rates observed during this study
were high and should be taken into consideration when using results from this method. When evaluating
wastewater using Method 1600, it is recommended that the false negative rate for each matrix be evaluated
through biochemical confirmation and results adjusted accordingly, especially if large numbers of atypical
colonies are observed in a particular matrix. If very few atypical colonies are observed in samples for a particular
matrix, the high false negative rates observed during this study may be less of a concern.
25 February 2004
-------
EPA Method 1600 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. 20th 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 6,4th
Edition, June 1994.
8.4 USEPA. 2002. EPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane-
Enterococcus Indoxyl-p-D Glucoside Agar (mEI), EPA-821-R-02-022, September 2002.
February 2004 26
-------
EPA Method 1600 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
27
February 2004
-------
-------
Appendix A:
Method 1600 Spiking Protocol
-------
-------
EPA Method 1600 Validation Study Results
Enterococci Spiking Protocol
Interlaboratory Wastewater Validation Study of Method 1600
(August 20,2003)
The purpose of this protocol is to provide laboratories with enterococci spiking procedures for the interlaboratory
wastewater validation study of Method 1600. 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 Enterococcus faecalis ATCC #19433 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% Azide Dextrose Broth. Prepare a 1% solution of azide dextrose broth by combining 99 mL of sterile
phosphate buffered saline (Method 1600, Section 7.4) and 1 mL of sterile single strength azide dextrose
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 Enterococcus faecalis ATCC #19433 in
Section 1.1, transfer a small loopful of growth to the 1% Azide dextrose broth 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 * 106 to 1.0 x 107 enterococci colony forming units (CFU) per mL. This is
referred to as the "undiluted spiking suspension." Note: During the Method 1600 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
of enterococci 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).
A-1 February 2004
-------
EPA Method 1600 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 1600,
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 dilution "A" (from
Section 2.1.1.1 above) to 99 mL of sterile phosphate buffered saline (Method 1600,
Section 7.4), cap, and mix by vigorously snaking 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 1600,
Section 7.4), cap, and mix by vigorously snaking 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 1600,
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 samplers')
2.1.2.1 To spike sample, add 3.0 mL of spiking suspen. n 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"* mL per 100 mL [(3.0
mL x 10"6 mL) per 100 mL of sample], which is referred to as V^^ ,„ 100 ^ ^^ in
Section 3.2 below. This is the "spiked" sample. Analyze the spiked sample according
to the instructions provided in Method 1600, 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 th^ agar surface is
dry, plates should be made several days in advance and stored inverted at re n 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 [W6 mL
(0.00000 l)of the original spiking suspension].
• Pipet 0.1 mL of dilution "D" (Section 2.1.1.4) onto surface of pre-dried TSA plate [ 10"7 mL
(0.000000 l)of the. original spiking suspension].
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
February 2004 A^2
-------
EPA Method 1600 Validation Study Results
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 Enterococci Percent Recovery
Spiked enterococci 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 Enterococci (CPU / mL) in Undiluted Spiking
Suspension
3.1.1 The number of enterococci (CFU / 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 CFU 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 enterococci (CFU / mL) in the undiluted spiking suspension
according to the following equation. (Example calculations are provided in Table 1 below.)
Enterococci undHutedspike = (CFU, + CFU2 + ... + CFUn) / (V, + V2 + ... + Vn)
Where,
Enterococci ^^^^ ^1*,, = Enterococci (CFU / mL) in undiluted spiking suspension
CFU = Number of colony forming units from TSA plates yielding
counts within the ideal range of 30 to 300 CFU per plate
V = Volume of undiluted sample on each TSA plate yielding
counts within the ideal range of 30 to 300 CFU per plate
n = Number of plates with counts within the ideal range
A-3 February 2004
-------
EPA Method 1600 Validation Study Results
TABLE 1.
EXAMPLE CALCULATIONS OF ENTEROCOCCI SPIKING SUSPENSION CONCENTRATION
Examples
Example
1
Example
2
CPU / plate (triplicate analyses) from
TSA plates in Section 2.2.5
10"5 mL plates
94, 106, 89
32, 55, 72
tO"6 mL plates
9,11,28
8,5,3
10-7mL plates
1,0.4
0,0,0
Enterococci CFU / mL in
undiluted
spiking suspension
(Enterococci ondllutad ^J*
(94+106+89) /(lO^+IO^+KT5) =
289 / (3.0 x 10*) = 9,633,333 =
9.6x106CFU/mL
(32+55+72) / (10*HO*+10*) =
159 / (3.0 x 10*) =5,300,000 =
5.3 x 10* CFU / mL
•Enterococci und,^,,*. 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 Enterococci (CFU /100 mL)
3.2.1 Calculate true concentration of spiked enterococci (CFU /100 mL) according to the following
equation. Example calculations are provided in Table 2 below.
'spiked Enterococci = (EnteFOCOCCI undj|utec| spike) X ( * spiked per 100 mL sample)
Where,
^ Spiked Enterococci
Enterococci
spiked perl 00 mL sample
Number of spiked Enterococci (CFU /100 mL)
Enterococci (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 Enterococci
Enterococci „„»,„*«, „*„
(Table 1 above)
9.6x106CFU/mL
5.3 x 106 CFU / ml
• spHwd par 100 mL nmpte
(Section 2.1.2.1 above)
3.0 XIO^mL per 100mL
of sample
S.OXIO^mLperlOOmL
of sample
• SplkKl Enterococci
(9.6 x 106 CFU / mL) x (3.0 x 10* ml / 100 mL) =
28.8 CFU /1 00 mL
(2.8 x 106 CFU / mL) x (3.0 x 10"6 mL / 100 mL) =
8.4 CFU / 100 mL
February 2004
A-4
-------
EPA Method 1600 Validation Study Results
3.3 Step 3: Calculate Percent Recovery
3.3.1 Calculate percent recovery (R) using the following equation.
Where,
R
N,
N_
1 Spiked Enterococci
Percent recovery
Enterococci (CFU /100 mL) in the spiked sample (Method 1600, Section
12)
Enterococci (CFU /100 mL) in the unspiked sample (Method 1600,
Section 12)
True spiked Enterococci (CFU / 100 mL) in spiked sample (Section 3.2,
above)
Note: During the validation study, Na (unspiked sample) is the mean enterococci (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/100mL)
42
34
16
10
Nu (CFU / 100 mL)
<1
10
<1
<1
Ts^edE-^occcc, (CFU/ 100 mL)
28.8
28.8
8.4
8.4
Percent recovery (R)
100 x (42 -1)7 28.8
= 142%
100 x (34-10)728.8
= 83%
100 x (16-1) 78.4
= 179%
100 x (10 -1)7 8.4
= 107%
4.0 BioBall™ Sample Spiking and Enumeration
During the validation study, each laboratory will enumerate enterococci 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
-------
EPA Method 1600 Validation Study Results
4.1 Sample spiking
4.1.1 Open BioBall™ vial by removing the crimp 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 1600, 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 ul of sterile phosphate buffered saline solution (Method 1600,
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 enterococci 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 Enterococci per BioBall™ and "True" Spiked Enterococci (CPU /
100mL)
The mean concentration of enterococci (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
enterococci per BioBall™ as the "true" spiked enterococci per 100 mL sample. For example,
Tspiked Enterococci (CFU /100 mL) = (30 + 38 + 28) / 3 = 32 per 100 mL
Where,
Tspikcd Enterococci = True spiked Enterococci (CFU /100 mL) in spiked sample
February 2004 A-6
-------
EPA Method 1600 Validation Study Results
5.2 Step 2: Calculate Percent Recovery
Calculate percent recovery (R) using the following equation.
R=100x
(N.-NJ
Where,
R
N,
N,,
T
x Spiked Enterococci
Percent recovery
Enterococci (CFU /100 mL) in the spiked sample (Method 1600, Section
12)
Enterococci (CFU /100 mL) in the unspiked sample (Method 1600, Section
12)
True spiked Enterococci (CFU / 100 mL) in spiked sample (Section 5.2,
above)
Note: During the validation study, Na (unspiked sample) is the mean enterococci (CFU /100 mL) of the 4
unspiked disinfected wastewater samples.
Example percent recovery calculations are provided in Table 4.
Table 4. Example Percent Recovery Calculations
N, (CFU / 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) 732 = 81%
A-7
February 2004
-------
-------
Appendix B:
Wastewater Laboratory Capabilities Checklist
-------
-------
EPA Method 1600 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@.dvncorr).coni.
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
LESEndo
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
February 2004
-------
EPA Method 1600 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 " Sn 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 dnd Enterococci
Access?
Example
/
Wastewater type
Primary treated
Raw
Primary treated
Secondary treated
Tertiary treated
Chemically
disinfected
Monitoring
frequency
1 per month
E. coli
Methods
SM9221B/F
Typical range
30x10*
Enterococci
Methods
SM 9230B
Typical range
12 x 10*
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 type(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
-------
EPA Method 1600 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
API 20E®
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.)
February 2004
-------
EPA Method 1600 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
------- |