United States
Environmental Protection
Agency
Results of the Interlaboratory Testing
Study for the Comparison of Methods
for Detection and Enumeration of
Enterococci and Escherichia coli in
Combined Sewer Overflows (CSOs)

September 2008

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U.S. Environmental Protection Agency
      Office of Water (4303T)
   1200 Pennsylvania Avenue, NW
      Washington, DC 20460
        EPA821-R-08-006

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                                                  Combined Sewer Overflow (CSO) Study Report
                                Acknowledgments

The contributions of the following persons and organizations to this study are gratefully acknowledged:

Volunteer Referee Laboratory

•  EPA Office of Research and Development, National Risk Management Research Laboratory: Mark
   C. Meckes and Laura Boczek

Volunteer Participant Laboratories

•  Lacey, Olympia, Turnwater, and Thurston (LOTT) Alliance: Paula Williamson and Paul Jue

•  Massachusetts Water Resources Authority (MWRA): Mariya Gofshteyn and Steve Rhode

•  Narragansett Bay Commission: Lisa Andrade, Walter Palm and John Motta

•  Pittsburgh Water and Sewer Authority: Jay Kuchta  and Stanley States

•  University of Iowa Hygienic Laboratory: Cathy Lord and Nancy Hall

•  Virginia Consolidated Laboratories: Debbie Paul, Bob Sulouff, Tom York, Jim Pearson and Grier
   Mills

•  Wisconsin State Laboratory of Hygiene (WSLH): Jeremy Olstadt, Linda Peterson, and Sharon
   Kluender

Volunteer Verification Laboratories

•  City of Los Angeles, Bureau of Sanitation, Environmental Monitoring Division: Gerald McGowen,
   Stan Asato, loannice Lee, Hung Pham, Pauline Nguyen, and Marieta Ravelo

•  Orange County Sanitation District (OCSD): Charlie McGee, Michael von Winckelman and Kim
   Patton

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Combined Sewer Overflow (CSO) Study Report
                                      Disclaimer

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 or OSTCWAMethods@epa.gov

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                                                   Combined Sewer Overflow (CSO) Study Report
                                 Table of Contents


Executive Summary	vii

Section 1.0 Background	1
  1.1   Summary of Methods	1
     1.1.1     Enterococci Test Methods	1
     1.1.2     E. coli Test Methods	1
Section 2.0 Study Objectives and Study Design	2

  2.1   Study Objectives	2
  2.2   Technical Approach	2
     2.2.1     Laboratory-Prepared Spike Stability Assessment	2
     2.2.2     Identification of Qualified Analytical Laboratories	3
      2.2.2.1   Referee Laboratory [EPA Office of Research and Development (ORD)/National Risk
               Management Research Laboratory (NRMRL)]	3
      2.2.2.2   Participant Laboratories	3
      2.2.2.3   Verification Laboratories	3
     2.2.3     Sample Collection	3
     2.2.4     Laboratory-Prepared Spiking Suspensions	4
     2.2.5     Validation Study Sample Analyses	4
      2.2.5.1   Preliminary Analyses	5
      2.2.5.2   Quality Control (QC) Analyses	5
      2.2.5.3   Assessment and Comparison of Method Sensitivity and Specificity	6
      2.2.5.4   Assessment and Comparison of Method Accuracy (Precision and Recovery)	6
      2.2.5.5   Development of Quantitative QC Criteria for Matrix Spikes (MS)	6
Section 3.0 Study Implementation	8

  3.1   Study Management	8
  3.2   Schedule	8
  3.3   Research and Participant Laboratories	8
Section 4.0 Data Reporting and Validation	9
  4.1   Data Reporting	9
  4.2   Data Validation	9
Section 5.0 Results	11
  5.1   Enterococci: Method 1106.1 and  1600 Unspiked Combined Sewer Overflow Sample Results. 11
  5.2   Enterococci: Method 1106.1 and  1600 Spiked Combined Sewer Overflow Sample Results	12
  5.3   E. coli: Method 1103.1 and 1603  Unspiked Combined Sewer Overflow Sample Results	13
  5.4   E. coli: Method 1103.1 and 1603  Spiked Combined Sewer Overflow Sample Results	15
  5.5   Enterococci: Method 1106.1 and  1600 Low- and High-Level Spiked PBS Sample Results	16
  5.6   E. coli: Method 1103.1 and 1603  Low-and High-Level Spiked PBS Sample Results	17
Section 6.0 Development of QC Acceptance Criteria	19
  6.1   Outlier Analyses	19
  6.2   Initial Precision and Recovery (IPR) and Ongoing Precision and Recovery (OPR)	19
Section 7.0 Assessment and Discussion of Method Performance	25
  7.1   Enterococci Methods	25
                                             in

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Combined Sewer Overflow (CSO) Study Report
    7.1.1     Method 1106.1	25
    7.1.2     Method 1600	25
    7.1.3     Comparison of Enterococci Method Performance	26
  7.2    E. co//Methods	28
    7.2.1     Method 1103.1	28
    7.2.2     Method 1603	28
    7.2.3     Comparison of E. coll Method Performance	29
Section 8.0 Conclusion	31

Section 9.0 References	32
Section 10.0 Acronyms	33
                                             IV

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                                                  Combined Sewer Overflow (CSO) Study Report
                                    List of Tables

Table 1.        Number of Sample Analyses per Laboratory for the Combined Sewer Overflow (CSO)
              Validation Study
Table 2.        Combined Sewer Overflow (CSO) Study Participant Laboratories
Table 3.        Summary of Valid, Enterococci Results for Unspiked Combined Sewer Overflow (CSO)
              Samples
Table 4.        Method 1106.1 Laboratory-Specific False Positive and False Negative Confirmation
              Rates for Unspiked Combined Sewer Overflow (CSO) Samples
Table 5.        Method 1600 Laboratory-Specific False Positive and False Negative Confirmation Rates
              for Unspiked  Combined Sewer Overflow (CSO) Samples
Table 6.        Summary of Valid, Enterococci Results for Spiked Combined Sewer Overflow (CSO)
              Samples
Table 7.        Summary of Valid, E. coll Results for Unspiked Combined Sewer Overflow (CSO)
              Samples
Table 8.        Method 1103.1 Laboratory-Specific False Positive and False Negative Confirmation
              Rates for Unspiked Combined Sewer Overflow (CSO) Samples
Table 9.        Method 1603  Laboratory-Specific False Positive and False Negative Confirmation Rates
              for Unspiked  Combined Sewer Overflow (CSO) Samples
Table 10.      Summary of Valid, E. coll Results for Spiked Combined Sewer Overflow (CSO)
              Samples
Table 11.      Summary of Valid, Enterococci Results for PBS Samples Spiked with Low-Level
              Spikes
Table 12.      Summary of Valid, Enterococci Results for PBS Samples Spiked with High-Level
              Spikes
Table 13.      Summary of Valid, E. coll Results for PBS Samples Spiked with Low-Level Spikes
Table 14.      Summary of Valid, E. coll Results for PBS Samples Spiked with High-Level Spikes
Table 15.      Calculated Initial Precision and Recovery (IPR) and Ongoing precision and Recovery
              (OPR) Acceptance Criteria for E.  coll Methods
Table 16.      Calculated Initial Precision and Recovery (IPR) and Ongoing precision and Recovery
              (OPR) Acceptance Criteria for Enterococci Methods
Table 17.      Calculated Matrix Spike Recovery Acceptance Criteria for E. coll Methods
Table 18.      Calculated Matrix Spike Recovery Acceptance Criteria for Enterococci Methods
Table 19.      Summary of Method 1106.1 and 1600 Enterococci Recoveries for Spiked PBS and
              Combined Sewer Overflow Samples
Table 20.      Comparison of Methods 1106.1 and 1600 False Positive and False Negative
              Confirmation Rates for Unspiked  CSO Matrices
Table 21.      Summary of Method 1103.1 and 1603 E. coll Recoveries for Spiked PBS and Combined
              Sewer Overflow Samples
Table 22.      Comparison of Methods 1103.1 and 1603 False Positive and False Negative
              Confirmation Rates for Unspiked  CSO Matrices

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Combined Sewer Overflow (CSO) Study Report
                              List of Appendices
Appendix A: Laboratory Capabilities Checklist	34
AppendixB: CSO Enterococci Spiking Protocol	41
Appendix C: CSOE. coli Spiking Protocol	49
                                          VI

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                                                   Combined Sewer Overflow (CSO) Study Report
                                Executive Summary

In 2001, EPA proposed several methods for both enterococci and Escherichia coll (E. coli) (FR 66:
45811) evaluation in ambient waters. During the comment period for this proposal, National Pollutant
Discharge Elimination System (NPDES) permit holders and others requested that EPA promulgate one or
more methods for these organisms for the evaluation of wastewater effluents (effluents) and combined
sewer overflows (CSOs). We promulgated Enterococci and E. coli methods for effluents in 2007 (FR
72:14220), but not for discharges that include CSOs. This report describes our evaluation of the
promulgated methods applied to CSO matrices. Previously there was no data on method performance in
CSO matrices.  Now we have data from five laboratories that may be used as a starting point for end
users. These data include quality control criteria for CSO matrices and PBS samples spiked at low and
high levels.

During rain events, a combined sewer overflow (CSO) event may occur. A CSO is defined as a discharge
from a combined sewer system (i.e., a wastewater collection system  owned by a State or municipality
which conveys sanitary wastewaters and storm water through a single-pipe system to a publicly owned
treatment works (POTW)) at a point prior to the POTW treatment plant.  CSOs are point sources that,
compared to typical wastewater effluent, often contain high levels of pathogenic microorganisms because
they contain stormwater, untreated human and industrial waste, toxic materials, and debris. These CSO
discharges are subject to NPDES permit requirements.

Method 1106.1 [Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin
Iron Agar (mE-EIA)], Method 1600 [Enterococci in Water by Membrane Filtration Using membrane-
Enterococcus Indoxyl-[3-D Glucoside Agar (mEI)], Method  1103.1 [Escherichia coli (E. coli)  in Water by
Membrane Filtration Using membrane-Thermotolerant&c/7en'c/7/a coli Agar (mTEC)], and Method 1603
[Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant
Escherichia coli Agar (Modified mTEC)] were followed during the study. The purposes of the Study
were to characterize method performance (precision and recovery) across multiple laboratories and
Combined Sewer Overflow matrices, and develop quantitative quality control (QC) acceptance criteria.

Seven volunteer participant laboratories, two verification laboratories, and one referee laboratory
participated in the Study which was conducted over a period of almost three years  from March 2005
through December 2007. Usable data sets were obtained from five of the seven labs.  During the study,
each laboratory spiked samples with laboratory-prepared Enterococcus faecalis (ATCC #19433) and
Escherichia coli (ATCC #11775) suspensions. High (500,000 E. faecalis or 3,000,000 E.  coli CFU/100
mL) and low (3000 CFU/100 mL) spike levels were used, depending on the CSO type: disinfected CSO
matrices were spiked with the low level spike, while total bypass CSO samples were spiked with the
high-level spike.  Samples were spiked in accordance with study-specific spiking protocols. Results from
unspiked and spiked CSO and PBS samples were used to assess method performance.

The mean percent recovery of enterococci for Method 1106.1 in disinfected wastewater and CSO's were
equivalent (86.3% and 86.8%, respectively). For the Enterococcus Method 1600, the mean percent
recovery was higher in disinfected wastewater (90.8%) than in CSO's (66.3%).  Recovery using either E.
coli method 1103.1 (57.8% in disinfected wastewater, 81% in CSO) or 1603 (67% in disinfected
wastewater, 91.7% CSO) was better when the method was used for disinfected wastewater than in CSO's.
                                             vn

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                                                    Combined Sewer Overflow (CSO) Study Report
Section 1.0      Background

Combined sewer overflow (CSO) events are discharges from municipal sewer systems or treatment plants
that occur when the volume of wastewater exceeds the system's capacity due to periods of heavy rainfall
or snow melt. During periods of increased flows treatment processes may be altered to maintain the
plants' integrity. Enterococci and Escherichia coll (E. coli) analyses are recommended as an indication of
recreational water quality. Epidemiological studies have led to the development of criteria which have
been used to establish recreational water standards based on established relationships between health
effects and water quality. Methods for monitoring these bacterial water quality indicators have recently
been approved for disinfected wastewaters and/or ambient/recreational waters.  National Pollutant
Discharge Elimination System (NPDES) permit holders and others have requested that EPA validate one
or more methods for the evaluation of enterococci and E. coli in CSO effluents (discharges). The
methods evaluated included EPA Methods 1106.1 (mE/EIA) and 1600 (mEI) and for enterococci, and
1103.1 (mTEC) and 1603 (modified mTEC) for E. coli (References 9.1- 9.4).

1.1     Summary of Methods

1.1.1   Enterococci Test Methods

In EPA Method 1106.1 (Reference 9.1), a water sample is  filtered through a 0.45 (im pore-size
membrane. Following filtration, the membrane is placed on a selective medium, mE agar, and incubated
at 41.0°C ± 0.5°C for 48 ± 3 hours.  Following incubation,  the filter is transferred to a differential
medium, EIA agar,  and incubated at 41.0°C ± 0.5°C for an additional 20-30 minutes. Pink to red colonies
that develop a black or reddish-brown precipitate on the underside of the filter are considered enterococci.

In EPA Method 1600 (Reference 9.2), a water sample is filtered through a 0.45 (im pore-size membrane.
After filtration, the membrane is placed on a selective medium, mEI agar, and incubated at 41.0°C ±
0.5 °C for 24 ± 2 hours.  All colonies greater than 0.5 mm in size that produce a blue halo (regardless of
color) are considered enterococci.

1.1.2   E. coli Test Methods

In EPA Method 1103.1 (Reference 9.3), a water sample is  filtered through a 0.45 (im pore-size
membrane. After filtration, the membrane is placed on a selective medium, mTEC, incubated at 35.0°C ±
0.5 °C for 2 ± 0.5 hours to resuscitate injured or stressed bacteria, and then incubated at 44.5 °C ± 0.2°C
for 22 ± 2 hours. Following incubation, the filter is transferred to a filter pad saturated with urea substrate
and left at room temperature for 15-20 minutes. All yellow, yellow-green or yellow-brown colonies are
considered E. coli.

In EPA Method 1603 (Reference 9.4), a water sample is filtered through a 0.45 (im pore-size membrane.
After filtration, the membrane is placed on a selective medium, modified mTEC agar, incubated at
35.0°C ± 0.5 °C for 2 ± 0.5 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.

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Combined Sewer Overflow (CSO) Study Report
Section 2.0      Study Objectives and Study Design


2.1    Study Objectives


The following objectives were established for the Study:

•   Characterize the stability of E. faecalis and E. coll laboratory-prepared spiking suspensions.

•   Characterize the sensitivity and specificity of each individual method across multiple laboratories and
    CSO matrices through the assessment of false positive and negative rates.

•   Characterize the accuracy (recovery and precision) of each individual method across multiple
    laboratories and CSO matrices.

•   Establish quantitative QC acceptance criteria for CSO matrix spike recoveries for each method.

•   Compare performance of the enterococci methods (1106.1 and 1600).

•   Compare performance of the E. coll methods (1103.1 and 1603).


The following data quality objectives were established for the Study:

•   Generate a minimum of 6 sets of valid data from participant laboratories for each method.

•   Data produced under this study must be generated according to the analytical and QA/QC procedures
    in each of the analytical methods or approved changes to these procedures to ensure that data is of
    known and reliable quality, and allows EPA to use the results of the study to identify any need for
    further revision of the method.


2.2    Technical Approach

Details on the technical approach for conducting this study are provided in Sections 2.2.1 to 2.2.5, below.

2.2.1  Laboratory-Prepared Spike Stability Assessment

Laboratory-prepared spiking suspension stability was assessed by the referee-laboratory prior to the CSO
validation study to determine viability of the suspensions over a four-day period. The purpose of this
assessment was to determine how frequently laboratories need to propagate laboratory-prepared spiking
suspensions in preparation for a potential CSO event. Assessment of laboratory-prepared spiking
suspension stability involved the enumeration of triplicate E. faecalis (ATCC #19433) and E. coll (ATCC
#11775) laboratory-prepared spiking suspensions at 24, 48, 72, and 96 hours after inoculation.

E. coli.  The laboratory enumerated three replicate laboratory-prepared E. coll spiking suspensions by the
following procedures.

    •  Spread plate technique using tryptic soy agar (TSA) plates (CSO validation study spiking
       protocol)
    •  EPA Method 1603 (modified mTEC)
    •  EPA Method 1103.1 (mTEC)

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                                                    Combined Sewer Overflow (CSO) Study Report
E.faecalis. The laboratory enumerated three replicate laboratory-prepared E. faecalls spiking
suspensions by the following procedures:

    •  Spread plate technique using TSA plates (CSO validation study spiking protocol)
    •  EPA Method 1600 (mEI)
    •  EPA Method 1106.1 (mE/EIA)

2.2.2  Identification of Qualified  Analytical Laboratories

Participant laboratories were chosen to be representative of the general user community, with experience
analyzing wastewater or ambient water samples for enterococci and E. coll using membrane filtration
techniques, and with access to representative CSO matrices. A detailed Laboratory Capabilities Checklist
was used to collect this information from laboratories and to screen potential participants to ensure that
laboratories were qualified.

2.2.2.1 Referee Laboratory [EPA Office of Research and Development (ORD)/National
       Risk Management Research Laboratory (NRMRL)]

Prior to the validation study the referee laboratory evaluated the stability of E. coll and E. faecalis. Based
on these results laboratories were instructed to propagate a "fresh" spiking suspension every fourth day.

2.2.2.2 Participant Laboratories

Participant laboratories analyzed samples to provide EPA with the data necessary to assess method
performance and develop QC acceptance criteria. Participant laboratories were representative of the
general user community, with some experience analyzing CSO samples using Methods 1600, 1106.1,
1603 and 1103.1.  Participants also needed to have access to a representative CSO matrix within driving
distance (2 hours) of the laboratory to ensure that holding times were met. A detailed Laboratory
Capabilities Checklist (Appendix A) was used to collect information from laboratories and screen
potential participants to ensure that laboratories were qualified. Laboratory availability was also
considered.

2.2.2.3 Verification Laboratories

Verification laboratories were required to have access to a Vitek® and experience characterizing either
isolates using the Vitek® system and gram-negative plus (GNI+) and/or gram-positive (GPI) cards.

To reduce cost, volunteer laboratories were recruited. To reduce the burden on participant laboratories
and to encourage volunteer participants, EPA provided the media, reagents, and disposable supplies
needed for stability assessment, validation and verification. Unlike other EPA method validation studies,
prepared plates (mE, EIA, mEI, mTEC, and modified mTEC) were provided to the laboratories to ensure
media was readily available when a CSO event occurred and to reduce the burden on the laboratories.
Use of dehydrated media would have required laboratories to prepare fresh media every two weeks.

2.2.3  Sample Collection

Samples were collected at the point of plant effluent discharge for this study. For untreated discharges, a
2-L bulk sample was collected and for disinfected discharges, a 3-L bulk sample was collected. Samples
were held at <10°C and above freezing prior to analysis and analyzed within 6 hours of sample collection.
Total bypass samples are those that were discharged without any treatment and secondary bypass
disinfected samples are those that went through primary treatment, disinfected, and then discharged.

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Combined Sewer Overflow (CSO) Study Report
2.2.4  Laboratory-Prepared Spiking Suspensions

Every fourth day that there was potential for a CSO event, participant laboratories propagated fresh
spiking suspensions of E. coll (ATCC #11775) and E. faecalis (ATCC #19433) to ensure a culture would
be available when a CSO event occurred. After incubation, laboratory-prepared suspensions were stored
in the refrigerator at <10°C and above freezing until a new suspension was propagated.

To determine the "true spike concentration" during the validation study, the participant laboratories were
directed to enumerate E. coli (ATCC #11775) and E. faecalis (ATCC #19433) laboratory-prepared
spiking suspensions on the same day that the validation study samples were spiked and analyzed.
Samples were spiked with laboratory-prepared spiking suspensions according to the CSO Spiking
Protocol (Appendix B).

    •  For disinfected CSO matrices, a 100-mL aliquot of each replicate was spiked with 3 x 10"4 mL of
       undiluted spiking suspension of E. faecalis (ATCC #19433) or with 3 xlO"5 mL of undiluted
       spiking suspension of E. coli (ATCC #11775), resulting in an approximate spike of 3000
       CFU/lOOmL.

    •  For total bypass CSO matrices, a 100-mL aliquot of each replicate was spiked with 5.0 x 10"2 mL
       of undiluted spiking suspension of E. faecalis (ATCC #19433) or with 3.0 x 10"2 mL of undiluted
       spiking suspension of E. coli (ATCC #11775), resulting in an approximate spike of 500,000 CPU
       or 3,000,000 CFU/100 mL, respectively.

       For sterile PBS samples spiked at a low-level, a 100-mL aliquot of each replicate was spiked with
       3 x 10"4 mL of undiluted spiking suspension ofE. faecalis (ATCC #19433) or with 3 x 1Q"5 mL of
       undiluted spiking suspension of E. coli (ATCC #11775), resulting in an approximate spike of
       3 000 CPU/100 mL.

    •  For sterile PBS samples spiked at a high-level, a 100-mL aliquot of each replicate was spiked
       with 5.0 x 10"2 mL of undiluted spiking suspension of E. faecalis (ATCC #19433) or with  3.0 x
       10"2 mL of undiluted spiking suspension ofE. coli (ATCC #11775), resulting in an approximate
       spike of 500,000 or 3,000,000 CFU/100 mL, respectively.

2.2.5  Validation Study Sample Analyses

During the validation study, all four methods (1103.1, 1106.1, 1600 and 1603) were used to analyze
unspiked and spiked CSO and PBS samples at multiple laboratories.  Table 1 summarizes the number and
type of samples that were evaluated to meet the objectives listed in Section 2.

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                                                   Combined Sewer Overflow (CSO) Study Report
Table 1.   Number of Sample Analyses per Laboratory and Method for the Combined
           Sewer Overflow (CSO) Validation Study
Matrix
Disinfected
Wastewater
Disinfected
Wastewater
Sterile PBS1
Sterile PBS1
CSO
CSO
Sterile PBS
Sterile PBS
Spiking
Description
Unspiked
Lab-prepared
BioBalls
Lab-prepared
Unspiked
Lab-prepared
(high or low spike
level, dependent on
CSO type)
Lab-prepared:
low-level spike
Lab-prepared:
high-level spike
Unspiked
No. of
Samples per
Method
1
1
4
1
4
4
3
3
1
Filters
per
Sample
5
5
1
3
3-5
3-5
3
5
1
Isolate
Verification
N/A
N/A
N/A
N/A
20 typical &
20 atypical
per sample
N/A
N/A
N/A
N/A
Purpose of Analysis
Preliminary analyses
Preliminary analyses
QC check for initial precision and
recovery (IPR)
Preliminary analyses
False positive and negative rates
Evaluation of ambient
background bacteria
concentrations
Assessment of method
performance and development of
QC criteria
Assessment of method and
laboratory performance
Assessment of method and
laboratory performance
QC check
 Phosphate buffered saline

2.2.5.1 Preliminary Analyses

Preliminary analyses were conducted using unspiked disinfected effluent, spiked disinfected effluent (low
level spike) and spiked PBS samples (low and high level spike) prior to the start of the validation study.
In addition, laboratories enumerated referee-prepared E. faecalis and E. coli spiking suspensions using
TSA plates and the spread plate technique.

2.2.5.2 Quality Control (QC) Analyses

Participating laboratories  completed the following QC analyses: media sterility checks, dilution water
sterility checks, filter sterility check, filtration blanks, positive controls, and negative controls.

    •  Methods 1600 and 1106.1. E. faecalis (ATCC #19433) served as the positive control and E. coli
       (ATCC #11775) as the negative control.
    •  Methods 1603 and 1103.1. E. coli (ATCC #11775) served as the positive control and E. faecalis
       (ATCC #19433) as the negative control.

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Combined Sewer Overflow (CSO) Study Report
2.2.5.3 Assessment and Comparison of Method Sensitivity and Specificity

The sensitivity and specificity of each method was assessed through the evaluation of false positive and
false negative rates.  Each of the participant laboratories evaluated four unspiked CSO samples for false
positive/negative results by submitting five typical and five atypical colonies from each of the four CSO
samples analyzed by each method to verification through biochemical evaluation.

The verification procedure is as follows:

    •  For each colony submitted to verification, the laboratory streaked the colony onto a TSA slant
       and incubated the slant at 35.0°C ± 0.5°C for 24 ± 2 hours. Tubes were labeled with sample
       identification information and colony type.  Sample identification, colony type, and morphology
       were recorded on the colony-specific tracking form provided to the laboratories.

    •  To prepare slants for shipping, participant laboratories wrapped the edges of the tubes with
       parafilm and wrapped the stack of tubes associated with each sample with bubble wrap. Tubes
       were placed into a cooler lined with a trash bag and were surrounded by blue ice. The cooler was
       sealed with shipping tape. FedEx shipping documents were provided and the cooler was shipped
       to the appropriate verification laboratory which conducted verifications using the Vitek®
       automated identification system.

    •  To minimize verification laboratory burden, verifications were conducted at two laboratories.
       One laboratory verified all E. coll isolates and the other verified all enterococci isolates. Each
       verification laboratory verified typical and atypical colonies for each method using the Vitek®.

   Note: The Vitek® automated identification system is a fully automated system that performs bacterial
   identification of isolates using fluorescent technology.  After primary isolation, an isolated colony is
   prepared at a known optical density in saline and inoculated into the Vitek® system. The gram
   negative card contains 41 fluorescent biochemical tests that are read every 15 minutes. Algorithms
   are used for organism identification.

The false positive rates were calculated as the percentage of positive results submitted to confirmation for
which the target organism was confirmed to not be present. The false negative rates were calculated as
the percentage of negative results submitted to confirmation for which the target organism was confirmed
to be present.

2.2.5.4 Assessment and Comparison of Method Accuracy (Precision and Recovery)

Method precision and recovery were evaluated through the analysis of CSO and PBS samples spiked with
laboratory-prepared spikes.  All laboratories spiked three PBS samples at each of two levels, high and
low, as described below. Each laboratory spiked four CSO samples at either a high or low level,
dependent on the type of discharge (e.g. secondary bypass disinfected, total bypass) and utility-specific
historical data.

Recoveries were assessed by comparing spike recovery (concentration in the  spiked samples minus the
ambient/unspiked concentration) to the  "true" spiked value. Precision was assessed based on the relative
standard deviation of the four replicate recoveries.

2.2.5.5 Development of Quantitative QC Criteria for Matrix Spikes (MS)

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                                                    Combined Sewer Overflow (CSO) Study Report
One of the goals of this study was to develop quantitative QC criteria for matrix spikes for use in
assessing CSO matrix interferences, for use in CSO samples. To collect the data necessary to develop
these criteria, each participant laboratory analyzed four CSO samples per method spiked with laboratory-
prepared spiking suspensions. The spiking approach was determined based on the type of CSO being
evaluated (e.g. secondary bypass disinfected, total bypass) and utility-specific historical data. Typically,
3-5 filters were analyzed per sample.

However, due to the limited number of valid data sets generated during this study, QC criteria were not
established.

(Note that for each method, the same four CSO samples spiked with laboratory-prepared spiking
suspensions were used to assess method accuracy and to obtain data to develop quantitative QC criteria
for matrix spike recoveries.)

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Combined Sewer Overflow (CSO) Study Report
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 was performed by
a contractor to EPA, the CSC Microbiology and Biochemistry Studies Group.

3.2    Schedule

Each laboratory analyzed initial precision and recovery (IPR) samples and met criteria for all four
methods prior to conducting Study analyses. Study analyses could not be scheduled because sample
collection/analyses were dependent on weather events. When laboratories analyzed for both analytes
(enterococci and E. coli), the CSO sample analyzed was from a single event.

The duration of the study was March 2005 to December 2007. After almost three years the study was
considered completed, as CSO events were so sporadic.

3.3    Research and Participant Laboratories

The participating laboratories involved in the Study are shown in Table 2.

Table 2.  Combined Sewer Overflow (CSO) Study Participant Laboratories
Lacey, Olympia, Turnwater, and Thurston (LOTT) Alliance
Paula Williamson and Paul Jue
500 Adams St, NE, Olympia, WA 98501
Narragansett Bay Commission
John Motta, Lisa Andrade, and Walter Palm
1 Service Road, Laboratory Building, Providence, Rl 02905
University of Iowa Hygienic Laboratory
Nancy Hall and Cathy Lord
Hygienic Laboratory 102, Oakdale Campus #1-1101 OH
Iowa City, IA 52242
Wisconsin State Laboratory of Hygiene
Sharon Kluender, Linda Peterson, and Jeremy Olstadt
435 Stovall Building 465 Henry Mall, Madison, Wl 53706
Massachusetts Water Resources Authority
Steve Rhode and Mariya Gofshteyn
190 Tafts Ave, Winthrop, MA 02152
Pittsburgh Water and Sewer Authority
Stanley States and Jay Kuchta
900 Freeport Rd, Pittsburgh, PA 15238
Virginia Consolidated Laboratories
Debbie Paul, Bob Sulouff, Tom York, Jim Pearson,
and Grier Mills
600 N 5th Street, Richmond, VA 23219

Referee Laboratory: EPA Office of Research and Development (ORD)
National Risk Management Research Laboratory (NRMRL)
Mark C. Meckes and Laura Boczek
26 West Martin Luther King Drive, Cincinnati, OH 45268
Verification Laboratory A: City of Los Angeles, Bureau of Sanitation, Environmental Monitoring Division
Gerald McGowen, Stan Asato, loannice Lee, Hung Pham, Pauline Nguyen, and
Marieta Ravelo
Hyperion Treatment Plant
12000 Vista del Mar, TSF Rm 452, Playa del Rey, CA 90293
Verification Laboratory B: Orange County Sanitation District (OCSD)
Charlie McGee, Michael von Winckelman and Kim Patton
10844 Ellis Ave, Fountain Valley, CA 92708-7018
a 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.

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                                                    Combined Sewer Overflow (CSO) Study Report
Section 4.0      Data Reporting and Validation

4.1    Data Reporting

Laboratories submitted the following data to CSC Microbiology and Biochemistry Studies Group for
review and validation:
•   Completed cover sheet with sample collection and QC information
•   Completed sample-specific reporting forms
•   Completed calculations spreadsheets
•   Colony-specific tracking forms for typical and atypical isolates
•   Printouts from Vitek® analyses to confirm colony identity
•   Documentation of any additional information that would assist in evaluating the data

4.2    Data Validation

The CSC Microbiology and Biochemistry Studies Group used data review checklists to ensure that each
data package was complete and 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 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 all procedures were performed according to each method and study-specific
    instructions
    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 described below were noted and considered either valid and acceptable or
invalid and unacceptable for inclusion in subsequent data analysis.

General Issues

In some instances replacement plates from the media vendor did not arrive prior to a CSO event due to
manufacturing issues (e.g., back orders, custom orders) requiring laboratories to use expired media to
analyze samples or wait for another event. In these cases QC checks (positive and negative controls)
were evaluated to determine if data was considered valid or invalid.  Please see below for specific data
validation issues including expired media.

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Combined Sewer Overflow (CSO) Study Report
Enterococci (Methods 1106.1 and 1600)

Laboratory 1

•   Although mE and EIA prepared plates exceeded the manufacturer's expiration date by 13 and 15
    days, respectively, all positive and negative controls exhibited appropriate responses, and thus data
    was considered valid and included in subsequent data analyses.

Laboratory 4

•   TSA plates were incubated for 46 hours, 22 hours longer than specified in the spiking protocol.
    Given that the TSA plate counts were consistent with previous results and other laboratories and
    many protocols using TSA require a 48 ± 3 hour incubation, TSA enumerations were considered
    valid and included in subsequent data analyses.
•   Although mE and mEI prepared plates exceeded the manufacturer's expiration date by 5 and 6 days,
    respectively, all positive and negative controls exhibited appropriate responses, and thus data was
    considered valid and included in subsequent data analyses.

Laboratory 7

•   The pH of PBS used (6.94) was below the accepted range for the methods (7.4 ± 0.2). However,
    positive and negative controls showed appropriate responses; therefore, data were considered valid
    and included in the subsequent data analyses.

E. coli (Methods 1103.1 and 1603)

Laboratory 1

•   Although modified mTEC prepared plates exceeded the manufacturer's expiration date by 13 days,
    all positive and negative controls exhibited appropriate responses, and thus data was considered valid
    and included in subsequent data analyses.

Laboratory 6

•   Dilutions evaluated for unspiked CSO samples did not produce reliable counts due to high levels of
    background organisms and therefore could not be used to accurately characterize target
    concentrations.  Therefore data was considered invalid and not included in subsequent data analyses.

Laboratory 7

•   The pH of the PBS used (6.94) was below the accepted range (7.4 ± 0.2).  However, positive and
    negative controls showed appropriate responses; therefore data were considered valid and included in
    the subsequent data analyses.
                                               10

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                                                   Combined Sewer Overflow (CSO) Study Report
Section 5.0      Results

This section includes results for Methods 1106.1 and 1600 unspiked (Section 5.1) and spiked (Section
5.2) Combined Sewer Overflow (CSO) matrices, Methods 1103.1 and 1603 unspiked (Section 5.3) and
spiked (Section 5.4) CSO matrices, and results for Methods 1106.1 and 1600 (Section 5.5) and Methods
1103.1 and 1603 (Section 5.6) for spiked PBS matrices.  Only valid results are included in this section; a
detailed description of data invalidation information is included in Section 4.

5.1     Enterococci: Method  1106.1 and 1600 Unspiked Combined Sewer Overflow
        Sample Results

Results from unspiked CSO sample analyses are provided in Table 3. The unspiked CSO data were used
to estimate the background concentration of enterococci in CSO samples.

Results of the verification analyses were used to assess method performance (see discussion in Section 6).
Laboratory-specific verification results are summarized in Tables 4 and 5. Any typical colony that was
identified as non-enterococci by the Vitek® was considered a false positive confirmation result. Any
atypical colony that was identified as enterococci by the Vitek® was  considered a false negative
confirmation result. Colonies that did not grow after streaking for growth onto TSA slants and isolates
that did  not grow at the verification laboratory were treated as if they had not been submitted to
verification and eliminated from subsequent data analyses.

Table 3.   Summary of Valid, Enterococci Results for Unspiked Combined Sewer
           Overflow (CSO)  Samples
Method
1106.1
1600
Lab
1
4
7
1
4
7
aCFU/100mL by Sample
1
200
340,000
1200
800
270,000
9000
2
1300
430,000
1500
630
240,000
16,000
3
1200
330,000
1800
1000
260,000
12,000
4
1500
350,000
2100
900
270,000
16,000
1106.1 Overall (n = 12)
1600 Overall (n = 12)
Mean
CFU/100 mL
1050
362,500
1650
833
260,000
13,250
121,733
91,361
SDb
580.2
45,734.7
387.3
157.8
14,142.1
3403.4
30,493.4 d
9697.8 d
RSD c (%)
55.3
12.6
23.5
19
5.4
25.7
40.9 e
22.6 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                              11

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Combined Sewer Overflow (CSO) Study Report
Table 4.   Method 1106.1 Laboratory-Specific False Positive and False Negative
          Confirmation Rates for Unspiked Combined Sewer Overflow (CSO) Samples
Laboratory
1
4
7
Overall
Typical
Colonies
Submitted
20
20
20
60
No. False
Positive
Colonies
0
3
0
3
False Positive
Confirmation
Rate (%)
0
15
0
5
Atypical
Colonies
Submitted
0
20
20
40
No. False
Negative
Colonies
0
7
0
7
False Negative
Confirmation
Rate (%)

35
0
17.5
Table 5.   Method 1600 Laboratory-Specific False Positive and False Negative
          Confirmation Rates for Unspiked Combined Sewer Overflow (CSO) Samples
Laboratory
1
4
7
Overall
Typical
Colonies
Submitted
18
19
20
57
No. False
Positive
Colonies
0
0
0
0
False Positive
Confirmation
Rate (%)
0
0
0
0
Atypical
Colonies
Submitted
0
13
19
32
No. False
Negative
Colonies
0
4
8
12
False Negative
Confirmation
Rate (%)

30.8
42.1
37.5
5.2    Enterococci: Method 1106.1 and 1600 Spiked Combined Sewer Overflow Sample
       Results
Results from CSO samples spiked with laboratory-prepared E. faecalis (ATCC #19433) suspensions
(Table 6) were used to assess method performance (see discussion in Section 6).

Table 6.   Summary of Valid, Enterococci Results for Spiked Combined Sewer Overflow
          (CSO) Samples
Method
1106.1
1600
Lab
1
4
7
1
4
7
Spike Level
(CFU/100 mL) a
2800
313,333
1,850,000
2800
313,333
1,850,000
Percent Recovery by Sample
5
16
108
108
24
109
107
6
9
12
130
17
77
102
7
9
172
119
17
109
97
8
5
235
119
24
13
102
1106.1 Overall (n = 12)
1600 Overall (n = 12)
Mean
Percent
Recovery
9.8
131.7
118.8
20.3
76.6
102
86.8
66.3
SDb
4.5
95.3
8.8
4.1
45.1
4.4
63.9
30.4
RSDC(%)
45.6
72.4
7.4
20.4
58.9
4.3
57.3
41.7
  Colony forming unit
b  Standard deviation
c  Relative standard deviation
d  Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e  Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                          12

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                                                    Combined Sewer Overflow (CSO) Study Report
5.3    E. co//: Method 1103.1 and 1603 Unspiked Combined Sewer Overflow Sample
       Results
Results from unspiked CSO sample analyses are provided in Table 7.  The unspiked CSO data were used
to estimate the background concentration of enterococci in CSO samples.

Results of the verification analyses were used to assess method performance (see discussion in Section 6).
Laboratory-specific verification results are summarized in Tables 8 and 9. Any typical colony that was
identified as non- E. coll by the Vitek® was considered a false positive confirmation result.  Any atypical
colony that was identified as E. coll by the Vitek® was considered a false negative confirmation result.
Colonies that did not grow after streaking for growth onto TSA slants and isolates that did not grow at the
verification laboratory were treated as if they had not been submitted to verification and eliminated from
subsequent data analyses.

Table 7.   Summary of Valid, E. co// Results  for Unspiked Combined Sewer Overflow
           (CSO) Samples
Method
1103.1
1603
Lab
1
4
5
7
1
4
5
7
aCFU/100mL by sample
1
13,200
420,000
15
30,000
20,400
570,000
8
30,000
2
10,700
430,000
6
10,000
13,200
640,000
5
10,000
3
11,900
430,000
15
30,000
12,700
680,000
8
30,000
4
12,100
380,000
16
20,000
15,300
630,000
13
20,000
1103.1 Overall (n = 16)
1603 Overall (n = 16)
Mean
CFU/100 mL
11,975
415,000
13
22,500
15,400
630,000
8.5
22,500
112,372
166,977
SDb
1024.3
23,804.8
4.7
9574.3
3518.5
45,460.6
3.3
9574.3
1 4,590.9 d
26,899.3 d
RSDC (%)
9
6
36
43
23
7
39
43
33 e
36 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                              13

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Combined Sewer Overflow (CSO) Study Report
Table 8.  Method 1103.1 Laboratory-Specific False Positive and False Negative
         Confirmation Rates for Unspiked Combined Sewer Overflow (CSO) Samples
Laboratory
4
5
7
Overall
Typical
Colonies
Submitted
15
20
20
55
No. False
Positive
Colonies
3
4
3
10
False Positive
Confirmation
Rate (%)
20
20
15
18.2
Atypical
Colonies
Submitted
20
20
20
60
No. False
Negative
Colonies
0
1
1
2
False
Negative
Confirmation
Rate (%)
0
5
5
3.3
Table 9.  Method 1603 Laboratory-Specific False Positive and False Negative
         Confirmation Rates for Unspiked Combined Sewer Overflow (CSO) Samples
Laboratory
1
4
7
Overall
Typical
Colonies
Submitted
20
20
20
60
No. False
Positive
Colonies
0
4
0
4
False Positive
Confirmation
Rate (%)
0
20
0
6.7
Atypical
Colonies
Submitted
20
20
20
60
No. False
Negative
Colonies
0
1
0
1
False
Negative
Confirmation
Rate (%)
0
5
0
1.7
                                      14

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                                                  Combined Sewer Overflow (CSO) Study Report
5.4    E. co//: Method 1103.1 and 1603 Spiked Combined Sewer Overflow Sample Results

Results from CSO samples spiked with laboratory-prepared E. coll (ATCC #11775) suspensions (Table
10) were used to assess method performance (see discussion in Section 6).

Table 10.  Summary of Valid, E. co// Results for Spiked Combined Sewer Overflow (CSO)
           Samples
Method
1103.1
1603
Lab
1
4
5
7
1
4
5
7
Spike Level
(CFU/100 mL) a
2010
1,630,000
3500
4,670,000
2010
1,630,000
3500
4,670,000
Percent Recovery by Sample
5
4379
79
88
117
6697
72
128
72
6
7364
85
71
113
2219
35
128
81
7
3832
67
51
79
8189
96
128
96
8
8857
30
88
104
2816
29
145
89
1103.1 Overall (n = 16)
1603 Overall (n = 16)
Mean
Percent
Recovery
6108.2
65
74.6
103.4
4980.1
58
132.6
84.6
1587.8
1313.8
SDb
2401.6
24.7
17.7
17.3
2918.4
31.8
8.6
10.3
1 386.7 d
1685.1 d
RSDC (%)
39.3
38
23.7
16.7
58.6
54.9
6.5
12.1
35.7e
47 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                             15

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Combined Sewer Overflow (CSO) Study Report
5.5    Enterococci: Method 1106.1 and 1600 Low- and High-Level Spiked PBS Sample
       Results
Results from PBS samples spiked with laboratory-prepared E.faecalis (ATCC #19433) suspensions
(Tables 11 ad 12) were used to assess method performance (see discussion in Section 6).

Table 11.  Summary of Valid, Enterococci Results for PBS Samples Spiked with Low-
           Level Spikes
Method
1106.1
1600
Lab
1
4
7
1
4
7
Spike Level
(CFU/100 mL) a
2800
1880
11,100
2800
1880
11,100
Percent Recovery by Sample
9
29
64
72
24
69
72
10
29
80
126
29
90
108
11
25
85
108
29
85
162
1106.1 Overall (n = 9 )
1600 Overall (n = 9 )
Mean
Percent
Recovery
27.4
76.2
102.1
27
81.6
114.1
68.6
74.2
SDb
2.1
11.1
27.5
2.7
11.1
45.3
21 d
33.1 d
RSDC (%)
7.5
14.5
27
9.9
13.6
39.7
22.3 e
30.5 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs

Table 12.  Summary of Valid, Enterococci Results for PBS Samples Spiked with High-
           Level Spikes
Method
1106.1
1600
Lab
1
4
7
1
4
7
Spike Level
(CFU/100 mL) a
466,667
313,333
1,850,000
466,667
313,333
1,850,000
Percent Recovery by Sample
12
21
105
108
30
109
108
13
19
80
114
41
73
70
14
24
86
108
21
112
119
1106.1 Overall (n = 9 )
1600 Overall (n = 9 )
Mean
Percent
Recovery
21.4
90.4
109
30.7
97.9
99.1
73.9
75.9
SDb
2.1
13.3
3.1
9.7
21.3
25.5
9.7 d
24.5 d
RSD c (%)
10
14.7
2.8
31.5
21.7
25.8
12.7 e
32.6 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                              16

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                                                   Combined Sewer Overflow (CSO) Study Report
5.6    E. co//: Method 1103.1 and 1603 Low- and High-Level Spiked PBS Sample Results

Results from PBS samples spiked with laboratory-prepared E. coll (ATCC #11775) suspensions (Tables
13 and 14) were used to assess method performance (see discussion in Section 6).

Table 13.  Summary of Valid, E. co// Results for PBS Samples Spiked with Low-Level
           Spikes
Method
1103.1
1603
Lab
1
4
5
6
7
1
4
5
6
7
Spike Level
(CFU/100 mL) a
2010
1630
3500
1620
4670
2010
1630
3500
1620
4670
Percent Recovery by Sample
9
6
86
60
62
94
6
61
149
43
84
10
6
55
49
68
90
5
49
146
123
109
11
7
80
89
130
120
5
86
114
68
71
1103.1 Overall (n =15)
1603 Overall (n = 15)
Mean
Percent
Recovery
6.6
73.6
65.7
86.4
101.4
5.8
65.4
136.2
78
87.8
66.8
74.6
SDb
0.8
16.2
20.6
37.6
16.2
0.6
18.7
19
41.4
19.6
26.6 d
29 d
RSD c (%)
11.5
22.1
31.4
43.5
16
9.9
28.6
14
53
22.4
33.5 e
36.4 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                             17

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Combined Sewer Overflow (CSO) Study Report
Table 14.  Summary of Valid, E. coli Results for PBS Samples Spiked with High-Level
           Spikes
Method
1103.1
1603
Lab
1
4
5
6
7
1
4
5
6
7
Spike Level
(CFU/100 mL) a
2,010,000
1,630,000
3,500,000
1,620,000
4,670,000
2,010,000
1,630,000
3,500,000
1,620,000
4,670,000
Percent Recovery by Sample
12
1
67
100
87
92
1
80
120
41
101
13
1
92
109
302
94
1
86
117
414
84
14
1
55
140
57
88
1
98
154
401
96
1103.1 Overall (n = 15)
1603 Overall (n =15)
Mean
Percent
Recovery
1.2
71.6
116.2
149
91.4
0.7
87.9
130.5
285.2
93.5
85.9
119.6
SDb
0.1
18.7
21.1
133.8
3.3
0.03
9.4
20.7
211.8
8.9
74.9 d
116.8 d
RSD c (%)
8.6
26.2
18.1
89.8
3.6
4.3
10.7
15.8
74.3
9.5
52.4 e
42.4 e
  Colony forming unit
b Standard deviation
c Relative standard deviation
d Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
  variances
e Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of the
  squared lab RSDs
                                               18

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                                                   Combined Sewer Overflow (CSO) Study Report
Section  6.0      Development of QC Acceptance Criteria

This section describes the development of quantitative QC acceptance criteria in a reference matrix (PBS)
and the matrix of interest (CSO) to support future assessments of laboratory and method performance.
All data analyses described below were performed using the results of PBS and CSO samples spiked with
laboratory-prepared suspensions.

6.1    Outlier Analyses

Valid results from samples spiked with laboratory-prepared suspensions were screened for outliers in
accordance with the procedures described in American Society for Testing and Materials (ASTM)
guidance D2777-98 (Reference 9.5).  Due to the small number of laboratories participating in this study,
the data could not be screened for outlying  laboratories. Grubbs test (Reference 9.5) was performed,
which evaluates individual sample results for outlying observations, as described below.

The PBS and CSO data were tested for the  presence of individual outlying recoveries using Grubbs test,
which was run separately for each matrix, method and spike level without performing any data
transformations. Application of Grubbs test resulted in the removal of a single outlying result. This
recovery (a high-spiked PBS result determined using Method  1103.1 by laboratory 6) was high-biased
compared to the other recoveries, and was not used in the development of QC criteria.

Outlier analyses were only performed for development of QC acceptance criteria (Section 6.2 and 6.3).
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 (i.e., outliers were not removed).

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.  Separate criteria were determined for low-level and
high-level PBS spikes.

The IPR and OPR recovery criteria were calculated based on within and between laboratory variance
components (Reference 9.6). 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 9.7).

Estimates of between laboratory variance and within laboratory variance were labeled s2L and s2w,
respectively.

The combined standard deviation for IPR samples (isc) is:
Where:
                                             19

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Combined Sewer Overflow (CSO) Study Report
       L =  number of laboratories for the given spike level and method
       nt  = number of PBS sample results for laboratory I for the given spike level and method
       nT = total number PBS sample results from all laboratories for the given spike level and
           method
Upper and lower limits for the mean recovery of four IPR samples were then calculated as:
                                 mean — 'o.975;z
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                                                    Combined Sewer Overflow (CSO) Study Report
                         odf=-
                                                OS
\( L \ 1
zx
1 1 !=1
2
_V )

* s2

2


-1- -
["( 0* 21
I1+^J 5w_
                                      L-l
nT - L
The precision criterion for IPR samples was calculated as a maximum relative standard deviation (RSD)
of four PBS sample results. The pooled RSD for each laboratory was calculated by dividing the pooled
within-laboratory standard deviation calculated previously by the overall mean recovery for that
laboratory and matrix, as shown below:
                                  RSD
                                      pool


Where:
       Sw andXmean are the pooled within-lab standard deviation and mean recovery calculated above

The maximum RSD was then calculated as:
                                                         pool
Where:
       L = the total number of laboratories for the given spike level and method.

The calculated IPR QC acceptance criteria are provided in Tables 15 and 16.
                                              21

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Combined Sewer Overflow (CSO) Study Report
Table 15.  Calculated Initial Precision and Recovery (IPR) and Ongoing precision and
	Recovery (OPR) Acceptance Criteria forE. coli Methods	
 Performance test
Method 1103.1  acceptance criteria
Method 1603 acceptance criteria
 Low level spike
 IPR
 •  Mean percent recovery
 •  Precision (as maximum relative
    standard deviation of 4 samples)
 OPR
 •  percent recovery
         detect1-156%
             63%

         detect-159%
        detect-194%
            62%

        detect-194%
 High level spike
 IPR
 •  Mean percent recovery
 •  Precision (as maximum relative
    standard deviation of 4 samples)
 OPR
 •  percent recovery2
          detect-184%
             43%

          detect -184%
        detect - 349%
            154%

        detect - 397%
  The term "detect" is used to indicate that the calculated lower limit was negative.
2 In cases where the OPR recovery criteria were calculated to be tighter than the IPR recovery criteria, the OPR
  criteria were set to the calculated IPR criteria
Table 16.  Calculated Initial Precision and Recovery (IPR) and Ongoing precision and
	Recovery (OPR) Acceptance Criteria for Enterococci Methods	
 Performance test
Method 1106.1  acceptance criteria
Method 1600 acceptance criteria
 Low level spike
 IPR
 •  Mean percent recovery
 •  Precision (as maximum relative
    standard deviation of 4 samples)
 OPR
 •  percent recovery 1
         detect1 - 200%
             55%

         detect - 200%
        detect-210%
            80%

        detect-210%
 High level spike
 IPR
 •  Mean percent recovery
 •  Precision (as maximum relative
    standard deviation of 4 samples)
 OPR
 •  percent recovery 1
          detect - 259%
             24%

          detect - 259%
        detect - 206%
            58%

        detect - 206%
  The term "detect" is used to indicate that the calculated lower limit was negative.
2 In cases where the OPR recovery criteria were calculated to be tighter than the IPR recovery criteria, the OPR
  criteria were set to the calculated IPR criteria
                                               22

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                                                    Combined Sewer Overflow (CSO) Study Report
Matrix Spike (MS) Recovery

QC acceptance criteria for matrix spikes (MS) were developed based on laboratory-spiked sample data
from the CSO matrices used in the validation study.  Separate QC acceptance criteria were calculated for
each method.

Recovery criteria were based on estimates of each variance component (between laboratory and within
laboratory) and were calculated using PROC MIXED from SAS version 9 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 9.7).  For each matrix/spike  level, between sample
variability could not be separated from between laboratory variability because each laboratory analyzed a
different CSO sample, and therefore the estimate of between laboratory variance also includes sample
variability.

Estimates of between laboratory variance and within laboratory variance were labeled s2L and s2w,
respectively.

The combined standard deviation for MS samples (sc) is:
1 L
1 IX
1
«y nT
Where:
       L =  number of laboratories for the given method
       nt  = number of CSO sample results for laboratory I for the given method
       nT = total number CSO sample results from all laboratories for the given method
Upper and lower limits for the recovery of MS samples were then calculated as:
                                -mean ~ I0.975;df)
Where:
       Xmean = the mean recovery of all CSO samples for the given method, and

       dfis calculated using Satterthwaite 's estimate as given below:
                             df=~
y L \
yn2
/_j i
1 1 i=i

nT
_v /
-


*52
•'L


2




	 L
7 1 A
lH 	 *5^
V wr y
                                         L-l
nT - L
                                              23

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Combined Sewer Overflow (CSO) Study Report
The calculated MS QC acceptance criteria are listed in Tables 17 and 18.

Table 17. Calculated Matrix Spike Recovery Acceptance Criteria for E. coli Methods
Performance test
Percent Recovery for MS samples
Method 1103.1 acceptance criteria
23-139%
Method 1603 acceptance criteria
detect1 - 206%
 The term "detect" is used to indicate that the calculated lower limit was negative.

Table 18. Calculated Matrix Spike Recovery Acceptance Criteria for Enterococci
          Methods
Performance test
Percent Recovery for MS samples
Method 1106.1 acceptance criteria
detect1 - 275%
Method 1600 acceptance criteria
detect -187%
 The term "detect" is used to indicate that the calculated lower limit was negative.
                                            24

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                                                  Combined Sewer Overflow (CSO) Study Report
Section 7.0      Assessment and Discussion  of Method
                     Performance

Method performance was evaluated through the evaluation of precision and recovery in CSO samples and
PBS samples spiked with laboratory-prepared spiking suspensions and through the assessment of false
positive and false negative rates in unspiked CSO samples.  Outlier analyses were not conducted for the
assessment of method performance, therefore all valid data were included in the assessment of method
performance.

7.1    Enterococci Methods

7.1.1   Method 1106.1

       Method 1106.1 Recovery and Precision
       Method 1106.1 recovery was characterized by mean laboratory-specific recoveries of enterococci
       from spiked CSO samples ranging from 9.8% to 131.7%, with an overall mean recovery of
       86.8%. Laboratory-specific RSDs for spiked CSO samples ranged from 7.4% to 72.4%, with a
       pooled, within-laboratory RSD of 57.3%.

       Mean laboratory-specific recoveries of enterococci from PBS spiked with low-level spikes ranged
       from 27.4% to 102.1%, with an overall mean recovery of 68.6%.  Laboratory-specific RSDs for
       PBS samples spiked with low-level spikes ranged from 7.5% to 27.0%, with a pooled, within-
       laboratory RSD of 22.3%.  Mean laboratory-specific recoveries of enterococci from PBS spiked
       with high-level spikes ranged from 21.4% to 109.9%, with an overall mean recovery of 73.9%.
       Laboratory-specific RSDs for PBS  samples spiked with high-level spikes ranged from 2.8% to
       14.7%, with a pooled, within-laboratory RSD of 12.7%.
           o  Method 1106.1 mean recoveries for Laboratory 1 were considerably lower for all three
              matrix/spike type combinations with mean recoveries of 9.8% (CSO), 27.4% (PBS low-
              level), and 21.4% (PBS high-level), skewing overall means.

       Method 1106.1 False Positive and Negative Assessment
       Laboratory-specific rates false positive confirmation rates ranged from 0% to 15.0%, with an
       overall false positive confirmation rate of 5%. In contrast, the false negative confirmation rates
       were higher ranging from 0% to 35%, with an overall false negative confirmation rate of 17.5%.
       It should be noted that all 7 of the atypical colonies that were identified as a false negative were
       from one lab, and therefore, from a single matrix. It should also be noted that  Laboratory 1 did
       not have any atypical colonies to submit to verification.

7.1.2   Method 1600

       Method 1600 Recovery and Precision
       Method 1600 recovery was characterized by mean laboratory-specific recoveries of enterococci
       from spiked CSO samples ranging from 20.3% to 102%, with an overall mean recovery of 66.3%.
       Laboratory-specific RSDs for spiked CSO samples ranged from 4.3% to 58.9%, with a pooled,
       within-laboratory RSD of 41.7%.

       Mean laboratory-specific recoveries of enterococci from PBS spiked with low-level spikes ranged
       from 27% to 114.1%, with  an overall mean recovery of 74.2%. Laboratory-specific RSDs for
       PBS samples spiked with spiked low-level spikes ranged from 9.9% to 39.7%, with a pooled,
       within-laboratory RSD of 30.5%. Mean laboratory-specific recoveries of enterococci from PBS
                                            25

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Combined Sewer Overflow (CSO) Study Report
       spiked with high-level spikes ranged from 30.7% to 99.1%, with an overall mean recovery of
       75.9%. Laboratory-specific RSDs for PBS samples spiked with high-level spikes ranged from
       21.7% to 31.5%, with a pooled, within-laboratory RSD of 32.6%.
           o  Method 1600 mean recoveries for Laboratory 1 were considerably lower for all three
              matrix/spike type combinations with mean recoveries of 20.3% (CSO),  27% (PBS low-
              level), and 30.7% (PBS high-level), skewing overall means.

       Method 1600 False Positive and Negative Assessment
       The false positive confirmation rate for all laboratories was 0%. In contrast, laboratory-specific
       false negative confirmation rates ranged from 30.8% to 42.1%. Eight of the twelve colonies that
       were identified as a false negative were from one lab, and therefore, from a single matrix.
       Laboratory 1 did not have any atypical colonies to submit to verification.

       Similar to the results observed  for unspiked secondary wastewater samples during the validation
       of Method 1600 in disinfected wastewater (2003), many of the false negatives (atypical colonies
       submitted to verification which identified as enterococci) were pink to red in color but simply
       lacked a blue halo (Reference 9.8).  The predecessor to EPA Method 1600 for enterococci is EPA
       Method 1106.1, which uses mE and EIA media. For EPA Method 1106.1, pink to red colonies
       on mE, which produce a brown precipitate after transfer to EIA are considered positive for
       enterococci.  Tetrazolium chloride (TTC), the reagent responsible for producing pink to red
       enterococci colonies on mE, is  also included as a reagent in mEI.

       When evaluating CSO matrices using Method 1600, some pink to red colonies without a  halo
       may be enterococci.  These colonies should be verified, especially if large numbers of these
       colonies are observed in a particular matrix. If very few pink to red colonies are observed in
       samples from a particular matrix, the high false negative rates observed during this study  may be
       less of a concern.

7.1.3  Comparison of Enterococci Method Performance

       Table 19 summarizes results of valid, spiked PBS and spiked CSO results for both Methods
       1106. land 1600.

       Table 19.  Summary of Method 1106.1 and 1600 Enterococci Recoveries for Spiked
                  PBS and Combined Sewer Overflow Samples
Method
1106.1
1600
PBS Low-level Spike
Mean
Recovery
(%)
68.6
74.2
SD a (%)
21
33.1
RSDb
(%)
22.3
30.5
PBS High-level Spike
Mean
Recovery
(%)
73.9
75.9
SD a (%)
40.9
38.1
RSDb
(%)
12.7
32.6
CSO
Mean
Recovery
(%)
86.7
66.3
SD a (%)
63.9
30.4
RSDb
(%)
57.3
41.7
         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
                                             26

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                                             Combined Sewer Overflow (CSO) Study Report
Comparison of Methods 1106.1 and 1600 Recovery
Mean recoveries of the two methods were compared using two-way ANOVA models fit
separately for each organism, matrix and spike type (i.e., spike source and level).  An F-test was
run first for each organism, matrix and spike type to test whether there was a significant
interaction between laboratory and method (i.e., whether the effect of method on mean recovery
differed significantly between laboratories). Where there was no significant laboratory-by-
method interaction, an overall F-test was used to assess whether there was a significant difference
in mean recovery between methods, controlling for variability between laboratories. Where there
was a significant laboratory-by-method interaction, a separate one-way ANOVA model was fit
for each laboratory.

There was no significant interaction between lab and method for any of the three matrix/spike
type combinations. Therefore, a single comparison of mean recoveries could be made for each
matrix and spike type. For all three matrix/spike type combinations, mean recovery did not differ
significantly between methods.

Comparison of Methods 1106.1 and 1600 Precision
Comparisons of recovery variability for samples spiked with laboratory-prepared E. faecalis
spiking suspensions observed for Methods  1106.1 and  1600 were evaluated using F-tests, based
on pooled within-laboratory variances. F-tests were performed separately for each matrix (PBS
and CSO) and spike type.  Based on these analyses, within-laboratory precision was significantly
better for Method  1600 (pooled within-laboratory standard deviation equaling 30.4%) than for
Method 1106.1 (pooled within-laboratory standard deviation equaling 63.9%) in CSOs. In
contrast the within-laboratory variability was better for Method 1106.1 (pooled within-laboratory
standard deviation equaling 9.7%) than for Method 1600 (pooled within-laboratory standard
deviation 24.2%) in PBS spiked with high-level spikes. The within laboratory variability did not
differ significantly for PBS spiked with low-level spikes.

Comparison of Methods 1106.1 and 1600 False Positive and False Negative Confirmation Rates
For CSO matrices, the false negative confirmation rates were higher for Method 1600 (37.5%)
compared to Method 1106.1 (17.5%).  In contrast, the false positive confirmation  rate was higher
for Method 1106.1 (5%) compared to Method 1600 i
Table 20.  Comparison of Methods 1106.1 and 1600 False Positive and False
           Negative Confirmation Rates for Unspiked CSO Matrices
Matrix
CSO
Method 11 06.1
False Positive
Confirmation Rate
(%)
5.0
False Negative
Confirmation Rate
(%)
17.5
Method 1600
False Positive
Confirmation Rate
(%)
0
False Negative
Confirmation Rate
(%)
37.5
                                       27

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Combined Sewer Overflow (CSO) Study Report
7.2    E. co// Methods

7.2.1   Method 1103.1

       Method 1103.1 Recovery and Precision
       Method 1103.1 was characterized by mean laboratory-specific recoveries of E. coli from spiked
       CSO samples ranging from 65% to 6108.2%, with an overall mean recovery of 1587.8%.
       Laboratory-specific RSDs for spiked CSO samples ranged from 16.7% to 39.3%, with a pooled,
       within-laboratory RSD of 35.7%.
       Mean laboratory-specific recoveries of E. coli from PBS spiked with low-level spikes ranged
       from 6.6% to 101.4%, with an overall mean recovery of 66.8%. Laboratory-specific RSDs for
       PBS samples spiked with low-level spikes ranged from 11.5% to 43.5%, with a pooled, within-
       laboratory RSD of 33.5%. Mean laboratory-specific recoveries of E. coli from PBS spiked with
       high-level spikes ranged from 1.2% to 149%, with an overall mean recovery of 85.9%.
       Laboratory-specific RSDs for PBS samples spiked with high-level spikes ranged from 3.6% to
       89.8%, with  a pooled, within-laboratory RSD of 52.4%.
           o  Method 1103.1 mean recoveries for Laboratory 1 were considerably lower for PBS
              spiked with low- and high-level spikes 6.6% and  1.2%, respectively, skewing overall
              means.
           o  Although Method 1103.1 overall mean recovery in CSO samples was very high,
              individual laboratory Method 1103.1 mean recoveries for 3 of 4 laboratories were
              considerably lower, ranging from 65% to 103.4%. Laboratory 1 observed a very high
              mean recovery of 6108.2%, skewing the overall recoveries. The high recoveries observed
              by Laboratory 1 could have been due to inaccurate determination of ambient
              concentrations or an error in  spiking the CSO samples.

       Method 1103.1 False Positive and Negative Assessment
       Laboratory-specific rates false positive confirmation rates ranged from 15% to 20%, with an
       overall false  positive confirmation rate of 18.2%.  In contrast, the false negative confirmation
       rates were lower ranging from 0% to 5%, with an overall  false negative confirmation rate of
       3.3%.

7.2.2   Method 1603

       Method 1603 Recovery and Precision
       Method 1603 recovery was characterized by mean laboratory-specific recoveries of E. coli from
       spiked CSO  samples ranging from 58% to 4980.1%,  with an overall mean recovery of 1313.8%.
       Laboratory-specific RSDs for spiked CSO samples ranged from 6.5% to 58.6%, with a pooled,
       within-laboratory RSD of 47%.

       Mean laboratory-specific recoveries ofE. coli from PBS spiked with low-level spikes ranged
       from 5.8% to 136.2%, with an overall mean recovery of 74.6%. Laboratory-specific RSDs for
       PBS samples spiked with low-level spikes ranged from 9.9% to 53%, with a pooled, within-
       laboratory RSD of 36.4%. Mean laboratory-specific recoveries ofE. coli from PBS spiked with
       high-level spikes ranged from 0.7% to 285.2%, with  an overall mean recovery of 119.6%.
       Laboratory-specific RSDs for PBS samples spiked with high-level spikes ranged from 4.3% to
       74.3%, with  a pooled, within-laboratory RSD of 42.4%.

           o  Method 1603 mean recoveries for Laboratory 1 were considerably lower for PBS spiked
              with low- and high-level spikes 5.8% and 0.7%, respectively, skewing overall means.
                                             28

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                                                   Combined Sewer Overflow (CSO) Study Report
           o  Although Method 1603 overall mean recovery in CSO samples was very high, individual
              laboratory Method 1603 mean recoveries for 3 of 4 laboratories were considerably lower,
              ranging from 58% to 132.6%. Laboratory 1 observed a very high mean recovery of
              4980.1%, skewing the overall recoveries. The high recoveries observed by Laboratory 1
              could have been due to inaccurate determination of ambient concentrations or an error in
              spiking the CSO samples.

       Method 1603 False Positive and Negative Assessment
       Laboratory-specific rates false positive confirmation rates ranged from 0% to 20%, with an
       overall false positive confirmation rate of 6.7%. In contrast, the  false negative confirmation rates
       were lower ranging from 0% to 5%, with an overall false negative confirmation rate of 1.7%.
7.2.3  Comparison of E. coli Method Performance

       Table 21 summarizes results of valid, spiked PBS and spiked CSO results for both Methods
       1103.land 1603.

       Table 21.  Summary of Method 1103.1 and 1603 E. coli Recoveries for Spiked PBS
                  and Combined Sewer Overflow Samples
Method
1103.1
1603
PBS Low-level Spike
Mean
Recovery
(%)
68.6
74.2
SD a (%)
21
33
RSDb
(%)
22.3
30.5
PBS High-level Spike
Mean
Recovery
(%)
73.9
75.9
SD a (%)
9.74
24.5
RSDb
(%)
12.7
32.6
CSO
Mean
Recovery
(%)
1587.8
1313.8
SD a (%)
6795.7
1685.1
RSDb
(%)
35.7
47
         Pooled within-lab standard deviation was determined by calculating the square root of the mean of the lab
         variances
       b Pooled within-lab relative standard deviation was determined by calculating the square root of the mean of
         the squared lab RSDs

       Comparison of Methods 1103.1 and 1603 Recovery
       Mean recoveries of the two methods were compared following the same methodology as used in
       comparing enterococci method performance (Section 6.1.3).

       There was a significant interaction between lab and method for high-level laboratory-prepared
       spiking suspensions in PBS samples. Therefore, a separate comparison of mean recoveries was
       performed for each of the five laboratories. Based on these separate comparisons, a significant
       difference in mean recovery between the methods was observed in samples analyzed by
       Laboratory  5, with mean recovery being significantly greater for samples analyzed by Method
       1603. No significant difference in mean recovery between methods was observed in samples
       analyzed by the other laboratories.

       For CSO samples and low-level laboratory-prepared spiking suspensions in PBS samples, no
       significant interaction between lab and method was observed. Therefore, a single comparison of
       mean recoveries could be made for these two matrix/spike type combinations. In each case, mean
       recovery did not differ significantly between methods.
                                             29

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Combined Sewer Overflow (CSO) Study Report
       Comparison of Methods 1103.1 and 1603 Precision
       Comparisons of recovery variability for samples spiked with laboratory-prepared E. coll spiking
       suspensions observed for Methods 1103.1 and 1603 were evaluated using F-tests, based on
       pooled within-laboratory variances. F-tests were performed separately for each matrix (PBS and
       CSO) and spike type. Based on these analyses, within-laboratory precision was not significantly
       different for any matrix/spike type combination.

       Comparison of Methods 1103.1 and 1603 False Positive and False Negative Confirmation Rates
       For CSO matrices, the false positive confirmation rates were higher for Method 1103.1 (18.2%)
       compared to Method 1603 (6.7%).  The false negative confirmation rate was slightly higher for
       Method 1103.1 (3.3%) compared to Method 1600 (1.7%).
       Table 22. Comparison of Methods 1103.1 and 1603 False Positive and False
                 Negative Confirmation Rates for Unspiked CSO Matrices
Matrix
CSO
Method 11 03.1
False Positive
Confirmation Rate
(%)
18.2
False Negative
Confirmation Rate
(%)
3.3
Method 1603
False Positive
Confirmation Rate
(%)
6.7
False Negative
ConfirmationRrate
(%)
1.7
                                            30

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                                                Combined Sewer Overflow (CSO) Study Report
Section 8.0     Conclusion

With data sets from five laboratories, quality control criteria for CSO matrices and PBS samples spiked at
low- and high-levels were developed. Previously there was no data on method performance in CSO
matrices.  Now we have data from five laboratories that may be used as a starting point for end users.
                                          31

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Combined Sewer Overflow (CSO) Study Report
Section 9.0      References


9.1 USEPA. 2002. EPA Method 1106.1: Enterococci in Water by Membrane Filtration Using
   membrane-Enterococcus-Esculin Iron Agar (mE-EIA), EPA-821-R-04-022, September 2002.

9.2 USEPA. 2002. EPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane-
   Enterococcus Indoxyl-fi-D-Glucoside Agar (mEI), EPA-821-R-02-022, September 2002.
9.3 USEPA. 2004. EPAMethod 1103.1: Escherichia coll (E. coll) in Water by Membrane Filtration
   Using membrane-Thermotolerant Escherichia coli Agar (mTEC), EPA-821-R-04-024, September
   2004.
9.4 USEPA. 2002. EPAMethod 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using
   Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC), EPA-821-R-02-023,
   September 2002.
9.5 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.
9.6 Neter, John, W. Wasserman, and M. H. Kutner. Applied Linear Statistical Models. 3rd Edition.
   Richard D. Irwin, Inc.  Burr Ridge, IL, 1990. Pages 619-620.
9.7 SAS Institute Inc.  1994. SAS/STAT User's Guide, Volume 2, GLM-VARCOMP. Version 6, 4th
   Edition, June 1994.
9.8 USEPA. 2005. Results of the Interlaboratory Validation of EPA Method 1600 (mEI) for Enterococci
   in Wastewater Effluent. EPA-821-R-04-019. February 2005.
                                           32

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                                               Combined Sewer Overflow (CSO) Study Report
Section 10.0    Acronyms

CPU      Colony forming unit
CSO      Combined sewer overflow
IPR       Initial precision and recovery
MS       Matrix spike
OPR      Ongoing precision and recovery
PBS       Phosphate buffered saline
QA       Quality assurance
QC       Quality control
RPD      Relative percent difference
RSD      Relative standard deviation
SD       Standard deviation
TNTC     Too numerous to count
                                          33

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Combined Sewer Overflow (CSO) Study Report
                                Appendix A:



                     Laboratory Capabilities Checklist
                                     34

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                                                          Combined Sewer Overflow (CSO) Study Report
                            Laboratory Capabilities Checklist
                    Combined Sewer Overflow (CSO) Method Validation Study

                                          (May 18, 2006)

EPA plans to invite 13 laboratories (12 participants and 1 verification) to participate in a study to validate
methods for the detection and enumeration of E. coll and enterococci in CSOs. The methods to be evaluated
include EPA Methods 1603 (modified mTEC) and 1103.1 (mTEC) for E. coli, and Methods 1600 (mEI) and
1106.1 (mE/EIA) for enterococci. Each of these methods will require filtration of diluted samples and subsequent
incubation on selective media prior to target analyte detection and enumeration. EPA will provide all media and
disposable materials for the study and will also cover all shipping costs. Volunteer laboratories and participants
will be acknowledged in the validation study reports and in the final versions of each method.  The study is
tentatively scheduled to begin late spring.

If your laboratory is interested in participating in the validation study as a participant or verification laboratory,
please provide the requested information below and fax the signed, completed checklist to Ruth Grunerud at
703.461.8056 by Friday, May 26th. In addition, please send the form electronically to Ruth Grunerud at
rgrunerud@csc.com. Ruth 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 ychambers(g), esc .com. Note: If it is more convenient for your laboratory, we are happy to contact
the wastewater facility directly, just let us know.

In  addition to  collecting information for the upcoming CSO validation study, EPA has also requested that a
limited amount of information on sanitary sewer overflows (SSOs) and storm runoff be collected.
Section 1. Laboratory Capabilities and Experience

a.  Please complete the requested capabilities and experience information below, if this information has not been
previously provided to CSC. The information requested in Table 1 pertains to experience with a given method,
regardless of matrix (e.g., surface water, wastewater) analyzed.

Table 1.   Analyst Experience
Analyst





Years of experience or estimated number of samples analyzed
Methods to be validated
modified
mTEC





mTEC





mE/EIA





mEI





Other membrane filter methods
mEndo or
LES Endo





mFC





NA-MUG





                                                 35
April 2008

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Combined Sewer Overflow (CSO) Study Report
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:
Table 2.   E. coli and Enterococci
Access?
Example
/








Wastewater type
Primary treated
Raw
Primary treated
Secondary treated
Tertiary treated
Disinfected
Combined Sewer
Overflow (CSO)
Sanitary Sewer
Overflow (SSO)
Storm Runoff
Monitoring
frequency
1 per month
















E. coli
Methods
SM9221B/F
















Typical range
30 x 105
















Enterococci
Methods
1106.1
















Typical range
12 x 103
















Section 2. Background Information

a.   Does your laboratory have access to CSO samples?
    Please indicate in Table 2, below.
Yes
No
b.  Has your laboratory ever participated in a wastewater and/or CSO study?
Yes
No
                                                36

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                                                           Combined Sewer Overflow (CSO) Study Report
    If your laboratory has experience analyzing wastewater/CSO/SSO/storm runoff samples for E. coll and/or
    enterococci, please place a check "/" next to the wastewater type(s) which you have analyzed and indicate
    the method(s) used for analysis and typical concentrations of each analyte (Table 2, above).  If your
    laboratory does not have experience analyzing wastewater/CSO samples for E. coll and/or enterococci, please
    complete Table 3, below.
d.   If your laboratory has experience analyzing wastewater/CSO/SSO/storm runoff samples for total coliforms,
    fecal coliforms, fecal streptococci, or other indicator organisms, please  place a check " /" next to the
    wastewater type(s) that you have access to and indicate the method(s) used for analysis and typical analyte
    ranges observed in Table 3, below.
Table 3. Other Indicator Organisms
Access?








Wastewater type
Raw
Primary treated
Secondary treated
Tertiary treated
Chemically disinfected
Combined Sewer
Overflow (CSO)
Sanitary Sewer
Overflow
Storm Runoff
Monitoring
frequency
















Other indicator organisms
Organism(s)
















Methods
















Typical range
















e.   If you indicated (Table 2 and Table 3) that your laboratory has access to and/or experience analyzing
    Combined Sewer Overflow (CSO) samples, please provide as much information regarding these matrices as
    possible (below). This information will assist in the evaluation matrix suitability.
    e. 1. Please characterize the composition of the CSO matrices that your laboratory has analyzed (e.g., %
       agricultural, % industrial, % residential, other- please specify)?
                                                  37
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Combined Sewer Overflow (CSO) Study Report
    e.2. CSC recognizes that your laboratory may have access to multiple facilities with potential CSO matrices.
       For each facility, please describe the treatment process (e.g., total bypass-no disinfection,
       primary/secondary bypass-disinfected, other-please describe)?  Please be as descriptive, as possible.

       Facility 1:
       Facility 2:
       Facility 3:
    e.3. What is the frequency of CSO events at each of these facilities?
    e.4. What amount of rainfall generally triggers a CSO event at each of these facilities?
    e.5. If available, please provide historical bacterial data (e.g., total coliforms, fecal coliforms, E. coll,
       enterococci, other) from the CSO matrices?
    e.6 Will your laboratory be prepared to collect and analyze CSO samples within the 8-hour holding time?
       Yes                   No
                                                   38

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                                                          Combined Sewer Overflow (CSO) Study Report
Q.I . Please provide as much information as possible regarding the potential treatment facilities where your
laboratory may obtain CSO samples:
Facility Name



Contact Name



Contact Phone



Contact Email



Address




Additional comments:
Section 3. General Information.

a.   How many membrane filtration funnels will be available for use during the study?
b.   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.)? 	

c.   How does your laboratory disinfect filtration assemblies/funnels between sample filtrations?
Section 4. Verification Laboratory

Is your laboratory potentially interested in verifying isolates from other laboratories?    Yes    No


Table 3. Verification Procedures
Verification procedure
API 20E®
VITEK®
BIOLOG
BBL Crystal™
Other (please describe below)*
Isolation medium





*If other, please describe:
                                                 39
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Combined Sewer Overflow (CSO) Study Report
I certify that the information provided above is accurate and complete:







Primary Analyst or Lab Manager (please print):	




Laboratory name:	
Signature:
Date:
                                               40

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                       Combined Sewer Overflow (CSO) Study Report
           Appendix B:



CSO Enterococci Spiking Protocol
                41                              April 2008

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                            Enterococci Spiking Protocol for
   Interlaboratory Validation  of Methods 1106.1  (mE-EIA) and  1600 (mEI) in
                            Combined Sewer Effluents (CSE)

                                         (April 11,2005)
The purpose of this protocol is to provide laboratories with enterococci spiking procedures for the interlaboratory
validation of Methods 1106.1 and 1600 in combined sewer effluents (CSE). The following sections are included
in this protocol:


     Section 1:  Preparation of Spiking Suspension
     Section 2:  Spiking Suspension Dilution and Enumeration
     Section 3:  Sample Spiking
     Section 4:  Calculation of Percent Recovery
1.0   Preparation of Spiking Suspension


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 (Methods 1106.1 and 1600, Section 7.5) 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 thoroughly.

1.3    Spiking Suspension (Undiluted).  From the stock culture of E. 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 x 106 to 1.0 x 107 enterococci colony forming units (CPU) per mL. This is
       referred to as the "undiluted spiking suspension".

Note: After incubation, the spiking suspension may be held at 6°C ± 2°Cfor a maximum of 72 hours. In
anticipation of a potential CSE event, spiking suspensions should be propagated every fourth day to ensure that a
viable suspension is available for sample spiking during the validation study.  (For example, if the suspension was
propagated on Monday, a new spiking suspension should be propagated on Friday.)
2.0   Dilution and Enumeration of Spiking Suspension

Since one of the objectives of spiking the sample is to assess 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 dilution (Section 2.1) and enumeration (Section 2.2) of the spiking suspension.

Note: Please be sure to thoroughly mix each of the spiking suspensions prior to performing the dilutions in the
steps below, as homogeneous suspensions are critical for accurate dilution and enumeration.

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2.1   Dilution of spiking suspension


Please note: The approach to diluting spiking suspensions was revised based on the practice week results.

        2.1.1   Mix the spiking suspension by vigorously shaking the bottle a minimum of 25 times.  Use a
               sterile pipette to transfer 11.0 mL of the undiluted spiking suspension (from Section 1.3 above) to
               99 mL of sterile phosphate buffered saline (PBS), 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"1 mL of the original undiluted spiking suspension.


        2.1.2   Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "A" (from Section 2.1.1
               above) to 99 mL of sterile PBS, 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 10~2 mL of
               the  original undiluted spiking suspension.


        2.1.3   Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution "B" (from Section 2.1.2
               above) to 99 mL of sterile PBS, 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"4 mL of
               the  original undiluted spiking suspension.


        2.1.4   Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" (from Section 2.1.3
               above) to 99 mL of sterile PBS, 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"5 mL of
               the  original undiluted spiking suspension
        2.1.5   Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "D" (from Section 2.1.4
               above) to 99 mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
               times. This is spiking suspension dilution "E". A 1.0-mL volume of dilution "E" is 10"6 mL of
               the original undiluted spiking suspension
2.2   Enumeration of undiluted spiking suspension (prepared in Section 1.3)


        2.2.1   Prepare tryptic soy agar (TSA) according to manufacturer's instructions, add 12 - 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 prior to use, plates should be made several days in advance and stored inverted at room
        temperature or dried using a laminar-flow hood.
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       2.2.2  Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread
              plates:
              •   Mix dilution "C" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of
                  dilution "C" (Section 2.1.3) onto surface of pre-dried TSA plate [10"5 mL (0.00001) of the
                  original spiking suspension].

              •   Mix dilution "D" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of
                  dilution "D" (Section 2.1.4) onto surface of pre-dried TSA plate [10"6 mL (0.000001) of the
                  original spiking suspension].

              •   Mix dilution "E" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of
                  dilution "E" (Section 2.1.5) onto surface of pre-dried TSA plate [10~7 mL (0.0000001) of the
                  original spiking suspension].

3.0  Sample Spiking
       PBS samples will be spiked at two levels, low (Section 3.1.1) and high (Section 3.1.2). For each spike
       level, three PBS samples will be spiked. Four CSE samples will be spiked at either a low or high spike
       level, dependent on whether the effluent sample is disinfected or untreated, as described in Section 3.2.

3.1  Spiked PBS  Samples
       3.1.1  Low Spiking Concentration

              •   Add 3.0 mL of spiking suspension dilution "C" (from Section 2.1.3, above) to each of three
                  100-mL sterile PBS samples and mix each sample by vigorously shaking the bottle a
                  minimum of 25 times.  The volume (mL) of undiluted spiking suspension added to each 100
                  mL sample is 3.0 mL x 10"4 mL per 100 mL, which is referred to as Vsplked per 100 mL in
                  Section 4.2 below.

              •   Filter the following aliquots from each spiked PBS sample prepared above: 10 mL, 1.0 mL,
                  and 0.1 mL and analyze according to the instructions provided in Methods 1106.1 and 1600,
                  Section 11.
       Note:  When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an  aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This
       will allow even distribution of the sample on the membrane.

     3.1.2 High Spiking Concentration
           •  Add 0.5 mL of spiking suspension dilution "A" (from Section 2.1.1) to each of three 100-mL
              sterile PBS samples and mix each sample by vigorously shaking the bottle a minimum of 25
              times.  The volume (mL) of undiluted spiking suspension added to each  100 mL sample is 5.0 *
              10"2 mL per 100 mL [(0.5 mL  x 10"1 mL) per 100 mL of sample],  which is referred to as Vsplked
              per 100 mL in Section 4.2 below.

           •  Prepare the following serial dilutions from each spiked PBS sample prepared above:  10"1, 10"2,
              10"3, and 10"4 mL. Labs may use 99 mL or 9 mL dilution  blanks to prepare the dilution series.
              Filter 1 mL of each serial dilution and analyze according to the instructions provided in Methods
              1106.1 and 1600, Section 11.
       Note:  When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an  aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This
       will allow even distribution of the sample on the membrane.
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                                                           Combined Sewer Overflow (CSO) Study Report
3.2   Spiked CSE Samples


       3.2.1   Low Spiking Concentration (only for use by laboratories that are spiking
               disinfected effluent)
               •   Add 3.0 mL of spiking suspension dilution "C" (from Section 2.1.3, above) to each of the
                  four 100-mL disinfected CSE samples and mix by vigorously shaking the bottle a minimum
                  of 25 times.  The volume (mL) of undiluted spiking suspension added to each 100 mL sample
                  is 3.0 x  10"4 mL per 100 mL, which is referred to as Vsplked per 100 mL in Section 4.2 below.

               •   Filter the following aliquots from each of the spiked disinfected CSE samples prepared
                  above: 10 mL, 1.0 mL, 0.1 (10"1) mL, and 0.01 (10~2) mL, and analyze according to the
                  instructions provided in Methods 1106.1 and 1600, Section 11.
       Note: When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This
       will allow even distribution of the sample on the membrane.

       3.2.2   High Spiking Concentration (only for use by laboratories that are spiking
               untreated effluent)
               •   Add 0.5 mL of spiking suspension dilution "A" (from Section 2.1.1) to each of four 100-mL
                  untreated CSE samples and mix by vigorously shaking the bottle a minimum of 25 times.
                  The volume (mL) of undiluted spiking suspension added to each 100 mL sample is 5.0 * 10"2
                  mL per  100 mL [(0.5  mL x 10"1 mL) per 100 mL of sample], which is referred to as Vsplked per
                  100 mL in Section 4.2 below.

               •   Prepare  the following serial dilutions from each of the spiked CSE samples prepared above:
                  10"3, 10"4, 10"5, 10"6, and 10"7 mL. Labs may use 99 mL or 9 mL dilution blanks to prepare the
                  dilution series. Filter 1 mL of each serial dilution and analyze according to the instructions
                  provided in Methods  1106.1 and 1600, Section  11.0.
       Note:  When analyzing smaller sample volumes (e.g,, <20 mL), 20-30 mL of PBS should be added to the
       funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This
       will allow even distribution of the sample on the membrane.

4.0   Calculation of Percent Recovery


Note: This section was added per laboratory  request and is for information purposes only. Laboratories are not
required to calculate percent recovery during this study.

Spiked enterococci percent recovery will be calculated in three steps as indicated in Sections 4.1 through 4.3
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.
                                                 45                                        April 2008

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4.1    Step 1 : Calculate Concentration of Enterococci (CPU / ml_) in Undiluted Spiking
       Suspension


       4.1 .1   The number of enterococci (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.

       4.1 .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).
       4.1 .3   Calculate the concentration of enterococci (CPU / mL) in the undiluted spiking suspension
              according to the following equation. (Example calculations are provided in Table 1 below.)
EnterOCOCCi undiluted spike =

Where,
           EnterOCOCCi undiluted spike

           CPU
                 V
                                             + CFU2 + ... + CFUn) / (^ + V2 + ... + Vn)
Enterococci (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
Table 1.   Example Calculations of Enterococci Spiking Suspension Concentration
Example
s

Example
1

Example
2
CPU / plate (triplicate analyses) from
TSA plates in Section 2.2.2
10'5mL plates

94, 106,89

169, 209, 304
KT6 mL plates

10,0,4

24, 30, 28
10~7 mL plates

0,0,0

0,2,0
Enterococci CPU / mL in
undiluted
spiking suspension
(EnterOCOCCi undiluted spike)*
(94+106+89) / (10'5+10-5+10-5)
289 / (3.0 x 10'5) = 9,633,333 =
ft /* .. A ft6 r*n i i .^.i
(169+209+30) / (10-5+10-5+1Q-6)
4087(2.1 x10'5) = 19,428,571
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                                                          Combined Sewer Overflow (CSO) Study Report
4.2    Step 2: Calculate "True" Spiked Enterococci (CPU /100 ml_)
       4.2.1  Calculate true concentration of spiked enterococci (CPU /100 mL) according to the following
              equation. Example calculations are provided in Table 2 below.

                I Spiked Enterococci ~ ^tntGrOCOCCI undiluted spike.) X (V spiked per 100 mL sample.)
               Where,
                 Spiked Enterococci
               EnterOCOCCi undiluted spike
                     =   Number of spiked enterococci (CPU /100 mL)
                     =   Enterococci (CPU / mL) in undiluted spiking suspension
                         (calculated in Section 4.1.3)
                » spiked perlOO mL sample

Table 2.    Example Calculations of Spiked Enterococci
                     =   mL of undiluted spiking suspension per 100 mL sample
                         (Section 3)
EnterOCOCCi undiluted
spike
(Table 1 above)
9.6x106CFU/ml_
1.9x107CFU/ml_
" spiked per 100 mL sample
3.0X10-4ml_per100
mL of sample (low)
5.0X10-2ml_per100
mL of sample (high)
I Spiked Enterococci
(9.6 x 106 CPU / mL) x (3.0 x 10'4 mL / 100 mL) =
2880 CPU/ 100 mL
(1.9 x 107 CPU / mL) x (5.0 x 10'2 mL / 100 mL) =
9.5 x 10s CPU/ 100 mL
4.3    Step 3: Calculate Percent Recovery
       4.3.1   Calculate percent recovery (R) using the following equation.
             R  =100 x
                                 T
Where,
  R
  Ns
  Nu
   Spiked Enterococci
Percent recovery
Enterococci (CPU / 100 mL) in the spiked sample (Methods 1106.1/1600, Section 12)
Enterococci (CPU / 100 mL) in the unspiked sample (Methods 1106.1/1600, Section 12)
True spiked Enterococci (CPU /100 mL) in spiked sample (Section 4.2, above)
Note: During the validation study, Nu (unspiked sample) is the mean Enterococci (CPU /100 mL) of the 4
unspiked effluent samples.
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       4.3.2   Example percent recovery calculations are provided in Table 3.
Table 3.    Example Percent Recovery Calculations
Ns
(CFU/100ml_)
3700
2600
1.6 x 1Q6
5.9 x 105
Nu
(CPU/ 100 ml_)
120
300
1.2 x 1Q3
5.5 x 1Q1
1 Spiked Enterococci
(CFU/100ml_)
2880
2880
9.5 x 105
9.5 x 105
Percent recovery (R)
1 00 x (3700- 120)72880
= 124%
1 00 x (2600 -300) 72880
= 80%
100 x (1.6 x 1Q6 - 1.2 x 103)/9.5x
105 = 168%
100 x (5.9 x 105-5.5x 101)/9.5x
105
= 62%
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        Appendix C:



CSO E. c0//Spiking Protocol
             49                              April 2008

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Combined Sewer Overflow (CSO) Study Report
                               E. co//Spiking Protocol for
             Interlaboratory Validation of Methods 1103.1  (mTEC) and
            1603 (modified mTEC)  in Combined  Sewer Effluents (CSE)
                                         (April 11, 2005)
The purpose of this protocol is to provide laboratories with E. coll spiking procedures for the interlaboratory
validation of Methods 1103.1 and 1603 in combined sewer effluents (CSE). The following sections are included
in this protocol:

     Section  1:   Preparation of Spiking Suspensions
     Section  2:   Spiking Suspension Dilution and Enumeration
     Section  3:   Sample Spiking
     Section  4:   Calculation of Percent Recovery

1.0  Preparation of Spiking Suspension

1.1    Stock Culture. Prepare a stock culture by inoculating a trypticase soy agar (TSA) slant (or other non-
       selective media) with Escherichia coll 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 (LIB). Prepare a 1% solution of LTB by combining 99 mL of sterile
       phosphate buffered saline (Methods 1103.1 and 1603, Section 7.5) and 1 mL of sterile single strength
       LTB in a sterile screw cap bottle or re-sealable dilution water container. Shake to mix.

1.3    Spiking  Suspension (Undiluted).  From the stock culture ofE. co//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 x
       107 to 1.0 x 10s E. coll colony forming units (CPU) per mL. This is referred to as the "undiluted spiking
       suspension."

Note: After the spiking suspension is incubated, it may be held at 6°C ± 2°Cfor a maximum of 72 hours. In
anticipation of a potential CSE event, spiking suspensions should be propagated every fourth day to ensure that a
viable suspension is available for sample spiking during the validation study. (For example, if the suspension was
propagated on Monday, a new spiking suspension should be propagated on Friday.)


2.0  Dilution and Enumeration of  Spiking Suspension

Since one of the objectives  of spiking the sample is to assess percent recovery, it is necessary to determine the
number of E. coll in the undiluted spiking suspension prepared in Section 1.3. This section provides instructions
for dilution (Section 2.1) and enumeration (Section 2.2) of the spiking suspension.

Note: Please  be sure to thoroughly mix  each of the spiking suspensions prior to performing the dilutions in the
steps below, as homogeneous suspensions are critical for accurate dilution and enumeration.
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2.1   Dilution of spiking suspension

       2.1.1  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 (PBS), 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.

       2.1.2  Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution "A" (from Section 2.1.1
              above) to 99 mL of sterile PBS, 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.

       2.1.3  Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" (from Section 2.1.2
              above) to 99 mL of sterile PBS, 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.

       2.1.4  Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" (from Section 2.1.3
              above) to 99 mL of sterile PBS, 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.


2.2    Enumeration of undiluted spiking suspension  (prepared in Section  1.3)

       2.2.1  Prepare tryptic soy agar (TSA) according to manufacturer's instructions, add 12 - 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 prior to use, 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:

              •   Mix dilution "B" by vigorously shaking the bottle a minimum of 25 times.  Pipet 0.1 mL of
                  dilution "B" (Section 2.1.2) onto surface of pre-dried TSA plate [10'5 mL (0.00001) of the
                  original spiking suspension].
              •   Mix dilution "C" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of
                  dilution "C" (Section 2.1.3) onto surface of pre-dried TSA plate [10'6 mL (0.000001) of the
                  original spiking suspension].
              •   Mix dilution "D" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of
                  dilution "D" (Section 2.1.4) onto surface of pre-dried TSA plate [10~7 mL (0.0000001) of the
                  original spiking suspension].
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3.0    Sample Spiking

       PBS samples will be spiked at two levels, low (Section 3.1.1) and high (Section 3.1.2). For each spike
       level, three PBS samples will be spiked. Four CSE samples will be spiked at either a low or high spike
       level, dependent on whether the effluent sample is disinfected or untreated, as described in Section 3.2.

Note: Please be sure to thoroughly mix each of the spiking suspensions prior to performing the dilutions in the
steps below, as homogeneous suspensions are critical for accurate dilution and enumeration.

3.1    Spiked PBS Samples
       3.1.1   Low Spiking Concentration
                  Add 3.0 mL of spiking suspension dilution "C" (from Section 2.1.3 above) to each of three
                   100-mL sterile PBS samples and mix each sample by vigorously shaking the bottle a
                  minimum of 25 times. The volume (mL) of undiluted spiking suspension added to each 100
                  mL sample is 3.0 x 10"5 mL per 100 mL, which is referred to as Vsplked per 100 mL in Section
                  4.2 below.
               •   Filter the following aliquots from each spiked PBS sample prepared above: 10 mL, 1.0 mL,
                  and 0.1 mL, and analyze according to the instructions provided in Methods 1103.1 and 1603,
                  Section 11.0.
       Note:  When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration.  This
       will allow even distribution of the sample on the membrane.

       3.1.2   High Spiking Concentration
               •   Add 3.0 mL of spiking suspension dilution "A" (from Section 2.1.1 above) to each of three
                   100-mL sterile PBS samples and mix by vigorously shaking the bottle a minimum of 25
                  times. The volume (mL) of undiluted spiking suspension added to each 100 mL sample is 3.0
                   x 10"2 mL per 100 mL, which is referred to as Vsplked per 100 mL in Section 4.2 below.
               •   Prepare the following serial dilutions from each spiked PBS sample prepared above: 10"1,
                   10"2, 10"3, and 10"4.  Labs may use 99 mL or 9 mL dilution blanks to prepare the dilution
                  series. Filter 1 mL of each serial dilution and analyze according to the instructions provided
                  in Methods 1103.1 and  1603, Section 11.0.
       Note:  When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration.  This
       will allow even distribution of the sample on the membrane.

3.2    Spiked CSE Samples

       3.2.1   Low Spiking Concentration (only for use by laboratories that are spiking disinfected
               effluent)
                  Add 3.0 mL of spiking suspension dilution "C" (from Section 2.1.3 above) to each of the
                  four, 100-mL disinfected CSE samples and mix by vigorously shaking the bottle a minimum
                  of 25 times. The volume (mL) of undiluted spiking suspension added to each 100 mL sample
                  is 3.0 x 10"5 mL per 100 mL, which is referred to as Vsplked per 100 mL in Section 4.2 below.
                  Filter the following aliquots from each of the spiked disinfected CSE samples prepared
                  above: 10 mL, 1.0 mL, 0.1  (10"1) mL, and 0.01 (10~2) mL, and analyze according to the
                  instructions provided in Methods 1103.1 and 1603, Section 11.0.
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                                                            Combined Sewer Overflow (CSO) Study Report
       Note:  When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This
       will allow even distribution of the sample on the membrane.

       3.2.2   High Spiking Concentration (only for use by laboratories that are spiking untreated
               effluent)
               •   Add 3.0 mL of spiking suspension dilution "A" (from Section 2.1.1 above) to each of four
                  100-mL untreated CSE samples and mix by vigorously shaking the bottle a minimum of 25
                  times.  The volume (mL) of undiluted spiking suspension added to each 100 mL sample is 3.0
                  x 10"2 mL per 100 mL, which is referred to as Vsplked per  100 mL in Section 4.2 below.
               •   Prepare the following serial dilutions from each of the spiked CSE samples prepared above:
                  10'3, 10'4,  10'5, 10'6, and 10'7 mL.  Labs may use 99 mL or 9 mL dilution blanks to prepare the
                  dilution series. Filter 1 mL of each serial dilution and analyze according to the instructions
                  provided in Methods  1103.1 and 1603, Section 11.0.
       Note:  When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the
       funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This
       will allow even distribution of the sample on the membrane.

4.0    Calculation of Percent Recovery

Note: This section was added per laboratory request and is for information purposes only.  Laboratories are not
required to calculate percent recovery during this study.

Spiked E. coli percent recovery will be calculated in three steps as indicated in Sections 4.1 through 4.3 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.

4.1    Step 1: Calculate Concentration of  E. coli (CPU / mL) in Undiluted  Spiking Suspension

       4.1.1   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.
       4.1.2   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).
       4.1.3   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.)
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Combined Sewer Overflow (CSO) Study Report
              E.  CO//
                      undiluted spike
              Where,
                  E. COli undiluted spike
                  CPU
                  V
CFU
CFUn)
V2
                                          Vn)
E. coll (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
Table 1.      Example calculations of E. co//'spiking suspension concentration
Example
s

Example
1

Example
2
CPU / plate (triplicate analyses) from
TSA plates (Section 2.2.2)
10'5mL plates

TNTC, TNTC,
TNTC

269, 289, 304
KT6 mL plates

94, 106,89

24, 30, 28
10~7 mL plates

10,0,4

0,2, 0
£. co// CPU / mL in undiluted
spiking suspension
(EC undiluted spike)
(94+106+89) / (10-6+10-6+1Q-6)
289 / (3.0 x 10'6) = 96,333,333
(269+289+30) / (10-5+10-5+10-6)
5887(2.1 x 10'5) = 28,000,000
*EC undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
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                                                          Combined Sewer Overflow (CSO) Study Report
4.2    Step 2: Calculate "True" Spiked E. coli (CPU /100 ml_)

       4.2.1   Calculate true concentration of spiked E. coli (CPU /100 mL) according to the following
               equation. Example calculations are provided in Table 2 below.

               Tspiked E. coli = (E. COll undiluted spike) x (V spiked per 100 mL sample)
               Where,

               1 Spiked E. coli

               E. COli undiluted spike
                                  Number of spiked E. coli (CPU / 100 mL)
                                  E. coli (CPU / mL) in undiluted spiking suspension
                                  (calculated in Section 4.1.3)
Table 2.
* spiked perlOO mL sample


Example Calculations of Spiked E. coli
                                                  mL of undiluted spiking suspension per 100 mL sample
                                                  (Section 3)
EL> undiluted spike
(Table 1 above)
9.6 x 107CFU/ml_
2.8 x 107CFU/ml_
v spiked per 100 mL sample
3.0 x 10-5ml_per100
mL of sample (low)
3.0 x 10-2ml_per100
mL of sample (high)
I Spiked E. coli
(9.6 x 107 CPU / mL) x (3.0 x 10'5 mL / 100 mL) =
2880 CPU /1 00 mL
(2.8 x 107 CPU / mL) x (3.0 x 10'2 mL / 100 mL) =
8.4 x 10s CPU/ 100 mL
4.3    Step 3: Calculate Percent Recovery
       4.3.1   Calculate percent recovery (R) using the following equation.
              R=WOx
              (Ns-Nu)
                    T
Where,
       R

       N,

       Nu

       TspikedE coli
          Percent recovery
          E. coli (CPU/ 100 mL) in the spiked sample (Methods 1103.1/1603, Section 13)

          E. coli (CPU / 100 mL) in the unspiked sample (Methods 1103.1/1603, Section 13)
          True spiked E. coli (CPU /100 mL) in spiked sample (Section 4.2, above)
Note: During the validation study, Nu (unspiked sample) is the mean E. coli (CFU /100 mL) of the 4 unspiked
effluent samples.
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4.3.2 Example percent recovery calculations are provided in Table 3.
Table 3. Example Percent Recovery Calculations
Ns
(CFU/100ml_)
3700
2600
1.6 x 1Q6
5.9 x 105
Nu
(CPU/ 100 ml_)
120
300
1.2 x 1Q3
5.5 x 1Q1
Tspiked £. co//
(CPU/ 100 ml_)
2880
2880
8.4 x 105
8.4 x 105
Percent recovery (R)
100x (3700- 120)72880
= 124%
100x (2600-300)72880
= 80%
100x(1.6x 106 - 1.2 x 103)/8.4x
105 =190%
100 x (5.9 x 105 - 5.5 x 1Q1) / 8.4 x 105
= 70%
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