PROTOCOL FOR ALTERNATE TEST PROCEDURES FOR COLIFORM BACTERIA
IN COMPLIANCE WITH WATER AND WASTEWATER REGULATIONS
QUANTITATIVE MEMBRANE FILTER METHODS
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C~ Version 1.0
^ December, 1995
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U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LABORATORY
CINCINNATI, OHIO 45268
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PROTOCOL FOR ALTERNATE TEST PROCEDURES FOR COLIFORM BACTERIA IN COMPLIANCE
WITH WATER AND WASTEWATER REGULATIONS FOR QUANTITATIVE MEMBRANE FILTER METHODS
I. INTRODUCTION
1.1 Regulatory Background
1.1.1 The Administrator, U.S. Environmental Protection Agency (EPA), approves
analytical methods for contaminants regulated under the Clean Water Act (CWA) and
the Safe Drinking Water Act (SDWA). When EPA publishes a regulation under these
Acts, which sets permit or maximum contaminant levels, it generally approves at
least one method for detection and/or quantification of that contaminant. After
a regulation is published, the Administrator may approve additional methods or
modifications of approved methods that have satisfactorily completed a
comparability study under the Alternate Test Procedure (ATP) Program.
1.1.2 Although the June 29, 1989 and January 8, 1991 regulations on the
microbiological characterization of finished drinking water samples require the
determination of the presence or absence of coliforms rather than their
quantitative enumeration.1'2 The quantitation of these organisms is still needed
for source water, other ambient waters and wastewaters under the Surface Water
Treatment Rule of the SDWA3 and the 304(h) regulations of the CWA4. The
quantitative MF methods for the evaluation of proposed quantitative
microbiological methods for total and fecal coliform bacteria are in this
protocol.
1.1.3 If the data evaluation demonstrates that the applicant's method performs
at least as well as the currently approved method, the National Exposure Research
Laboratory at Cincinnati (NERL-Cincinnati) will recommend approval to the Office
of Water, which begins the regulation development process. Regulation
development includes a Federal Register notice proposing to approve an ATP,
public comment on the proposed method, and depending on public comment, a final
rule published in the Federal Register that approves the method. The regulation
development process can take one year or more.
1.2 Comparability Determination
1.2.1 This protocol describes the method description and Comparability Study
data which EPA needs to evaluate an ATP for quantitative membrane filter (MF)
methods in microbiology. The ATP program is intended to be flexible,
consequently, EPA's NERL-Cincinnati may modify the study design for a particular
proposed method. For this reason, before beginning the comparability testing,
the applicant is required to contact the ATP Coordinator, Ecological Exposure
Research Division, NERL-Cincinnati, U.S. Environmental Protection Agency, 26 West
Martin Luther King Drive, Cincinnati, Ohio 45268, for approval of the test
design.
1.2.2 Generally the reference method selected by EPA for use in the
Comparability Study will be the same type of test as the proposed method.
Consequently, if an MF method were proposed for the target organism, the
corresponding MF reference method for the target organism would be used in the
Comparability Study. However, for ATP applications in which a new technology is
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proposed and for which there is no reference method counterpart, the ATP Program
will use professional judgment in determining which other method would be the
most appropriate as the reference method for the ATP Comparison Study.
2. APPLICATION FOR ATP
2.1 General Requirements
2.1.1 The general requirements for an application for nationwide approval of a
new or revised method for total coliforms, fecal coliforms, and/or E. coli
currently include the name and address of the applicant and/or authorized
representative; the microbiological analyte and EPA regulation for which the new
procedure is proposed; justification for the proposed new method; the title,
company identification number, the date of submission, and a complete, stand-
alone description of the proposed method in the required format (see Section 2.3
below).
2.1.2 If the applicant believes the proposed method is very similar to an Agency
method, and/or represents a minor optional change to an Agency method, the
applicant should also prepare a two-column side-by-side description of the
sections of the Agency reference method and the proposed method and highlight
differences between the methods. If the method is a proposed commercial version
of a previously approved method, differences in reagents, interferences, test
conditions, etc. should be presented with available performance data from the
proposed and reference methods.
2.1.3 NERL-Cincinnati will judge the proposed method to be: 1) an acceptable
version of or an optional minor modification of a previously promulgated method,
which does not require approval as an ATP or 2) a significantly different method
which requires an application for an ATP approval.
2.2 Every application for approval of a method must be made in triplicate
(original + 2 copies) and forwarded to the Director, Ecological Exposure Research
Division, NERL-Cincinnati, USEPA, Cincinnati, OH 45268. Upon receipt of the
application, the ATP Coordinator will assign it an identification number, which
should be used in all future communications. NERL-Cincinnati staff will initiate
its technical reviews. The initial review will concentrate on the clarity and
completeness of the description of the proposed method, the applicability of the
proposed microbiological principles and reactions and the performance
characteristics described for the method. The ATP staff will evaluate the
submitted information and advise the applicant whether or not a Comparability
Study is required.
2.3 Method Description
2.3.1 Each method description must include the following topics, listed in the
EMMC method format5, in the order given. The purpose of the description is to:
1) permit a fair comparison of the proposed and reference methods and 2) provide
a clean, clear description of the method that can be easily used by laboratories.
The method should read like a scientific paper.
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2.3.1.1 Scope and Application
Include target organisms, type of test, e.g., membrane filter, chromogenic
test, fluorogenic test, etc. and the sample types to which it is applicable.
2.3.1.2 Summary of Method
Include a brief outline of the method that describes its essential features
without extraneous details.
2.3.1.3 Definitions
Include special terms or unique usage of terms. Do not include common
microbiological terms.
2.3.1.4 Interferences
Include information and data generated by applicant during method
development using typical samples containing a specific quantity of an
interference such toxic materials, particulates, non-target organisms, etc.
2.3.1.5 Safety
Refer to good laboratory practices and use of a hood, goggles, and/or
protective clothing, if appropriate. Emphasize any special procedure or
precaution.
2.3.1.6 Instrumentation, Equipment and Supplies
Describe the necessary instrumentation, equipment and supplies and reference
applicable manuals.
2.3.1.7 Reagents, Standards and Media
Describe reagent, standard and media formulations and preparation. Indicate
shelf life of packaged materials and special storage requirements.
2.3.1.8 Sample Collection, Dechlorination, Preservation, Shipment, and Storage
Detafl sample collection and handling requirements. Consider the sample
collector, sample containers, dechlorination (if applicable), sample holding
times and temperature as specified in Standard Methods*.
2.3.1.9 Quality Control (QC)
Indicate the specific QC procedures and the frequency of performance
required for the proposed method. They should include sterility checks, positive
and negative controls, verification/confirmation of the target organism, media
performance checks, duplicate analyses, etc. Document that a general QA/QC
program is in operation, that routine QC checks are recorded and that actions are
taken if a problem is indicated7'*.
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2.3.1.10 Calibration and Standardization
If applicable, include the calibration steps that are performed on pH meter,
analytical balance, thermometer, autoclave, etc.
2.3.1.11 Procedure
Detail the sample preparation and analytical steps in the proposed method
write-up. Exceptions are those routine microbiological procedures, such as
membrane filtration of samples, that are known to professionals and that may be
incorporated by reference.
2.3.1.12 Data Analyses and Calculations
Describe the procedures for data analyses, calculations, interpretation and
reporting of results.
2.3.1.13 Method Performance Characteristics (sensitivity, specificity, recovery,
and precision)
Provide available information on the performance characteristics of the
proposed method and the procedures by which they were determined. Specificity
data should demonstrate the ability of the proposed method to recover and
distinguish the target organism from other organisms in the sample. The proposer
should have data on method sensitivity, the variability of replicate analyses and
data on recoveries of known numbers of target and non-target organisms by the
proposed method. Summaries of these evaluative data should be included.
(However, in addition to the data provided above in the method description,
a separate formal Specificity Study will be required as part of the ATP process.
The method for determining the specificity of a method is presented in the
Appendix.)
2.3.1.14 Pollution Prevention
Pollution prevention is any technique that reduces or eliminates the
quantity or toxicity of waste at the point of generation. It is the
environmental management tool preferred over waste disposal or recycling. When
feasible, laboratory staff should use a pollution prevention technique such as
preparation of the smallest practical volumes of reagents, standards and media
or downsizing of the test units in a method.
If the proposed method prevents or reduces exposure to toxicity, pollution
of the laboratory or the general environment including reduced generation of
wastes, cite here. Also indicate non-applicability.
2.3.1.15 Waste Management
The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable rules and
regulations. Excess reagents and samples and method process wastes should be
characterized and disposed of in an acceptable manner. The Agency urges
laboratories to protect the air, water, and land by minimizing and controlling
all releases from hoods and bench operations, complying with the letter and
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spirit of any waste discharge permit and regulations, and by complying with all
solid and hazardous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For further information on
waste management consult "The Waste Management Manual for Laboratory Personnel,"
available from the American Chemical Society.
Describe the proper disposal methods for waste reagents, materials, supplies
and samples.
2.3.1.16 References
Cite those source documents and publications which are necessary sources of
information to properly perform the method.
2.3.1.17 Tables, Diagrams, Flowcharts, and Validation Data
Provide as needed.
2.3.1.18 Proprietary Information
Mark proprietary information in the proposed method description as
"Confidential". EPA staff will treat proprietary information according to the
regulations outlined in Subparts A and B in Part 2 of Title 40 of the CFR.
2.4 Study Approval
2.4.1 Method comparability data and quality control data will be required for
each application of a new or significantly modified method. The applicant is
urged not to initiate the Comparability Study until EPA has completed an
evaluation of the method description and the preliminary performance
characteristic information and has approved the Comparability Study design.
3. COMPARABILITY STUDY DESIGN
3.1 Summary of Study Design
3.1.1 Typically, the Comparability Study data are generated from replicate
analyses of ten or more ambient waters and/or wastewaters from geographically-
dispersed areas, which contain the target organism or organisms in varying
numbers and different water or wastewater compositions. To simplify the tests,
preliminary screening analyses are recommended to establish approximate numbers
and reduce the number of dilutions and analyses in the formal study. The volumes
or dilutions of water and wastewater samples tested must generate data within the
acceptable range of the Agency reference method.
3.1.2 A minimum of twenty (20) replicate analyses are performed by each method
on the 3 or more dilutions selected for each of the 10 samples. The replicate
filtrations on the three or more dilutions of each sample must be performed on
the same day for both the reference and proposed methods.
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3.1.3 See the following example of a Comparability Study design.
Example of ATP Study Design
Natural Sources No. Samples/
~ of Organisms Source
Reference Method
and 10 1
Proposed Method
Replicate MF Minimum Comparability
Filtrations per Organism* Results Required
and Sample Dilution per Method
Reference Method 20 200
Proposed Method 20 200
*Total Coliform, Fecal Coliform, etc.
3.2 Laboratory Participation
3.2.1 Since the purpose of the study is to compare the proposed method to the
reference method with minimal variability due to individual analyst error, the
number of laboratories participating in the study should be minimized. It is
strongly recommended that only one laboratory perform the analyses. This
laboratory must be certified under the drinking water laboratory certification
program. An applicant having a vested interest in the method, instrumentation,
apparatus, reagents, media, or associated kits, may not perform the Comparability
Study analyses in the applicant's laboratory.
3.3 Quality Control Data
3.3.1 Conduct QC checks on each day of analyses including temperature of
incubators, analyses of known positive and negative response cultures, sterility
controls and verification of colonies. Maintain records of QC checks performed.
See the Manual for the Certification of Laboratories Analyzing Drinking Water7
and the Intralaboratory Quality ControlGuidelinesin Standard Methods8 for
general guidance.
3.4 Sample Collection and Handling
3.4.1 Each sample should be collected and held in a single sterile, wide-mouth
bottle of non-toxic heat-resistant plastic or borosilicate glass with a leak-
proof screw cap or ground-glass stopper. The container must be resistant to the
solvent action of water and survive sterilization without deformity or production
of toxic materials. Screw caps must not produce bacteriostatic, toxic or
nutritive products during sterilization. Bottles and closures should be checked
for these effects before use in the study*.
3.4.2 Samples should be maintained at 1-4 C during transit and holding time.
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4. PREPARATION FOR COMPARABILITY STUDY
4.1 Selection and Collection of Water and Uastewater Samples
4.1.1 The reason for requiring ten different water/wastewater samples is to
obtain, as roach as is practical, a good presentation of the wide range of water
types with their even wider range of target organisms, to which a method must
respond appropriately. The ten samples can be collected over time as needed to
complete the Comparability Study, with the understanding that all testing of a
water or wastewater must be completed in a single day for both the reference and
proposed methods.
4.1.2 Collect each water or wastewater sample in sufficient volume to complete
all replicate analyses of sample or dilution volumes by both the reference and
proposed methods.
4.2 Sample Volumes and Dilutions for the Comparability Study
4.2.1 Since the study is intended to compare the performance of the proposed
method to the reference method, the data for the comparison must be from the same
sample volumes or dilutions for both methods based on those sample volumes or
dilutions that produce plate counts within the appropriate range for the
reference method.
4.2.2 The sample volumes or dilutions are prepared by one of two options:
Option A utilizes preliminary analyses to establish target organism
densities before beginning the Comparability Study. This approach reduces the
number of dilutions needed to assure a bracketing of the range count but requires
the additional effort of these analyses and a 24 h holding period for samples.
Option B proceeds directly with the Comparability Study. The samples are
not held for 24 h and this option does not require preliminary analyses.
Instead, the analyst gambles on her/his ability to "guess-timate" the sample
volumes or dilutions necessary to bracket the reference method's counting range.
This usually requires an expanded series of dilutions.
4.3 Option A. Determination of Bacterial Density
4.3.1 After collecting a water or wastewater sample(s), determine the density
of the target organism using the MF procedure and media designated for the target
organism:
Target Organism
Total col i forms
Fecal col i forms
TABLE 1
Method
SM 9222B10
SM 92220"
Medium
M-Endo LES Agar
M-FC
4.3.2 Count the colonies of target organisms after 24 h to determine their
approximate density in each water or wastewater sample. Use these results to
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establish the sample volumes and/or dilution series needed to bracket the desired
counting range for the reference method(s) in the Comparative Study. For the
total coliform method using M-Endo LES agar, the range is 20-80 colonies/plate
and for the fecal coliform method using M-FC agar, the range is 20-60 colonies/
plate. Sample volumes of less than 1 ml^njust be added as a dilution. Filtration
volumes of tess than 10 ml must be proceeded in the filtration process by
approximately 10 ml of sterile dilution water.
5. COMPARABILITY STUDY
5.1 Test Methods
5.1.1 The description of the reference methods and guidelines for use of the
proposed method follow. For valid comparisons, the samples, sample volumes and
dilutions must be the same for both methods and be based on the appropriate
dilutions for the reference method. The spiked samples must be stirred
continuously while portions are withdrawn for filtration and preparation of
dilutions.
5.1.2 Include the testing of pure cultures of organisms of known positive and
negative responses for total coliform and fecal coliform cultures to insure a
proper interpretation of the results.
5.2 Reference Methods
5.2.1 The current reference methods that will be used in the comparison studies
are listed in Table 2.
Table 2
Target Organism Reference Method Medium
Total coliforms SM 9222B10/9221B12 M-Endo LES/LTB/BGLB
Fecal coliforms SM 9222B10/9221E.11J M-FC/LTB/EC
5;3 Reference Method forTotal Coliform Bacteria
5.3.1 Filter the appropriate volumes of water or wastewater and/or dilutions to
yield 20-80 total coliform bacteria on M-Endo LES agar.
5.3.2 Incubate at 35 C for 22-24 h. Golden green metallic sheen colonies are
coliforms. Count and record sheen colonies.
5.3.3 For each sample, randomly pick 10 sheen colonies from each of 20 replicate
plates, into LTB tubes. Incubate for 24-48 h at 35 C and examine. Growth,
growth and gas, or growth and acid are positive presumptive tests.
5.3.4 Transfer growth from positive tubes into BGLB tubes and incubate at 35 C
for 24-48 h. Growth and gas verify total coliform bacteria. Adjust original
count based on verified count, and record.
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5.4 Reference Method for Fecal Coliform Bacteria
5.4.1 Filter the appropriate volumes of water or wastewater and/or dilutions to
yield 20-60 fecal coliform bacteria on M-FC agar plates.
5.4.2 Incubate M-FC agar plates for 24 h at 44.5 C. Count and record blue
colonies as fecal coliform bacteria.
5.4.3 From each sample randomly pick 10 blue colonies on each of the 20
replicate plates into LIB tubes. Incubate for 24-48 h at 35 C. Growth, growth
with gas, or growth and acid are presumptive positive tests.
5.4.4 Transfer growth from the positive LIB tubes into EC tubes and incubate at
44~5 C for 24 h. Growth and gas in EC medium verify fecal coliform bacteria.
(Mjustj)rifljina 1 count based on pps1t1_ye_ver1f1cat1on and record results^.
5.5 Proposed Method
5.5.1 Filter the appropriate amounts of water or wastewater and/or dilutions and
follow developer's directions for analyses.
5.5.2 Incubate tests units as directed. Follow developer's instructions for
identifying and enumerating target colonies.
5.5.3 Complete verification steps according to the developer's directions.
5.6 Recording Results
Record the colony counts for all volumes tested by the reference and
proposed methods.
6. DATA REPORTING
6.1 All the data from the comparability study, i.e., the replicate observations
of the samples by the EPA reference method and the proposed method and the
quality control observations should be forwarded to the Director, Ecological
Exposure Research Division, National Exposure Research Laboratory - Cincinnati
(address on page 1).
6.2 The results from the analyses of the ten samples should be recorded in the
formats suggested in Attachments 1 and 2. "Note that the forms have only one
-entry space for LTB and BGLB verification results for each pick from M-Endo LES
j of M-FC agar. The values entered should be the sums of the 24 and 48 h
< _resppjis.e_s_.j The evaluation of the application can be accomplished more quickly
by the EERD, NERL-Cincinnati ATP program if the information is also forwarded on
disks compatible with an IBM-PC computer. The text on the disks should be
presented in the latest version of WordPerfect (currently 5.1) and the data
presented in WordPerfect or in ASCII. Confirm the current formats with EPA
before generating study data.
7. DATA REVIEW
7.1 Upon receipt of an applicant's data sets, NERL-Cincinnati staff will
initiate its technical and statistical reviews. Appropriate criteria will be
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used to determine the acceptability of the reference method data as a basis in
the evaluation of the analyses by the proposed method. If this evaluation is
favorable, the proposed method will be recommended for approval by the program
office and the applicant so notified. If the results of the analyses by the
approved method are not satisfactory, the applicant will be notified that the
application -has not been approved.
7.2 The statistical review will consist of two parts: an examination of the
data including descriptive statistics and a comparability analysis of the data.
First, the data will be screened for outliers and unusual patterns. Descriptive
statistics, such as the mean, standard deviation and coefficient of variation,
will then be. calculated for each set ofjreplicates._'The "second part of the
statistical review will compare the precision and-recovery of the proposed method
with that of the reference method using appropriate statistical techniques. The
^rec4sit>rrTTf~^enP^^ that of the reference
method by means of a nonparameteric statistical procedure attributable to
Scheffe14. An analysis of variance (ANOVA) will be used to compare the recovery
of the proposed method to that of the approved method.
8. METHOD RECOMMENDATION AND APPROVAL
8.1 After completion of the technical and statistical reviews, NERL-Cincinnati
will prepare its recommendation for approval/disapproval of the new or
significantly revised method. It will notify the applicant of its
recommendation, and forward the recommendation to the Office of Water (OW), which
has the responsibility for proposing the method in the Federal Register.
Following a three-month public comment period, OW will review submitted comments
and prepare the final nationwide approval/disapproval decision and promulgation
notice in the Federal Register.
8.2 Upon approval of the method, the applicant will be responsible for the
publication and distribution of the approved method to anyone requesting a copy.
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9. REFERENCES
1. U.S. Environmental Protection Agency. Drinking Water; National Primary
Drinking Water Regulations; Total Coliforms (Including Fecal Coliforms and
f. co7/); Final Rule. Federal Register 54(124):27562-27568, June 29,
1989. -
2. U.S. Environmental Protection Agency. National Primary Drinking Water
Regulations; Analytical Techniques; Colifonn Bacteria, Federal Register
56(5):636-643, January 8, 1991.
3. 40 CFR Part 141 and 142, Drinking Water: National Primary Drinking Water
Regulations; Filtration, Disinfection, Turbidity, Giardia lamblia, Viruses,
LegioneTla and Heterotrophic Bacteria; Final Rule June 29, 1989, pp. 27530-
27531.
4. 40 CFR Part 136, Guidelines Establishing Test Procedures for the Analysis
of Pollutants Under the Clean Water Act; Final Rule and Interim Final Rule
and Proposed Rule, October 26, 1984, p. 19.
5. Villa, 0. and L. Reed: Co-Chairs, EMMC Methods Integration Panel. Final
Version of Approved EMMC Format (Memorandum to Members of EMMC Steering
Committee, Methods Integration Panel, and Work Group, Tri-Chairs). U.S.
Environmental Protection Agency, February 14, 1992. pp. 1-2.
6. Standard Methods for the Examination of Water and Wastewater. 18th Ed.,
APHA, Washington, DC, 1992, Section 9060A and 9060B, pp. 9-18 thru 9-20.
7. U.S. Environmental Protection Agency. Manual for the Certification of
Laboratories Analyzing Drinking Water: Criteria and Procedures, Quality
Assurance, 3rd ed. EPA/570-9-90-008A. Chapter 5, Microbiology (revised).
Office of Drinking Water, Washington, D.C., October 1991, pp. 37-48.
8. Standard Methods Section 9020B, pp. 9-3 thru 9-12.
9. Standard Methods Section 9020B, p. 9-5.
10. Standard Methods Section 9222B, pp. 9-54 thru 9-58.
11. Standard Methods Section 9222D, pp. 9-60 thru 9-61.
12. Standard Methods.... Section 9221B, pp. 9-46 to 9-50.
13. Standard Methods Section 9221E.1, pp. 9-52 and 9-53.
14. Scheffe, Henry. The Analysis of Variance. John Wiley and Sons, New York,
NY, 1959.
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ATTACHMENT 1. EXAMPLE FORMAT
RESULTS OF ATP COMPARABILITY STUDY OF QUANTITATIVE MF METHOD FOR TOTAL COUFORM BACTERIA
Title of Proposed Method: Lab Nome & Location:
Analyst: Supervisor's Signature:
RECORD COLONY COUNTS and VERIFIED COUNTS by REFERENCE and PROPOSED METHODS
Sample # and Description:
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ATTACHMENT 2. EXAMPLE FORMAT
RESULTS OF ATP COMPARABILITY STUDY OF QUANTITATIVE MF METHOD FOR FECAL COUFORM BACTERIA
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