[Federal Register Volume 66, Number 169 (Thursday, August 30, 2001)]
[Proposed Rules]
[Pages 45811-45829]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 01-21813]


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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 136

[FRL-7045-6]
RIN 2040-AD08


Guidelines Establishing Test Procedures for the Analysis of 
Pollutants; Analytical Methods for Biological Pollutants in Ambient 
Water; Proposed Rule

AGENCY: Environmental Protection Agency (EPA).

ACTION: Proposed rule.

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SUMMARY: This proposed regulation would amend the ``Guidelines 
Establishing Test Procedures for the Analysis of Pollutants'' under 
section 304(h) of the Clean Water Act (CWA), by adding several 
analytical test procedures for enumerating the bacteria, Escherichia 
coli (E. coli) and enterococci, and the protozoans, Cryptosporidium and 
Giardia, in ambient water to the list of Agency-approved methods.
    This proposal would make available a suite of Most Probable Number 
(MPN) (i.e. multiple-tube, multiple-well) and membrane filter (MF) 
methods for enumerating E. coli and enteroccoci bacteria in ambient 
water. Both culture-based and enzyme-substrate techniques are included. 
Some test methods are also applicable to total coliform determinations 
when these are the preliminary or concurrent steps for E. coli 
enumeration. Similarly, this document proposes new methods for 
detecting Cryptosporidium and Giardia in ambient water. Regulators may 
use these test procedures to assess Cryptosporidium and Giardia 
concentrations in ambient waters.

DATES: Comments must be postmarked, delivered by hand, or 
electronically mailed on or before October 29, 2001. Comments provided 
electronically will be considered timely if they are submitted 
electronically by 11:59 p.m. Eastern Time (ET) on October 29, 2001.

ADDRESSES: Send written comments on the proposed rule to ``Part 136 
Biological Methods'' Comment Clerk (W-99-14); Water Docket (4101); U. 
S. Environmental Protection Agency; Ariel Rios Building; 1200 
Pennsylvania Avenue, NW., Washington, DC 20460. Hand deliveries should 
be delivered to: EPA's Water Docket at 401 M Street, SW., East Tower 
Basement (Room EB 57), Washington, DC 20460. If you wish to hand-
deliver your comments, please call (202) 260-3027 between 9 a.m. and 4 
p.m., Monday through Friday, excluding Federal holidays, to obtain the 
room location for the Docket. Comments also may be submitted 
electronically to: [email protected]. 

FOR FURTHER INFORMATION CONTACT: For regulatory information regarding 
this proposal, contact Maria Gomez-Taylor, Ph.D.; Engineering and 
Analysis Division (4303); Office of Science and Technology; Office of 
Water; U.S. Environmental Protection Agency; Ariel Rios Building; 1200 
Pennsylvania Avenue, NW.; Washington, DC 20460, or call (202) 260-1639.
    For technical information regarding analytical methods proposed in 
today's rule, contact Robin Oshiro; Office of Science and Technology 
(4304); Office of Water; U.S. Environmental Protection Agency; Ariel 
Rios Building; 1200 Pennsylvania Avenue, NW.; Washington, DC 20460, or 
call (202) 260-7278.

SUPPLEMENTARY INFORMATION:

Potentially Affected/Regulated Entities

    EPA Regions, as well as States, Territories, and Tribes are 
authorized to implement the water quality standards program and the 
National Pollutant Discharge Elimination System (NPDES) program, and to 
issue permits that comply with the technology-based and water quality-
based requirements of the Clean Water Act (CWA). In doing so, 
permitting authorities, including authorized States, Territories, and 
Tribes, make discretionary choices when writing permits, including the 
selection of pollutants to be measured and monitoring requirements. If 
EPA has ``approved'' (i.e., promulgated through rulemaking) 
standardized testing procedures for a given pollutant, the permit must 
specify one of the approved testing procedures or an approved alternate 
test procedure. Although EPA proposes to include test methods for four 
biological pollutants in section 136.3, it recommends their use only 
for ambient water quality monitoring. EPA does not propose to approve 
these test methods for effluent matrices.
    EPA has developed ambient water quality criteria for E. coli and 
enteroccoci bacteria and is considering criteria for Cryptosporidium 
and Giardia. The States, Territories, and Tribes may adopt these 
criteria into their water quality standards and may issue water 
quality-based permits that require monitoring for these pollutants in 
ambient waters. Therefore, discharges with water quality-based permits 
could be affected by the standardization of testing procedures in this 
rulemaking in instances where the permitting

[[Page 45812]]

authority requires that such permits incorporate ambient water 
monitoring. EPA does not require inclusion of ambient water monitoring 
for NPDES permits. In addition, when a State, Territory, or authorized 
Tribe provides certification of Federal licenses under the CWA section 
401, and when such certification requires measurement of waste 
constituents specified in 40 CFR 136, then such measurements must be in 
accordance with approved testing procedures if such procedures are 
available. 40 CFR 136.1(c). Categories and entities that ultimately may 
be affected/regulated include:

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                                               Examples of potentially
                 Category                    affected/regulated entities
------------------------------------------------------------------------
Regional, State, and Territorial            States, Territories, and
 Governments and Indian Tribes.              Tribes authorized to
                                             administer the water
                                             quality standards programs;
                                             States, Territories, and
                                             Tribes providing
                                             certification under Clean
                                             Water Act section 401;
                                             Governmental permittees.
Municipalities............................  Publicly-owned treatment
                                             works with water quality-
                                             based permits.
Industry..................................  Industrial facilities with
                                             water quality-based
                                             permits.
------------------------------------------------------------------------

    This table is not intended to be exhaustive, but rather provides 
guidance for readers regarding entities likely to be affected/regulated 
by this action. This table lists the types of entities that EPA is now 
aware could potentially be affected/regulated by this action. Other 
types of entities not listed in the table also could be affected/
regulated. If you have questions regarding the applicability of this 
action to a particular entity, consult one of the persons listed in the 
FOR FURTHER INFORMATION CONTACT section.

Record and Commenting Procedures

    The record for this rulemaking has been established under docket 
number W-99-14. A copy of the supporting documents cited in this 
proposal are available for review at EPA's Water Docket. The record is 
available for inspection from 9 a.m. to 4 p.m. EST, Monday through 
Friday, excluding Federal holidays at EPA's Water Docket, 401 M Street 
SW., East Tower Basement (Room EB 57), Washington, DC 20460. For access 
to docket materials, please call (202) 260-3027 to schedule an 
appointment.
    Commenters are requested to submit any references cited in their 
comments. Commenters also are requested to submit an original and three 
copies of their written comments and enclosures, and to clearly 
identify the specific pollutant and method to which the comment 
applies. Commenters that want a confirmed receipt of their comments 
should include a self-addressed, stamped envelope. All comments must be 
postmarked or delivered by hand. No facsimiles (faxes) will be 
accepted.
    Electronic comments must be submitted as a Word Perfect for Windows 
5/6/7/8 file or an ASCII file, avoiding the use of special characters 
and any form of encryption. Comments and data also will be accepted on 
disks in Word Perfect 5/6/7/8 or ASCII file format. Electronic comments 
on this notice may be filed online at many Federal Depository 
Libraries. All electronic comments must be identified by docket number. 
Electronic comments will be transferred into a paper version for the 
official record. EPA will attempt to clarify electronic comments if 
there is an apparent error in transmission.

Information on Internet Access

    This Federal Register document has been placed on the Internet for 
public review and downloading at the following location: http//
www.epa.gov/fedrgstr.

Availability and Sources for Methods

    Copies of analytical methods published by EPA are available for a 
nominal cost through the National Technical Information Service (NTIS); 
U.S. Department of Commerce; 5285 Port Royal Road; Springfield, VA 
22161, or call (800) 553-6847. Copies of the EPA methods cited in this 
proposal may be obtained from Robin Oshiro; Office of Science and 
Technology (4304); Office of Water; U.S. Environmental Protection 
Agency; Ariel Rios Building; 1200 Pennsylvania Avenue, NW.; Washington, 
DC 20460, or call (202) 260-7278. Copies of several of the EPA methods 
cited in this proposal may also be downloaded from the EPA Office of 
Research and Development; National Exposure Research Laboratory (NERL)-
Cincinnati Microbiology home page at www.epa.gov/microbes/. Copies of 
published journal articles for selected EPA methods are available in 
the public domain. All other methods must be obtained from the 
publisher. Publishers (with contact information) for all methods are 
included in the References section of today's rule. Copies of all 
methods are also available in the public record for this proposal.

Outline of Preamble

I. Statutory Authority
II. Regulatory Background
III. Explanation of Today's Action
    A. Methods for Bacterial Pollutants
    1. Most Probable Number (MPN) and Membrane Filtration (MF) 
Methods
    2. Selection of Proposed Methods
    3. Methods for E. coli
    4. Methods for Enterococci
    5. Request for Comment and Available Data
    B. Methods for Protozoa
    1. Cryptosporidium and Giardia
    2. Request for Comment and Available Data
IV. Administrative Requirements
    A. Executive Order 12866--Regulatory Planning and Review
    B. Unfunded Mandates Reform Act
    C. Regulatory Flexibility Act (RFA), as amended by the Small 
Business Regulatory Enforcement Act of 1996 (SBREFA), 5 U.S.C. 601 
et seq.
    D. Paperwork Reduction Act
    E. National Technology Transfer and Advancement Act
    F. Executive Order 13045--Protection of Children From 
Environmental Health Risks and Safety Risks
    G. Executive Order 13175--Consultation and Coordination With 
Indian Tribal Governments
    H. Executive Order 13132--Federalism
    I. Executive Order 13211--Actions Concerning Regulations That 
Significantly Affect Energy Supply, Distribution, or Use
V. Media Acronyms
VI. References

I. Statutory Authority

    Today's proposal is pursuant to the authority of sections 303(c), 
304(a), 304(h) and 501(a) of the Clean Water Act (CWA), 33 U.S.C. 
1313(c), 1314(a), 1314(h), 1361(a) (the ``Act''). Section 303(c) of the 
Act establishes the basis for the current water quality standards 
program. This section requires EPA to review and approve or disapprove 
State-adopted water quality standards. Section 304(a) of the Act 
requires the EPA Administrator to conduct non-regulatory scientific 
assessments of ecological and public health effects to support the 
development of water quality criteria associated with specific ambient 
water uses. When these criteria are adopted as State water quality 
standards under section 303, they become the enforceable maximum 
acceptable levels of pollutants in ambient waters. Section 304(h) of 
the Act requires the EPA Administrator to ``promulgate guidelines 
establishing test procedures for the analysis of pollutants that shall 
include the factors which must be provided in any certification 
pursuant to section 401 of this Act or permit applications pursuant to 
section 402 of this Act.'' Section 501(a) of the Act authorizes the 
Administrator to ``prescribe such regulations as are necessary to carry 
out this function under this Act.''

[[Page 45813]]

II. Regulatory Background

    To fulfill the CWA's mandate to maintain ``fishable and swimmable'' 
waters, EPA is required to develop ambient water quality criteria based 
on a scientific assessment of the relationship between pollutant 
concentrations and environmental and human health effects. Ambient 
water refers to any fresh, marine, or estuarine surface water used for 
recreation; propagation of fish, shellfish, or wildlife; agriculture; 
industry; navigation; or as source water for drinking water facilities. 
These ambient water quality criteria become enforceable water quality 
standards when adopted by State, Territorial, Tribal, and local 
governments implementing a water-quality based approach to pollution 
control. For bacterial pollution in ambient water, EPA has developed 
bacteriological ambient water quality criteria recommendations for E. 
coli in freshwater and enterococci in freshwater and marine waters (51 
FR 8012, March 7, 1986). There are a number of zoonotic diseases of 
concern to humans (diseases transferred from animals to humans) if 
recreational or other waters are contaminated with fecal material from 
non-human animal species. E. coli species are a subset of the coliform 
bacteria group that is part of the normal intestinal flora of humans 
and animals and is, therefore, a direct indicator of fecal 
contamination from these sources in water. Enterococci, which include 
Enterococcus faecalis and Enterococcus faecium, are enteric bacteria 
used to indicate fecal contamination and the possible presence of 
pathogens, in water. Based on previous EPA guidance, total and fecal 
coliform bacteria currently have been included in many water quality 
standards as indicators of bacterial contamination (USEPA, 1976). 
However, more recent epidemiological studies described in Ambient Water 
Quality Criteria for Bacteria--1986 (USEPA, 1986a), indicate that E. 
coli and enterococci show a direct correlation with swimming-associated 
gastrointestinal illness rates, while fecal coliforms do not. As the 
concentration of E. coli and/or enterococci increase(s), the illness 
rates also increase. These indicators are used as part of the bacterial 
water quality criteria and standards to enhance the protection of human 
health and the environment.
    In addition to bacterial pollution, EPA is concerned about 
waterborne parasites and has developed test methods for Cryptosporidium 
and Giardia. These waterborne parasites are responsible for cases of 
severe and widespread human illness when present in drinking water 
supplies as a result of contamination of source waters. To support 
future regulation of these organisms in drinking water, the Safe 
Drinking Water Act Amendments of 1996 required the EPA to evaluate the 
risk to public health associated with Cryptosporidium and Giardia 
contamination. To implement these requirements, EPA plans to assess 
Cryptosporidium and Giardia occurrence in freshwater surface water 
bodies. Because one of the designated uses of some ambient waters may 
include the use of the waterbody as a drinking water source, EPA may 
develop ambient water quality criteria for Cryptosporidium and Giardia 
in the future. EPA plans to use the test methods discussed in this 
notice to support these assessments. By doing so, EPA desires to 
promote consistency on the methods used for these assessments to ensure 
that the data collected are of good quality and comparable. EPA also 
wishes to make these methods available for use by the States and for 
general use for risk assessments.
    In today's notice, EPA is proposing test methods for E. coli, 
enterococci, Cryptosporidium, and Giardia. Proposal of the bacterial 
methods supports the use of E. coli and enterococci as indicators in 
place of the total and fecal coliform indicators in State, Territorial, 
Tribal, and local water quality-based monitoring programs. Proposal of 
test methods for Cryptosporidium and Giardia supports the use of these 
methods in evaluating surface water occurrence of these organisms and 
the associated watershed vulnerability levels of concern for 
waterbodies designated as potential drinking water sources under the 
water quality standards program. EPA proposes to approve the use of 
test methods for E. coli, enterococci, Cryptosporidium, and Giardia for 
ambient water quality monitoring only. Although EPA believes that these 
methods are appropriate for ambient water quality monitoring, the 
Agency has not determined that these methods are acceptable for 
application to other matrices.
    This proposal was initiated in response to national directives that 
seek to improve and assist in State, Territorial, Tribal, and local 
implementation of water quality standards, ambient water monitoring 
programs, and public notification programs to reduce public health 
risks posed by biological pollutants in ambient water. The primary 
initiatives that served as impetus for today's proposal include the 
Beaches Environmental Assessment Closure and Health (BEACH) Program; 
the Beach Action Plan (EPA-600-R-98-079); the Beach Watch Program; the 
Beaches Environmental Monitoring for Public Access and Community 
Tracking (EMPACT) Program; and the Water Quality Criteria and Standards 
Plan. Additionally, this rule is expected to satisfy requests by State, 
Territorial, Tribal, and local governments, regulated entities, and 
environmental laboratories that EPA publish analytical test procedures 
for enumerating E. coli, enterococci, Cryptosporidium, and Giardia in 
ambient water that were evaluated through interlaboratory validation or 
extensive intralaboratory comparison with previously approved methods.

III. Explanation of Today's Action

A. Methods for Bacterial Pollutants

    This proposal would make available a suite of Most Probable Number 
(MPN) (i.e., multiple-tube, multiple-well), and membrane filter (MF) 
methods for enumerating (i.e., determining organism density) E. coli 
and enteroccoci in ambient water as part of State, Territorial, Tribal, 
and local water quality monitoring programs. Multiple-tube, multiple-
well, and MF formats include culture and enzyme-substrate techniques. 
Culture methods use lactose fermentation (E. coli), presence of 
turbidity (enterococci), colony formation, or color to detect the 
target organism. Enzyme-substrate tests use chromogenic (e.g., indoxyl-
-D-glucuronide) or fluorogenic (e.g., 4-methylumbelliferyl-
-D-glucuronide, [MUG]) substrates that react with specific 
enzymes (generally, -glucuronidase in E. coli and -
glucosidase in enterococci) to produce color changes or fluorescence to 
detect the target organism. The methods included in this proposal were 
developed by EPA, voluntary consensus standards bodies (VCSBs) (i.e., 
American Public Health Association [APHA], American Water Works 
Association [AWWA], and Water Environment Foundation [WEF] who jointly 
publish Standard Methods for the Examination of Water and Wastewater, 
referred to as ``Standard Methods;'American Society for Testing and 
Materials [ASTM]; Association of Official Analytical Chemists 
International [AOAC]), and commercial vendors with methods submitted to 
the EPA Office of Water (OW) Alternate Test Procedure (ATP) process. 
For several procedures, an EPA method, VCSB method, and/or a 
commercially available method (submitted to the ATP program) are 
proposed.

[[Page 45814]]

    Although there are several methods (not yet approved by EPA) that 
are applicable to simultaneous determination of total coliform and E. 
coli, EPA is proposing to approve methods for analysis of E. coli only. 
EPA made this choice because at present there are no EPA-approved 
methods for E. coli, whereas EPA-approved methods are already available 
for the determination of total coliform. There is a request for comment 
on the expansion of today's rule to include total coliforms in Section 
III.A.5. Several of the total coliform test methods (or selected 
procedural steps) have already been approved by EPA (see Table IA at 40 
CFR 136.3) or have been proposed for approval for the Clean Water Act 
or Safe Drinking Water Act compliance monitoring programs (66 FR 3526, 
January 16, 2001).
    Proposed methods were selected based on data generated by EPA 
laboratories, submissions to the ATP program and VCSBs, published peer-
reviewed journal articles, and/or publicly available study reports that 
indicate their applicability to quantitative analysis of the target 
organisms in ambient water. Since data were generated in multiple 
studies using different method versions and different statistical 
analyses, the test procedures in today's rule must be evaluated against 
the end-users' needs based on data quality objectives. End-users should 
compare any new proposed alternate method with the relevant EPA-
recommended method(s) before adopting it for that matrix to ensure that 
the proposed method generates data of comparable quality. EPA-
recommended methods for matrices in which they were tested are 
summarized in Tables 3 and 5. A media acronym table is provided in 
Section V. Full citations for methods and data reports are provided in 
the References section and are included in the docket for today's 
proposed rulemaking. At the time of final rulemaking, EPA plans to 
issue a draft protocol for determining the comparability of alternative 
test methods to those promulgated in the final rule. In addition, EPA 
will issue draft guidance on acceptable characteristics of methods for 
determining equivalency (e.g., acceptable range of false positives/
false negatives). There is a request for comment in Section III.A.5 
inviting suggestions on acceptable characteristics of methods and on 
method comparability criteria to support the equivalency testing 
protocol.
1. Most Probable Number (MPN) and Membrane Filtration (MF) Methods
    In Most Probable Number tests, the number of tubes/wells producing 
a positive reaction provides an estimate of the original, undiluted 
density (i.e., concentration) of target organisms in the sample. This 
estimate of target organisms, based on probability formulas, is termed 
the Most Probable Number. MPN tests can be conducted in multiple-tube 
fermentation (MTF), multiple-tube enzyme substrate, or multiple-well 
enzyme substrate formats. In multiple-tube tests, serial dilutions may 
be used to obtain estimates over a range of concentrations, with 
replicate tubes analyzed at each ten-fold dilution/volume. The numbers 
of replicate tubes and sample dilutions/volumes are selected based on 
the expected quality of the water sample. Generally, for non-potable 
water samples, five replicate tubes at a minimum of three dilutions/
volumes are used. Tubes are incubated, and positive results are 
reported and confirmed. Positive results are determined under specified 
conditions by the presence of acid and/or the production of gas using 
MTF tests, or by color change or fluorescence using enzyme substrate 
tests. Tests also may be conducted in a multiple-well format to 
determine MPN, using commercially prepared substrate media, multiple-
well trays, and MPN tables provided by the manufacturer. Target 
organism density is estimated by comparing the number of positive tubes 
or wells with MPN tables. The MPN tables relate the number of positive 
tubes or wells to an estimate of the mean target organism density based 
on probability formulas. Results in both types of tests are generally 
reported as MPN per 100 mL.
    The multiple-tube fermentation methodology is useful for detecting 
low concentrations of organisms (100/100 mL), particularly in samples 
containing heavy particulate matter, toxic compounds (e.g. metals), or 
injured or stressed organisms. Multiple-tube tests are applicable to 
freshwater, estuarine, and marine ambient waters. Since MPN tables 
assume a Poisson distribution, samples must be adequately shaken to 
break up any clumps and provide even distribution of bacteria. If the 
sample is not gently shaken, the MPN value may underestimate the actual 
bacterial density. The overall precision of each multiple-tube test 
depends on the number of tubes used and sample dilutions/volumes 
tested. Unless a large number of tubes are used (five tubes per 
dilution/volume or more), the precision of multiple-tube tests can be 
very poor. Precision is improved when the results from several samples 
from the same sampling event are processed, estimated separately, and 
then mathematically combined using the geometric mean. Further 
background information on multiple-tube tests is available in the 20th 
Edition of Standard Methods for the Examination of Water and Wastewater 
(APHA, 1998).
    Membrane filtration is a direct-plating method in which sample 
dilutions/volumes are filtered through 0.45 m membrane filters 
that are subsequently transferred to petri plates containing selective 
primary isolation agar or an absorbent pad saturated with selective 
broth. A second substrate medium is used in two-step MF procedures to 
confirm and/or differentiate the target organisms. The total sample 
volume to be analyzed may be distributed among multiple filters and 
diluted as needed, based on the anticipated water sample type, quality, 
and character (e.g., organism density, turbidity). The goal is to 
obtain plates with counts within the acceptable counting range of the 
method. The acceptable counting range of membrane filter tests depends 
on the specific analytical technique and the target organism under 
study. Plates are incubated and target colonies are counted. A 
percentage of the target colonies may then be verified as specified by 
the method. Target colonies are detected by observing the presence of 
colonies that meet a specific morphology, color, or fluorescence under 
specified conditions. Colonies may be counted with the aid of a 
fluorescent light, magnifying lens or dissecting microscope, or long-
wavelength (366-nm) ultraviolet (UV) light source. Results generally 
are reported as colony-forming units (CFU) per 100 mL. Organism density 
is determined by dividing the number of target CFU by the volume (mL) 
of undiluted sample that is filtered and multiplying by 100. If 
verification steps are performed, the initial target colony count is 
adjusted based upon the percentage of positively verified colonies and 
reported as a ``verified count per 100 mL'' (APHA, 1998).
    Membrane filtration is applicable to most freshwater, estuarine, 
and marine ambient waters, with limitations where an underestimation of 
organism density is likely: water samples with high turbidity, toxic 
compounds, or large numbers of non-coliform (background) bacteria, and 
organisms damaged by chlorine or toxic compounds. To minimize these 
interferences, replicates of smaller sample dilutions/volumes may be 
filtered and the results combined. When the MF method has not been used 
previously on an

[[Page 45815]]

individual water type, parallel tests should be conducted with a MTF to 
demonstrate applicability, lack of interferences, and at least 
comparable recovery. For example, colonies from samples containing 
high-background levels or stressed organisms should be verified. If the 
MTF results are consistently higher than those obtained in MF tests, or 
there is an indication of suboptimal recovery, use an appropriate 
recovery enhancement technique and demonstrate that the recovery 
enhancement technique is comparable to MTF. Further background 
information on MF tests is available in Standard Methods for the 
Examination of Water and Wastewater (APHA,1998).
    A statistical comparison of results obtained by the multiple-tube 
and MF methods showed that the MF method is more precise in enumerating 
target organisms than the MPN test, but differences in recovery were 
generally not statistically significant. However, based on 
susceptibility to interferences, MF tests may underestimate the number 
of viable bacteria, and the MPN method may overestimate the 
concentration because of the built-in positive bias of the method 
(Thomas, 1955). Tables with 95% confidence limits are available for 
both methods, based on the assumption that bacteria exhibit a Poisson 
distribution. Because of susceptibility of some MF tests to 
interferences, verification of some MF results with multiple-tube tests 
is critical. Additionally, some MPN tests require confirmation tests 
because of the false positive/false negative rates of the particular 
media. In general, although numerical results may not be identical, 
data from each method yield similar water quality information based on 
performance.
2. Selection of Proposed Methods
    A variety of methods for E. coli and enteroccoci are being proposed 
in today's rule because a range of techniques are routinely used for 
different applications by regulatory authorities, permitees, 
laboratories, researchers, and others. The methods presented have been 
evaluated based on different study designs and statistical analyses. 
The variety of waters subject to monitoring, the selection of an 
appropriate method, number of tubes, sample dilutions/volumes, and 
other analytical design decisions may be made based on the available 
information on the type, quality, character, consistency of results, 
anticipated target organism density, and designated use of the water to 
be monitored.
3. Methods for E. coli
    EPA is proposing several methods for enumerating E. coli in ambient 
water. Brief descriptions of the proposed multiple-tube, multiple-well, 
and MF methods are provided. Method performance data is summarized in 
Table 3.
    In Table 1, methods in the same row use the same technique, but are 
published by different entities. For example, ONPG-MUG is published in 
the ``Standard Methods'' manual and in the Association of Official 
Analytical Chemists (AOAC) manual, and is also available as a 
commercial product. Voluntary Consensus Standards (VCS) Methods are 
those developed or adopted by domestic and international voluntary 
consensus standard bodies. The American Public Health Association 
(APHA), American Water Works Association (AWWA), and Water Environment 
Foundation (WEF) jointly publish methods approved by a methods approval 
program in Standard Methods for the Examination of Water and Wastewater 
(``Standard Methods''). The American Society for Testing and Materials 
(ASTM) are methods that have met the requirements of the ASTM methods 
approval program. The Association of Official Analytical Chemists also 
publishes methods that have met the requirements of the AOAC methods 
approval program. EPA methods are those that have been developed by the 
US EPA.

                                                 Table 1.--Proposed Methods for E. coli Enumeration 1,2
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                            VCS methods
           Technique                 Method 1,2          EPA method     ---------------------------------------------------      Commercial example
                                                                            Standard methods          ASTM          AOAC
--------------------------------------------------------------------------------------------------------------------------------------------------------
Most Probable Number (MPN).....  LTBEC-MUG  ..................  9221B.1/9221F          ...............
                                 ONPG-MUG..........                      9223B                                      991.15  Colilert 3,5
                                 ONPG-MUG..........  ..................  9223B                  ...............  .........  Colilert-18 3,6
                                 CPRG-MUG..........  ..................  9223B                  ...............  .........  ColisureTM 3,5
Membrane Filter (MF)...........  mENDONA-M  ..................  9222B/9222G            ...............  .........  ............................
                                  UG.
                                 LES-ENDON  ..................  9222B/9222G            ...............             ............................
                                  A-MUG.
                                 mFCNA-MUG  ..................  9222D/9222G            ...............             ............................
                                 mTEC agar.........  1103.1............  9213D                  D5392-93         .........  ............................
                                 Modified mTEC agar  Modified 1103.1...  .....................  ...............             ............................
                                 MI agar...........  EPA-600-R-013 \7\.  .....................  ...............             ............................
                                 m-ColiBlue24 broth  ..................  .....................  ...............  .........  m-ColiBlue24 4,5
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ A media acronym table is provided in Section V.
\2\ Tests must be conducted in a format that provides organism enumeration.
\3\ Manufactured by IDEXX.
\4\ Manufactured by Hach Company.
\5\ Method currently approved for determining presence/absence of total coliform and E. coli in drinking water.
\6\ Acceptable version of method approved as a drinking water ATP.
\7\ Membrane Filter Method for the Simultaneous Detection of Total Coliforms and Escherichia coli in Drinking Water.

Most Probable Number Tests for E. coli
a. LTBEC-MUG (Standard Methods 9221B.1/9221F)
    The multiple-tube fermentation method for enumerating E. coli in 
water uses multiple-tubes and dilutions/volumes in a two-step procedure 
to determine E. coli concentrations (APHA, 1998). In the first step, or 
``presumptive phase,'' a series of tubes containing lauryl tryptose 
broth (LTB) are inoculated with undiluted samples and/or dilutions/
volumes of the samples and mixed. Inoculated tubes are incubated for 24 
 2 h at 35  0.5  deg.C. Each tube then is 
swirled gently and examined for growth (i.e., turbidity) and

[[Page 45816]]

production of gas in the inner Durham tube. If there is no growth or 
gas, tubes are re-incubated for 24  2 h at 35  
0.5  deg.C and re-examined. Production of growth and gas within 48 
 3 h constitutes a positive presumptive test for coliforms, 
which include E. coli.
    After enrichment in the presumptive medium, positive tubes are 
subjected to a second step for enumeration of E. coli. Presumptive 
tubes are agitated, and growth is transferred using a sterile loop or 
applicator stick to tubes containing EC broth supplemented with 4-
methylumbelliferyl--D-glucuronide (MUG). Inoculated tubes are 
incubated at 44.5  0.2  deg.C for 24  2 h in a 
water bath. All tubes exhibiting growth and gas production are examined 
for bright blue fluorescence under long-wavelength UV light (366-nm) 
indicating a positive test for E. coli. The density of E. coli in MPN/
100 mL is then calculated from the number of positive EC-MUG tubes, 
using MPN tables or formulas.
b. ONPG-MUG (Standard Methods 9223B, AOAC 991.15, Colilert, 
Colilert-18, and Autoanalysis Colilert)
    ONPG-MUG tests are chromogenic/fluorogenic enzyme substrate tests 
for the simultaneous determination of total coliforms and E. coli in 
water. These tests use commercially available media containing the 
chromogenic substrate ortho-nitrophenyl--D-galactopyranoside 
(ONPG), to detect total coliforms and the fluorogenic substrate 4-
methylumbelliferyl--D-glucuronide (MUG), to detect E. coli. 
All tests must be conducted in a format that provides quantitative 
results for ambient water. Colilert-18 should be used for 
testing marine waters with a minimum of a 10-fold dilution with sterile 
freshwater. Media formulations are available in disposable tubes for 
the multiple-tube procedure or packets for the multiple-well procedure. 
Appropriate preweighed portions of media for mixing and dispensing into 
multiple-tubes and wells are also available. The use of commercially 
prepared media is required for quality assurance and uniformity.
    For the multiple-tube procedure, a well-mixed sample and/or sample 
dilution/volume is added to tubes containing predispensed media. Tubes 
are then capped and mixed vigorously to dissolve the media. 
Alternatively, this procedure can be performed by adding appropriate 
amounts of substrate media to a bulk diluted sample (with appropriate 
dilutions for enumeration), then mixing and dispensing into multiple-
tubes. The number of tubes, and number of dilutions/volumes are 
determined based on the type, quality, and character of the water 
sample. A multiple-well procedure may be performed with sterilized 
disposable packets. The commercially available Quanti-Tray or 
Quanti-Tray/2000 multiple-well tests uses Colilert 
or Colilert-18 media to determine E. coli (IDEXX, 1999b,c). 
In these tests, the packet containing media is added to a 100-mL sample 
(with appropriate dilutions for enumeration). The sample is then mixed 
and poured into the tray. A tray sealer separates the sample into 51 
wells (Quanti-Tray) or 96 wells (Quanti-tray/2000) and seals the 
package which is subsequently incubated at 35  0.5  deg.C 
for 18 h when using Colilert-18 or 24 h when using 
Colilert. If the response is questionable after the specified 
incubation period, the sample is incubated for up to an additional 4 h 
at 35  0.5  deg.C for both Colilert tests.
    After the appropriate incubation period, each tube or well is 
compared to the reference color ``comparator'' provided with the media. 
If the sample has a yellow color greater or equal to the comparator, 
the presence of total coliforms is verified, and the tube or well is 
then checked for fluorescence under long-wavelength UV light (366-nm). 
The presence of fluorescence greater than or equal to the comparator is 
a positive test for E. coli. If water samples contain humic acid or 
colored substances, inoculated tubes or wells should also be compared 
to a sample water blank. The concentration in MPN/100 mL is then 
calculated from the number of positive tubes or wells using MPN tables 
provided by the manufacturer.
c. CPRG-MUG (Standard Methods 9223B, ColisureTM)
    CPRG-MUG is a chromogenic/fluorogenic enzyme substrate test for the 
simultaneous determination of total coliforms and E. coli in water. 
These tests use a commercially available medium containing the 
chromogenic substrate chlorophenol red--D-galactopyranoside 
(CPRG) to detect total coliforms, and the fluorogen MUG to detect E. 
coli. The sample is incubated for 24  2 h at 35 
 0.5  deg.C. If results are negative after 24 h, the sample 
is incubated up to an additional 4 h before calculating results. If the 
sample has changed from a yellow color to a red or magenta color, the 
presence of total coliforms is verified and the tube or well is then 
checked for fluorescence. The presence of blue fluorescence under a 
long-wavelength UV light (366-nm) is a positive test for E. coli. The 
concentration in MPN/100 mL is then calculated from the number of 
positive tubes or wells using MPN tables provided by the manufacturer. 
ColisureTM is a commercially available format of this method 
and uses the same quantitative formats (multiple-tube and multiple-
well) available for the Colilert tests. ColisureTM 
is subject to the same interferences and procedural cautions listed for 
the Colilert tests.
Membrane Filter (MF) Tests for E. coli
a. mEndo, LES-Endo, or mFC followed by transfer to NA-MUG media 
(Standard Methods 9222B/9222G or 9222D/9222G)
    These membrane filter methods for enumerating E. coli are two-step 
incubation procedures (APHA, 1998). First, a sample is filtered through 
a 0.45 m filter, then the filter is placed on a pad saturated 
with mEndo broth or a plate containing mEndo or LES-Endo agar and 
incubated for 23  1 h at 35  0.5  deg.C. Pink 
to red colonies with a metallic (golden-green) sheen on the filter are 
considered to be total coliforms. If initial determination of fecal 
coliforms is desired or non-potable water samples are analyzed, mFC 
media can be substituted for mEndo/LES-Endo. Following initial 
isolation of total coliforms (or fecal coliforms), the filter is 
transferred to nutrient agar containing 4-methylumbelliferyl--
D-glucuronide (NA-MUG) and incubated for 4 h at 35  0.5  
deg.C. Sheen colonies on mEndo that fluoresce under a long-wavelength 
UV light (366-nm) are positive for E. coli.
b. mTEC Agar (EPA Method 1103.1, Standard Methods 9212D, ASTM D5392-93)
    The mTEC agar method is a two-step procedure that provides a direct 
count of E. coli in water based on the development of colonies on the 
surface of a membrane filter when placed on a selective nutrient and 
substrate media (USEPA, 1985a). This method originally was developed by 
EPA to monitor the quality of recreational water. This method was also 
used in health studies to develop the bacteriological ambient water 
quality criteria for E. coli. In this method, a water sample is 
filtered through a 0.45m; membrane filter, the filter is 
placed on mTEC agar (a selective primary isolation medium), and the 
plate is incubated first at 35  0.5  deg.C for 2 h to 
resuscitate injured or stressed bacteria and then at 44.5  
0.2  deg.C for 23  1 h in a water bath. Following 
incubation, the filter is transferred to a filter pad saturated with 
urea substrate medium. After 15 minutes, all yellow or yellow-brown 
colonies (occasionally yellow-green) are

[[Page 45817]]

counted as positive for E. coli using a fluorescent lamp and either a 
magnifying lens or a stereoscopic microscope.
c. Modified mTEC Agar (Modified EPA Method 1103.1)
    The modified mTEC agar method is a single-step MF procedure that 
provides a direct count of E. coli in water based on the development of 
colonies on the surface of a filter when placed on selective modified 
mTEC media (USEPA, 2000a). This is a modification of the standard mTEC 
media that eliminates bromcresol purple and bromphenol red from the 
medium, adds the chromogen 5-bromo-6-chloro-3-indoyl--D-
glucuronide (Magenta Gluc), and eliminates the transfer of the filter 
to a second substrate medium. In this method, a water sample is 
filtered through a 0.45m membrane filter, the filter is placed 
on modified mTEC agar, incubated at 35  0.5  deg.C for 2 h 
to resuscitate injured or stressed bacteria, and then incubated for 23 
 1 h in a 44.5  0.2  deg.C water bath. 
Following incubation, all red or magenta colonies are counted as E. 
coli.
d. MI Agar
    The MI agar method is a single-step procedure used to 
simultaneously enumerate total coliforms and E. coli (Brenner, 1993). 
In this EPA-developed method, a water sample is filtered through a 
0.45m membrane filter, the filter is placed on an MI agar 
plate, and the medium is incubated at 35  0.5  deg.C for 24 
h. As with NA-MUG and modified mTEC, the MI agar MF procedure is based 
on the ability of E. coli to produce the enzyme -
glucuronidase, which hydrolyzes Indoxyl--D-glucuronide (IBDG) 
to form a blue color (indigo). E. coli colonies exhibit a blue color 
and may also be fluorescent under a long-wavelength UV light (366-nm). 
If desired, the plates can also be observed under long-wavelength UV 
light (366-nm) for the presence of fluorescent total coliform species. 
Because the blue color from the breakdown of IBDG can mask 
fluorescence, non-fluorescent blue colonies are included in the total 
coliform count. Water samples with high turbidity can clog the membrane 
filter, interfering with filtration and potentially interfering with 
the identification of target colonies. However, E. coli colonies on MI 
agar can be counted on plates from waters containing high particulate 
or background/non-coliform concentrations, chlorine-stressed organisms 
or nutrient-deprived organisms, temperature-sensitive E. coli, and/or 
anaerogenic strains that may not be recovered by other multiple-tube or 
membrane filter tests.
e. m-ColiBlue24 Broth
    This broth method is a single-step MF test for enumerating total 
coliforms and E. coli. As with NA-MUG, modified mTEC, and MI media, the 
selective identification of E. coli is based on the detection of the 
-glucuronidase enzyme. The test medium includes the chromogen 
5-bromo-4-chloro-3-indoxyl--D-glucuronide (BCIG or X-Gluc). 
The chromogen BCIG is hydrolyzed by -glucuronidase, releasing 
an insoluble indoxyl salt that produces blue colonies. M-ColiBlue24 
broth is a commercially available format of this method and contains a 
nutritive lactose-based medium containing inhibitors to eliminate the 
growth of non-coliforms. With m-ColiBlue24 broth, a water sample is 
filtered through a 0.45m membrane filter, and the filter is 
transferred to a plate containing an absorbent pad saturated with m-
ColiBlue24 broth. The filter is incubated at 35  0.5  deg.C 
for 24 h and examined for colony growth (Hach, 1999). The presence of 
E. coli is indicated by blue colonies.

                                  Table 2.--Analytes Detected by Proposed Media
----------------------------------------------------------------------------------------------------------------
                                                               Total  coliform  Fecal  coliform
              Technique                        Media                 \1\              \1\            E. coli
----------------------------------------------------------------------------------------------------------------
Most Probable Number (MPN)..........  LTBEC-MUG.....           X \2\   ...............               X
                                      ONPG-MUG...............               X   ...............               X
                                      CPRG-MUG...............               X   ...............               X
                                      mFCNA-MUG.....  ...............               X                X
                                      mENDONA-MUG...               X   ...............               X
                                      LES-ENDONA-MUG               X   ...............               X
Membrane Filter (MF)................  mTEC...................  ...............  ...............               X
                                      Modified mTEC..........  ...............  ...............               X
                                      MI.....................               X   ...............               X
                                      m-ColiBlue24 broth.....               X   ...............              X
----------------------------------------------------------------------------------------------------------------
\1\ Detection of total coliform or fecal coliform are included because their detection may be preliminary steps
  required for E. coli enumeration and are part of the E. coli method.
\2\ LTB is the presumptive test for total coliforms. It is necessary to transfer sample to BGLB for confirmation
  to determine total coliform density.

Method Comparison Studies

    To confirm the applicability and comparability of results obtained 
with individual methods, parallel quantitative comparison tests with 
multiple-tube or MF tests, and positive and negative control tests 
should be conducted for each site-specific sample in accordance with 
analytical quality control procedures in Standard Methods for the 
Examination of Water and Wastewater. Performance data for E. coli 
multiple-tube, multiple-well, and MF methods are provided in Table 3.

[[Page 45818]]



                             Table 3.--Study Comparisons of E. coli Proposed Methods
----------------------------------------------------------------------------------------------------------------
                                                    Study design/
  Methods compared or tested      Water type(s)       number of             Results \1\         Reference(s) \2\
                                     tested            samples
----------------------------------------------------------------------------------------------------------------
MI agar compared to mEndo Overall            Brenner, 1993.
 arrow>NA-MUG and/or mTEC agar.  spiked drinking   laboratory        differences not            Brenner, 1996a.
                                 water, and non-   studies (23       statistically significant  Brenner, 1996b.
                                 potable water.    samples and 51    MI agar:
                                                   samples) and an   Specificity 95.7%;
                                                   interlaboratory   MI agar: False
                                                   study (19 labs,   Positive (FP) = 4.3%;
                                                   6 samples each).   MI agar: False
                                                                     Negative (FN) = 4.3%
Colilert compared to  Surface water...  ................   No significant     Cowburn, 1994.
 multiple-tube fermentation                                          difference in recovery of  Edberg, 1988.
 and membrane filtration.                                            E. coli                    Edberg, 1989.
                                                                     Correlation        Ellgas, undated.
                                                                     Coefficient (r) for
                                                                     Colilert ranged
                                                                     from 0.706 to 0.89
                                                                                                Fricker, 1995.
                                                                                                Fricker, 1996a.
                                                                                                Palmer, 1993.
Colilert compared to  Surface water...  47 split samples  Colilert found    Edberg, 1990.
 LTBEC-MUG.                                                 to be equally sensitive
                                                                     to LTBEC-MUG
mTEC agar compared to modified  Surface water...  Single-            E. coli recovery   EPA, 1999b.
 mTEC agar.                                        laboratory, 43    rates were not
                                                   split-samples.    statistically different
                                                                     mTEC agar: FP =
                                                                     6%; FN = 5%
                                                                     modified mTEC
                                                                     agar: FP = 0%; FN = 4%
mTEC agar compared to modified  Beach water       70 samples from    No statistically   Francy, 1999.
 mTEC agar, MI agar, and         (recreational).   three Lake Erie   significant difference
 Colilert.                               beaches.          between MI agar and mTEC
                                                                     agar. Statistically
                                                                     significant differences
                                                                     between modified mTEC
                                                                     agar and/or Colilert and
                                                                     standard method
                                                                     Modified mTEC      ................
                                                                     agar: r = 0.966*; FP =
                                                                     0%*; FN = 11%*
                                                                     MI agar: r =
                                                                     0.983*; FP = 3%*; FN =
                                                                     4%*
                                                                     Colilert: r =
                                                                     0.946*; FP = 5%*; FN =
                                                                     9%*
                                                                     *Based on
                                                                     reference method (mTEC
                                                                     agar)
m-ColiBlue24 broth, mEndo Overall agreement  Grant, 1997.
 arrow>NA-MUG, and mTEC agar.    non-chlorinated   samples, 3 non-   with the reference
                                 wastewater,       chlorinated       methods was 98.8% for m-
                                 wastewater        wastewaters, 2    ColiBlue24 broth and
                                 spiked drinking   wastewater        92.1% for mTEC agar
                                 water, finished   spiked drinking  m-ColiBlue24 broth: FP =
                                 drinking water.   water, and 1      2.5%; FN = 0%;
                                                   finished         Sensitivity = 100%;
                                                   drinking water.   Specificity = 97.7%
Colilert, Colilert-   Fresh             204                No statistically   IDEXX, 1999d.
 18, and mTEC agar.    recreational      (Colilert r = 0.905 and
                                                   18)     0.921 respectively
                                                   samples.
Colilert, most        Marine water,     22 laboratories   All three techniques        Noble, 1999.
 probable number, and membrane   seawater spiked   using 13 common   provided comparable
 filtration.                     with sewage       samples plus 2    results on marine samples
                                 effluent.         external QC
                                                   samples.
Colilert-18 and       Untreated         6 rivers           Both techniques    Ostensvik, 2000.
 membrane filtration.            surface water.    draining into     provided comparable
                                                   drinking water    results
                                                   reservoirs.
Colisure TM compared to EC-MUG  Primary effluent  21 samples from    Colisure TM: FP =  59 FR 35891,
 (multiple-tube fermentation)                      7 different       4.3%; FN = 2.4%             1994.
 and method for detection of                       geographical      Detection of
 chlorine-injured E. coli.                         locations and     chlorine-injured E. coli:
                                                   31 samples from   Colisure TM had an
                                                   6 different       average of 1.76 times
                                                   locations (for    more E. coli-positive
                                                   detection of      results after 28 hours
                                                   chlorine-         than the standard method
                                                   injured E.
                                                   coli).
----------------------------------------------------------------------------------------------------------------
\1\ Methods of determining false positive and false negative rates were not standardized for all comparison
  studies.
\2\ Complete reference information is provided in Section VI.

4. Methods for Enterococci
    EPA is proposing several methods for enumerating enterococci in 
water. Brief descriptions of the proposed MPN and MF methods are 
provided below. In Table 4, methods in the same horizontal row use the 
same technique, but are published by different entities.

[[Page 45819]]



                                                                 Table 4.--Proposed Methods for Enterococci Enumeration.\1\, \2\
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                                                        VCS method \4\
            Methodology                               Method \3\                      EPA    -------------------------------------------------------------------       Commercial  example
                                                                                    method       Standard Methods              ASTM                  AOAC
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Most Probable......................  Azide dextrose/PSE/BHI.....................  ..........  9230B
Number (MPN).......................  MUG media..................................  ..........  ......................  D6503-99                .................  Enterolert \TM4\
Membrane Filter....................  mEEIA agar........................      1106.1  9230C                   D5259-92
(MF)...............................  mEI agar...................................     1600
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ Complete reference information is provided in Section VI.
\2\ A media acronym table is provided in Section V.
\3\ Tests must be conducted in a format that provides organism enumeration.
\4\ Manufactured by IDEXX.

Most Probable Number (MPN) Tests for Enterococci

a. Azide Dextrose/PSE/BHI (Standard Methods 9230B)
    The Azide Dextrose/PSE/BHI technique for enumerating enterococci in 
water uses multiple-tubes and dilutions/volumes in a three-step 
procedure (presumptive fecal streptococcus, confirmed fecal 
streptococcus, and enterococcus) to determine enterococci 
concentrations (APHA, 1998). In the presumptive phase, multiple-tubes 
containing azide dextrose are inoculated with sample and mixed with the 
broth by gentle agitation. Inoculated tubes are incubated for 24 
 2 h at 35 deg.C  0.5  deg.C. Each tube then is 
swirled and examined for turbidity. If turbidity is absent, tubes are 
incubated for an additional 24 h and re-examined. Production of 
turbidity within 48  3 h constitutes a positive presumptive 
reaction for fecal streptococci.
    After enrichment during the presumptive phase, positive azide 
dextrose tubes are subjected to a confirmation step for fecal 
streptococci. A portion of growth from each positive azide dextrose 
tube is streaked on Pfizer selective Enterococcus (PSE) agar using a 
sterile loop. Inverted plates are incubated at 35  deg.C  
0.5  deg.C for 24  2 h and observed for the presence of 
brownish-black colonies with a brown halo. Such colonies are confirmed 
as fecal streptococci.
    Target colonies from the PSE medium can be transferred to a tube of 
brain-heart infusion (BHI) broth and incubated at 45  deg.C 
 0.5  deg.C for 48 h. Simultaneously, these colonies can be 
transferred to BHI broth containing 6.5% NaCl and incubated at 35  
deg.C  0.5  deg.C for 48 h. Growth at both 45  deg.C in BHI 
medium and in BHI medium containing 6.5% NaCl at 35  deg.C is 
indicative of the Enterococcus bacterial group. The concentration in 
MPN/100 mL is then calculated from the number of positive 6.5% NaCl 
broth tubes using MPN tables or formulas.
b. 4-methylumbelliferyl--D-glucoside (MUG) Medium (ASTM D6503-
99, EnterolertTM)
    This method utilizes a medium contaning the fluorogenic substrate 
4-methylumbelliferyl--D-glucoside (MUG) to determine 
enterococci concentrations. EnterolertTM is a commercially 
available test that utilizes this substrate test for the determination 
of enterococci in water (IDEXX, 1999f). EnterolertTM tests 
are incubated for 24 h at 41  0.5  deg.C and may use the 
same quantitative formats available for the Colilert tests, 
cited earlier in Section III-A. After incubation, the presence of blue/
white fluorescence is a positive result for enterococci. The 
concentration in MPN/100 mL is then calculated from the number of 
positive tubes or wells using MPN tables provided by the manufacturer. 
EnterolertTM is subject to the same interferences and 
cautions listed for the Colilert tests. In addition, marine 
water samples must be diluted at least tenfold with sterile, non-
buffered freshwater (EnterolertTM is already buffered).

Membrane Filter (MF) Tests for Enterococci

a. mEEIA Agar (EPA 1106.1, Standard Methods 9230C, ASTM D5259-
92)
    The mEI agar method is a two-step MF procedure that provides a 
direct count of bacteria in water, based on the development of colonies 
on the surface of a membrane filter when placed on a selective nutrient 
medium (USEPA, 1985b). A water sample is filtered through a 
0.45m membrane filter, and the filter is placed on a plate 
containing selective mE agar. After the plate is incubated at 41 
 0.5  deg.C for 48 h, the filter is transferred to an 
Esculin iron agar (EIA) plate and incubated at 41  0.5  
deg.C for 20-30 min. After incubation, all pink to red colonies on mE 
agar that form a black or reddish-brown precipitate on the underside of 
the filter when placed on EIA are counted as enterococci. Organism 
density is reported as enterococci per 100 mL.
b. mEI Agar (EPA Method 1600)
    The mEI agar method is a single-step MF procedure that provides a 
direct count of bacteria in water, based on the development of colonies 
on the surface of a filter when placed on selective mEI agar (USEPA, 
1997). This medium, a modification of the mE agar in EPA Method 1106.1, 
contains a reduced amount of 2-3-5-triphenyltetrazolium chloride, and 
an added chromogen, Indoxyl--D-glucoside. The transfer of the 
filter to EIA is eliminated, thereby providing results within 24 h. In 
this method, a water sample is filtered, and the filter is placed on 
mEI agar and incubated at 41  0.5  deg.C for 24 h. 
Following incubation, all colonies with a blue halo, regardless of 
colony color, are counted as enterococci. Results are reported as 
enterococci per 100 mL.

Method Comparison Studies

    To confirm the applicability and comparability of results obtained 
with individual methods, parallel quantitative comparison tests with 
multiple-tube or MF tests, and positive and negative control tests 
should be conducted for each site-specific sample in accordance with 
analytical quality control procedures in Standard Methods for the 
Examination of Water and Wastewater. Performance data for enterococci 
multiple-tube, multiple-well, and MF methods are provided in Table 5.

[[Page 45820]]



                           Table 5.--Study Comparisons of Enterococci Proposed Methods
----------------------------------------------------------------------------------------------------------------
                                  Water type(s)      Study design/
  Methods compared or tested          tested       Number of samples     Results \1\         Reference(s) \2\
----------------------------------------------------------------------------------------------------------------
Enterolert TM compared to mE Data      Abbott, 1998.
 arrow>EIA agar.                 bathing water,                        indicated a
                                 tidal lagoons,                        strong linear
                                 water from                            correlation (r =
                                 marinas,                              0.927) and no
                                 untreated                             significant
                                 effluents, and                        difference
                                 marine water                          between the two
                                 from stormwater-                      methods (p =
                                 drainage sites.                       0.39).
                                                                       Enterole
                                                                       rt TM: False
                                                                       Positive (FP) =
                                                                       2.4%; False
                                                                       Negative (FN) =
                                                                       0.3%;
                                                                       Sensitivity =
                                                                       99.8%;
                                                                       Specificity =
                                                                       97.0%.
EnterolertTM compared to mE When      Budnick, 1996.
 arrow>EIA agar.                 freshwater         Connecticut        analyzing the
                                 recreational       Department of      entire sample
                                 bathing samples.   Public Health.     population,
                                                                       there were no
                                                                       significant
                                                                       differences
                                                                       between the two
                                                                       methods.
                                                                       Results
                                                                       classified by
                                                                       sample type
                                                                       (freshwater v.
                                                                       marine) showed a
                                                                       greater
                                                                       difference
                                                                       between the two
                                                                       methods.
                                                                       Enterole
                                                                       rtTM FP = 5.1%;
                                                                       FN = 0.4%.
EnterolertTM compared to mE Correlat  Chen, 1996.
 arrow>EIA agar.                 freshwater,                           ion coefficient
                                 marine water,                         (r) of 0.91
                                 and untreated                         between the two
                                 effluents.                            methods.
                                                                       Enterole
                                                                       rtTM: FP = 4.9%;
                                                                       FN = 0.6%.
EnterolertTM compared to mE r = 0.91  Fricker, 1996.
 arrow>EIA agar.                 (323), partially  Thames Water        Overall
                                 treated effluent   Utilities.         EnterolertTM
                                 (516), treated                        detected
                                 effluents (620),                      enterococci in
                                 and finished                          more samples and
                                 drinking water                        had fewer false
                                 (1012).                               positives, but
                                                                       these
                                                                       differences were
                                                                       not
                                                                       statistically
                                                                       significant.
                                                                       Enterole
                                                                       rtTM: FP = 4.5%.
                                                                       mE-EIA:
                                                                       FP = 6.2%.
mEEIA agar compared    Freshwater and     176 samples         No        Liebman, 1999.
 to mEI agar.                    marine water.      (including 44      significant
                                                    duplicates).       difference
                                                   Single-laboratory   between the two
                                                    study.             methods.
                                                     ...............   mE mEI
                                                                       agar: FP = 2%;
                                                                       FN = 7%; RPD =
                                                                       45.2%.
mEEIA agar compared    Surface water,     Single-laboratory   No        Messer, 1998.
 to mEI agar.                    non-chlorinated    study.             significant
                                 primary           Samples analyzed    difference
                                 effluent,          in duplicate.      between the two
                                 chlorinated                           methods.
                                 secondary                             mEI
                                 effluent, and                         agar: FP = 6%;
                                 marine waters.                        FN = 6.5%.
Azide Dextrose/PSE/BHI, mE Methods   Noble, 1999.
 arrow>EIA agar, and             seawater spiked    using 13 common    provide
 EnterolertTM.                   with sewage        samples plus 2     comparable
                                 effluent.          external QC        results.
                                                    samples.           Average
                                                                       difference among
                                                                       methods was less
                                                                       than 6%.
Azide Dextrose/PSE/BHI, mE Idexx     Noble, 2000a.
 arrow>EIA agar, mEI agar, and   from randomly      side-by-side       vs. Standard
 EnterolertTM.                   selected sites.    analyses on        Method: r = 0.1;
                                                    approximately      correspondence =
                                                    280 samples.       88%*.
                                                                       mEI agar
                                                                       vs. Standard
                                                                       Method: r = 0.9
                                                                       correspondence =
                                                                       99%.
                                                                       mEI agar
                                                                       vs.
                                                                       EnterolertTM: r
                                                                       = 0.89
                                                                       correspondence =
                                                                       97%.
                                                                       Enterole
                                                                       rtTM produced
                                                                       concentrations
                                                                       above the State
                                                                       threshold while
                                                                       standard methods
                                                                       produced results
                                                                       below for all
                                                                       samples with
                                                                       contradictory
                                                                       results.
mEEIA agar, mEI agar,  Seawater samples   6 labs performed    Enterole  Noble, 2000b.
 and EnterolertTM.               from 79 randomly   side-by-side       rtTM`` vs. mEI
                                 selected sites     split sample       agar: r = 0.93.
                                 (31 open beach     analyses on        Enterole
                                 sites and 48       approximately 48   rtTM vs. mEEIA agar:
                                 meters of a                           r = 0.94.
                                 freshwater
                                 outlet).
----------------------------------------------------------------------------------------------------------------
\1\ Methods of determining false positive and false negative rates were not standardized for all comparison
  studies.
\2\ Complete reference information is provided in Section VI.


[[Page 45821]]

5. Request for Comment and Available Data
    EPA requests public comments on the proposed methods for the 
bacterial indicators of fecal contamination. EPA invites comments on 
the technical merit, applicability, and implementation of the proposed 
E. coli and enterococci methods for ambient water monitoring. EPA also 
requests public comments on whether E. coli methods that are also 
applicable to total coliforms should be approved for determination of 
total coliforms in the final rule. Commenters should specify the method 
and bacteria/organisms to which the comment applies. EPA encourages 
commenters to provide copies of supporting data or references cited in 
comments. EPA also requests public comments on acceptable 
characteristics of these test methods for specific matrix applications, 
on comparability criteria to determine equivalency of alternative test 
methods, supporting data, and examples of any available alternative 
equivalency testing protocols. Additionally, EPA requests comments on 
any other applicable methods for analyzing E. coli and enterococci in 
ambient water not included in today's proposal. Method descriptions and 
supporting data may be submitted for additional test procedures that 
are applicable to enumerating these bacteria in ambient water.

B. Methods for Protozoa

    EPA developed and validated two methods for determination of 
protozoan concentrations in ambient waters to support ongoing voluntary 
monitoring of ambient waters used as source waters for drinking water 
treatment plants. EPA validated Method 1622 for the determination of 
Cryptosporidium in ambient water in August 1998 and issued a validated 
draft method in January 1999. EPA validated Method 1623 for the 
simultaneous determination of Cryptosporidium and Giardia in ambient 
water in February 1999 and issued a validated draft method in April 
1999. Methods 1622 and 1623 were revised and updated as a result of 
revised quality control criteria and the development of equivalent 
filters for use with the methods (USEPA, 2001c). The updates to Method 
1622 (EPA-821-R-01-026) and Method 1623 (EPA-821-R-01-025) are proposed 
in today's rule.
1. Cryptosporidium and Giardia
    Discussions of Methods 1622 and 1623 are combined for today's rule 
since all use essentially the same methodology: filtration, 
concentration, immunomagnetic separation of oocysts and cysts from 
captured material, immunofluorescence assay to determine presumptive 
concentrations, and confirmation through vital dye staining and 
differential interference contrast (DIC) microscopy for the detection 
of Cryptosporidium oocysts and Giardia cysts.
    A 10- to 50-L volume of water is filtered and the oocysts, cysts, 
and extraneous materials are retained on the filter. Elution of the 
materials on the filter is accomplished with an aqueous buffered salt 
and detergent solution. The oocysts and cysts are concentrated through 
centrifugation, and the supernatant fluid is aspirated. Oocysts and 
cysts are captured by the attachment of magnetic beads conjugated to 
anti-Cryptosporidium and anti-Giardia antibodies. The oocysts and cysts 
are magnetically separated from the extraneous materials, and the 
extraneous materials are discarded. The magnetic beads are then 
detached from the oocysts and cysts. The oocysts and cysts are prepared 
on well slides and stained with fluorescently-labeled monoclonal 
antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample 
is examined using fluorescence and differential interference contrast 
(DIC) microscopy. Qualitative analysis is performed by carefully 
scanning each slide well for objects that have the size, shape, and 
fluorescence characteristics of Cryptosporidium oocysts or Giardia 
cysts. Potential oocysts or cysts are confirmed through DAPI staining 
characteristics and DIC microscopy. Oocysts and cysts are identified 
when the size, shape, color, and morphology agree with specified 
criteria and examples in a photographic library. Quantitative analysis 
is performed by counting the total number of objects confirmed as 
oocysts or cysts on the slide.
    The Method 1622 interlaboratory validation study (EPA-821-R-01-027) 
was conducted in August 1998 and involved 12 laboratories that analyzed 
spiked reagent water and raw surface water samples. Eleven laboratories 
participated in the Method 1623 interlaboratory validation study (EPA-
821-R-028) conducted in 1999. Both the interlaboratory validation 
studies for Methods 1622 and 1623 followed the same approach for 
preparing spiked suspensions for single-blind test samples. The 
Cryptosporidium results obtained during the Method 1623 study were not 
statistically different from the Cryptosporidium results obtained 
during the Method 1622 interlaboratory validation study.
2. Request for Comment and Available Data
    EPA requests public comments on the proposed methods for the 
protozoan pollutants. EPA invites comments on the technical merit, 
applicability, and implementation of the proposed Cryptosporidium and 
Giardia methods for ambient water monitoring. Commenters should specify 
the method and pollutant to which the comment specifies. EPA encourages 
commenters to provide copies of supporting data or references cited in 
comments. Additionally, EPA requests comments on any other applicable 
methods for analyzing for Cryptosporidium and Giardia in ambient water 
not included in today's proposal. Method descriptions and supporting 
data may be submitted for additional test procedures that are 
applicable to enumerating these protozoa in water.

IV. Administrative Requirements

A. Executive Order 12866: Regulatory Planning and Review

    Under Executive Order 12866, [58 FR 51735; (October 4, 1993)], the 
Agency must determine whether a regulatory action is ``significant'' 
and therefore subject to Office of Management and Budget (OMB) review 
and the requirements of the Executive Order. The Executive Order 
defines ``significant regulatory action'' as one that is likely to 
result in a rule that may:
    (1) Have an annual effect on the economy of $100 million or more or 
adversely affect in a material way the economy, a sector of the 
economy, productivity, competition, jobs, the environment, public 
health or safety, or State, local, or tribal governments or 
communities.
    (2) Create a serious inconsistency or otherwise interfere with an 
action taken or planned by another agency.
    (3) Materially alter the budgetary impact of entitlements, grants, 
user fees, or loan programs or the rights and obligations of recipients 
thereof.
    (4) Raise novel legal or policy issues arising out of legal 
mandates, the President's priorities, or the principles set forth in 
the Executive Order.''
    It has been determined that this rule is not a ``significant 
regulatory action'' under the terms of Executive Order 12866 and is 
therefore not subject to OMB review.

B. Unfunded Mandates Reform Act

    Title II of the Unfunded Mandates Reform Act of 1995 (UMRA), Pub. 
L.

[[Page 45822]]

104-4, establishes requirements for Federal agencies to assess the 
effects of their regulatory actions on State, Tribal, and local 
governments and the private sector. Under section 202 of the UMRA, EPA 
generally must prepare a written statement, including a cost-benefit 
analysis, for proposed and final rules with ``Federal mandates'' that 
may result in expenditures to State, Tribal, and local governments, in 
the aggregate, or to the private sector, of $100 million or more in any 
one year.
    Before promulgating an EPA rule for which a written statement is 
needed, section 205 of the UMRA generally requires EPA to identify and 
consider a reasonable number of regulatory alternatives and adopt the 
least costly, most cost-effective or least burdensome alternative that 
achieves the objectives of the rule. The provisions of section 205 do 
not apply when they are inconsistent with applicable law. Moreover, 
section 205 allows EPA to adopt an alternative other than the least 
costly, most cost-effective or least burdensome alternative if the 
Administrator publishes with the final rule an explanation of why that 
alternative was not adopted. Before EPA establishes any regulatory 
requirements that may significantly or uniquely affect small 
governments, including Tribal governments, it must have developed under 
section 203 of the UMRA a small government agency plan. The plan must 
provide for the notification of potentially affected small governments, 
enabling officials of affected small governments to have meaningful and 
timely input in the development of EPA regulatory proposals with 
significant Federal intergovernmental mandates, and informing, 
educating, and advising small governments on compliance with the 
regulatory requirements.
    Today's proposed rule contains no Federal mandates (under the 
regulatory provisions of title II of the UMRA) for State, Tribal, or 
local governments or the private sector that may result in expenditures 
of $100 million or more in any one year. This rule makes available 
testing procedures for E. coli, enterococci, Cryptosporidium, and 
Giardia that may be used by a State, Territorial, Tribal or local 
authority for compliance with water quality standards or ambient 
monitoring requirements when testing is otherwise required by these 
regulatory authorities. Thus, today's rule is not subject to the 
requirements of sections 202 and 205 of the UMRA.
    EPA has also determined that this rule contains no regulatory 
requirements that might significantly or uniquely affect small 
governments. As discussed below, under the Regulatory Flexibility Act, 
the economic impact on small entities is anticipated to be small. It 
would not significantly affect them because any incremental costs 
incurred are small and it would not uniquely affect them because it 
would affect entities of all sizes depending upon whether testing for 
these bacteria or protozoa is otherwise required by a regulatory 
authority. Further, monitoring for small entities is generally expected 
to be less frequent than monitoring for larger entities. Thus, today's 
rule is not subject to the requirements of sections 203 of UMRA.

C. Regulatory Flexibility Act (RFA),as amended by the Small Business 
Regulatory Enforcement Act of 1996 (SBREFA), 5 U.S.C. 601 et seq.

    The RFA generally requires an agency to prepare a regulatory 
flexibility analysis of any rule subject to notice and comment 
rulemaking requirements under the Administrative Procedure Act or any 
other statute unless the agency certifies that the rule will not have a 
significant economic impact on a substantial number of small entities. 
Small entities include small businesses, small organizations, and small 
governmental jurisdictions.
    For purposes of assessing the impacts of today's rule on small 
entities, small entity is defined as: (1) A small business as defined 
by the Small Business Administration definition of small business; (2) 
a small governmental jurisdiction that is a government of a city, 
county, town, school district or special district with a population of 
less that 50,000; and (3) a small organization that is any not-for-
profit enterprise which is independently owned and operated and is not 
dominant in its field.
    After considering the economic impacts of today's proposed rule on 
small entities, I certify that this action will not have a significant 
economic impact on a substantial number of small entities. This 
proposed regulation would approve testing procedures for the 
measurement of E. coli and enterococci bacteria, and Cryptosporidium 
and Giardia protozoa in ambient water. EPA anticipates that the methods 
will be used by State regulatory authorities for evaluating attainment 
of water quality standards or ambient monitoring requirements. EPA 
NPDES regulations do not require monitoring of ambient water conditions 
in NPDES permits. In a few instances, ambient water monitoring 
requirements may be included in an EPA-issued permit where site-
specific circumstances warrant. EPA regulations do, require NPDES 
permittees to use EPA-approved test methods for all monitoring data 
reported to the Agency (40 CFR 122.21). Consequently, to the extent 
that an NPDES permit requires monitoring and reporting of ambient water 
for E. coli, enterococci, Cryptosporidium, or Giardia (and NPDES 
regulations require the use of EPA-approved methods for all 
monitoring), EPA approval of these test methods arguably may impose 
costs on NPDES permit holders, including small entities. EPA is 
unaware, however, of any EPA-issued NPDES permits that currently 
require monitoring of ambient water for such pollutants. Hence, EPA 
does not expect approval of these methods to impose any additional 
costs as a result of their applicability to EPA-issued permits. As 
noted above, EPA's NPDES regulations do not require monitoring of 
ambient water conditions. Consequently, to the extent that a State 
requires such monitoring, those requirements are imposed under State, 
rather than federal, authority. Because States have the discretion not 
to require such monitoring, any increased costs to small entities 
arising from use of the methods proposed for approval by EPA today that 
are imposed as a result of State law are not attributable to this 
regulation.
    Nonetheless, EPA evaluated these potential costs to determine 
whether EPA approval of the methods will have a significant impact on a 
substantial number of small entities. As previously noted, States may 
require ambient water monitoring to evaluate attainment of water 
quality standards. A few States currently require NPDES permit holders 
to monitor ambient water. Thus, some NPDES permittees are already 
testing ambient water for these parameters. Hence, the impact of using 
EPA-approved methods for such dischargers may represent little or no 
increased burden.
    The small entities that might be affected by this rule include 
small governmental jurisdictions that have publically-owned treatment 
works (POTWs) and small businesses with water quality-based discharge 
permits. EPA looked first at the potential cost of the E. coli and 
enterococci methods proposed today. EPA conducted a survey of State, 
municipal, and commercial laboratories that routinely conduct bacterial 
analysis of water to compare the incremental analytical costs for 
existing total and fecal coliform methods already employed by many 
water quality monitoring programs with the methods proposed here. The 
mean analytical costs for total and fecal coliform were $22 ($15-48) 
and $21

[[Page 45823]]

($15-$35), respectively. The mean analytical costs for E. coli and 
enterococci were $22 ($10-$35) and $32 ($25-$50), respectively. The 
similarity of costs for total and fecal coliform versus E. coli and 
enterococci methods is expected since the analytical procedures used to 
determine these pollutants generally employ similar techniques, media, 
equipment, and require comparable laboratory time and effort to 
complete analysis. Some States are already using the proposed test 
methods for E. coli and enterococci in State ambient water quality 
monitoring programs (indeed, EPA is proposing to approve consensus 
methods for enumerating E. coli and enterococci in ambient waters. See 
section IV.E, below) and thus this rule would formalize current 
practice in those States. Furthermore, EPA expects that any modest 
potential increase in costs for enterococci analyses will be reduced 
once the proposed methods are broadly implemented by environmental 
laboratories and State water quality monitoring programs.
    Next, EPA looked at the costs for testing for Cryptosporidium and 
Giardia. The range in cost for Methods 1622 and 1623 analysis of 
Cryptosporidium and Giardia is between $400-$500 for each method. As 
stated in section IV.E. below, EPA is not aware of any other acceptable 
test methods currently available for monitoring these pollutants. 
Methods 1622 and 1623 have been previously used for monitoring of 
various drinking water plant source waters to establish a national 
estimate of Cryptosporidium and Giardia occurrence. Because of the 
relatively high costs, EPA does not anticipate that these test methods 
will be used for daily or ongoing monitoring, but may be used program-
specific occurrence assessments.
    The purpose of this rule is only to make these methods available to 
States, Tribal and municipalities that may want to use them for ambient 
water monitoring. As noted above, the costs associated with 
Cryptosporidium and Giardia analysis would not be a Federally-mandated 
cost, but rather would flow from a State's adoption of ambient 
monitoring requirements. The inclusion of these test methods in section 
136.3 is intended to make these test methods available to States and 
others for use in water quality monitoring programs. EPA is not 
establishing any compliance monitoring requirements for these 
pollutants.

D. Paperwork Reduction Act

    This action does not impose an information collection burden under 
the provisions of the Paperwork Reduction Act, 44 U.S.C. 3501 et seq. 
This rule proposes to make available new test methods for E. coli, 
enterococci, Cryptosporidium and Giardia for use in ambient water 
monitoring programs but EPA would not require the use of these test 
methods.
    Burden means the total time, effort, or financial resources 
expended by persons to generate, maintain, retain, or disclose or 
provide information to or for a Federal agency. This includes the time 
needed to review instructions; develop, acquire, install, and utilize 
technology and systems for the purposes of collecting, validating, and 
verifying information, processing and maintaining information, and 
disclosing and providing information; adjust the existing ways to 
comply with any previously applicable instructions and requirements; 
train personnel to be able to respond to a collection of information; 
search data sources; complete and review the collection of information; 
and transmit or otherwise disclose the information.
    An Agency may not conduct or sponsor, and a person is not required 
to respond to a collection of information unless it displays a 
currently valid OMB control number. The OMB control numbers for EPA's 
regulations are listed in 40 CFR part 9 and 48 CFR chapter 15.

E. National Technology Transfer and Advancement Act

    Section 12(d) of the National Technology Transfer and Advancement 
Act (NTTAA) of 1995, Public Law 104-113, section 12(d) (15 U.S.C. 272 
note), directs EPA to use voluntary consensus standards in its 
regulatory activities unless to do so would be inconsistent with 
applicable law or otherwise impractical. Voluntary consensus standards 
are technical standards (e.g., material specifications, test methods, 
sampling procedures, business practices) that are developed or adopted 
by voluntary consensus standard bodies. The NTTAA directs EPA to 
provide Congress, through the Office of Management and Budget (OMB), 
explanations when the EPA decides not to use available and applicable 
voluntary consensus standards. This rulemaking involves technical 
standards. Therefore, the Agency conducted a search to identify 
potentially applicable voluntary consensus standards. EPA's search of 
the technical literature revealed several consensus methods appropriate 
for enumerating E. coli and enterococci in ambient waters. Accordingly, 
methods for E. coli and enterococci published by Standard Methods for 
the Examination of Water and Wastewater, ASTM, and AOAC are included in 
this proposal and are listed in Table IA at the end of this notice (see 
footnotes 4,10, and 11, respectively, for the complete citations). No 
voluntary consensus standards were found for Cryptosporidium or 
Giardia. EPA welcomes comments on this aspect of the proposed 
rulemaking and, specifically, invites the public to identify 
potentially applicable voluntary consensus standards for enumerating E. 
coli, enterococci, Cryptosporidium, and Giardia in ambient waters, and 
to explain why such standards should be used in this regulation.

F. Executive Order 13045--Protection of Children From Environmental 
Health Risks and Safety Risks

    Executive Order 13045, (62 FR 19885, April 23, 1997) applies to any 
rule that: (1) Is determined to be ``economically significant'' as 
defined under Executive Order 12866, and (2) concerns an environmental 
health or safety risk that EPA has reason to believe may have a 
disproportionate effect on children. If the regulatory action meets 
both criteria, the Agency must evaluate the environmental health or 
safety effects of the planned rule on children, and explain why the 
planned regulation is preferable to other potentially effective and 
reasonably feasible alternatives considered by the Agency.
    This proposed rule is not subject to the Executive Order because it 
is neither economically significant as defined in Executive Order 
12866, nor does it concern an environmental health or safety risk that 
EPA has reason to believe may have a disproportionate effect on 
children.

G. Executive Order 13175: Consultation and Coordination With Indian 
Tribal Governments

    Executive Order 13175, entitled ``Consultation and Coordination 
with Indian Tribal Governments'' (65 FR 67249, November 6, 2000), 
requires EPA to develop an accountable process to ensure ``meaningful 
and timely input by tribal officials in the development of regulatory 
policies that have tribal implications.'' ``Policies that have tribal 
implications'' is defined in the Executive Order to include regulations 
that have ``substantial direct effects on one or more Indian tribes, on 
the relationship between the Federal government and the Indian tribes, 
or on the distribution of power and responsibilities between the 
Federal government and Indian tribes.''
    This proposed rule does not have tribal implications. It will not 
have substantial direct effects on tribal

[[Page 45824]]

governments, on the relationship between the Federal government and 
Indian tribes, or on the distribution of power and responsibilities 
between the Federal government and Indian tribes, as specified in 
Executive Order 13175. This proposed rule makes available test methods 
that may be used by a regulatory authority to demonstrate compliance 
with ambient water quality monitoring or water quality standards. 
However, Federal regulations do not require the use of these test 
methods. Thus, Executive Order 13175 does not apply to this rule.
    In the spirit of Executive Order 13175, and consistent with EPA 
policy to promote communications between EPA and tribal governments, 
EPA specifically solicits additional comment on this proposed rule from 
tribal officials.

H. Executive Order 13132: Federalism

    Executive Order 13132, entitled ``Federalism'' (64 FR 43255, August 
10, 1999), requires EPA to develop an accountable process to ensure 
``meaningful and timely input by State and local officials in the 
development of regulatory policies that have federalism implications.'' 
``Policies that have federalism implications'' is defined in the 
Executive Order to include regulations that have ``substantial direct 
effects on the States, on the relationship between the national 
government and the States, or on the distribution of power and 
responsibilities among the various levels of government.''
    This proposed rule does not have federalism implications. It will 
not have substantial direct effects on the States, on the relationship 
between the national government and the States, or on the distribution 
of power and responsibilities among the various levels of government, 
as specified in Executive Order 13132. This proposed rule makes new 
analytical methods available for conducting analysis of ambient water 
for enumeration of E.coli, enterococci, Cryptosporidium, or Giardia. 
EPA does not, however, propose to require use of these methods under 
this rule. Thus, Executive Order 13132 does not apply to this rule.
    Although Section 6 of Executive Order 13132 does not apply to this 
rule, EPA did consult with representatives of State and local 
governments in developing the proposed regulation. In fact, it was 
State representatives who requested that EPA include test methods for 
these biological pollutants in section 136.3 because they want to use 
EPA-approved test methods for ambient water monitoring. EPA is 
proposing this action in response to these requests. EPA included a 
number of test methods currently being used by States for these 
pollutants in today's proposed rulemaking. No significant concerns were 
raised about these methods.
    In the spirit of Executive Order 13132, and consistent with EPA 
policy to promote communications between EPA ans State and local 
governments, EPA specifically solicits comment on this proposed rule 
from State and local officials.

I. Executive Order 13211: Actions Concerning Regulations That 
Significantly Affect Energy Supply, Distribution, or Use

    Executive Order 13211, ``Actions Concerning Regulations That 
Significantly Affect Energy Supply, Distribution, or Use'' (66 FR 28355 
(May 22, 2001)), provides that agencies shall prepare and submit to the 
Administrator of the Office of Information and Regulatory Affairs, 
Office of Management and Budget, a Statement of Energy Effects for 
certain actions identified as ``significant energy actions.'' Section 
4(b) of Executive Order 13211 defines ``significant energy actions'' as 
``any action by an agency (normally published in the Federal Register) 
that promulgates or is expected to lead to the promulgation of a final 
rule or regulation, including notices of inquiry, advance notices of 
proposed rulemaking, and notices of proposed rulemaking: (1)(i) that is 
a significant regulatory action under Executive Order 12866 or any 
successor order, and (ii) is likely to have a significant adverse 
effect on the supply, distribution, or use of energy; or (2) that is 
designated by the Administrator of the Office of Information and 
Regulatory Affairs as a significant energy action.''
    We have not prepared a Statement of Energy Effects for this 
proposed rule because this rule is not a significant energy action, as 
defined in Executive Order 13211. This is not a significant regulatory 
action under Executive Order 12866, and it does not have a significant 
adverse effect on the supply, distribution, or use of energy.

V. Media Acronyms

BHI--brain-heart infusion agar
BGLB--brilliant green lactose bile broth
CPRG--chlorophenol red--D-galactopyranoside
DAPI--4',6-diamidino-2-phenylindole
DIC--differential interference contrast
EC--E. coli
EIA--esculin iron agar
LES-Endo--Lawrence Experimental Station--Endo Agar
LTB--lauryl tryptose broth
mEI--membrane-Enterococcus iron agar
mFC--membrane-Fecal coliform agar
mTEC--membrane-Thermotolerant E. coli agar
MUG--4-methylumbelliferyl--D-glucuronide
NA--nutrient agar
ONPG--ortho-nitorphenyl--D-galactopyranoside
PSE--Pfizer selective Enterococcus agar

VI. References

Abbott, S., et al. 1998. ``Evaluation of Enterolert for the 
Enumeration of Enterococci in the Marine Environment.'' New Zealand 
Journal of Marine and Freshwater Research. 32:505-513.
AOAC. 1995. Official Methods of Analysis of AOAC International, 16th 
Edition, Volume I, Chapter 17. AOAC International. 481 North 
Frederick Avenue, Suite 500, Gaithersburg, Maryland 20877-2417.
APHA. 1998. Standard Methods for the Examination of Water and 
Wastewater. 20th Edition. American Public Health Association. 1015 
15th Street, NW, Washington, DC 20005.
ASTM. 2000. Annual Book of ASTM Standards--Water and Environmental 
Technology. Section 11.02. ASTM. 100 Barr Harbor Drive, West 
Conshohocken, PA 19428.
Brenner, K.P., et al. 1993. ``New Medium for the Simultaneous 
Detection of Total Coliforms and Escherichia coli in Water.'' 
Applied and Environmental Microbiology. 59:3534-3544.
Brenner, K.P., et al.1996a. ``Comparison of the Recoveries of 
Escherichia coli and Total Coliforms from Drinking Water by the MI 
Agar Method and the U.S. Environmental Protection Agency-Approved 
Membrane Filter Method.'' Applied and Environmental Microbiology. 
62:203-208.
Brenner, K.P., et al. 1996b. ``Interlaboratory Evaluation of MI Agar 
and the U.S. Environmental Protection Agency-Approved Membrane 
Filter Method for the Recovery of Total Coliforms and Escherichia 
coli from Drinking Water.'' Journal of Microbiological Methods. 
67:111-119.
Budnick, G.E., et al. 1996. ``Evaluation of Enterolert for 
Enumeration of Enterococci in Recreational Waters.'' Applied and 
Environmental Microbiology. 62:3881-3884.
Chen, C.M.., et al. 1996. ``Enterolert--A Rapid Method for Detection 
of Enterococci spp.'' In: ASM abstracts, no. Q448. 96th American 
Society of Microbiology general meeting. New Orleans. 464 p.
Cowburn, J.K., et al. 1994. A Preliminary Study of the use of 
Colilert for Water Quality Monitoring. Lett. Appl. 
Microbiol. 19:50-52.
Edberg, S.C., et al. 1990. ``Enumeration of Total Coliforms and 
Escherichia coli from Source Water by Defined Substrate 
Technology.'' Applied and Environmental Microbiology. 56(2):366-369.
Edberg, S.C., et al. 1989. ``National Field Evaluation of a Defined 
Substrate

[[Page 45825]]

Method for the Simultaneous Detection of Total Coliforms and 
Escherichia coli from Drinking Water: Comparison with Presence/
Absence Techniques.'' Applied and Environmental Microbiology. 55: 
1003-1008.
Edberg, S.C., et al. 1988. ``National Field Evaluation of a Defined 
Substrate Method for the Simultaneous Enumeration of Total Coliforms 
and Escherichia coli from Drinking Water: Comparison with Standard 
Multiple-tube Fermentation Method.'' Applied and Environmental 
Microbiology. 54:1595-1601.
Ellgas, W.M., et al. Undated. ``Evaluation of Autoanalysis 
Colilert In Wastewater.''
Francy, D.S., et al. 2000. ``Comparison of Methods for Determining 
Escherichia coli Concentrations in Recreational Waters.'' Water 
Research. 34:2770-2778.
Fricker, E.J., et al. 1996a. ``Use of Two Presence/Absence Systems 
for the Detection of E. coli and Coliforms from Water.'' Water 
Research. 30:2226-2228.
Fricker, E.J., et al. 1996b. ``Use of Defined Substrate Technology 
and a Novel Procedure for Estimating the Numbers of Enterococci in 
Water.'' Journal of Microbiological Methods. 27: 207-210.
Fricker, E.J., et al. 1995. ``Quantitative Procedures for the 
Detection of E. coli, Coliforms, and Enterococci in Water using 
Quanti-Tray and Enterolert.'' p. 2031-2036. In Proceedings of the 
Water Quality Technology Conference, New Orleans. American Water 
Works Association, Denver, CO.
Grant, M.A. 1997. ``A New Membrane Filtration Medium for 
Simultaneous Detection and Enumeration of Escherichia coli and Total 
Coliforms.'' Applied and Environmental Microbiology. 63:3526-3530.
Hach Company, Inc. 1999. m-ColiBlue24 Method is available from Hach 
Company, 100 Dayton Ave., Ames, IA 50010.
Hach Company, Inc. 1998. ``Comparability Study Using The Protocol 
for Alternate Test Procedures for Microbiology in Compliance With 
Drinking Water Regulations--m-ColiBlue 24, Membrane Filter Method 
For Isolation of Total Coliforms and E. coli in Finished Water.'' 
Morehead State University Water Testing Laboratory. Dr. Ted Pass II.
IDEXX Laboratories, Inc. 1999a. Description of Colilert, 
Colilert-18, Quanti-Tray, Quanti-Tray/
2000, ColisureTM, and EnterolertTM methods may 
be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, 
Westbrook, Maine 04092.
IDEXX Laboratories, Inc. 1999b. ``Quanti-TrayTM: A Simple 
Method for Quantitation of Bacterial Density in Liquid Samples.''
IDEXX Laboratories, Inc. 1999c. ``Quanti-Tray/2000TM: 
Detection and Enumeration of Bacteria from High Bacterial Density 
Liquid Samples Without Dilution.''
IDEXX Laboratories, Inc. 1999d. Recreational Water Study, State of 
Illinois.
IDEXX Laboratories, Inc. 1999e. Recreational Water Study, Oakland 
County Health Department, Pontiac, Michigan.
Levin, M.A., et al. 1975. ``Membrane Filter Technique for 
Enumeration of Enterococci in Marine Waters.'' Applied Microbiology. 
30:66-71.
Liebman, M. 1999. ``Short Summary of Statistical Results Comparing 
the New Methods 1600 vs. the Standard Method for Enterococci.'' EPA 
Region 1 Draft Report.
Messer, J.W., et al. 1998. ``A Rapid, Specific Membrane Filtration 
Procedure for Enumeration of Enterococci in Recreational Water.'' 
Applied and Environmental Microbiology. 64:678-680.
Noble, R.T., et al. 1999. Southern California Bight 1998 Regional 
Monitoring Program: Summer Shoreline Microbiology. Southern 
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CA.
Noble, R.T., et al. 2000a. Southern California Bight 1998 Regional 
Monitoring Program: Winter Shoreline Microbiology. Southern 
California Coastal Water Research Project Project. Westminster, CA.
Noble, R.T., et al. 2000b. Southern California Bight Regional 
Monitoring Program: Storm Event Shoreline Microbiology. Southern 
California Coastal Water Research Project Project. Westminster, CA.
Ostensvik, O. 2000. ``Coliform Bacteria and Escherichia coli in 
Norwegian Drinking Water Sources--Comparison of Methods Based on the 
Fermentation of Lactose and Methods Based on the Activity of 
Specific Enzymes.'' In Proceedings of the Water Quality Technology 
Conference, Salt Lake City. American Water Works Association. 
Denver, CO.
Palmer, C.J. et al. 1993. ``Evaluation of Colilert-marine Water for 
Detection of Total Coliforms and Escherichia coli in the Marine 
Environment.'' Applied and Environmental Microbiology. 59:786-790.
Thomas, H.A., et al. 1955. ``Estimation of Coliform Density by the 
Membrane Filter and the Fermentation Tube Methods.'' American 
Journal of Public Health. 45(11): 1431-1437.
USEPA. 2001a. Method 1622: Cryptosporidium in Water by Filtration/
IMS/FA. U.S. Environmental Protection Agency. Office of Water. 
Washington, DC. EPA-821-R-01-026.
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Filtration/IMS/FA. U.S. Environmental Protection Agency. Office of 
Water. Washington, DC. EPA-821-R-01-025.
USEPA. 2001c. Implementation and Results of the Information 
Collection Rule Supplemental Surveys. U.S. Environmental Protection 
Agency. Office of Water. Washington, DC. EPA 815-R-01-003.
USEPA. 2001d. Interlaboratory Validation Study Results for 
Cryptosporidium Precision and Recovery for U.S. EPA Method 1622. 
U.S. Environmental Protection Agency. Office of Water. Washington, 
DC. EPA-821-R-01-027.
USEPA. 2001e. Results of the Interlaboratory Method Validation Study 
for Determination of Cryptosporidium and Giardia Using U.S. EPA 
Method 1623. U.S. Environmental Protection Agency. Office of Water. 
Washington, DC. EPA-821-R-01-028.
USEPA. 2000a. Improved Enumeration Methods for the Recreational 
Water Quality Indicators: Enterococci and Escherichia coli. U.S. 
Environmental Protection Agency. Office of Science and Technology. 
Washington, DC. EPA/821/R-91/004.
USEPA. 2000b. Membrane Filter Method for the Simultaneous Detection 
of Total Coliforms and Escherichia coli in Drinking Water. U.S. 
Environmental Protection Agency, Office of Research and Development, 
Cincinnati, OH. EPA 600-R-00-013.
USEPA. 1999a. Action Plan for Beaches and Recreational Waters. U.S. 
Environmental Protection Agency. Office of Research and Development. 
Office of Water. EPA/600/R-98/079.
USEPA. 1999b. ``A Modified mTEC Medium for Monitoring Recreational 
Waters.'' Presented at American Society for Microbiology Annual 
Meeting, Miami Beach, FL. May 1997.
USEPA. 1998. Clean Water Action Plan: Restoring and Protecting 
America's Waters. U.S. Environmental Protection Agency and U.S. 
Department of Agriculture. February 14, 1998. EPA-840-R-98-001.
USEPA. 1997. Method 1600: Membrane Filter Test Method for 
Enterococci in Water. U.S. Environmental Protection Agency. Office 
of Water. Washington, DC. EPA-821-R-97-004.
USEPA. 1986a. Ambient Water Quality Criteria for Bacteria--1986. 
Office of Water Regulations and Standards Division. Washington, DC. 
EPA-440-5-84-002.
USEPA. 1986b. Memo from T.H. Ericksen, HERL, Microbiology Branch to 
Participants in the EMSL-HERL Study for the Interlaboratory Testing 
of mE and mTEC Media for the Enumeration of Enterococci and E. coli. 
Four data summary tables attached.
USEPA. 1985a. ``Test Method 1103.1: Escherichia coli In Water By The 
Membrane Filter Procedure'' Included in: Test Methods For 
Escherichia coli and Enterococci In Water By the Membrane Filter 
Procedure. U.S. Environmental Protection Agency, Office of Research 
and Development, Environmental Monitoring Support Laboratory, 
Cincinnati, OH. EPA-600-4-85-076.
USEPA. 1985b. ``Test Method 1106.1: Enterococci In Water By The 
Membrane Filter Procedure'' Included in: Test Methods For 
Escherichia coli and Enterococci In Water By the Membrane Filter 
Procedure. U.S. Environmental Protection Agency, Office of Research 
and Development, Environmental

[[Page 45826]]

Monitoring Support Laboratory, Cincinnati, OH. EPA-600-4-85-076.
USEPA. 1976. ``Quality Criteria for Water.'' U.S. Environmental 
Protection Agency. Washington, DC. EPA-600-3-76-079.

List of Subjects in 40 CFR Part 136

    Environmental protection, Reporting and recordkeeping requirements, 
Water pollution control.

    Dated: August 23, 2001.
Christine Todd Whitman,
Administrator.

    For the reasons set out in the preamble, title 40, chapter I of the 
Code of Federal Regulations, is proposed to be amended as follows:

PART 136--GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANALYSIS 
OF POLLUTANTS

    1. The authority citation for Part 136 continues to read as 
follows:

    Authority: Secs. 301, 304(h), 307, and 501(a) Pub. L. 95-217, 91 
Stat. 1566, et seq. (33 U.S.C. 1251, et seq.) (The Federal Water 
Pollution Control Act Amendments of 1972 as amended by the Clean 
Water Act of 1977.)

    2. Section 136.3 is amended:
    a. In paragraph (a) by revising Table IA.
    b. By revising paragraphs (b)(10) and (b)(11), adding and reserving 
paragraphs (b)(44) to (b)(50), and adding paragraphs (b)(51) through 
(b)(60).
    c. In paragraph (e) by revising the entries in Table II for Table 
IA and adding a new footnote 17.


Sec. 136.3  Identification of test procedures.

    (a) * * *

                                                                         Table IA.--List of Approved Biological Methods
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                    Standard methods
       Parameter and units              Method \1\                 EPA            18th, 19th, 20th Ed.           ASTM               AOAC              USGS                      Other
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria:
  1. Coliform (fecal), number per  Most Probable Number  p.132 \3\..............  9221C E \4\.........
   100 mL.                          (MPN), 5 tube.         .....................    ..................
                                   3 dilution, or        p. 124 \3\.............  9222D \4\...........                                       B-0050-85 \5\
                                    Membrane filter
                                    (MF) \2\, single
                                    step.
  2. Coliform (fecal) in presence  MPN, 5 tube, 3        p. 132 \3\.............  9221C E \4\.........
   of chlorine, number per 100 mL.  dilution, or.        p. 124 \3\.............  9222D \4\...........
                                   MF, single step \6\.
  3. Coliform (total), number per  MPN, 5 tube, 3        p. 114 \3\.............  9221B \4\...........
   100 mL.                          dilution, or.        p. 108 \3\.............  9222B \4\...........                                       B-0025-85 \5\
                                   MF \2\, single step
                                    or two step.
  4. Coliform (total), in          MPN, 5 tube, 3        p. 114 \3\.............  9221B \4\...........
   presence of chlorine, number     dilution, or.        p. 111 \3\.............  9222(B+B.5c) \4\....
   per 100 mL.                     MF2 with enrichment.
  5. E. coli, number per 100 mL    MPN 7,9,15..........  .......................  9221B.1/9221F
   \29\.                                                                           4,12,14.
                                                                                  9223B 4,13..........  ......................    11 991.15  ......................  Colilert 13,18
                                                                                                                                                                     Colilert-
                                                                                                                                                                      13,16,18
                                                                                                                                                                     Colisure TM 13,17,18
                                   MF 2,6,7,8,9........  .......................  9222B/9222G 4,20....
                                                         1103.1 \21\............  921D \4\............  53592-93 \10\
                                                         1103.1M 22.............
                                                         MI agar \23\...........
                                                         .......................  ....................                                       ......................  mColiBlue24 \19\
  6. Fecal streptococci, number    MPN, 5 tube, 3        p. 139 \3\.............  9230B \4\...........
   per 100 mL.                      dilution,.           p. 136 \3\.............  9230C \4\...........                                       B-0055-85 \5\
                                   mf \2\, or..........  p. 143 \3\.............
                                   Plate count.........
  7. Enterococci, number per 100   MPN 7,9.............  .......................  9230B \4\...........
   mL \29\.
                                                                                                        D6503-99 \10\                        ......................  Enterolert TM 13,24
                                   MF 2,6,7,8,9........  1106.1 \25\............  9230C \4\...........  D5259-92 \10\
                                                         1600 \26\..............
                                   Plate count.........  p. 14 \33\.............
Protozoa:
  8. Cryptosporidium \29\........  Filtration/IMS/FA...  1622 \27\
                                                         1623 \28\..............
  9. Giardia \29\................  Filtration/IMS/FA...  1623 \28\..............
Aquatic Toxicity:

[[Page 45827]]

 
  10. Toxicity, acute, fresh       Daphnia,              Sec. 9 \30\............
   water organisms, LC50, percent   Ceriodaphnia,
   effluent.                        Fathead Minnow,
                                    Rainbow Trout,
                                    Brook Trout, or
                                    Bannerfish Shiner
                                    mortality.
  11. Toxicity, acute, estuarine   Mysid, Sheepshead     Sec. 9 \30\............
   and marine organisms, LC50,      Minnow, or Menidia
   percent effluent.                spp. mortality.
  12. Toxicity, chronic, fresh     Fathead minnow        1000.0 \31\
   water organisms, NOEC or IC25,   larval survival and    .....................
   percent effluent.                growth.              1001.0 \31\............
                                   Fathead minnow          .....................
                                    embryo-larval          .....................
                                    survival and         1002.0 \31\............
                                    teratogenicity.
                                   Ceriodaphnia
                                    survival and
                                    reproduction.
                                   Selenastrum growth..  1000.0 \32\............
  13. Toxicity, chronic,           Sheepshead minnow     1004.0 \31\
   estuarine and marine             larval survival and    .....................
   organisms, NOEC or IC25,         growth.                .....................
   percent effluent.               Sheepshead minnow     1005.0 \32\............
                                    embryo-larval          .....................
                                    survival and         1006.0 \32\............
                                    teratogenicity.        .....................
                                   Menidia beryllina     1007.0 \32\............
                                    larval survival and    .....................
                                    growth.                .....................
                                   Mysidopsis bahia      1008.0 \32\............
                                    survival, growth,      .....................
                                    and fecundity.       1009.0 \32\ ...........
                                   Arbacia punctulata
                                    fertilization.
                                   Champia parvula
                                    reproduction
                                    1004.032.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Notes to Table IA:
\1\ The method must be specified when results are reported.
\2\ A 0.45 m membrane filter (MF) or other pore size certified by the manufacturer to fully retain organisms to be cultivated and to be free of extractables which could interfere with
  their growth.
\3\ USEPA. 1978. Microbiological Methods for Monitoring the Environment, Water, and Wastes. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati,
  Ohio. EPA/600/8-78/017.
\4\ APHA. 1998, 1995, 1992. Standard Methods for the Examination of Water and Wastewater. American Public Health Association. 20th, 19th, and 18th Editions. Amer. Publ. Hlth. Assoc.,
  Washington, DC.
\5\ USGS. 1989. U.S. Geological Survey Techniques of Water-Resource Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and Analysis of Aquatic Biological and
  Microbiological Samples, U.S. Geological Survey, U.S. Department of Interior, Reston, Virginia.
\6\ Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Most Probable Number method will be required to resolve any controversies.
\7\ Tests must be conducted to provide organism enumeration (density). Select the appropriate configuration of tubes/filtrations and dilutions/volumes to account for the quality, character,
  consistency, and anticipated organism density of the water sample.
\8\ When the MF method has not been used previously to test ambient waters with high turbidity, large number of noncoliform bacteria, or samples that may contain organisms stressed by
  chlorine, a parallel test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of results.
\9\ To assess the comparability of results obtained with individual methods, it is suggested that side-by-side tests be conducted across seasons of the year with the water samples routinely
  tested in accordance with the most current Standard Methods for the Examination of Water and Wastewater or EPA alternate test procedure (ATP) guidelines.
\10\ ASTM. 2000, 1999, 1998. Annual Book of ASTM Standards--Water and Environmental Technology. Section 11.02. ASTM. 100 Barr Harbor Drive, West Conshohocken, PA 19428.
\11\ AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. AOAC International. 481 North Frederick Avenue, Suite 500, Gaithersburg, Maryland 20877-
  2417.
\12\ The multiple-tube fermentation test is used in 9221B.1. Lactose broth may be used in lieu of lauryl tryptose broth (LTB), if at least 25 parallel tests are conducted between this broth
  and LTB using the water samples normally tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total coliform using lactose broth is less than 10
  percent. No requirement exists to run the completed phase on 10 percent of all total coliform-positive tubes on a seasonal basis.
\13\ These tests are collectively known as defined enzyme substrate tests, where a substrate is used to detect the enzyme -glucuronidase produced by E. coli.
\14\ After prior enrichment in a presumptive medium for total coliform using 9221B.1, all presumptive tubes or bottles showing any amount of gas, growth or acidity within 48 h  3 h
  of incubation shall be submitted to 9221F. Commercially available EC-MUG media or EC media supplemented in the laboratory with 50 g/mL of MUG may be used.

[[Page 45828]]

 
\15\ Samples shall be enumerated by the multiple-tube or multiple-well procedure. Using multiple-tube procedures, employ an appropriate tube and dilution configuration of the sample as needed
  and report the Most Probable Number (MPN). Samples tested with Colilert and ColisureTM tests may be enumerated with the multiple-well procedures, Quanti-Tray or Quanti-
  Tray 2000, and the MPN calculated from the table provided by the manufacturer.
\16\ Colilert-18'' is an optimized formulation of the Colilert'' for the determination of total coliforms and E. coli that provides results within 18 h of incubation at 35  deg.C rather than
  the 24 h required for the Colilert'' test and is recommended for marine water samples.
\17\ Colisure must be incubated for 28 h before examining the results. If an examination of the results at 28 h is not convenient, then results may be examined at any time between 28
  h and 48 h.
\18\ Descriptions of the Colilert, Colilert-18, Quanti-Tray, Quanti-Tray/2000, and ColisureTM may be obtained from IDEXX Laboratories, Inc., One IDEXX
  Drive, Westbrook, Maine 04092.
\19\ A description of the mColiBlue24 test is available from Hach Company, 100 Dayton Ave., Ames, IA 50010.
\20\ Subject total coliform positive samples determined by 9222B or other membrane filter procedure to 9222G using NA-MUG media.
\21\ USEPA. 1985. Test Method 1103.1: Escherichia coli In Water By The Membrane Filter Procedure included in: Test Methods For Escherichia coli and Enterococci In Water By the Membrane Filter
  Procedure. U.S. Environmental Protection Agency, Office of Research and Development, Environmental Monitoring Support Laboratory, Cincinnati, OH. EPA-600-4-85-076.
\22\ USEPA. 2000. Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli. U.S. Environmental Protection Agency. Office of Science and
  Technology. Washington, DC. EPA/821/R-91/004.
\23\ Preparation and use of MI agar with a standard membrane filter procedure is set forth in the article, Brenner et al. 1993. ``New Medium for the Simultaneous Detection of Total Coliform
  and Escherichia coli in Water.'' Appl. Environ. Microbiol. 59:3534-3544 and electronic document, EPA-600-R-00-013.
\24\ A description of the Enterolert test may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092.
\25\ USEPA. 1985. Test Method 1106.1: Enterococci In Water By The Membrane Filter Procedure included in: Test Methods For Escherichia coli and Enterococci In Water By the Membrane Filter
  Procedure. U.S. Environmental Protection Agency, Office of Research and Development, Environmental Monitoring Support Laboratory, Cincinnati, OH. EPA-600-4-85-076.
\26\ USEPA. 1997. Method 1600: Membrane Filter Test Method for Enterococci in Water. U.S. Environmental Protection Agency. Office of Water. Washington, DC. EPA-821-R-97-004.
\27\ Method 1622 uses filtration, concentration, immunomagnetic separation of oocysts from captured material, immunofluorescence assay to determine concentrations, and confirmation through
  vital dye staining and differential interference contrast microscopy for the detection of Cryptosporidium.
\28\ Method 1623 uses filtration, concentration, immunomagnetic separation of oocysts and cysts from captured material, immunofluorescence assay to determine concentrations, and confirmation
  through vital dye staining and differential interference contrast microscopy for the simultaneous detection of Cryptosporidium and Giardia oocysts and cysts.
\29\ Recommended for enumeration of target organism in ambient water only. Applicability to other matrices must be demonstrated.
\30\ USEPA. 1993. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. Fourth Edition. U.S. Environmental Protection Agency, Environmental Monitoring
  Systems Laboratory, Cincinnati, Ohio. EPA/600/4-90/027F.
\31\ USEPA. 1994. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Third Edition. U.S. Environmental Protection Agency,
  Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. EPA/600/4-91/002.
\32\ Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. Second Edition. U.S. Environmental Protection Agency,
  Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. EPA/600/4-91/003. These methods do not apply to marine waters of the Pacific Ocean.

* * * * *
    (b) * * *

    (10) Annual Book of ASTM Standards, Water, and Environmental 
Technology, Section 11, Volumes 11.01 and 11.02, 1994, 1999, and 2000 
in 40 CFR 136.3, Table IA.
    (11) AOAC. 1995. Official Methods of Analysis of AOAC 
International, 16th Edition, Volume I, Chapter 17. AOAC International. 
481 North Frederick Avenue, Suite 500, Gaithersburg, Maryland 20877-
2417. Table IA.

* * * * *

    (51) IDEXX Laboratories, Inc. 1999. Description of 
Colilert, Colilert-18, Quanti-Tray, 
Quanti-Tray/2000, ColisureTM, and 
EnterolertTM methods are available from IDEXX Laboratories, 
Inc., One IDEXX Drive, Westbrook, Maine 04092. Table IA, Notes 18 and 
24.
    (52) Hach Company, Inc. 1999. m-ColiBlue24 Method is available from 
Hach Company, 100 Dayton Ave., Ames, IA 50010. Table IA, Note 19.
    (53) USEPA. 1985. Test Method 1103.1: Escherichia coli In Water By 
The Membrane Filter Procedure included in: Test Methods For Escherichia 
coli and Enterococci In Water By the Membrane Filter Procedure. U.S. 
Environmental Protection Agency, Office of Research and Development, 
Environmental Monitoring Support Laboratory, Cincinnati, OH. EPA-600-4-
85-076. Table IA, Note 21.
    (54) USEPA. 1985. Test Method 1106.1: Enterococci In Water By The 
Membrane Filter Procedure included in: Test Methods For Escherichia 
coli and Enterococci In Water By the Membrane Filter Procedure. U.S. 
Environmental Protection Agency, Office of Research and Development, 
Environmental Monitoring Support Laboratory, Cincinnati, OH. EPA-600-4-
85-076. Table IA, Note 25.
    (55) USEPA. 2000. ``Improved Enumeration Methods for the 
Recreational Water Quality Indicators: Enterococci and Escherichia 
coli.'' U.S. Environmental Protection Agency, Office of Science and 
Technology, Washington, DC. EPA/821/R-91/004. Table IA, Note 22.
    (56) Brenner et al. 1993. ``New Medium for the Simultaneous 
Detection of Total Coliform and Escherichia coli in Water.'' Appl. 
Environ. Microbiol. 59:3534-3544. Table IA, Note 23.
    (57) USEPA 2000. ``Membrane Filter Method for the Simultaneous 
Detection of Total Coliforms and Escherichia coli in Drinking Water.'' 
February 2000. U.S. Environmental Protection Agency, Office of Research 
and Development, Cincinnati, OH 45268. EPA 600-R-00-013. Table IA, Note 
23.
    (58) USEPA. 1997. ``Method 1600: Membrane Filter Test Method for 
Enterococci in Water.'' U.S. Environmental Protection Agency, Office of 
Water, Washington, DC. EPA-821-R-97-004. Table IA, Note 26.
    (59) USEPA. 2001. ``Method 1622: Cryptosporidium in Water by 
Filtration/IMS/FA.'' U.S. Environmental Protection Agency, Office of 
Water, Washington, DC. EPA-821-R-01-026. Table IA, Note 27.
    (60) USEPA. 2001. ``Method 1623: Cryptosporidium and Giardia in 
Water by Filtration/IMS/FA.'' U.S. Environmental Protection Agency, 
Office of Water, Washington, DC. EPA-821-R-01-025. Table IA, Note 28.

* * * * *

    (e) * * *

                   Table II.--Required Containers, Preservation Techniques, and Holding Times
----------------------------------------------------------------------------------------------------------------
                                                                                                      Maximum
                                                                                                   holding time
           Parameter No./name                   Container \1\              Preservation 2,3       \4\ (in hours)
 
----------------------------------------------------------------------------------------------------------------
Table IA--Bacteria Tests:
    1-5 Coliform, total, fecal, and E.   PP, G                        Cool, 10  deg.C, 0.0008%                 6
     coli.                                                             Na2S2O3 5.
    6 Fecal streptococci...............  PP, G                        Cool, 10  deg.C, 0.0008%                 6
                                                                       Na2S2O3 5.
    7 Enterococci......................  PP, G                        Cool, 10  deg.C, 0.0008%                 6
                                                                       Na2S2O3 5.

[[Page 45829]]

 
Table IA--Protozoa Tests:
    8 Cryptosporidium..................  LDPE                         0-8  deg.C................         \17\ 72
    9 Giardia..........................  LDPE                         0-8  deg.C................         \17\ 72
Table IA--Aquatic Toxicity Tests:
    10-13 Toxicity, acute and chronic..  P, G                         Cool, 4  deg.C \16\.......              36
 
*                  *                  *                  *                  *                  *
                                                        *
----------------------------------------------------------------------------------------------------------------
\1\ Polyethylene (P) or glass (G). For bacteria, plastic sample containers must be made of sterilizable
  materials (polypropylene [PP] or other autoclavable plastic). For protozoa, plastic sample containers must be
  made of low-density polyethylene (LDPE).
\2\ Sample preservation should be performed immediately upon sample collection. For composite chemical samples,
  each aliquot should be preserved at the time of collection. When use of an automated sampler makes it
  impossible to preserve each aliquot, then chemical samples may be preserved by maintaining at 4  deg.C until
  compositing and sample splitting is completed.
\3\ When any sample is to be shipped by common carrier or sent through the United States Mails, it must comply
  with the Department of Transportation Hazardous Materials Regulations (49 CFR part 172). The person offering
  such material for transportation is responsible for ensuring such compliance. For the preservation
  requirements of Table II, the Office of Hazardous Materials, Transportation Bureau, Department of
  Transportation, has determined that the Hazardous Materials Regulations do not apply to the following
  materials: Hydrochloric acid (HCl) in water solutions at concentrations of 0.04% by weight or less (pH about
  1.96 or greater); Nitric acid (HNO3) in water solutions of 0.15% by weight or less (pH about 1.62 or greater);
  Sulfuric acid (H2SO4) in water solutions of concentrations of 0.35% by weight or less (pH about 1.15 or
  greater); and Sodium hydroxide (NaOH) in water solutions at concentrations of 0.080% by weight or less (pH
  about 12.30 or less).
\4\ Samples should be analyzed as soon as possible after collection. The times listed are the maximum times that
  samples may be held before analyses and still be considered valid. Samples may be held for longer periods only
  if the permittee, or monitoring laboratory, has data on file to show that for the specific types of samples
  under study, the analytes are stable for the longer time, and has received a variance from the Regional
  Administrator under Sec.  136.3(e). Some samples may not be stable for the maximum time period given in the
  table. A permittee or monitoring laboratory is obligated to hold the samples for a shorter time if knowledge
  exists to show that this is necessary to maintain sample stability. See Sec.  136.3(e) for details. The term
  ``analyze immediately'' usually means within 15 minutes or less of sample collection.
\5\ Should only be used in the presence of residual chlorine.
*                  *                  *                  *                  *                  *
   *
\16\ Sufficient ice should be placed with the samples in the shipping container to ensure that ice is still
  present when samples arrive at the laboratory. However, even if ice is present when the samples arrive, it is
  necessary to immediately measure the temperature of the samples and confirm that the 4  deg.C temperature
  maximum has not been exceeded. In the isolated cases where it can be documented that this holding temperature
  can not be met, the permittee can be given the option of on-site testing or can request a variance. The
  request for a variance should include supportive data which show that the toxicity of the effluent samples is
  not reduced because of the increased holding temperature.
\17\ Holding time is calculated from time of sample collection to the completion of centrifugation.

* * * * *
[FR Doc. 01-21813 Filed 8-29-01; 8:45 am]
BILLING CODE 6560-50-P