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

Note: EPA no longer updates this information, but it may be useful as a reference or resource.

[Federal Register: July 21, 2003 (Volume 68, Number 139)]
[Rules and Regulations]
[Page 43271-43283]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr21jy03-27]

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ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 136
[FRL-7529-7]
RIN 2040-AD71

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

AGENCY: Environmental Protection Agency (EPA).
ACTION: Final rule.

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SUMMARY: By today's action, EPA approves test methods for the analysis
of Escherichia coli (E. coli), enterococci, Cryptosporidium and Giardia
in fresh ambient water matrices. In addition, EPA approves test methods
for the analysis of enterococci in marine ambient water matrices. The
test methods approved in today's rule have been published by the
following organizations: EPA, American Public Health Association,
American Water Works Association, Water Environment Federation,
Association of Official Analytical Chemists International, and American
Society for Testing and Materials, or commercial vendors. EPA's
approval of these methods will help States, Tribes, communities, and
environmental laboratories better assess public health risks from
microbiological pollutants.

DATES: This regulation is effective August 20, 2003. The incorporation
by reference of these methods is approved by the Director of the
Federal Register on August 20, 2003. For judicial review purposes, this
final rule is promulgated as of 1 p.m. (Eastern time) on August 4, 2003
as provided at 40 CFR 23.2.

FOR FURTHER INFORMATION CONTACT: Robin K. Oshiro, Engineering and
Analysis Division (4303T), 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) 566-1075
or E-mail at oshiro.robin@epa.gov.

SUPPLEMENTARY INFORMATION:

A. Potentially Regulated Entities

EPA Regions, as well as States, Tribes, and Territories authorized
to implement the National Pollutant Discharge Elimination System
(NPDES) program, issue permits to implement the technology-based and
water quality-based requirements of the Clean Water Act (CWA). Forty
five States and one Territory are currently authorized to issue NPDES
permits. EPA retains permit issuance authority in non-authorized
jurisdictions. NPDES permitting authorities make a number of
discretionary choices associated with permit writing, including the
selection of pollutants to be measured and, in many cases, limited in
permits. If EPA has ``approved'' (i.e., promulgated through rulemaking)
standardized testing procedures for a given pollutant, the NPDES
permitting authority must specify one of the approved testing
procedures or an approved alternate test procedure for the measurements
required under the permit. Although EPA is including test methods for
four biological pollutants in 40 CFR 136.3, it recommends their use for
ambient water quality monitoring only. EPA is not approving these test
methods for effluent matrices. Therefore, EPA expects entities
operating under an NPDES permit would be affected by the promulgation
of these ambient methods only where their permit specifies ambient
monitoring requirements for the specified parameters.
EPA developed and recommended ambient recreational water quality
criteria for E. coli and enterococci 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. If the NPDES permitting authority
requires ambient water monitoring in the permit for the specified
parameters, dischargers could be affected by the standardization of
testing procedures in this rulemaking. Generally, the permitting
authority requires the use of methods approved at 40 CFR part 136 for
compliance with such monitoring requirements. If no approved methods
are available at 40 CFR part 136, then the permitting authority has
discretion to specify the use of suitable methods.
In addition, when a State, Territory, or authorized Tribe provides
certification of Federal licenses under the CWA section 401, approved
testing procedures generally must be used where applicable. Categories
and entities that may be regulated include:

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Examples of potentially regulated
Category entities
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State, Territorial and Indian States, Territories, and Tribes
Tribal Governments. authorized to administer the NPDES
permitting program.
Municipalities.................... Publicly-owned treatment works with
ambient monitoring requirements for
the specified parameters in their
NPDES permits.
Industry.......................... Industrial facilities with ambient
monitoring requirements for the
specified parameters in their NPDES
permits.
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This table is not intended to be exhaustive, but rather provides a
guide for readers regarding entities likely to be regulated by this
action. This table lists the types of entities that EPA is now aware
could potentially be regulated by this action. Other types of entities
not listed in the table could also be regulated. To determine whether
your facility or organization is regulated by this action, you should
carefully examine the applicability criteria in parts 122 and 136 of
title 40 of the Code of Federal Regulations. If you have questions
regarding the applicability of this action to a particular entity,
consult the person listed in the preceding FOR FURTHER INFORMATION
CONTACT section.

B. How Can I Get Copies of This Document and Other Related Information?

1. Docket. EPA has established an official public docket for this
action under Docket ID No. OW-2002-0010. The official public docket
consists of the documents specifically referenced in this action, any
public comments received, and other information related to this action.
Although a part of the official docket, the public docket does not
include Confidential Business Information (CBI) or other information
whose disclosure is restricted by statute. The official public docket
is the collection of materials that is available for public viewing at
the Water Docket in the EPA Docket Center, EPA West, Room B102, 1301
Constitution Avenue, NW., Washington, DC. The EPA Docket Center Public
Reading Room is open from 8:30 a.m. to 4:30 p.m. Eastern Time, Monday
through Friday, excluding legal holidays. The telephone number for the
Public Reading Room is

[[Page 43273]]

202-566-1744, and the telephone number for the Water Docket is 202-566-
2426.
2. Electronic Access. You may access this Federal Register document
electronically through the EPA Internet under the Federal Register
listings at http://www.epa.gov/fedrgstr/.
An electronic version of the public docket is available through
EPA's electronic public docket and comment system, EPA Dockets. You may
use EPA Dockets at http://www.epa.gov/edocket/ to view public comments,
access the index listing of the contents of the official public docket,
and to access those documents in the public docket that are available
electronically. Once in the system, select ``search,'' then key in the
appropriate docket identification number. Although not all docket
materials may be available electronically, you may still access any of
the publicly available docket materials through the docket facility
identified in section B.1.
3. Copies of Consensus Standards. Copies of the consensus standards
may be obtained from the Docket (see section B.1.). Copies of the
consensus standards may also be obtained from the following sources,
depending on the standard. Copies of final methods published by
American Society for Testing and Materials (ASTM) are available for a
nominal cost through ASTM International, 100 Barr Harbor Drive, West
Conshohocken, PA 19428-2959. Copies of ``Standard Methods'' are
available for a nominal cost from the American Public Health
Association, 1015 Fifteenth Street, NW., Washington, DC 20005. Copies
of Association of Official Analytical Chemists International (AOAC)
methods are available for a nominal cost from the Association of
Official Analytical Chemists International, 481 N. Frederick Ave.,
Suite 500, Gaithersburg, MD 28077.

I. Statutory Authority

Today's rule is promulgated pursuant to the authority of sections
303(c), 304(a), 304(h), and 501(a) of the Clean Water Act (CWA or ``the
Act''), 33 U.S.C. 1314(a), 1314(h), 1361(a). 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 develop and publish water quality criteria
associated with specific ambient water uses. When these criteria are
adopted as State water quality standards under section 303(c), 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 his functions under this
Act.'' EPA publishes CWA analytical method regulations at 40 CFR part
136.

II. Background

A. The Role of Methods for Biological Pollutants

To fulfill the CWA's mandate to maintain ``fishable and swimmable''
waters, EPA develops 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.
Ambient water quality criteria become enforceable water quality
standards when adopted by State, Territorial, Tribal, and local
governments and approved by EPA.
For bacterial pollution in ambient water designated for
recreational use, EPA has developed water quality criteria for E. coli
in freshwater and for enterococci in both 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
ambient 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 are direct indicators 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 are included
in many water quality standards as indicators of bacterial
contamination (EPA, 1976). More recent epidemiological studies (Cabelli
1983, Dufour 1984) described in Ambient Water Quality Criteria for
Bacteria--1986 (EPA, 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. Thus,
using these indicators as part of the bacterial water quality standards
will enhance the protection of human health and the environment.
In addition to bacterial pollution, EPA is concerned about
waterborne parasites and developed test methods for Cryptosporidium and
Giardia in freshwater. These waterborne parasites have been found to be
the causative agent of human gastroenteritis in some contaminated
waters and are responsible for cases of severe and widespread human
illness when present in drinking water supplies as a result of
contamination of source waters. Because one of the designated uses of
some ambient waters may be use of the water body as a drinking water
source, EPA may develop ambient water quality criteria for
Cryptosporidium and Giardia in the future. EPA would expect to use the
test methods discussed in this action to support these future criteria.
By doing so, EPA desires to promote consistency in the methods used for
these future criteria to ensure that the data collected are of good
quality and are comparable for all freshwater. EPA also wishes to make
these methods available for use by the States for general risk
assessments.
By today's action, EPA is promulgating test methods for E. coli,
enterococci, Cryptosporidium, and Giardia for use in freshwaters, and
enterococci for use in marine waters. Promulgation of the bacterial
methods supports the use of E. coli and enterococci as indicators of
fecal contamination in addition to fecal coliform indicators in State,
Territorial, Tribal, and local water quality-based monitoring. States
may use the test methods for Cryptosporidium and Giardia for different
monitoring purposes, such as evaluating surface water occurrence of
these organisms and the associated watershed vulnerability for
waterbodies designated as potential drinking water sources.
This rule provides uniform methodology to assist 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. Today's rule supports several EPA initiatives: The
Beaches Environmental Assessment Closure and Health (BEACH) Program,
the Beach Action Plan (EPA-600-R-98-079), the Beach Watch Program, the

[[Page 43274]]

Beaches Environmental Monitoring for Public Access and Community
Tracking (EMPACT) Program (EPA 905-R-98-002), and the Water Quality
Criteria and Standards Plan (EPA-822-R-98-003). Additionally, this rule
is expected to satisfy requests from governments, regulated entities,
and environmental laboratories that EPA publish analytical test
procedures that were evaluated through interlaboratory validation for
enumerating E. coli, enterococci, Cryptosporidium, and Giardia in
ambient waters.
As previously noted, EPA developed water quality criteria for
enterococci in both freshwater and in marine waters. Today's action
approves methods for measuring enterococci in both freshwater and
marine waters. EPA has not developed marine criteria for E. coli,
Cryptosporidium, and Giardia because these pollutants do not generally
survive in marine conditions. Thus, EPA has not identified any
programmatic need to promulgate methods for these pollutants in marine
waters.
EPA is aware of the importance of having methods for measuring
these pollutants in wastewater effluent. The Agency does not currently
have validated methods for use in this matrix and thus was unable to
propose any such methods with the methods for ambient waters. The
Agency is currently in the process of trying to validate E. coli and
enterococci methods for use with wastewater effluent and plans to
propose them by the end of 2004.

B. Summary of Proposed Rule

EPA published a proposed rule in the Federal Register on August 30,
2001 (66 FR 45811) to amend 40 CFR part 136, ``Guidelines Establishing
Test Procedures for the Analysis of Pollutants,'' by approving several
analytical test procedures for enumerating the bacteria Escherichia
coli (E. coli) and enterococci and the protozoans Cryptosporidium and
Giardia in ambient water. The proposal described a suite of Most
Probable Number (MPN) (i.e., multiple-tube, multiple-well) and membrane
filter (MF) methods for enumerating E. coli and enterococci bacteria in
ambient water, and improved filtration/immunomagnetic separation/
fluorescent antibody methods for Cryptosporidium and Giardia
protozoans. These test methods were proposed for use by States,
Territories, and Tribes, for use in water quality monitoring programs.
A summary of the major comments to the proposal is presented in
Section V.

III. Summary of Final Rule

EPA is approving the use of test methods for E. coli, enterococci,
Cryptosporidium, and Giardia for ambient fresh water quality
monitoring. In addition, EPA is approving the use of test methods for
enterococci for ambient marine water quality monitoring. 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 matrices other than ambient waters.
Today's action promulgates the test methods described in the
proposed rule (66 FR 45811, August 30, 2001) for the analysis of E.
coli, enterococci, Cryptosporidium, and Giardia in ambient water. For
E. coli, approved methods include most probable number methods
(LTB→EC-MUG, ONPG-MUG) and membrane filtration methods
(mENDO→NA-MUG, LES-ENDO→NA-MUG, mFC→NA-MUG, mTEC agar,
Modified mTEC agar, MI agar, m-ColiBlue 24 broth). For enterococci,
approved methods include most probable number methods (Azide-Dextrose/
PSE/BHI, MUG) and membrane filtration methods (mE→EIA agar, mEI
agar). For Cryptosporidium, EPA approves Methods 1622 and 1623. For
Giardia, EPA approves Method 1623.
The proposed rule indicated that EPA intended to issue guidance on
the assessment of method comparability in conjunction with the final
rule. In the record for today's rule, EPA is making available the
latest version of the guidance document, EPA Microbiological Alternate
Test Procedure (ATP) Protocol for Drinking Water, Ambient Water, and
Wastewater Monitoring Methods, Guidance (EPA-821-B-03-004). The
guidance is a result of the Agency's desire to develop a guidance
document to describe the process for seeking EPA approval of alternate
test procedures (ATPs) for microbiological methods or new
microbiological methods for use in monitoring drinking water, ambient
water, and wastewater. Under EPA's ATP program, any person may apply
for approval of the use of an ATP or new method to test for a regulated
analyte. EPA anticipates that the standardized ATP procedures described
in the guidance should generally expedite the approval of ATPs and
encourage the development of innovative methods for compliance
monitoring under the National Pollution Discharge Elimination System
(NPDES) permit program. In addition to the ATP process, the guidance
describes the process for conducting side-by-side method comparisons
and for conducting quality control (QC) acceptance criteria-based
method studies for EPA-designated reference methods with QC acceptance
criteria. The guidance document serves as a supplement to the ATP
program requirements specified at 40 CFR 136.4, 136.5, and 141.27. The
guidance document may be revised in the future based on comments
received from persons using the guidance, as appropriate.

IV. Changes From the Proposed Rule

A. Revision of Method Titles

To ensure consistency with other EPA microbiological methods, EPA
revised some of the EPA methods' titles and added some method numbers.
The technical content of these methods did not change from the versions
of the methods included in the proposed rule. Specifically, EPA adopted
the following modified titles:
? Method 1103.1: Escherichia coli (E. coli) in Water by
Membrane Filtration using membrane-Thermotolerant Escherichia coli Agar
(mTEC)
? Method 1106.1: Enterococci in Water by Membrane Filtration
using membrane-Enterococcus-Esculin Iron Agar (mE-EIA)
? Method 1600: Enterococci in Water by Membrane Filtration
using membrane-Enterococcus Iron Agar (mEI)
? Method 1603: Escherichia coli (E. coli) in Water by
Membrane Filtration using Modified Membrane-Thermotolerant Escherichia
coli Agar (Modified mTEC)
? Method 1604: Total Coliforms and Escherichia coli (E. coli)
in Water by Membrane Filtration using a Simultaneous Detection
Technique (MI Medium)

B. Colisure

EPA included this method in the proposal because it anticipated
that new validation data for ambient waters would be provided to the
Agency prior to this final rule. EPA requested such data from the
manufacturer, but the manufacturer declined to conduct the study.
Therefore EPA declines to approve this method and did not include it in
today's final rule.

C. Table II Protozoan Test Holding Time

The proposal incorrectly indicated that the maximum sample holding
time for the protozoan tests (Cryptosporidium and Giardia) was 72
hours. This has been changed to the correct holding time of 96 hours,
as indicated in the Methods, which were included in the docket for the
proposal. The correct

[[Page 43275]]

holding time of 96 hours is clearly indicated in the Methods and can be
found on page 10, section 8.2.1 of the April 2001 versions of Method
1622 and Method 1623.
Although footnote 17 of the proposal inaccurately stated the
technique for calculating holding time, the underlying methods
themselves described this technique correctly. The footnote has been
corrected to indicate that holding time is properly calculated from the
time of sample collection to elution for samples shipped to the
laboratory in bulk and calculated from the time of sample filtration to
elution for samples filtered in the field.

V. Response to Major Comments

EPA encouraged public participation in this rulemaking and
requested comments on the methods proposed for E. coli, enterococci,
Cryptosporidium, and Giardia. EPA also requested any data that would
support comments on specific test methods. Fourteen stakeholders
provided comments addressing over 25 issues. These stakeholders
included four laboratories, seven regulatory authorities, and three
industries/industry groups.
The following sections summarize major comments received on the
proposed rule and EPA's response. The complete Response to Comments
document can be found in the Docket for today's final rule.

A. E. coli and Enterococci Methods for Wastewater Analysis

Several commenters requested that the methods for E. coli and
enterococci be approved for the analysis of wastewater samples. Since
these methods were not validated in wastewater, they are not approved
for use in that matrix. EPA is in the process of validating methods for
the analysis of E. coli and enterococci in wastewater and plans to
propose test methods for these bacterial indicators by the end of 2004.

B. Cryptosporidium and Giardia Methods for Wastewater and Biosolids
Analysis

Several comments advocated the use of EPA Method 1622 and 1623 for
the analysis of wastewater and biosolids samples; other comments
requested that EPA modify and approve the methods for use in those
matrices. EPA has not validated these methods for those uses. Thus this
final rule applies only to ambient water. If EPA develops water quality
criteria for Cryptosporidium and Giardia at a future time, EPA may
validate EPA Methods 1622 and/or 1623 for use in the NPDES Program.

C. Limitations of Determinative Technique of Proposed Cryptosporidium
and Giardia Methods and Potential for False Positives

Several comments expressed concern regarding the subjectivity and
limitations of the immunofluorescence assay (IFA)-based determination
procedure in EPA Methods 1622 and 1623 and the related potential for
false positives. EPA acknowledges that IFA relies on analyst training
and experience for reliable results. However, EPA Methods 1622 and 1623
provide the analyst with three microscopy tools to aid in the
identification of potential target particulates during microscopic
examination. The methods provide detailed, progressive criteria for
determining whether a particulate is a Cryptosporidium oocyst or a
Giardia cyst based on the use of these tools and include the use of
immunomagnetic separation (IMS) as the sample cleanup procedure to
minimize the transfer of non-target particulates to the slide.
Nonetheless, the inherent technical judgement involved in the
determinative step in EPA Methods 1622 and 1623, combined in some cases
with interfering materials and/or cross-reactivity of the antibody
stain, may still lead to false positives or false negatives. Although
other determinative techniques that are currently under development
have the promise of providing less-subjective assessments of the
presence of Cryptosporidium oocysts and Giardia cysts in a sample,
these techniques are not yet validated and are therefore not yet
appropriate for EPA approval for ambient water monitoring. Extensive
details on the performance of EPA Methods 1622 and 1623, including
inter- and intra-laboratory precision and recovery of the methods at
multiple laboratories and on a variety of ambient water types (i.e.,
validation), are provided in the Results of the Interlaboratory
Validation Study of EPA Method 1622 (EPA-821-R-01-027), the Results of
the Interlaboratory Validation Study of EPA Method 1623 (EPA-821-R-01-
028) and the Implementation and Results of the Information Collection
Rule Supplemental Surveys (EPA-815-R-01-003), which were included in
the docket for the proposal. Given the robustness of the validation
procedure, the Agency is confident that although the IFA technique
requires specialized training, overall, the methods will provide for
valid Cryptosporidium and Giardia precision and recovery for use in
ambient waters.

D. Application of Performance-Based Measurement System (PBMS) Concept
to EPA Methods 1622 and 1623

Several commenters recommended that the performance of alternate
antibody reagents be evaluated for EPA Methods 1622 and 1623 using a
quantitative PBMS approach. EPA agrees with the comments, and considers
the PBMS Tier 2 validation approach described in Methods 1622 and 1623,
Section 9, to be appropriate for antibody stains and IMS. However, EPA
does not believe that the PBMS Tier 2 validation approach is adequate
to assess the comparability of methods with different determinative
techniques, such as comparing a polymerase chain reaction (PCR)-based
method to an IFA-based method. Use of a different determinative
technique is generally considered to be a different method, rather than
a modified version of a method because it is usually very difficult to
compare methods that use different determinative techniques. For
example, the filtration/IMS/IFA technique employed in Methods 1622 and
1623 differs considerably from genetic tests because the former
measures the infective form of Cryptosporidium and Giardia, while the
latter measures genetic material (DNA or RNA). Similarly, the membrane
filtration method for bacteria differs from an MPN method for bacteria
because the former is a direct quantitative method, whereas the latter
employs a qualitative statistical index rather than an actual
enumeration of the number of organisms present in the sample. An
appropriate approach for these comparisons would be to perform side-by-
side tests. This approach is outlined in the draft guidance document,
EPA Microbiological Alternate Test Procedure (ATP) Protocol for
Drinking Water, Ambient Water, and Wastewater Monitoring Methods,
Guidance (EPA-821-B-03-004).

VI. Statutory and Executive Order Reviews

A. Executive Order 12866: Regulatory Planning and Review

Under Executive Order 12866 (58 FR 51735 (October 4, 1993)), the
Agency must determine whether the 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

[[Page 43276]]

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; or
(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. 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 action promulgates new test methods for E. coli, enterococci,
Cryptosporidium, and Giardia for use in ambient water monitoring
programs. If the regulating authority replaces the indicator organism
from fecal coliforms to one of the bacterial organisms (E. coli or
enterococci) and the relevant NPDES permit requires ambient water
monitoring, then the permittee would be required to use one of these
approved methods for these organisms. Currently, permittees generally
are not required to monitor for Cryptosporidium or Giardia because EPA
has not developed water quality criteria for these protozoans. Burden
means that 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 purpose 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.

C. Regulatory Flexibility Act

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 U.S. Small Business Administration definitions at 13 CFR
121.201; (2) a small governmental jurisdiction that is a government of
a city, county, town, school district or special district with a
population of less than 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 final rule on
small entities, I certify that this action will not have a significant
economic impact on a substantial number of small entities. This
regulation promulgates 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 some
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 however, 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, 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 approved 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 waters. Thus, some NPDES permittees are already
testing ambient water for these parameters. The impact of using EPA
approved methods for such dischargers may represent little or no
increased burden since, for these permittees, the replacement of fecal
coliforms with E. coli or enterococci would simply require different
methods.
The small entities that might be affected by this rule include
small governmental jurisdictions that have publicly-owned treatment
works (POTWs) and small businesses with water quality-based discharge
permits. The average costs for total and fecal coliform were comparable
to those for E. coli and enterococci ($35) because the analytical
procedures generally employ similar techniques, media, equipment, and
require comparable laboratory time and effort. Some States are already
using the methods for E. coli and enterococci in State ambient water
quality monitoring programs. 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
promulgated methods are broadly implemented by environmental
laboratories and State water quality monitoring programs.
EPA also reviewed the costs for testing for Cryptosporidium and
Giardia. The costs for Methods 1622 and 1623 analysis of
Cryptosporidium and Giardia range from $400 to $500 for each sample
(with matrix spikes being assessed as individual samples) for each
method. Because of the relatively high costs, EPA does not anticipate
that these test methods will be used for daily or ongoing monitoring,
but they may be used for program-specific occurrence assessments.

[[Page 43277]]

The purpose of this rule is only to make these methods available to
States, Tribes, and municipalities that may want to use them for
ambient water monitoring. The costs associated with Cryptosporidium and
Giardia analysis would not be a Federally-mandated cost, but rather
would emanate from a State's adoption of ambient monitoring
requirements or other identified needs such as evaluation of Best
Management Practices (BMPs) or downstream impacts of wastewater
treatment plant effluents or other identified needs. The inclusion of
these test methods in 40 CFR 136.3 is intended to make these test
methods available to States and others for use in water quality
monitoring programs. While monitoring for these protozoans may be
beneficial since these organisms may be ingested from recreational and
source waters, EPA is not establishing any compliance monitoring
requirements for these pollutants. Therefore, EPA believes that this
rule will not have a significant economic impact on a substantial
number of small entities.

D. Unfunded Mandates Reform Act

Title II of the Unfunded Mandates Reform Act of 1995 (UMRA), Public
Law 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.
EPA has determined that this rule does not contain a Federal
mandate for State, Tribal, and local governments or the private sector
that may result in expenditures of $100 million or more for State,
Tribal, and local governments, in the aggregate, or the private sector
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 above, 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 also is not subject to the requirements of section 203 of the
UMRA.

E. 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 final 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. Today's rule promulgates new
analytical methods for conducting analysis of ambient water for
enumeration of E. coli, enterococci, Cryptosporidium, or Giardia. EPA
does not, however, require use of these methods under this rule. Thus,
Executive Order 13132 does not apply to this rule.
Although 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 40 CFR 136.3 because they want to use EPA
approved test methods for ambient water monitoring. EPA included a
number of test methods currently being used by States for these
pollutants in today's rulemaking. In the spirit of Executive Order
13132, and consistent with EPA policy to promote communications between
EPA and State and local governments, EPA specifically solicited comment
on the proposed rule from State and local officials. No significant
concerns were raised by commenters about these methods.

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

Executive Order 13175, entitled ``Consultation and Coordination
with Indian Tribal Governments'' (59 FR 22951, November 9, 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 the Indian tribes.''
This final rule does not have tribal implications. It will not have
substantial direct effects on tribal 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.
Today's rule promulgates new analytical methods for conducting analysis
of

[[Page 43278]]

ambient water for enumeration of E. coli, enterococci, Cryptosporidium,
or Giardia. EPA does not, however, require use of these methods under
this rule. Thus, Executive Order 13175 does not apply to this rule.
Moreover, in the spirit of Executive Order 13175, and consistent with
EPA policy to promote communications between EPA and tribal
governments, EPA specifically solicited comment on the proposed rule
from tribal officials. EPA did not receive comments from Tribal
officials. Thus, Executive Order 13175 does not apply to this rule.

G. 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 rule is
not subject to the Executive Order because it is not ``economically
significant'' as defined in Executive Order 12866. Further, it does not
concern an environmental health or safety risk that EPA has reason to
believe may have a disproportionate effect on children.

H. Executive Order 13211: Actions That Significantly Affect Energy
Supply, Distribution, or Use

This rule is not subject to Executive Order 13211, ``Actions
Concerning Regulations That Significantly Affect Energy Supply,
Distribution, or Use'' (66 FR 28355 (May 22, 2001)) because it is not a
significant regulatory action under Executive Order 12866.

I. National Technology Transfer and Advancement Act

As noted in the proposed rule, section 12(d) of the National
Technology Transfer and Advancement Act of 1995, (``NTTAA''), 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, and business
practices) that are developed or adopted by voluntary consensus
standards bodies. The NTTAA directs EPA to provide Congress, through
the Office of Management and Budget (OMB), explanations when the Agency
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-International are included for
promulgation and are listed in Table IA at the end of this document
(see footnotes 4, 10, and 11, respectively, for the complete
citations). No voluntary consensus standards were found for
Cryptosporidium or Giardia.

J. Congressional Review Act

The Congressional Review Act, 5 U.S.C. 801 et seq., as added by the
Small Business Regulatory Enforcement Fairness Act of 1996 (SBREFA),
generally provides that before a rule may take effect, the agency
promulgating the rule must submit a rule report, which includes a copy
of the rule, to each House of the Congress and to the Comptroller
General of the United States. EPA will submit a report containing this
rule and other required information to the U.S. Senate, the U.S. House
of Representatives, and the Comptroller General of the United States
prior to publication of the rule in the Federal Register. A major rule
cannot take effect until 60 days after it is published in the Federal
Register. This action is not a ``major rule'' as defined by 5 U.S.C.
804(2). This rule will be effective on August 20, 2003.

List of Subjects in 40 CFR Part 136

Environmental protection, Incorporation by reference, Reporting and
recordkeeping requirements, Water pollution control.

Dated: July 11, 2003.
Linda J. Fisher,
Acting Administrator.

? For the reasons set out in the preamble, title 40, chapter I of the
Code of Federal Regulations, is 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. In paragraph (b) by revising references (10), (34), (38) and (39)
and adding references (52) through (62).
? c. In Table II to paragraph (e) by revising entries to the Section
labeled ``Table IA--Bacteria Tests,'' to read as follows:
* * * * *
(a) * * *

Table IA.--List of Approved Biological Methods
--------------------------------------------------------------------------------------------------------------------------------------------------------
Standard methods 18th,
Parameter and units Method \1\ EPA 19th, 20th Ed. ASTM AOAC USGS Other
--------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria:
1. Coliform (fecal), number Most Probable Number p. 132 9221C E \4\
per 100 mL. (MPN), 5 tube 3 \3\
dilution, or
Membrane filter (MF) p. 124 9222D \4\ ........... ........... B-0050-85
\2\, single step. \3\ \5\

[[Page 43279]]

2. Coliform (fecal) in MPN, 5 tube, 3 p. 132 9221C E \4\ ........... ........... ...........
presence of chlorine, number dilution, or \3\
per 100 mL.
MF, single step \6\. p. 124 9222D \4\ ........... ........... ........... ....................
\3\
3. Coliform (total), number MPN, 5 tube, 3 p. 114 9221B \4\
per 100 mL. dilution, or \3\
MF \2\, single step p. 108 9222B \4\ ........... ........... B-0025-85 ....................
or two step. \3\ \5\
4. Coliform (total), in MPN, 5 tube, 3 p. 114 9221B \4\ ........... ........... ........... ....................
presence of chlorine, number dilution, or \3\
per 100 mL.
MF \2\ with p. 111 9222(B+B.5c) \4\
enrichment. \3\
5. E. coli, number per 100 mL MPN \7,9,15\, ......... 9221B.1/9221F \4,12,14\
\28\. multiple tube,.
multiple tube/ ......... 9223B \4,13\ ........... 991.15 \11\ ........... Colilert[reg]
multiple well, \13,17\
Colilert-18[reg]
\13,16,17\
MF \2,6,7,8,9\ two ......... 9222B/9222G \4,19\
step, or
1103.1 9213D \4\ D5392-93
\20\ \10\
single step......... 1603 \21\
1604 \22\
......... ....................... ........... ........... ........... mColiBue 24 \18\
6. Fecal streptococci, number MPN, 5 tube, 3 p. 139 9230B \4\
per 100 mL. dilution, \3\
MF \2\, or.......... p. 136 ....................... ........... B-0055-85 ...........
\3\ \5\
Plate count......... p. 143
\4\
7. Enterococci, number per MPN \7, 9\ multiple ......... 9230B \4\
100 mL. tube.
multiple tube/ ......... ....................... D6503-99 ........... ........... Enterolert[reg]\13,2
multiple well. \10\ 3\
MF \2,6,7,8,9\ two 1106.1 9230C \4\ D5259-92
step. \24\ \10\
single step, or..... 1600 \25\
Plate count......... p. 143
\3\
Protozoa:
8. Cryptosporidium \28\...... Filtration/IMS/FA... 1622 \26\
1623 \27\
9. Giardia \28\.............. Filtration/IMS/FA... 1623 \27\
Aquatic Toxicity:
10. Toxicity, acute, fresh Ceriodaphnia dubia 2002.0
water organisms, LC50, acute. \29\
percent effluent.
Daphnia puplex and 2021.0
Daphnia magna acute. \29\

[[Page 43280]]

Fathead Minnow, 2000.0
Pimephales \29\
promelas, and
Bannerfin shiner,
Cyprinella leedsi,
acute.
Rainbow Trout, 2019.0
Oncorhynchus \29\
mykiss, and brook
trout, Salvelinus
fontinalis, acute.
11. Toxicity, acute, Mysid, Mysidopsis 2007.0
estuarine and marine bahia, acute. \29\
organisms of the Atlantic
Ocean and Gulf of Mexico,
LC50, percent effluent.
Sheepshead Minnow, 2004.0
Cyprinodon \29\
variegatus, acute.
Silverside, Menidia 2006.0
beryllina, Menidia \29\
menidia, and
Menidia peninsulae,
acute.
12. Toxicity, chronic, fresh Fathead minnow, 1000.0
water organisms, NOEC or Pimephales \30\
IC25, percent effluent. promelas, larval
survival and growth.
Fathead minnow, 1001.0
Pimephales \30\
promelas, embryo-
larval survival and
teratogenicity.
Daphnia, 1002.0
Ceriodaphnia dubia, \30\
survival and
reproduction.
Green alga, 1003.0
Selenastrum \30\
capricornutum,
growth.
13. Toxicity, chronic, Sheepshead minnow, 1004.0
estuarine and marine Cyprinodon \31\
organisms of the Atlantic variegatus, larval
Ocean and Gulf of Mexico, survival and growth.
NOEC or IC25, percent
effluent.

[[Page 43281]]

Sheepshead minnow, 1005.0
Cyprinodon \31\
variegatus, embryo-
larval survival and
teratogenicity.
Inland silverside, 1006.0
Menidia beryllina, \31\
larval survival and
growth.
Mysid, Mysidopsis 1007.0
bahia, survival, \31\
growth, and
fecundity.
Sea urchin, Arbacia 1008.0
punctulata, \31\
fertilization.
--------------------------------------------------------------------------------------------------------------------------------------------------------
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, D.C.
\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, 1996. Annual Book of ASTM Standards--Water and Environmental Technology. Section 11.02. American Society for Testing and
Materials. 100 Barr Harbor Drive, West Conshohocken, PA 19428.
\11\ AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. Association of Official Analytical Chemists
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, for example, a substrate is used to detect the enzyme
[beta]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 [mu]g/mL of MUG may be used.
\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[reg]
may be enumerated with the
multiple-well procedures, Quanti-Tray[reg]
or Quanti-Tray[reg]
2000, and the MPN calculated from the table provided by the manufacturer.
\16\ Colilert-18 [reg]
is an optimized formulation of the Colilert[reg]
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[reg]
test and is recommended for marine water samples.
\17\ Descriptions of the Colilert[reg], Colilert-18[reg], Quanti-Tray[reg], and Quanti-Tray[reg]/2000 may be obtained from IDEXX Laboratories, Inc., One
IDEXX Drive, Westbrook, Maine 04092.
\18\ A description of the mColiBlue24'' test, Total Coliforms and E. coli, is available from Hach Company, 100 Dayton Ave., Ames, IA 50010.
\19\ Subject total coliform positive samples determined by 9222B or other membrane filter procedure to 9222G using NA-MUG media.
\20\ USEPA. 2002. Method 1103.1: Escherichia coli (E. coli) In Water By Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC).
U.S. Environmental Protection Agency, Office of Water, Washington D.C. EPA-821-R-02-020.
\21\ USEPA. 2002. Method 1603: Escherichia coli (E. coli) In Water By Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (
modified mTEC). U.S. Environmental Protection Agency, Office of Water, Washington D.C. EPA-821-R-02-023.
\22\ 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 in USEPA. 2002. Method 1604:
Total Coliforms and Escherichia coli (E. coli) in Water by Membrane Filtration by Using a Simultaneous Detection Technique (MI Medium). U.S.
Environmental Protection Agency, Office of Water, Washington DC. EPA 821-R-02-024.
\23\ A description of the Enterolert [reg]
test may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092.
\24\ USEPA. 2002. Method 1106.1: Enterococci In Water By Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA). U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA-821-R-02-021.
\25\ USEPA. 2002. Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-[beta]-D-Glucoside Agar (mEI). U.S.
Environmental Protection Agency, Office of Water, Washington, DC. EPA-821-R-02-022.

[[Page 43282]]

\26\ 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.
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.
\27\ 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. 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.
\28\ Recommended for enumeration of target organism in ambient water only.
\29\ USEPA. October 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition.
U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA/821/R-02/012.
\30\ USEPA. October 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth
Edition. U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA/821/R-02/013.
\31\ USEPA. October 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms.
Third Edition. U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA/821/R-02/014.

* * * * *
(b) * * *
REFERENCES, SOURCES, COSTS, AND TABLE CITATIONS
* * * * *
(10) Annual Book of ASTM Standards, Water, and Environmental
Technology, Section 11, Volumes 11.01 and 11.02, 1994, 1996, 1999, and
Volume 11.02, 2000 in 40 CFR 136.3, Tables IA, IB, IC, ID, and IE.
* * * * *
(34) USEPA. October 2002. Methods for Measuring the Acute Toxicity
of Effluents and Receiving Waters to Freshwater and Marine Organisms.
Fifth Edition. U.S. Environmental Protection Agency, Office of Water,
Washington, DC EPA 821-R-02-012. Available from: National Technical
Information Service, 5285 Port Royal Road, Springfield, Virginia 22161,
Pub. No. PB2002-108488. Table IA, Note 29.
* * * * *
(38) USEPA. October 2002. Short-Term Methods for Measuring the
Chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisms. Fourth Edition. U.S. Environmental Protection Agency, Office
of Water, Washington, DC EPA-821-R-02-013. Available from: National
Technical Information Service, 5285 Port Royal Road, Springfield,
Virginia 22161, Pub. No. PB2002-108489. Table IA, Note 30.
(39) USEPA. October 2002. Short-Term Methods for Measuring the
Chronic Toxicity of Effluents and Receiving Waters to Marine and
Estuarine Organisms. Third Edition. U.S. Environmental Protection
Agency, Office of Water, Washington, DC EPA-821-R-02-014. Available
from: National Technical Information Service, 5285 Port Royal Road,
Springfield, Virginia 22161, Pub. No. PB2002-108490. Table IA, Note 31.
* * * * *
(52) IDEXX Laboratories, Inc. 2002. Description of Colilert[reg],
Colilert-18'', Quanti-Tray[reg], Quanti-Tray[reg]/2000, Enterolert[reg]
methods are available from IDEXX Laboratories, Inc., One Idexx Drive,
Westbrook, Maine 04092. Table IA, Notes 17 and 23.
(53) Hach Company, Inc. Revision 2, 1999. Description of m-
ColiBlue24[reg]
Method, Total Coliforms and E. coli, is available from
Hach Company, 100 Dayton Ave., Ames, IA 50010. Table IA, Note 18.
(54) USEPA. 2002. Method 1103.1: Escherichia coli (E. coli) in
Water by Membrane Filtration Using membrane-Thermotolerant Escherichia
coli Agar (mTEC). U.S. Environmental Protection Agency, Office of
Water, Washington D.C. September 2002, EPA-821-R-02-020. Available at
NTIS, PB2003-100125. Table IA, Note 20.
(55) USEPA. 2002. Method 1106.1: Enterococci in Water by Membrane
Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA). U.S.
Environmental Protection Agency, Office of Water, Washington D.C.
September 2002, EPA-821-R-02-021. Available at NTIS, PB2003-100126.
Table IA, Note 24.
(56) USEPA. 2002. Method 1603: Escherichia coli (E. coli) in Water
by Membrane Filtration Using Modified membrane-Thermotolerant
Escherichia coli Agar (Modified mTEC). U.S. Environmental Protection
Agency, Office of Water, Washington, DC September 2002, EPA-821-R-02-
023. Available at NTIS, PB2003-100128. Table IA, Note 21.
(57) Brenner et al. 1993. New Medium for the Simultaneous Detection
of Total Coliforms and Escherichia coli in Water. Appl. Environ.
Microbiol. 59:3534-3544. Available from the American Society for
Microbiology, 1752 N Street NW., Washington, DC 20036. Table IA, Note
22.
(58) USEPA. 2002. Method 1604: Total Coliforms and Escherichia coli
(E. coli) in Water by Membrane Filtration using a Simultaneous
Detection Technique (MI Medium). U.S. Environmental Protection Agency,
Office of Water, Washington D.C.. September 2002, EPA 821-R-02-024.
Available from NTIS, PB2003-100129. Table IA, Note 22.
(59) USEPA. 2002. Method 1600: Enterococci in Water by Membrane
Filtration using membrane-Enterococcus Indoxyl-[beta]-D-Glucoside Agar
(mEI). U.S. Environmental Protection Agency, Office of Water,
Washington D.C. September 2002, EPA-821-R-02-022. Available from NTIS,
PB2003-100127. Table IA, Note 25.
(60) USEPA. 2001. Method 1622: Cryptosporidium in Water by
Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of
Water, Washington, DC April 2001, EPA-821-R-01-026.
Available from NTIS, PB2002-108709. Table IA, Note 26.
(61) USEPA. 2001. Method 1623: Cryptosporidium and Giardia in Water
by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of
Water, Washington, DC April 2001, EPA-821-R-01-025. Available from
NTIS, PB2002-108710. Table IA, Note 27.
(62) 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, Note 11.
* * * * *
(e) * * *

[[Page 43283]]

Table II.--Required Containers, Preservation Techniques, and Holding Times
----------------------------------------------------------------------------------------------------------------
Maximum holding time
Parameter No./name Container \1\ Preservation \2\,\3\ \4\ (hours)
----------------------------------------------------------------------------------------------------------------
Table IA--Bacteria Tests:
1-5 Coliform, total, fecal, and PP, G................. Cool, <10[deg]C, 0.008% 6
E. coli. Na₂S₂O₃5.
6 Fecal streptococci........... PP, G................. Cool, <10[deg]
0.008% 6
Na₂S₂O₃5.
7 Enterocci.................... PP, G................. Cool, <10[deg]
0.008% 6
Na₂S₂O₃5.
Table IA--Protozoa Tests:
8 Cryptosporidium.............. LDPE.................. 0-8[deg]C.................. 96 \17\
9 Giardia...................... LDPE.................. 0-8[deg]C.................. 96 \17\

* * * * * * *
----------------------------------------------------------------------------------------------------------------
\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 elution for samples shipped to the laboratory
in bulk and calculated from the time of sample filtration to elution for samples filtered in the field.

* * * * *
[FR Doc. 03-18155 Filed 7-18-03; 8:45 am]
BILLING CODE 6560-50-P

Notices For
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