[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]
-----------------------------------------------------------------------
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.
-----------------------------------------------------------------------
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:
------------------------------------------------------------------------
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
California Coastal Water Research Project Authority. Westminster,
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.
USEPA. 2001b. 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.
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