[Federal Register Volume 70, Number 157 (Tuesday, August 16, 2005)]
[Proposed Rules]
[Pages 48256-48268]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 05-16195]
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Part III
Environmental Protection Agency
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40 CFR Part 136
Guidelines Establishing Test Procedures for the Analysis of Pollutants;
Analytical Methods for Biological Pollutants in Wastewater and Sewage
Sludge; Proposed Rule
Federal Register / Vol. 70, No. 157 / Tuesday, August 16, 2005 /
Proposed Rules
[[Page 48256]]
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ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 136
[OW-2004-0014; FRL-7952-7]
RIN 2040-AE68
Guidelines Establishing Test Procedures for the Analysis of
Pollutants; Analytical Methods for Biological Pollutants in Wastewater
and Sewage Sludge; Proposed Rule
AGENCY: Environmental Protection Agency (EPA).
ACTION: Proposed rule.
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SUMMARY: This proposed regulation would amend the ``Guidelines
Establishing Test Procedures for the Analysis of Pollutants'' under
section 304(h) of the Clean Water Act (CWA), by adding analytical test
procedures for enumerating the bacteria, Escherichia coli (E. coli) and
enterococci, in wastewater; and by adding analytical test procedures
for enumerating fecal coliforms and Salmonella in sewage sludge to the
list of Agency-approved methods. Specifically, EPA is proposing both
membrane filter (MF) and multiple-tube fermentation (MTF, i.e.,
multiple-tube, multiple-well) methods for E. coli and enterococci
bacteria in wastewater, and MTF methods for fecal coliforms and
Salmonella in sewage sludge. EPA's approval of these methods will help
Regions, States, communities, and environmental laboratories better
assess public health risks from microbiological pollutants.
DATES: Comments must be received on or before October 17, 2005.
ADDRESSES: Submit your comments, identified by Docket ID No. OW-2004-
0014, by one of the following methods:
I. Federal eRulemaking Portal: http://www.regulations.gov. Follow
the on-line instructions for submitting comments.
II. Agency Web site: http://www.epa.gov/edocket. EDOCKET, EPA's
electronic public docket and comment system, is EPA's preferred method
for receiving comments. Follow the on-line instructions for submitting
comments.
III. E-mail: [email protected], Attention Docket ID No. OW-
2004-0014.
IV. Mail: Water Docket, Environmental Protection Agency, Mailcode:
4101T, 1200 Pennsylvania Ave., NW., Washington, DC 20460.
V. Hand Delivery: EPA Water Center, EPA West Building, Room B102,
1301 Constitution Avenue, NW., Washington, DC, Attention Docket ID No.
OW-2004-0014. Such deliveries are only accepted during the Docket's
normal hours of operation, and special arrangements should be made for
deliveries of boxed information.
Instructions: Direct your comments to Docket ID No. OW-2004-0014.
EPA's policy is that all comments received will be included in the
public docket without change and may be made available on-line at
http://www.epa.gov/edocket, including any personal information
provided, unless the comment includes information claimed to be
Confidential Business Information (CBI) or other information whose
disclosure is restricted by statute. Do not submit information that you
consider to be CBI or otherwise protected through EDOCKET,
regulations.gov, or e-mail. The EPA EDOCKET and the Federal
regulations.gov Web sites are ``anonymous access'' systems, which means
EPA will not know your identity or contact information unless you
provide it in the body of your comment. If you send an e-mail comment
directly to EPA without going through EDOCKET or regulations.gov, your
e-mail address will be automatically captured and included as part of
the comment that is placed in the public docket and made available on
the Internet. If you submit an electronic comment, EPA recommends that
you include your name and other contact information in the body of your
comment and with any disk or CD-ROM you submit. If EPA cannot read your
comment due to technical difficulties and cannot contact you for
clarification, EPA may not be able to consider your comment. Electronic
files should avoid the use of special characters, any form of
encryption, and be free of any defects or viruses. For additional
information about EPA's public docket visit EDOCKET on-line or see the
Federal Register of May 31, 2002 (67 FR 38102).
Docket: All documents in the docket are listed in the EDOCKET index
at http://www.epa.gov/edocket. Although listed in the index, some
information is not publicly available, i.e., CBI or other information
whose disclosure is restricted by statute. Certain other material, such
as copyrighted material, is not placed on the Internet and will be
publicly available only in hard copy form. Publicly available docket
materials are available either electronically in EDOCKET or in hard
copy at the Water Docket, EPA/DC, EPA West, Room B102, 1301
Constitution Ave., NW., Washington, DC. The Public Reading Room is open
from 8:30 a.m. to 4:30 p.m., Monday through Friday, excluding legal
holidays. The telephone number for the Public Reading Room is (202)
566-1744, and the telephone number for the Water Docket is (202) 566-
2426.
FOR FURTHER INFORMATION CONTACT: Robin K. Oshiro, Office of Science and
Technology (4303-T); Office of Water, U.S. Environmental Protection
Agency, Ariel Rios Building, 1200 Pennsylvania Avenue, NW., Washington,
DC 20460, (202) 566-1075 (e-mail: [email protected]).
SUPPLEMENTARY INFORMATION:
A. Does This Action Apply to Me?
EPA Regions, as well as States, Territories and Tribes authorized
to implement the National Pollutant Discharge Elimination System
(NPDES) program, issue permits that must comply with the technology-
based and water quality-based requirements of the Clean Water Act
(CWA). In doing so, NPDES permitting authorities, including States,
Territories, and Tribes, make several discretionary choices when they
write a permit. These choices include the selection of pollutants to be
measured, monitoring requirements, permit conditions (e.g., triggers),
and, in many cases, limits in permits. EPA's NPDES regulations
(applicable to all authorized State NPDES programs) require monitoring
results to be reported at the intervals specified in the permit, but in
no case less frequently than once per year. Monitoring results must be
conducted according to test procedures approved under 40 CFR part 136
(see 40 CFR 122.41(j)(4), 122.44(i)(1)(iv) and 122.44(i)(2)).
Therefore, entities with NPDES permits may potentially be regulated by
actions proposed in this rulemaking. In addition, when an authorized
State, Territory, or Tribe certifies Federal licenses under CWA section
401, they must use the standardized analysis and sampling procedures.
Categories and entities that could potentially be regulated include:
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Examples of potentially regulated
Category entities
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Federal, State, Territorial, Federal, State, Territorial, and Tribal
and Indian Tribal entities authorized to administer the
Governments. NPDES permitting program; Federal,
State, Territorial, and Tribal entities
providing certification under Clean
Water Act section 401.
Industry..................... Facilities that must conduct monitoring
to comply with NPDES permits.
[[Page 48257]]
Municipalities............... POTWs that must conduct monitoring to
comply with NPDES permits.
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This table is not intended to be exhaustive, but rather provides a
guide for readers regarding entities likely to be regulated by this
action. This table lists types of entities that EPA is now aware could
potentially be regulated by this action. Other types of entities not
listed in the table could also be regulated. To determine whether your
facility is regulated by this action, you should carefully examine the
applicability language at 40 CFR 122.1, (NPDES purpose and scope), 40
CFR 136.1 (NPDES permits and CWA), 40 CFR 503.32 (Sewage sludge and
pathogens). If you have questions regarding the applicability of this
action to a particular entity, consult the appropriate person listed in
the preceding FOR FURTHER INFORMATION CONTACT section.
B. What Should I Consider as I Prepare My Comments for EPA?
1. Submitting CBI. Do not submit this information to EPA through
EDOCKET, regulations.gov or e-mail. Clearly mark the part or all of the
information that you claim to be CBI. For CBI information in a disk or
CD-ROM that you mail to EPA, mark the outside of the disk or CD-ROM as
CBI and then identify electronically within the disk or CD-ROM the
specific information that is claimed as CBI. In addition to one
complete version of the comment that includes information claimed as
CBI, a copy of the comment that does not contain the information
claimed as CBI must be submitted for inclusion in the public docket.
Information so marked will not be disclosed except in accordance with
procedures set forth in 40 CFR part 2.
2. Tips for Preparing Your Comments. When submitting comments,
remember to:
I. Identify the rulemaking by docket number and other identifying
information (subject heading, Federal Register date and page number).
II. Follow directions--The agency may ask you to respond to
specific questions or organize comments by referencing a Code of
Federal Regulations (CFR) part or section number.
III. Explain why you agree or disagree; suggest alternatives and
substitute language for your requested changes.
IV. Describe any assumptions and provide any technical information
and/or data that you used.
V. If you estimate potential costs or burdens, explain how you
arrived at your estimate in sufficient detail to allow for it to be
reproduced.
VI. Provide specific examples to illustrate your concerns, and
suggest alternatives.
VII. Explain your views as clearly as possible, avoiding the use of
profanity or personal threats.
VIII. Make sure to submit your comments by the comment period
deadline identified.
3. Docket Copying Costs. 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 K.
Oshiro; Office of Science and Technology (4303-T); Office of Water;
U.S. Environmental Protection Agency; Ariel Rios Building; 1200
Pennsylvania Avenue, NW., Washington, DC 20460, or call (202) 566-1075.
Copies of several of the EPA methods cited in this proposal may also be
downloaded from the EPA Office of Water, Office of Science and
Technology, home page at http://www.epa.gov/waterscience/methods/.
Copies of all methods are also available in the public record for this
proposal.
Table of Contents
I. Statutory Authority
II. Explanation of Today's Action
A. Methods for NPDES Compliance Monitoring
B. Request for Public Comment and Available Data
C. Editorial Revision and Clarification to 40 CFR Part 136
D. Sampling, Sample Preservation, and Holding Times for NPDES
Compliance Monitoring: Revisions to 40 CFR Part 136, Table II
III. Statutory and Executive Order Reviews
A. Executive Order 12866: Regulatory Planning and Review
B. Paperwork Reduction Act
C. Regulatory Flexibility Act
D. Unfunded Mandates Reform Act
E. Executive Order 13132: Federalism
F. Executive Order 13175: Consultation and Coordination With
Indian Tribal Governments
G. Executive Order 13045: Protection of Children From
Environmental Health & Safety Risks
H. Executive Order 13211: Actions That Significantly Affect
Energy Supply, Distribution, or Use
I. National Technology Transfer Advancement Act
IV. References
I. Statutory Authority
EPA is proposing this action pursuant to the authority of sections
301(a), 304(h), 405(d) and (e), and 501(a) of the Clean Water Act
(``CWA'' or the ``Act''), 33 U.S.C. 1311(a), 1314(h), 1361(a). Section
301(a) of the Act prohibits the discharge of any pollutant into
navigable waters unless, among other things, the discharge complies
with a National Pollutant Discharge Elimination System (NPDES) permit
issued under section 402 of the Act. Section 304(h) of the Act requires
the Administrator of the EPA 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 application 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 [the Act].'' EPA generally codifies
its test procedures in the Code of Federal Regulations (including
analysis and sampling requirements) for CWA programs at 40 CFR part
136, though some specific requirements are in other sections (e.g., 40
CFR 503.8).
II. Explanation of Today's Action
A. Methods for NPDES Compliance Monitoring
This proposal would make available membrane filter (MF) methods and
a suite of Multiple Tube Fermentation (MTF) methods (i.e., multiple-
tube, multiple-well) including culture and enzyme-substrate techniques
available for enumerating (i.e., determining organism density) E. coli
and enteroccoci in wastewaters and fecal coliforms and Salmonella in
sewage sludge as part of State, Territorial, Tribal, and local water
quality and sewage sludge monitoring programs.
EPA selected the methods based on data generated by EPA
laboratories, or submissions to the ATP program. Since multiple studies
using different method versions and different statistical analyses
generated the EPA laboratory data, the test procedures in today's rule
must be evaluated against the end-users' needs based on data quality
objectives. EPA recommends that all new proposed
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alternative methods be compared to the appropriate EPA approved
reference method before adopting it for that matrix to ensure that the
proposed method generates data of comparable quality. For full details
regarding alternative microbial methods, see the EPA Microbiological
Alternate Test Procedure (ATP) Protocol for Drinking Water, Ambient
Water, and Wastewater Monitoring Methods (EPA 821-B-03-004). Full
citations for methods and validation data reports are provided in the
References section and are included in the docket for today's proposed
rulemaking.
1. Membrane Filtration (MF) and Multiple Tube Fermentation (MTF)
Methods
Membrane filtration is a direct-plating method in which sample
dilutions/volumes are filtered through 0.45 [mu]m membrane filters that
are subsequently transferred to petri plates containing selective
primary isolation agar or an absorbent pad saturated with selective
broth. 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. 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'' (Standard Methods for the Examination of Water and Wastewater,
1998).
Membrane filtration is applicable to most tertiary treated
wastewaters but has limitations where an underestimation of organism
density is likely, such as water samples with high turbidity, toxic
compounds, large numbers of non-coliform (background) bacteria. In
addition, membrane filtration may have limitations where organisms are
damaged by chlorine or toxic compounds, such as can be found in primary
and some secondary treated wastewaters. 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 individual water type, parallel tests should be
conducted with a Multiple Tube Fermentation (MTF) to demonstrate
applicability, lack of interferences, and at least comparable (e.g.,
equivalent or better) 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,
the user should use an appropriate recovery enhancement technique that
the tester demonstrates is comparable to MTF. Further background
information on MF tests is available in Standard Methods for the
Examination of Water and Wastewater (1998).
In Multiple Tube Fermentation (MTF) 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 Multiple Tube Fermentation. MTF tests may be
conducted in multiple-tube fermentation, 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 MTF, 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),
injured or stressed organisms, or high levels of heterotrophic plate
count bacteria (HPC). The membrane filtration technique may be more
appropriate in instances where the toxins are water soluble; in such
cases, the toxin may be eliminated while the organisms are retained on
the filter. Multiple-tube tests are applicable to sewage sludge
analysis. 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 Standard Methods
for the Examination of Water and Wastewater (1998).
A statistical comparison of results obtained by the MF and MTF
methods showed that the MF method is more precise in enumerating target
organisms than the MTF 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 MTF method may overestimate the concentration because
of the built-in positive bias of the method (Thomas, 1955). Because of
susceptibility of some MF tests to interferences, verification of some
MF results with confirmatory multiple-tube tests is critical.
Additionally, some MTF 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.
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2. Methods for E. coli in Wastewater
EPA is proposing several methods for enumerating E. coli in
wastewater. 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 Association of Official Analytical Chemists
(AOAC) also publishes methods that have met the requirements of the
AOAC methods approval program. EPA methods are those that have been
developed and validated by the US EPA.
Table 1.--Proposed Methods for E. coli Enumeration in Wastewater
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VCS methods
EPA ----------------------
Technique Method \1\ method Standard Commercial example
methods AOAC
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Membrane Filter (MF)............ Modified mTEC agar. 1603 ......... .........
Multiple Tube Fermentation (MTF) ONPG-MUG........... ........ 9223B 991.15 Colilert[supreg] \2\
ONPG-MUG........... ........ 9223B ......... Colilert-18[supreg] \2\
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\1\ Tests must be conducted in a format that provides organism enumeration.
\2\ Manufactured by IDEXX.
a. Membrane Filter (MF) Test for E. coli: Modified mTEC Agar (EPA
Method 1603). 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, 2004a). 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-[beta]-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 0.45 [mu]m 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.
b. Multiple Tube Fermentation Tests for E. coli: ONPG-MUG (Standard
Methods 9223B, AOAC 991.15, Colilert[supreg], Colilert-18[supreg]).
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-[beta]-D-galactopyranoside (ONPG), to
detect total coliforms and the fluorogenic substrate 4-
methylumbelliferyl-[beta]-D-glucuronide (MUG), to detect E. coli. All
tests must be conducted in a format that provides quantitative results
for ambient water. Colilert-18[supreg] should be used for testing
marine waters with a minimum of a 10-fold dilution with sterile non-
buffered, oxidant-free water. 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[supreg] or
Quanti-Tray[supreg]/2000 multiple-well tests uses Colilert[supreg] or
Colilert-18[supreg] media to determine E. coli (IDEXX, 1999a,b,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[supreg] or 24 h when using
Colilert[supreg]. 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[supreg] 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.
3. Methods for Enterococci for Wastewater
EPA is proposing several methods for enumerating enterococci in
wastewater. Brief descriptions of the proposed MF and MTF methods are
provided below. In Table 2, methods in the same horizontal row use the
same technique, but are published by different entities.
[[Page 48260]]
Table 2.--Proposed Methods for Enterococci in Wastewater.
----------------------------------------------------------------------------------------------------------------
VCS methods
Methodology Method \1\ EPA --------------------- Commercial example
method ASTM AOAC
----------------------------------------------------------------------------------------------------------------
Membrane Filter (MF)............. mEI agar............ 1600 .......... ....... ........................
Multiple Tube Fermentation (MTF). MUG media........... ........ D6503-99 ....... Enterolert\TM\ \2\
----------------------------------------------------------------------------------------------------------------
\1\ Tests must be conducted in a format that provides organism enumeration.
\2\ Manufactured by IDEXX.
a. Membrane Filter (MF) Test for Enterococci: mEI Agar (EPA Method
1600). The mE-EIA 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 filter when placed on selective mE agar
(USEPA, 2004b). 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-[beta]-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 that are greater than 0.5 mm in diameter, are counted
as enterococci. Results are reported as enterococci per 100 mL.
b. Multiple Tube Fermentation (MTF) Tests for Enterococci: 1. 4-
methylumbelliferyl-[beta]-D-glucoside (MUG) Medium (ASTM D6503-99,
EnterolertTM). This method utilizes a medium containing the
fluorogenic substrate 4-methylumbelliferyl-[beta]-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, 1999a).
EnterolertTM tests are incubated for 24 h at 41
0.5 [deg]C and may use the same quantitative formats available for the
Colilert[supreg] tests, cited earlier in Section III-A. After
incubation, the presence of blue/white fluorescence, as viewed using a
6-watt, 365 nm, UV light, 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[supreg] tests. In addition, marine
water samples must be diluted at least tenfold with sterile, non-
buffered oxidant-free water (EnterolertTM is already
buffered).
4. Methods for Fecal Coliforms in Sewage Sludge
EPA is proposing methods for enumerating fecal coliforms in sewage
sludge (Table 3). Brief descriptions of the proposed MTF methods are
provided below.
Table 3.--Proposed Methods for Fecal Coliforms in Sewage Sludge
------------------------------------------------------------------------
EPA
Methodology Method \1\ method
------------------------------------------------------------------------
Multiple Tube Fermentation (MTF)....... LT-EC................ 1680
A-1.................. 1681
------------------------------------------------------------------------
\1\ Tests must be conducted in a format that provides organism
enumeration.
a. Multiple Tube Fermentation (MTF) Tests for Fecal Coliforms:
1. LT-EC Medium (EPA Method 1680). The multiple-tube fermentation
method for enumerating fecal coliforms in sewage sludge uses multiple-
tubes and dilutions/volumes in a two-step procedure to determine fecal
coliform concentrations (USEPA, 2004c). 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 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. Failure to
produce gas is a negative reaction and indicates fecal coliform
bacteria are not present. Turbidity without gas indicates an invalid
test that requires repeat analysis.
Results of the MTF procedure using LTB/EC media are reported in
terms of MPN/g dry weight calculated from the number of positive EC
tubes and percent total solids (dry weight basis).
2. A-1 Medium (EPA Method 1681). The multiple-tube fermentation
method for enumerating fecal coliforms in sewage sludge uses multiple-
tubes and dilutions/volumes in a procedure to determine fecal coliform
concentrations (USEPA 2004d). It should be noted that the Triton X-100
(polyethylene glycol p-isoloctylphenyl ether) is extremely volatile,
and thus the medium must be used within one week (and preferably on the
day of) preparation. In the first step, a series of tubes containing A-
1 broth are inoculated with undiluted samples and/or dilutions/volumes
of the samples and mixed. Inoculated tubes are incubated for 3 h at 35
0.5 [deg]C, then transferred to a water bath at 44.5
[deg]C 0.2 [deg]C. After 21 2 h, tubes are
examined for growth (i.e., turbidity) and production of gas in the
inner Durham tube. Production of growth and gas within 24
4 h constitutes the presence of fecal coliforms. Failure to produce
both turbidity and gas is a negative reaction and indicates fecal
coliform bacteria are not present.
Results of the MTF procedure using A-1 media are reported in terms
of MPN/g calculated from the number of positive A-1 tubes and percent
total solids (dry weight basis).
5. Methods for Salmonella in Sewage Sludge
EPA is also proposing methods for enumerating Salmonella in sewage
sludge (Table 4). Brief descriptions of the proposed MTF method are
provided below.
Table 4.--Proposed Methods for Salmonella in Sewage Sludge
------------------------------------------------------------------------
EPA
Methodology Method \1\ method
------------------------------------------------------------------------
Multiple Tube Fermentation (MTF)....... Modified MSRV........ 1682
------------------------------------------------------------------------
\1\ Tests must be conducted in a format that provides organism
enumeration.
a. Multiple Tube Fermentation (MTF) Tests for Salmonella in Sewage
Sludge: Multiple Tube Fermentation (MTF) Test for Salmonella (EPA
Method 1682). The multiple-tube fermentation method for enumerating
Salmonella in sewage sludge uses multiple-tubes and dilutions/volumes
in a multiple-step procedure to determine Salmonella concentrations
(USEPA 2004e). In the selective phase, a series of tubes
[[Page 48261]]
containing tryptic soy broth (TSB) are inoculated with undiluted
samples and/or dilutions/volumes of the samples and mixed. Inoculated
tubes are incubated for 24 2 h at 36 1.5
[deg]C. After incubation, six discrete, 30-[mu]L drops from each TSB
tube are spotted onto the selective Rappaport-Vassiliadis agar medium
semisolid modification (MSRV). The drops are allowed to absorb into the
agar for approximately 1 hour at room temperature, then incubated,
inoculated side up, at 42 [deg]C 0.5 [deg]C for 16 to 18
hours in a humidity-controlled hot air incubator.
The plates are examined for the appearance of motility surrounding
inoculations, as evidenced by a ``whitish halo'' of growth
approximately 2 cm from the center of the spot. Growth from the outer
edge of the halo is streaked onto labeled XLD plates for isolation with
a sterile inoculating needle or loop. Two halos and chosen are stabbed
using an inoculating loop into the halo's outer edge, which is then
streaked onto individual XLD plates (one spot per XLD plate) that are
then incubated for 18 to 24 hours at 36 [deg]C 1.5 [deg]C.
After incubation, one of the plates is submitted to biochemical
confirmation (the other is refrigerated for reference). Pink to red
colonies with black centers on XLD plates are considered Salmonella.
In the confirmatory phase, isolated colonies exhibiting Salmonella
morphology (pink to red colonies with black centers) are picked and
inoculated into triple sugar iron agar (TSI) slants, lysine iron agar
(LIA) slants, and urease broth, all of which are incubated for 24
2 hours at 36 [deg]C 1.5 [deg]C. A positive
TSI reaction is an acid butt (yellow in color) and an alkaline slant
(red in color) with or without H2S gas production. A
positive LIA reaction is an alkaline butt (purple in color) and an
alkaline slant (purple in color) with or without H2S gas
production. When H2S gas production is present, the butts of
both the LIA and TSI may be black, which would be considered a positive
reaction for Salmonella. Urease is an orange medium and will change to
pink or deep purplish-red if positive. A negative urease test is one
that exhibits no color change after inoculation. Salmonella are
negative for urease.
To confirm cultures via polyvalent O antiserum, growth on the slant
portion of TSI (regardless of whether TSI is positive or negative) is
emulsified using sterile physiological saline, and two discrete drops
of emulsified growth are placed onto a slide. One drop of polyvalent O
antiserum is to be added to the first drop of emulsified growth, and
one drop of sterile saline is added to the second drop of emulsified
growth as a visual comparison. The slide is observed under
magnification for an agglutination reaction which indicates a positive
result. In order for the original TSB tube to be considered positive
for Salmonella, the associated inoculations should be MSRV positive,
XLD positive, either TSI or LIA positive, urease negative, and
polyvalent-O positive. Failure in any of these test constitutes a
negative Salmonella reaction.
A total solids determination is performed on a representative
sewage sludge sample and is used to calculate MPN/g dry weight.
Salmonella density is reported as MPN / 4 g dry weight.
B. Request for Comment and Available Data
EPA is not proposing the use of EPA Method 1103.1 (mTEC) for E.
coli or EPA Method 1106.1 (mE-EIA) for enterococci for use in
wastewater because the validation test results for these methods showed
that the false positive and false negative rates for these methods were
unacceptably high. Specifically, the validation of Method 1103.1 had
laboratory-specific rates combined over unspiked disinfected/secondary
results ranging from 14.4% to 22.9% for false positives and from 8.9%
to 16.9% for false negatives (USEPA 2004f). Additionally, the
validation of Method 1106.1 had laboratory-specific rates combined over
unspiked disinfected/secondary results ranging from 0.0% to 18.0% for
false positives and from 55.4% to 60.5% for false negatives (USEPA
2004g).
EPA is not proposing to extend the holding time from 6 hours to 24
hours for fecal coliforms using Method 1680 (LTB/EC) from Class A
aerobically digested sewage sludge or for Salmonella using Method 1682
(MSRV) from Class B thermophilically digested sewage sludge because the
holding time studies for these methods showed significant differences
in concentrations of these organisms using these methods after 24 hours
holding time (USEPA 2004h).
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 wastewater monitoring, and for
fecal coliform and Salmonella methods for sewage sludge monitoring.
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 wastewater and for fecal coliforms
and Salmonella in sewage sludge and for holding times for the proposed
methods in sewage sludge 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
wastewater and sewage sludge, respectively.
C. Editorial Revision and Clarification to 40 CFR Part 136
40 CFR part 136, Table I currently includes microbial (bacterial,
and protozoan) methods for use in both wastewater and ambient waters.
For clarification purposes, EPA proposes to move those methods which
are applicable to ambient waters to a new Table IG.
D. Sampling, Sample Preservation, and Holding Times for NPDES
Compliance Monitoring: Revisions to 40 CFR Part 136, Table II
40 CFR part 136, Table II specifies sampling, preservation, and
holding time requirements. This proposal would make additions to these
tables for sewage sludge methods added to Table IA. In addition,
clarification is provided for the holding time for bacterial tests.
III. Statutory and Executive Order Reviews
A. Executive Order 12866: Regulatory Planning and Review
Under Executive Order 12866 (58 FR 51735 (October 4, 1993)), the
Agency must determine whether the regulatory action is ``significant''
and therefore subject to 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;
[[Page 48262]]
(3) Materially alter the budgetary impact of entitlements, grants,
user fees, or loan programs or the rights and obligations of recipients
thereof; or
(4) Raise novel legal or policy issues arising out of legal
mandates, the President's priorities, or the principles set forth in
the Executive Order.
It has been determined that this proposed rule is not a
``significant regulatory action'' under the terms of Executive Order
12866 and is therefore not subject to Executive Order 12866 review.
B. Paperwork Reduction Act
This action does not impose an information collection burden under
the provisions of the Paperwork Reduction Act, 44 U.S.C. 3501 et. seq.
This rule proposes to make available new test methods for E. coli and
enterococci for use in wastewater monitoring programs, and new test
methods for fecal coliform and Salmonella for use in sewage sludge
monitoring programs, but EPA would not require the use of these test
methods. This rule does not impose any information collection,
reporting, or record keeping requirements.
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 purpose of collecting, validating, and
verifying information, processing and maintaining information, and
disclosing and providing information; adjust the existing ways to
comply with any previously applicable instructions and requirements;
train personnel to be able to respond to a collection of information;
search data sources; complete and review the collection of information;
and transmit or otherwise disclose the information.
An Agency may not conduct or sponsor, and a person is not required
to respond to a collection of information unless it displays a
currently valid OMB control number. The OMB control numbers for EPA's
regulations in 40 CFR are listed in 40 CFR part 9.
C. Regulatory Flexibility Act
The Regulatory Flexibility Act (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 this rule on small
entities for methods under the Clean Water Act, small entity is defined
as: (1) A small business that meets RFA default definitions (based on
SBA size standards) found in 13 CFR 121.201; (2) a small governmental
jurisdiction that is a government of a city, county, town, school
district or special district with a population less than 50,000; and
(3) a small organization that is any not-for-profit enterprise which is
independently owned and operated and is not dominant in its field.
After considering the economic impacts of today's proposed rule on
small entities, I certify that this action will not have a significant
economic impact on a substantial number of small entities. In
determining whether a rule has a significant economic impact on a
substantial number of small entities, the impact of concern is any
significant adverse economic impact on small entities, since the
primary purpose of the regulatory flexibility analyses is to identify
and address regulatory alternatives ``which minimize any significant
economic impact of the rule on small entities.'' 5 U.S.C. 603 and 604.
Thus, an agency may certify that a rule will not have a significant
economic impact on a substantial number of small entities if the rule
relieves regulatory burden, or otherwise has a positive economic effect
on all of the small entities subject to the rule.
This proposed regulation would approve testing procedures for the
measurement of E. coli and enterococci bacteria in wastewater, and
fecal coliforms and Salmonella bacteria in sewage sludge. The inclusion
of these test methods in 40 CFR 136.3 is intended to make these test
methods available to States and others for use in wastewater and sewage
sludge monitoring programs. EPA is not establishing any compliance
monitoring requirements for these pollutants.
EPA analyzed the annualized cost estimates to regulated entities
(small governmental jurisdictions that have publically-owned treatment
works (POTWs) and small businesses with water quality-based discharge
permits) for adoption of the newly proposed test methods for
Escherichia coli (E. coli) and enterococci in wastewater and found that
all incremental costs results are negative (a cost savings) to
regulated firms. The cost savings for the adoption of wastewater
testing procedures are as follows.
The savings for facilities to shift from fecal coliform testing to
E. coli Method 1603 will range from $36 million to $226 million. The
savings to shift to E. coli Method 1103.1 will range from $35 million
to $220 million. The savings for facilities to shift from fecal
coliform testing to enterococci Method 1600 will range from
approximately $36 million to $225 million. The savings to those
currently employing E. coli Method 1103.1 and shifting to E. coli
Method 1603 will range from approximately $0.9 million to $5.8 million,
and those currently shifting from enterococci Method 1106.1 to
enterococci Method 1600 will range from $7,000 to $48,000.
We continue to be interested in the potential impacts of the
proposed rule on small entities and welcome comments on issues related
to such impacts.
D. Unfunded Mandates Reform Act
Title II of the Unfunded Mandates Reform Act of 1995 (UMRA), Public
Law 104-4, establishes requirements for Federal agencies to assess the
effects of their regulatory actions on State, tribal, and local
governments and the private sector. Under section 202 of the UMRA, EPA
generally must prepare a written statement, including a cost-benefit
analysis, for proposed and final rules with ``Federal mandates'' that
may result in expenditures to State, local, and tribal 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
[[Page 48263]]
intergovernmental mandates, and informing, educating, and advising
small governments on compliance with the regulatory requirements.
This rule contains no Federal mandates (under the regulatory
provisions of Title II of UMRA) for State, local, or tribal governments
or the private sector. The rule imposes no enforceable duty on any
State, local, or tribal governments or the private sector. In fact,
this rule should (on the whole) save money for governments and the
private sector by increasing method flexibility, and allowing these
entities to reduce monitoring costs by taking advantage of innovations.
Thus, today's rule is not subject to the requirements of sections 202
and 205 of the UMRA.
EPA has determined that this rule contains no regulatory
requirements that might significantly or uniquely affect small
governments. This rule makes available testing procedures for E. coli,
enterococci, fecal coliform, and Salmonella that may be used by a
State, Territorial, Tribal or local authority for compliance with water
quality standards (E. coli, enterococci) or sewage sludge (fecal
coliforms, Salmonella) monitoring requirements when testing is
otherwise required by these regulatory authorities. Thus, today's rule
is not subject to the requirements of section 203 of UMRA.
E. Executive Order 13132: Federalism
Executive Order 13132, entitled ``Federalism'' (64 FR 43255, August
10, 1999), requires EPA to develop an accountable process to ensure
``meaningful and timely input by State and local officials in the
development of regulatory policies that have federalism implications.''
``Policies that have federalism implications'' is defined in the
Executive Order to include regulations that have ``substantial direct
effects on the States, on the relationship between the National
Government and the States, or on the distribution of power and
responsibilities among the various levels of government.''
This 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 rule makes available
testing procedures for E. coli and enterococci in wastewater, and for
fecal coliforms and Salmonella in sewage sludge. There is no cost to
State and local governments and the rule does not preempt State law.
Thus, Executive Order 13132 does not apply to this rule.
In the spirit of Executive Order 13132, and consistent with EPA
policy to promote communications between EPA and State and local
governments, EPA specifically solicits comment on this proposed rule
from State and local officials.
F. 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 9, 2000),
requires EPA to develop an accountable process to ensure ``meaningful
and timely input by tribal officials in the development of regulatory
policies that have tribal implications.''
``Policies that have tribal implications'' is defined in the
Executive Order to include regulations that have ``substantial direct
effects on one or more Indian tribes, on the relationship between the
Federal Government and the Indian tribes, or on the distribution of
power and responsibilities between the Federal Government and the
Indian tribes.''
This proposed rule does not have tribal implications. It will not
have substantial direct effects on Tribal governments, on the
relationship between the Federal Government and Indian tribes, or on
the distribution of power and responsibilities between the Federal
Government and Indian tribes, as specified in Executive Order 13175.
This rule makes available testing procedures for E. coli and
enterococci in wastewater, and for fecal coliforms and Salmonella in
sewage sludge. The costs to Tribal governments will be minimal (in
fact, governments may see a cost savings), and the rule does not
preempt State law. 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 comment on this proposed rule from Tribal
officials.
G. Executive Order 13045: Protection of Children From Environmental
Health Risks and Safety Risks
Executive Order 13045: ``Protection of Children from Environmental
Health Risks and Safety Risks'' (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 not economically significant as defined in Executive Order 12866,
and because the Agency does not have reason to believe the
environmental health or safety risks addressed by this action present a
disproportionate risk to children. This action makes available testing
procedures for E. coli and enterococci in wastewater, and for fecal
coliforms and Salmonella in sewage sludge.
H. Executive Order 13211: Actions That Significantly Affect Energy
Supply, Distribution, or Use
This rule is not subject to Executive Order 13211, ``Actions
Concerning Regulations That Significantly Affect Energy Supply,
Distribution, or Use'' (66 FR 28355 (May 22, 2001)) because it is not a
significant regulatory action under Executive Order 12866.
I. National Technology Transfer and Advancement Act
Section 12(d) of the National Technology Transfer and Advancement
Act of 1995, (``NTTAA''), Public Law 104-113, section 12(d) (15 U.S.C.
272 note), directs EPA to use voluntary consensus standards in its
regulatory activities unless to do so would be inconsistent with
applicable law or otherwise impractical. Voluntary consensus standards
are technical standards (e.g., material specifications, test methods,
sampling procedures, and business practices) that are developed or
adopted by voluntary consensus standard bodies. The NTTAA directs EPA
to provide Congress, through the OMB, explanations when the Agency
decides not to use available and applicable voluntary consensus
standards.
This proposed 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 wastewaters. 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 1A at the end of this notice. No voluntary consensus
standards were found for fecal coliforms
[[Page 48264]]
or Salmonella in sewage sludge. 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 or enterococci in wastewaters, and fecal coliforms
and Salmonella in sewage sludge, and to explain why such standards
should be used in this regulation.
IV. References
IDEXX. 1999a. Description of Colilert[reg], Colilert-18[supreg],
Quanti-Tray[supreg], Quanti-Tray[supreg]/2000,
EnterolertTM methods are available from IDEXX
Laboratories, Inc., One Idexx Drive, Westbrook, Maine 04092.
IDEXX. 1999b. ``Quanti-Tray[supreg]: A Simple Method for
Quantitation of Bacterial Density in Liquid Samples.''
IDEXX. 1999c. ``Quanti-Tray/2000[supreg]: Detection and Enumeration
of Bacteria from High Bacterial Density Liquid Samples Without
Dilution.''
USEPA. 2004a. Method 1603: Escherichia coli (E. coli) in Water by
Membrane Filtration Using Modified membrane-Thermotolerant
Escherichia coli Agar (Modified mTEC). December 2004. U.S.
Environmental Protection Agency, Office of Water, Washington DC EPA-
821-04-025.
USEPA. 2004b. Method 1600: Enterococci in Water by Membrane
Filtration Using membrane-Enterococcus Indoxyl-[beta]-D-Glucoside
Agar (mEI). December 2004. U.S. Environmental Protection Agency,
Office of Water, Washington DC EPA-821-04-023.
USEPA. 2004c. Method 1680: Fecal Coliforms in Sewage Sludge by
Multiple-Tube Fermentation Using Lauryl-Tryptose E. coli (LT-EC)
Broth. December 2004. U.S. Environmental Protection Agency, Office
of Water, Washington DC EPA-821-04-026.
USEPA. 2004d. Method 1681: Fecal Coliforms in Sewage Sludge by
Multiple-Tube Fermentation Using A-1 Broth. December 2004. U.S.
Environmental Protection Agency, Office of Water, Washington DC EPA-
821-04-027.
USEPA. 2004e. Method 1682: Salmonella in Sewage Sludge by Multiple-
Tube Fermentation Using Modified Semisolid Rappaport-Vassiliadis
(MSRV) Medium. December 2004. U.S. Environmental Protection Agency,
Office of Water, Washington DC EPA-821-04-028.
USEPA. 2004f. Results of the Interlaboratory Validation of EPA
Method 1103.1 (mTEC) for E. coli in Wastewater Effluent. December
2004. U.S. Environmental Protection Agency, Office of Water,
Washington DC EPA-821-04-02.
USEPA 2004g. Results of the Interlaboratory Validation of EPA Method
1106.1 (mE-EIA) for E. coli in Wastewater Effluent. December 2004.
U.S. Environmental Protection Agency, Office of Water, Washington DC
EPA-821-04-02.
USEPA. 2004h. Assessment of the Effects of Holding Time on Fecal
Coliform and Salmonella Concentrations in Biosolids. December 2004.
U.S. Environmental Protection Agency, Office of Water, Washington DC
EPA-821-04-029.
List of Subjects in 40 CFR Part 136
Environmental protection, Incorporation by reference, Reporting and
recordkeeping requirements, Water pollution control.
Dated: August 10, 2005.
Stephen L. Johnson,
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 as follows:
a. In paragraph (a) by revising Table IA.
b. In paragraph (a) by adding Table IG after the footnotes of Table
IF.
c. In paragraph (b) by revising references 54, 55, 56 and 59, and
adding references 63 through 65.
d. In paragraph (e) by revising the entry for Table IA and adding
an entry for Table IG in Table II.
Sec. 136.3 Identification of test procedures.
(a) * * *
Table IA.--List of Approved Biological Methods
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Standard methods 18th, Standard methods on-line
Parameter and units Method \1\ EPA 19th, 20th ed.\4\ \4\ AOAC, ASTM, USGS Other
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria: 1. Coliform (fecal), Multiple Tube p. 132 \3\, 1680 22 24, 9221C E.................. 9221C E-99............... ......................... .........................
number per 100 mL. Fermentation (MTF), 1681 23 24.
5 tube 3 dilution,
or.
Membrane filter (MF) p. 124 \3\............... 9222D.................... 9222D-97................. B-0050-85 \5\............ .........................
\2\, single step.
2. Coliform (fecal) in presence of MTF, 5 tube, 3 p. 132 \3\............... 9221C E.................. 9221C E-99............... ......................... .........................
chlorine, number per 100 mL. dilution, or.
MF 12 16 single step p. 124 \3\............... 9222D.................... 9222D-97................. ......................... .........................
\6\.
3. Coliform (total), number per MTF, 5 tube, 3 p. 114 \3\............... 9221B.................... 9221B-99................. ......................... .........................
100 mL. dilution, or.
MF \2\, single step p. 108 \3\............... 9222B.................... 9222B-97................. B-0025-85 \5\............ .........................
or two step.
4. Coliform (total), in presence MTF, 5 tube, 3 p. 114 \3\, p. 111 \3\... 9221B, 9222(B+B.5c)...... 9221B-99, 9222(B+ B.5c)- ......................... .........................
of chlorine, number per 100 mL. dilution, or MF \2\ 97.
with enrichment.
5. E. coli, number per 100 mL..... MTF, multiple tube/ ......................... 9223B \12\............... 9223B-97 \12\............ 991.15 \11\.............. Colilert[reg] 12 14,
multiple well,. Colilert-18[reg] 12 13
14
MF 2 6 7 8 9, single 1603 16 25............... ......................... ......................... ......................... .........................
step.
6. Fecal streptococci, number per MTF, 5 tube, 3 p. 139 \3\............... 9230B.................... 9230B-93................. ......................... .........................
100 mL. dilution,.
MF \2\, or........... p. 136 \3\............... 9230C.................... 9230C-93................. B-0055-85 \5\............ .........................
Plate count.......... p. 143 \3\............... ......................... ......................... ......................... .........................
7. Enterococci, number per 100 mL. MTF, multiple tube/ ......................... ......................... ......................... D6503-99 \10\............ Enterolert [reg] 12 17
multiple well.
[[Page 48265]]
MF 2 6 7 8 9 single 1600 18 25............... ......................... ......................... ......................... .........................
step.
8. Salmonella, number per 100 mL.. MTF multiple tube.... 1682 24 26............... ......................... ......................... ......................... .........................
Aquatic Toxicity:
9. Toxicity, acute, fresh water Ceriodaphnia dubia 2002.0 \19\.............. ......................... ......................... ......................... .........................
organisms, LC50, percent effluent. acute.
Daphnia puplex and 2021.0 \19\.............. ......................... ......................... ......................... .........................
Daphnia magna acute.
Fathead Minnow, 2000.0 \19\.............. ......................... ......................... ......................... .........................
Pimephales promelas,
and Bannerfin
shiner, Cyprinella
leedsi, acute.
Rainbow Trout, 2019.0 \19\.............. ......................... ......................... ......................... .........................
Oncorhynchus mykiss,
and brook trout,
Salvelinus
fontinalis, acute.
10. Toxicity, acute, estuarine and Mysid, Mysidopsis 2007.0 \19\.............. ......................... ......................... ......................... .........................
marine organisms of the Atlantic bahia, acute.
Ocean and Gulf of Mexico, LC50,
percent effluent.
Sheepshead Minnow, 2004.0 \19\.............. ......................... ......................... ......................... .........................
Cyprinodon
variegatus, acute.
Silverside, Menidia 2006.0 \19\.............. ......................... ......................... ......................... .........................
beryllina, Menidia
menidia, and Menidia
peninsulae, acute.
11. Toxicity, chronic, fresh water Fathead minnow, 1000.0 \20\.............. ......................... ......................... ......................... .........................
organisms, NOEC or IC25, percent Pimephales promelas,
effluent. larval survival and
growth.
Fathead minnow, 1001.0 \20\.............. ......................... ......................... ......................... .........................
Pimephales promelas,
embryo-larval
survival and
teratogenicity.
Daphnia, Ceriodaphnia 1002.0 \20\.............. ......................... ......................... ......................... .........................
dubia, survival and
reproduction.
Green alga, 1003.0 \20\.............. ......................... ......................... ......................... .........................
Selenastrum
capricornutum,
growth.
12. Toxicity, chronic, estuarine Sheepshead minnow, 1004.0 \21\.............. ......................... ......................... ......................... .........................
and marine organisms of the Cyprinodon
Atlantic Ocean and Gulf of variegatus, larval
Mexico, NOEC or IC25, percent survival and growth.
effluent.
Sheepshead minnow, 1005.0 \21\.............. ......................... ......................... ......................... .........................
Cyprinodon
variegatus, embryo-
larval survival and
teratogenicity.
Inland silverside, 1006.0 \21\.............. ......................... ......................... ......................... .........................
Menidia beryllina,
larval survival and
growth.
[[Page 48266]]
Mysid, Mysidopsis 1007.0 \21\.............. ......................... ......................... ......................... .........................
bahia, survival,
growth, and
fecundity.
Sea urchin, Arbacia 1008.0 \21\.............. ......................... ......................... ......................... .........................
punctulata,
fertilization.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ The method must be specified when results are reported.
\2\ A 0.45-[mu]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,
OH. 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 http://www.standardmethods.org.
\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, VA.
\6\ Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Multiple Tube Fermentation method will be required to resolve any controversies.
\7\ Tests must be conducted to provide organism enumeration (density). Select the appropriate configuration of tubes/filtrations and dilutions/volumes to account for the quality, character,
consistency, and anticipated organism density of the water sample.
\8\ When the MF method has not been used previously to test ambient waters with high turbidity, large number of noncoliform bacteria, or samples that may contain organisms stressed by
chlorine, a parallel test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of results.
\9\ To assess the comparability of results obtained with individual methods, it is suggested that side-by-side tests be conducted across seasons of the year with the water samples routinely
tested in accordance with the most current Standard Methods for the Examination of Water and Wastewater or EPA alternate test procedure (ATP) guidelines.
\10\ ASTM. 2000, 1999, 1996. Annual Book of ASTM Standards--Water and Environmental Technology. Section 11.02. American Society for Testing and Materials. 100 Barr Harbor Drive, West
Conshohocken, PA 19428.
\11\ AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. Association of Official Analytical Chemists International. 481 North Frederick Avenue,
Suite 500, Gaithersburg, MD 20877-2417.
\12\ These tests are collectively known as defined enzyme substrate tests, where, for example, a substrate is used to detect the enzyme [beta]-glucuronidase produced by E. coli.
\13\ Colilert-18[reg] is an optimized formulation of the Colilert[reg] for the determination of total coliforms and E. coli that provides results within 18 h of incubation at 35[deg]C rather
than the 24 h required for the Colilert[reg] test and is recommended for marine water samples.
\14\ Descriptions of the Colilert[reg], Colilert-18[reg], Quanti-Tray[reg], and Quanti-Tray[reg]/2000 may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, ME 04092.
\15\ Subject total coliform positive samples determined by 9222B or other membrane filter procedure to 9222G using NA-MUG media.
\16\ USEPA. 2004. Method 1603: Escherichia coli (E. coli) In Water By Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar ( modified mTEC). U.S. Environmental
Protection Agency, Office of Water, Washington, DC EPA-821-R-04-025.
\17\ A description of the Enterolert[reg] test may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, ME 04092.
\18\ USEPA. 2004. Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-[beta]-D-Glucoside Agar (mEI). U.S. Environmental Protection Agency, Office of
Water, Washington, DC EPA-821-R-04-023.
\19\ USEPA. October 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. U.S. Environmental Protection Agency,
Office of Water, Washington, DC EPA/821/R-02/012.
\20\ USEPA. October 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. U.S. Environmental Protection
Agency, Office of Water, Washington, DC EPA/821/R-02/013.
\21\ USEPA. October 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC EPA/821/R-02/014.
\22\ USEPA. December 2004. Method 1680: Fecal Coliforms in Sewage Sludge by Multiple-Tube Fermentation Using Lauryl-Tryptose E. coli (LT-EC) Broth. December 2004. U.S. Environmental Protection
Agency, Office of Water, Washington DC EPA-821-R-04-026.
\23\ USEPA. December 2004. Method 1681: Fecal Coliforms in Sewage Sludge by Multiple-Tube Fermentation Using A-1 Broth. December 2004. U.S. Environmental Protection Agency, Office of Water,
Washington DC EPA-821-R-04-027.
\24\ Recommended for enumeration of target organism in sewage sludge.
\25\ Recommended for enumeration of target organism in wastewater effluent.
\26\ USEPA. December 2004. Method 1682: Salmonella in Sewage Sludge by Multiple-Tube Fermentation Using Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium December 2004. U.S. Environmental
Protection Agency, Office of Water, Washington DC EPA-821-R-04-028.
* * * * * * *
Table IG.--List of Approved Microbiological Methods for Ambient Water
--------------------------------------------------------------------------------------------------------------------------------------------------------
Standard methods
Parameter and units Method \1\ EPA 18th, 19th, 20th Standard methods AOAC, ASTM, USGS Other
ed.\4\ on-line \4\
--------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria:...................... MTF 6 8 14 ................... 9221B.1 / 9221F 11 9221B.1 / 9221F-
multiple tube. 13. g599 11 13.
1. E. coli, number per 100 mL. multiple tube/ ................... 9223B \12\........ 9223B-97 \12\..... 991.15 \10\....... Colilert[supreg]
multiple well. 12 16 Colilert-
18[supreg] 12 15
16
MF 2 5 6 7 8, two 1103.1 \19\........ 9222B / 9222G 9222B / 9222G-97 D5392-93 \9\...... ..................
step. \18\, 9213D. \18\.
[[Page 48267]]
single step....... 1603 \20\, 1604 .................. .................. .................. mColiBlue-24 \17\
\21\.
7. Enterococci, number per 100 MTF 6 8 multiple ................... 9230B............. 9230B-93.......... .................. Entero-
mL. tube. ................... .................. .................. D6503-99 \9\...... lert[supreg] 12
multiple tube/ 22
multiple well.
MF 2 5 6 7 8 two 1106.1 \23\........ 9230C............. 9230C-93.......... D5259-92 \9\...... ..................
step,.
single step, or 1600 \24\, p. 143 .................. .................. .................. ..................
Plate count. \3\.
Protozoa:
8. Cryptosporidium......... Filtration/IMS/FA. 1622 25, 1623 26... .................. .................. .................. ..................
9. Giardia................. Filtration/IMS/FA. 1623 \26\.......... .................. .................. .................. ..................
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ The method must be specified when results are reported.
\2\ A 0.45-[mu]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, OH. 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 http://www.standardmethods.org
\5\ Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Multiple Tube Fermentation method will be
required to resolve any controversies.
\6\ 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.
\7\ 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.
\8\ 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.
\9\ ASTM. 2000, 1999, 1996. Annual Book of ASTM Standards--Water and Environmental Technology. Section 11.02. American Society for Testing and
Materials. 100 Barr Harbor Drive, West Conshohocken, PA 19428.
\10\ AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. Association of Official Analytical Chemists
International. 481 North Frederick Avenue, Suite 500, Gaithersburg, MD 20877-2417.
\11\ 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.
\12\ These tests are collectively known as defined enzyme substrate tests, where, for example, a substrate is used to detect the enzyme [beta]-
glucuronidase produced by E. coli.
\13\ After prior enrichment in a presumptive medium for total coliform using 9221B.1, all presumptive tubes or bottles showing any amount of gas, growth
or acidity within 48 h 3 h of incubation shall be submitted to 9221F. Commercially available EC-MUG media or EC media supplemented in the
laboratory with 50 [mu]g/mL of MUG may be used.
\14\ 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[supreg] may be enumerated with
the multiple-well procedures, Quanti-Tray[supreg] or Quanti-Tray[supreg] 2000, and the MPN calculated from the table provided by the manufacturer.
\15\ Colilert-18[supreg] is an optimized formulation of the Colilert[supreg] 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[supreg] test and is recommended for marine water samples.
\16\ Descriptions of the Colilert[supreg], Colilert-18[supreg], Quanti-Tray[supreg], and Quanti-Tray[supreg]/2000 may be obtained from IDEXX
Laboratories, Inc., One IDEXX Drive, Westbrook, ME 04092.
\17\A description of the mColiBlue24[supreg] test, Total Coliforms and E. coli, is available from Hach Company, 100 Dayton Ave., Ames, IA 50010.
\18\ Subject total coliform positive samples determined by 9222B or other membrane filter procedure to 9222G using NA-MUG media.
\19\ USEPA. 2004. Method 1103.1: Escherichia coli (E. coli) In Water By Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC).
U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA-821-R-04-024.
\20\ USEPA. 2004. Method 1603: Escherichia coli (E. coli) In Water By Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (
modified mTEC). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA-821-R-04-025.
\21\ Preparation and use of MI agar with a standard membrane filter procedure is set forth in the article, Brenner et al. 1993. ``New Medium for the
Simultaneous Detection of Total Coliform and Escherichia coli in Water.'' Appl. Environ. Microbiol. 59:3534-3544 and in USEPA. 2002. Method 1604:
Total Coliforms and Escherichia coli (E. coli) in Water by Membrane Filtration by Using a Simultaneous Detection Technique (MI Medium). U.S.
Environmental Protection Agency, Office of Water, Washington, DC EPA 821-R-02-024.
\22\ A description of the Enterolert[supreg] test may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, ME 04092.
\23\ USEPA. 2004. Method 1106.1: Enterococci In Water By Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA). U.S. Environmental
Protection Agency, Office of Water, Washington, DC EPA-821-R-04-022.
\24\ USEPA. 2004. Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-[beta]-D-Glucoside Agar (mEI). U.S.
Environmental Protection Agency, Office of Water, Washington, DC EPA-821-R-04-023.
\25\ Method 1622 uses filtration, concentration, immunomagnetic separation of oocysts from captured material, immunofluorescence assay to determine
concentrations, and confirmation through vital dye staining and differential interference contrast microscopy for the detection of Cryptosporidium.
USEPA. 2001. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA-821-
R-01-026.
\26\ Method 1623 uses filtration, concentration, immunomagnetic separation of oocysts and cysts from captured material, immunofluorescence assay to
determine concentrations, and confirmation through vital dye staining and differential interference contrast microscopy for the simultaneous detection
of Cryptosporidium and Giardia oocysts and cysts. USEPA. 2001. Method 1623. Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S.
Environmental Protection Agency, Office of Water, Washington, DC EPA-821-R-01-025.
[[Page 48268]]
(b) * * *
REFERENCES, SOURCES, COSTS, AND TABLE CITATIONS:
* * * * *
(54) USEPA. 2004. Method 1103.1: Escherichia coli (E. coli) in
Water by Membrane Filtration Using membrane-Thermotolerant Escherichia
coli Agar (mTEC). U.S. Environmental Protection Agency, Office of
Water, Washington, DC December 2004, EPA-821-R-04-024. Table IG, Note
19.
(55) USEPA. 2004. Method 1106.1: Method 1600: Enterococci in Water
by Membrane Filtration using membrane-Enterococcus-Esculin Iron Agar
(mE-EIA). December 2004. U.S. Environmental Protection Agency, Office
of Water, Washington DC EPA-821-R-04-022. Table IG, Note 23.
(56) USEPA. 2004. Method 1603: Escherichia coli (E. coli) in Water
by Membrane Filtration Using Modified membrane-Thermotolerant
Escherichia coli Agar (Modified mTEC). U.S. Environmental Protection
Agency, Office of Water, Washington, DC December 2004, EPA-821-R-04-
025. Table IA, Note 16, and Table IG, Note 20.
* * * * *
(59) USEPA. 2004. Method 1600: Enterococci in Water by Membrane
Filtration using membrane-Enterococcus Indoxyl-[beta]-D-Glucoside Agar
(mEI). December 2004. U.S. Environmental Protection Agency, Office of
Water, Washington, DC EPA-821-R-04-023. Table IA, Note 18, and Table
IG, Note 24.
* * * * *
(63) USEPA. 2004. Method 1680: Fecal Coliforms in Sewage Sludge by
Multiple-Tube Fermentation Using Lauryl-Tryptose E. coli (LT-EC) Broth.
December 2004. U.S. Environmental Protection Agency, Office of Water,
Washington DC EPA-821-R-04-026. Table IA, Note 22.
(64) USEPA. 2004. Method 1681: Fecal Coliforms in Sewage Sludge by
Multiple-Tube Fermentation Using A-1 Broth. December 2004. U.S.
Environmental Protection Agency, Office of Water, Washington DC EPA-
821-R-04-027. Table IA, Note 23.
(65) USEPA. 2004. Method 1682: Salmonella in Sewage Sludge by
Multiple-Tube Fermentation Using Modified Semisolid Rappaport-
Vassiliadis (MSRV) Medium. December 2004. U.S. Environmental Protection
Agency, Office of Water, Washington DC EPA-821-R-04-028. Table IA, Note
26.
* * * * *
(e) * * *
Table II.--Required Containers, Preservation Techniques, and Holding Times
----------------------------------------------------------------------------------------------------------------
Parameter No./name Container \1\ Preservation 2 3 17 Maximum holding time 4 17
----------------------------------------------------------------------------------------------------------------
Tables lA, IG--Bacteria Tests:
1-5 Coliform, total, fecal, and PP,G............. Cool, < 10 [deg]C 18 6 hours 19, 24 hours 20
E. coli. 0.0008% Na2S2O3 5 18.
6 Fecal streptococci........... PP,G............. Cool, < 10 [deg]C, 6 hours 19
0.0008% Na2S2O3 5.
7 Enterococci.................. PP,G............. Cool, < 10 [deg]C, 6 hours 19
0.0008% Na2S2O3 5.
8 Salmonella................... PP,G............. Cool, < 10 [deg]C 18.. 619 or 24 hours 21
Table lG--Protozoa Tests:
9 Cryptosporidium.............. LDPE............. 0-8 [deg]C............ 96 hours 17
10 Giardia..................... LDPE............. 0-8 [deg]C............ 96 hours 17
* * * * * * *
----------------------------------------------------------------------------------------------------------------
\1\ Polyethylene (P) or glass (G). For microbiology, plastic sample containers must be made of sterilizable
materials (polypropylene or other autoclavable plastic).
\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 samples make it 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 at concentrations of 0.15% by weight or less (pH about
1.62 or greater); Sulfuric acid (H2SO4) in water solutions at 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 analysis 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 sample 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 presence of residual chlorine.
* * * * * * *
\17\ Holding time is calculated from time of sample collection to elution for samples shipped to the laboratory
in bulk and calculated from the time of sample filtration to elution for samples filtered in the field.
\18\ Sewage sludge samples collected for fecal coliform and Salmonella analysis do not require the addition of
0.0008% Na2S2O3.
\19\ Holding time for bacterial tests is 6 hours for transport of the sample to the laboratory, and an
additional 2 hours to process the sample in the laboratory.
\20\ An extended holding time of 24 hours is limited to sewage sludge Class A composted samples to be analyzed
for fecal coliforms using either EPA Method 1680 (LTB/EC) or EPA Method 1681 (A-1) and Class B aerobically
digested samples using EPA Method 1681 (A-1) only. Initial analysis of the sample in the laboratory must
commence within 24 hours of sample collection.
\21\ An extended holding time of 24 hours is limited to sewage sludge Class A composted samples to be analyzed
for Salmonella using EPA Method 1682 (MSRV) only. Initial analysis of the sample in the laboratory must
commence within 24 hours of sample collection.
[FR Doc. 05-16195 Filed 8-15-05; 8:45 am]
BILLING CODE 6560-50-P