[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]



[[Page 48255]]

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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:

------------------------------------------------------------------------
                                   Examples of potentially regulated
           Category                             entities
------------------------------------------------------------------------
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.
------------------------------------------------------------------------

    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

[[Page 48258]]

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.

[[Page 48259]]

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
----------------------------------------------------------------------------------------------------------------
                                                                      VCS methods
                                                          EPA   ----------------------
            Technique                  Method \1\       method    Standard                Commercial  example
                                                                  methods      AOAC
----------------------------------------------------------------------------------------------------------------
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\
----------------------------------------------------------------------------------------------------------------
\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