[Federal Register Volume 60, Number 199 (Monday, October 16, 1995)]
[Rules and Regulations]
[Pages 53528-53544]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-25348]



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[[Page 53529]]


ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 136

[WH-FRL-5308-7]
RIN 2040-AC54


Whole Effluent Toxicity: Guidelines Establishing Test Procedures 
for the Analysis of Pollutants

AGENCY: Environmental Protection Agency (EPA).

ACTION: Final rule.

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SUMMARY: This final rule amends the ``Guidelines Establishing Test 
Procedures for the Analysis of Pollutants,'' 40 CFR part 136, to add 
whole effluent toxicity (WET) testing methods to the list of Agency 
approved methods in Tables IA and II, under the Clean Water Act. This 
action amends 40 CFR 136.3 (Tables 1A and II) by adding methods for 
measuring the acute and short-term chronic toxicity of effluents and 
receiving waters.
    This rulemaking was initiated at the request of the States. The 
overall benefit of today's rulemaking is that it will reduce costs and 
eliminate the confusion caused by the multiple versions of any one test 
method currently in use. For example, currently, an industry with 
facilities in six different states may be required to conduct six 
different versions of the same test method. EPA estimates that 
standardizing these approved methods could save the regulated community 
up to 20% of the current test method costs, which range from $160.00-
$2240.00, depending upon the test method. This rulemaking will also 
reduce the current resource burden in the States because they will no 
longer need to justify the inclusion of WET monitoring or WET limits in 
National Pollution Discharge Elimination System (NPDES) permits on a 
case-by-case basis.
    This rule incorporates three technical documents, by reference, 
thereby dramatically reducing the number of pages included in today's 
Federal Register. A listing of these documents and where they can be 
viewed or obtained can be found in section VIII of the preamble.
    Methods for measuring mutagenicity (changes in genes or 
chromosomes)or for monitoring viruses in wastewaters and sludges that 
were included in the December 1989 proposal are not included in this 
final rule. When better scientific methods for measuring mutagenicity 
and viruses become available, the Agency will evaluate them for 
possible inclusion in 40 CFR part 136. Finally, the methods for marine 
chronic toxicity in today's rule do not apply to discharges into marine 
waters of the Pacific Ocean. Methods addressing such discharges will be 
proposed at a later date.

EFFECTIVE DATE: This final rule becomes effective November 15, 1995. 
The incorporation by reference of certain publications listed in this 
regulation is approved by the Director of the Office of Federal 
Register on November 15, 1995.
    In accordance with 40 CFR 23.2, this rule shall be considered 
issued for the purposes of judicial review October 26, 1995, at 1 p.m. 
eastern daylight time. Under section 509(b)(1) of the Clean Water Act, 
judicial review of these amendments can be obtained only by filing a 
petition for review in the United States Court of Appeals within 120 
days after they are considered issued for the purposes of judicial 
review. Under section 509(b)(2) of the Clean Water Act, the 
requirements of these amendments may not be challenged later in civil 
or criminal proceedings to enforce these requirements.

ADDRESSES: The public record and all supporting materials pertinent to 
the development of this final rule, including response to comments 
received on the December 1989 proposal, are available for inspection at 
the Water Docket located at the U.S. Environmental Protection Agency, 
401 M Street SW., Washington, DC 20460. For access to the Docket 
materials, call (202) 260-3027 between 9 a.m. and 3:30 p.m. A listing, 
of where to view or obtain copies of the three manuals incorporated by 
reference in today's rulemaking, can be found in section VIII of the 
preamble.

FOR FURTHER INFORMATION CONTACT: Ms. Margarete A. Heber, Health and 
Ecological Criteria Division, Office of Science and Technology, (Mail 
Code 4304) U.S. Environmental Protection Agency, 401 M St. SW., 
Washington, DC 20460 or call (202) 260-0658; or Ms. Teresa Norberg-
King, Environmental Research Laboratory, U.S. Environmental Protection 
Agency, 6201 Congdon Boulevard, Duluth, MN 55804.

SUPPLEMENTARY INFORMATION

Table of Contents

I. Authority
II. Regulatory Background
    A. Analytical Methods under 40 CFR part 136
    B. Toxicity Testing
    C. EPA's Whole Effluent Toxicity (WET) Policy
    D. Proposed Rule Published December 4, 1989
III. Biological Methods Included in the Final Rule
    A. Basis for Approval
    B. Summary of Methods to Measure the Toxicity of Effluents and 
Receiving Waters to Freshwater and Marine Organisms
    1. Methods to Measure the Acute Toxicity of Effluent and 
Receiving Waters To Freshwater, Estuarine and Marine Organisms
    2. Short-Term Methods to Estimate the Chronic Toxicity of 
Effluents and Receiving Waters to Freshwater Estuarine and Marine 
Organisms
    (a) Short-Term Chronic Toxicity Test Methods for Freshwater 
Organisms
    (b) Short-Term Chronic Toxicity Test Methods for Estuarine and 
Marine Organisms
IV. Summary of Response to Comments for Aquatic Toxicity Tests
    A. Summary of Changes
    B. Effluent and Receiving Water Toxicity Tests with Fish and 
Aquatic Life
    1. Test Variability
    2. Quality Assurance/Quality Control (QA)/(QC)
    a. Existence of QA Guidelines for Toxicity Tests
    b. Reference Toxicant Tests
    3. Sample Collection, Holding Time and Temperature
    a. Sample Containers
    b. Sample Holding Time and Temperature
    4. Toxicity Testing Species
    a. Addition of the MICROTOXR Test System
    b. Indigenous (Feral) Test Organisms
    c. Supplemental Species
    5. Test Conditions
    6. Applicability of Tests
    a. Criteria for Test Selection
    b. Ceriodaphnia Test
    c. Test Validation in Receiving Waters
    d. Stage of Development of Toxicity Test Methods
    e. Ability of Laboratories to Perform the Arbacia and Champia 
tests
    C. Statistical Analysis of Results of Toxicity Tests with Fish 
and Other Aquatic Life
    D. Implementation and Miscellaneous Issues
VI. Regulatory Analysis
    A. Unfunded Mandate Reform Act of 1995
    B. Regulatory Flexibility Act
    C. Paperwork Reduction Act
    D. Executive Order 12866
VII. Materials to be Incorporated by Reference into 40 CFR Part 136
VIII. Public Availability of Materials Incorporated by Reference
IX. References

I. Authority

    EPA is promulgating this rule under the authority of sections 301, 
304(h), and 501(a) of the Clean Water Act (``CWA'' or the ``Act''), 33 
U.S.C. 1251 et seq., 33 U.S.C. 1311, 1314(h), 1361(a). Section 301 of 
the Act prohibits the discharge of any pollutant into navigable waters 
unless the discharge complies with certain requirements of the Act, 
including a requirement for a National Pollutant Discharge 

[[Page 53530]]
Elimination System (``NPDES'') permit issued pursuant to CWA section 
402. Section 304(h) of the Act requires the Administrator to 
``promulgate guidelines establishing test procedures for the analysis 
of pollutants that shall include the factors which must be provided in 
any certification pursuant to (CWA section 401) or permit applications 
pursuant to (CWA section 402).'' 33 U.S.C. 1314(h). Section 501(a) 
authorizes the Administrator ``to prescribe such regulations as are 
necessary to carry out his function under the Act.'' 33 U.S.C. 1361(a).

II. Regulatory Background

A. Analytical Methods Under 40 CFR Part 136

    The CWA establishes two principal bases for the incorporation of 
effluent limitations in NPDES permits. Effluent limitations implement 
both technology-based and water quality-based requirements of the Act. 
Technology-based limitations represent the degree of control that can 
be achieved using various levels of pollution control technology. In 
addition to the technology-based effluent limitations, the Act directs 
the states, with federal approval and oversight, to establish water 
quality-based standards to assure protection of the quality of state 
waters. The state standards designate uses for navigable waters and 
establish water quality criteria to protect such uses. If necessary to 
achieve compliance with applicable water quality standards, NPDES 
permits must contain water quality-based limitations more stringent 
than the applicable technology-based standards.
    To ensure compliance with these effluent limitations, EPA has 
promulgated regulations providing nationally-approved testing 
procedures in 40 CFR part 136. Approved analytical test procedures also 
must be used for the analysis of pollutants in permit applications, 
discharge monitoring reports, state certification under CWA section 
401, as well as determining compliance with pretreatment standards 
issued under CWA section 307. Test procedures have previously been 
approved for 262 different parameters (Table 1, 40 CFR 136.3). Approved 
test procedures apply to the analysis of bacteriological, inorganic 
(metal, non-metal, mineral, nutrient, demand, residue) and physical, 
non-pesticide organic, pesticide, and radiological parameters. Today's 
rule adds methods to the list of nationally-approved methods. 
Regulations also provide a mechanism for the approval of alternate 
analytical methods at 40 CFR 136.4. Under this regulation, the 
Administrator may approve alternate test procedures developed and 
proposed by dischargers or other persons.
    Finally, there may be discharges that require limitations for 
certain parameters using test procedures not yet approved under 40 CFR 
part 136. Under 40 CFR 122.41(j)(4) and 122.44(i)(1)(iv) permit writers 
may include, through permit proceedings, parameters requiring the use 
of test procedures that are not approved part 136 methods. EPA also may 
include such parameters in accordance with the provisions prescribed at 
40 CFR 401.13, ``Test Procedures for Measurements.'' Many of the whole 
effluent toxicity testing methods, incorporated by reference in today's 
rulemaking, have been included in NPDES permits utilizing the 
provisions in 40 CFR 122.41(j)(4). Today's rulemaking will relieve the 
NPDES permit writers of having to include these test methods on a case-
by-case-basis. By the same token, the test methods standardized in 
today's rule will replace unapproved test methods (or variations 
thereof) for NPDES permits issued after the effective date of today's 
rule. Existing NPDES permits need not be re-opened to include test 
methods from today's rule.

B. Toxicity Testing

    Until recently, EPA programs for the control of toxic discharges 
were based largely on effluent limitations for individual chemicals. 
EPA has developed water quality criteria for many pollutants based on 
comprehensive testing and evaluation that, unlike whole-effluent 
testing, considers a variety of toxic endpoints, including human health 
impact and bioaccumulation. Once a water quality criterion is 
developed, it can be used to develop a state numeric criteria within a 
water quality standard (40 CFR 131.11(b)) and/or permit limit to ensure 
that the level of that toxicant in the discharge does not exceed the 
water quality standard (40 CFR 122.44(d)(1)(iii) & (iv)).
    Data on the toxicity of substances to aquatic organisms, however, 
are available for only a limited number of elements and compounds. 
Effluent limitations on specific compounds, therefore, do not 
necessarily provide adequate protection for aquatic life when the 
toxicity of effluent components is not known, effects of effluent 
components are additive, synergistic, or antagonistic, and/or when an 
effluent has not been chemically characterized. In such situations, EPA 
and the States can use biological methods to examine the whole effluent 
toxicity, rather than attempt to identify all toxic pollutants, 
determine the effects of each pollutant individually, and then attempt 
to assess their collective effect.
    When whole effluent toxicity testing is used, toxicity itself is a 
pollutant parameter. The toxicants creating that toxicity need not be 
specifically identified to limit the effluent's toxicity. An analogy 
between effluent toxicity and biochemical oxygen demand (BOD) can be 
drawn. Both are measurements of a biological effect. Both can be 
quantified. In neither case are the causative agents of the biological 
effect specifically identified. Thus, whole effluent toxicity is like 
BOD in that it is a useful parameter for characterizing an undesirable 
effect caused by the discharge of a complex mixture of waste materials.
    The Declaration of Goals and Policy at Section 101(a)(3) of the Act 
states that ``it is the national goal that the discharge of toxic 
pollutants in toxic amounts be prohibited.'' Section 502 (13), 
describes toxic pollutants as ``* * * those pollutants, or combinations 
of pollutants, including disease-causing agents, which after discharge 
and upon exposure, ingestion, inhalation or assimilation into any 
organism, either directly from the environment or indirectly by 
ingestion through food chains, will, on the basis of information 
available to the Administrator, cause death, disease, physiological 
malfunctions, behavioral abnormalities, physical deformation, birth 
defects, genetic mutations, and cancer.'' Today's rule establishes 
procedures to measure some of these effects. Owners or operators of 
NPDES facilities may be required as a permit application or permit 
condition to perform one or more of these tests methods to assure 
compliance with relevant water quality standards. Both the D.C. and 
Ninth Circuit Courts of Appeals have recently upheld EPA's authority to 
set and measure limits on toxicity without regulating specific toxic 
pollutants (NRDC v. EPA 859 F.2d 156 (D.C. Cir. 1988); NRDC v. EPA 863 
F.2d 1426 (9th Cir. 1988).

C. EPA's Whole Effluent Toxicity (WET) Policy

    To achieve the goals of the Federal water pollution control 
legislation, extensive effluent toxicity screening programs were 
conducted during the 1970s by the EPA regional and state programs and 
permittees. Acute toxicity tests (USEPA, 1975, Methods for Acute 
Toxicity Tests with Fish, Macroinvertebrates, and Amphibians, National 
Water Quality Research 

[[Page 53531]]
Laboratory, Duluth, Minnesota; USEPA, 1978, Environmental Monitoring 
and Support Laboratory, USEPA, Cincinnati, Ohio, EPA/600/4-78/012) were 
used to measure effluent toxicity and to estimate the effects of toxic 
effluents on aquatic life in receiving waters. During this period, 
short-term inexpensive methods were not available to detect the more 
subtle, low-level, long-term (chronic), adverse effects (such as 
reduction in growth and reproduction, and occurrence of terata) of 
effluents on aquatic organisms. Rapid developments in toxicity test 
methods since 1980, however, have resulted in the availability of 
several methods that permit detection of the low-level, adverse effects 
(chronic toxicity) of effluents to freshwater and marine organisms in 
nine days or less.
    As a result of the increased awareness of the value of effluent 
toxicity test data for toxics control in the water quality program and 
the NPDES permit program, EPA issued a national policy statement 
entitled, ``Policy for the Development of Water Quality-Based Permit 
Limitations for Toxic Pollutants,'' in the Federal Register (49 FR 
9016, Mar. 9, 1984). This policy statement was updated in a document 
entitled, ``Whole Effluent Toxicity (WET) Control Policy,'' published 
by EPA in July 1994 (EPA 833-B-94-002).
    The policy recommended the use of toxicity data to assess and 
control the discharge of toxic pollutants to the nation's waters 
through the NPDES permits program. The policy stated: ``Biological 
testing of effluents is an important aspect of the water quality-based 
approach for controlling toxic pollutants. Effluent toxicity data, in 
conjunction with other data, can be used to establish control 
priorities, assess compliance with state water quality standards, and 
set permit limitations to achieve those standards.''
    The policy also addressed the technical approach for assessing and 
controlling the discharge of toxic pollutants to the nation's waters 
through the NPDES permit program, and discussed the application of 
chemical and biological methods for assuring the regulation of effluent 
discharges in accordance with federal and state requirements. The 
policy stated that ``EPA will use an integrated strategy consisting of 
both biological and chemical methods to address toxic and non-
conventional pollutants from industrial and municipal sources. In 
addition to enforcing specific discharge limits for toxic pollutants, 
EPA and the States will use biological techniques and available data on 
the biological effects of chemicals to assess toxicity impacts and 
human health hazards based on the general standards of `no toxic 
materials in toxic amounts'.''
    Additional guidance on the implementation of biomonitoring and the 
use of effluent and receiving water toxicity data is available in a 
technical support document published by the EPA Office of Water 
(``Technical Support Document for Water Quality-Based Toxics Control,'' 
March 1991, EPA/505/2-90/001; PB91-127415).
    Since the l984 Agency policy, the use of effluent toxicity tests 
has increased steadily within the EPA and State NPDES programs to 
identify toxic discharges, and by permittees as a self-monitoring tool 
(USEPA, 1979, Interim NPDES Compliance Biomonitoring Inspection Manual, 
Washington, DC). Regulatory authorities must now establish whole 
effluent toxicity limits where necessary to meet the requirements of 40 
CFR 122.44(d) (54 FR 23868, Jun. 2, 1989). The 1989 rule, which 
clarified EPA's Surface Water Toxics Control Program, defined ``whole 
effluent toxicity'' and described procedures for determining whether an 
NPDES permit must include a water quality-based effluent limitation. 
The regulation also addressed procedures for deriving effluent limits 
from state narrative or numeric water quality criteria. At that time, 
EPA noted that protocols and guidance documents used to perform 
toxicity tests were only recommended. With today's rule, when NPDES 
permits require whole effluent toxicity limits, testing must be 
conducted according to the toxicity test protocols described in the 
test manuals cited in Table IA, 40 CFR part 136, as amended (except for 
chronic toxicity limitation for discharges into marine waters of the 
Pacific Ocean).
    The Environmental Monitoring Systems Laboratory--Cincinnati (EMSL-
Cincinnati) developed standard test procedures and published 
standardized acute and chronic toxicity tests methods to minimize 
intralaboratory and interlaboratory variability in toxicity tests 
conducted by EPA regional and state programs and NPDES permittees.

D. Proposed Rule Published December 4, 1989

    On December 4, l989, EPA proposed at 54 FR 50216 to add the 
following methods to Table IA, 40 CFR part 136: (1) Methods to measure 
the acute toxicity of effluents and receiving waters to freshwater and 
marine organisms, (2) short-term methods to estimate the chronic 
toxicity of effluents and receiving waters to freshwater, estuarine, 
and marine organisms, (3) methods to measure the mutagenicity 
(genotoxicity) of wastewaters, sludges, and surface waters, and (4) 
methods to recover, enumerate, and identify human enteric viruses in 
wastewater, sludges, and surface waters. Changes were also proposed for 
Table II, on sample preservation and holding times. EPA provided a 60-
day public comment period.
    In response to the Proposed Rule, comments were received from a 
broad cross-section of public and private agencies, including major 
trade organizations, large industries, large environmental consulting 
firms, universities, state and interstate water pollution control 
agencies, and other Federal agencies. A summary of the major comments 
concerning acute and chronic testing for freshwater and marine 
organisms, and EPA's responses to them, are addressed below. Responses 
to the remainder of the comments are contained in the Supplementary 
Information Document (SID) portion of the rulemaking record. The entire 
Water Docket is available for inspection from 9 to 3:30 p.m. at 401 M 
St SW., Washington DC 20460. Call (202) 260-3027 for an appointment.
    In addition, the Agency decided not to finalize the test methods 
proposed to measure the mutagenicity (genotoxicity) of wastewaters, 
sludges, and surface waters; and methods to recover, enumerate, and 
identify human enteric viruses in wastewater, sludges, and surface 
waters. In the mid 1980s, the Agency believed that a simple test like 
the Ames test could be used as a predictor of chronic health effects 
(i.e. carcinogenicity). However, this test produces many false results, 
and, thus, could potentially confuse or mislead regulators. Presently, 
the Agency is working on different methods to recover, enumerate, and 
identify human enteric viruses, and so the methods proposed are no 
longer representative of the best available science.

III. Biological Methods Included in the Final Rule

A. Basis for Approval

    Many of the comments received on the proposed rule were helpful in 
identifying ambiguities and minor inconsistencies in the aquatic 
toxicity test methods which had been published at different times 
during the seven years preceding the proposal. This was particularly 
true with regard to the comment received from numerous commenters to 
reformat the three manuals to make them both consistent with each other 
and easier to use. The biological methods added to Table IA, 40 CFR 
part 136, in this final rule are 

[[Page 53532]]
described below, and are included in the rulemaking docket.
    The tests have been validated in a number of studies conducted by 
EPA, state programs, and universities. The methods are well established 
and are currently being implemented in a number of NPDES permits. 
Furthermore, each of the methods has extensive guidance on quality 
assurance and routine quality control activities.
    Information on the single laboratory precision of the methods is 
included in the respective short-term test manuals in the rulemaking 
docket. The methods in this rule have precision profiles comparable to 
previously established part 136 methods. The Agency stands behind the 
conclusion that the biological methods in this rule are applicable for 
use in NPDES permits.

B. Summary of Methods to Measure the Toxicity of Effluents and 
Receiving Waters to Freshwater and Marine Organisms

    The three aquatic toxicity test manuals cited at 54 FR 50216 have 
been revised as a result of public comment on the proposed rule. The 
revised editions, discussed below, are as follows: (1) USEPA. 1993. 
Methods for Measuring the Acute Toxicity of Effluents and Receiving 
Waters to Freshwater and Marine Organisms, Fourth Edition, EPA/600/4-
90/027F; (2) USEPA. 1994. Short-term Methods to Estimate the Chronic 
Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 
Third Edition, July 1994, EPA/600/4-91/002; and (3) USEPA. 1994. Short-
term Methods to Estimate the Chronic Toxicity of Effluents and 
Receiving Waters to Estuarine and Marine Organisms, Second Edition, 
July 1994, EPA/600/4-91/003.
1. Methods to Measure the Acute Toxicity of Effluents and Receiving 
Waters to Freshwater Estuarine and Marine Organisms
    This rule includes methods to measure the acute toxicity of 
effluents and receiving waters to freshwater and marine fish and 
invertebrates, as described in the EPA methods manual, Methods for 
Measuring the Acute Toxicity of Effluents and Receiving Waters to 
Freshwater and Marine Organisms (EPA/600/4-90/027F). This methods 
manual represents the fourth edition of the acute toxicity test manual 
first published by EMSL-Cincinnati in 1978 (EPA/600/4-78/012). The 
methods, developed with the assistance of the Agency's Toxicity 
Assessment Subcommittee of the Biological Advisory Committee, are 
periodically updated, expanded, and republished. Any such changes, 
however, will be published in the Federal Register prior to their 
effective date for regulatory purposes. The most recent (third) edition 
was published in 1985 (EPA/600/4-85/013).
    The current manual (EPA/600/4-90/027F) describes tests for 
effluents and receiving waters, and includes guidelines for the 
following areas: Laboratory safety; quality assurance; facilities and 
equipment; effluent sampling and holding times; dilution water; test 
species selection, culturing, and handling; data collection, 
interpretation and utilization; report preparation; and dilutor and 
mobile toxicity test laboratory design.
    The acute toxicity tests in the manual generally involve exposure 
of any of 20 test organisms to each of five effluent concentrations and 
a control water. The test duration depends on the objectives of the 
test and the test species, and ranges from 24-96 hours. The manual 
includes a list of freshwater and marine test organisms, and specified 
test conditions for 10 commonly used freshwater and marine organisms--
Ceriodaphnia dubia, Daphnia magna, Daphnia pulex, fathead minnows 
(Pimephales promelas), rainbow trout (Oncorhynchus mykiss), brook trout 
(Salvelinus fontinalis), mysids (Mysidopsis bahia and Holmesimysis 
costata), Bannerfish shiners (Notropis leedsi), sheepshead minnows 
(Cyprinodon variegatus), and silversides (Menida menidia, M. beryllina, 
and M. peninsulae). The organisms and test conditions are selected by 
the user (e.g. permitting authority for NPDES permits) depending on the 
objectives of the test and the effluent and receiving water 
characteristics.
    The tests are used to determine the effluent concentration, 
expressed as a percent volume, that within the prescribed test period 
causes death in 50% of the organisms (LC50), or whether survival in a 
given (single) concentration of effluent, or in receiving water, is 
significantly different than in controls. Where death is not easily 
detected, e.g., with some invertebrates like Ceriodaphnia and Daphnia, 
immobilization is considered equivalent to death. Procedures for 
determining the LC50 include the graphical method, the Probit method 
and the trimmed Spearman-Karber method. Where survival in a single 
effluent concentration or in receiving water is compared to survival in 
the control to determine if they are significantly different, a 
hypothesis test, Dunnett's Test, is used. Copies of computer programs 
for statistical analysis of the data referred to in the manual are 
available from EMSL-Cincinnati.
    End-of-the-pipe effluent toxicity data are used to predict 
potential acute and chronic toxicity of effluents in the receiving 
water, based on the LC50 and appropriate dilution, application, and 
persistence factors. The tests can be conducted as a part of self-
monitoring permit requirements, compliance evaluation inspections, 
compliance biomonitoring inspections, compliance sampling inspections, 
toxics sampling inspections, performance audit inspections, and special 
investigations. The tests can be performed in a central test laboratory 
or on-site by the regulatory agency or the permittee. Acute toxicity 
tests can be used in toxicity reduction evaluations to identify toxic 
waste streams within plants, to aid in the development and 
implementation of toxicity reduction plans, and also can be used to 
compare and control the effectiveness of various treatment technologies 
for a given type of industry, irrespective of the receiving water (49 
FR 9016, Mar. 9, 1984).
    Several types of acute toxicity tests are described, including 
static non-renewal, static renewal, and flow-through. The selection of 
the test type will depend upon the objectives of the test, available 
resources, requirements of the test organisms, and effluent 
characteristics, such as fluctuations in effluent toxicity. Special 
environmental requirements of some organisms (such as flowing water, or 
fluctuating water levels) may preclude the use of static tests.
    Static tests include: (1) Non-renewal tests in which the test 
organisms are exposed to the same effluent solution or receiving water 
for the duration of the test, and, (2) renewal tests in which the 
organisms are exposed to a fresh test solution every 24 hours or other 
prescribed interval, either by transferring the test organisms from one 
test chamber to another or by replacing all or a portion of the 
effluent solution in the test chambers. Sample renewal reduces some of 
the possible effects of factors which may affect the apparent toxicity 
of the effluent, such as toxicant adsorption on the walls of the test 
chambers, biodegradation and/or chemical transformation of the 
toxicants, volatilization, and uptake and metabolism of toxicants by 
test organisms.
    Two types of flow-through tests are described: (1) Effluent is 
pumped continuously from the sampling point directly to the dilutor 
system; and (2) effluent grab or composite samples are collected 
periodically, placed in a tank adjacent to the test laboratory, and 

[[Page 53533]]
pumped continuously from the tank to the dilutor system. The flow-
through method employing continuous effluent sampling is the preferred 
method for on-site tests. Because of the large volume (often 400 L/day) 
of effluent normally required, flow-through tests are generally 
considered too costly and impractical to conduct at off-site 
laboratories.
    Parameters and Units:
    The results of the test are reported as the LC50 (Lethal 
Concentration--50), which is the concentration of effluent causing 
death (or immobilization, or other adverse effect) in 50% of the test 
organisms or, in the case of single concentration tests, a 
statistically significant increase in lethality in the effluent sample 
as compared to the control.
    Precision:
    Data on single laboratory precision (intra-) and multi-laboratory 
(inter-) precision from tests with reference toxicants are provided in 
the manual (EPA/600/4-90/027F).
2. Short-Term Methods to Estimate the Chronic Toxicity of Effluents and 
Receiving Waters to Freshwater, Estuarine, and Marine Organisms
    Today's rule includes two sets of short-term chronic toxicity test 
methods: (1) Four methods for freshwater organisms and (2) six methods 
for estuarine and marine organisms, found in the EPA methods manuals, 
Short-term Methods for Estimating the Chronic Toxicity of Effluents and 
Receiving Waters to Freshwater Organisms, Third Edition (EPA/600/4-91/
002) July 1994, and Short-term Methods for Estimating the Chronic 
Toxicity of Effluents and Receiving Waters to Estuarine and Marine 
Organisms, Second Edition (EPA/600/4-91/003) July 1994, respectively. 
The tests are used to estimate one or more of the following: (1) The 
chronic toxicity of effluents collected at the end of the discharge 
pipe and tested with a standard dilution water; (2) the chronic 
toxicity of effluents collected at the end of the discharge pipe and 
tested with dilution water consisting of receiving water collected 
upstream or beyond the influence of the outfall, or with other 
uncontaminated surface water or standard dilution water having 
approximately the same hardness or salinity as the receiving water, 
depending on the nature of the receiving water (fresh or saline) and 
test organisms; (3) the toxicity of diluted effluent in the receiving 
water downstream or at increasing distance from the outfall; and (4) 
the effects of multiple discharges on the quality of the receiving 
water. The tests may also be useful in developing site-specific water 
quality criteria.
    The use of short-term, subchronic, and chronic toxicity tests in 
the NPDES Program is recommended in the 1984 EPA policy on water-
quality based permit limits, and subsequently can be required under 40 
CFR 122.44(d). The short-term chronic methods are more effective 
analytical tools because they provide a more comprehensive prediction 
of the effects of toxic effluents on aquatic life in receiving waters 
than is provided by acute toxicity tests, at a greatly reduced level of 
effort compared to earlier chronic toxicity test methods (i.e. fish 
full-life-cycle chronic and 30-day early life-stage tests, and the 21- 
to 28-day invertebrate life-cycle tests). The endpoints generally used 
in chronic tests are survival, growth, and reproduction. The effects 
include the synergistic, antagonistic, and additive effects of all the 
chemical, physical, and biological components that adversely affect the 
physiological and biochemical functions of the test organisms.
    (a) Short-Term Chronic Toxicity Test Methods for Freshwater 
Organisms. The approved toxicity test methods for freshwater organisms 
are found in the manual, Short-Term Methods for Estimating the Chronic 
Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 
Third Edition (EPA/600/4-91/002) July 1994. The manual describes four- 
to seven-day methods for estimating the chronic toxicity of effluents 
and receiving waters to three species: (1) The fathead minnow, 
Pimephales promelas; (2) the cladoceran, Ceriodaphnia dubia; and (3) 
the alga, Selenastrum capricornutum.
    Guidelines are also included on laboratory safety, quality 
assurance, facilities and equipment, dilution water, effluent sampling 
and holding, data analysis, report preparation, and organism culturing 
and handling. Copies of computer programs for statistical analysis of 
the data referred to in the manual, are available from EMSL-Cincinnati. 
The approved short-term chronic tests are:

METHOD 1000.0:

    Fathead Minnow (Pimephales promelas) Larval Survival and Growth 
Test. Larvae (preferably less than 24 hours old) are exposed in a 
static renewal system to a control water and at least five 
concentrations of effluent, or to receiving water for seven days. Test 
results are determined on the survival and weight of the larvae in test 
solutions, compared to the controls.

METHOD 1001.0:

    Fathead Minnow (Pimephales promelas) Embryo-larval Survival and 
Teratogenicity Test. Fathead minnow embryos are exposed in a static 
renewal system to a control water and at least five different 
concentrations of effluent, or to receiving water, from shortly after 
egg fertilization to hatch, and the larvae are exposed an additional 
four days posthatch (total of eight days). Test results are determined 
on the combined frequency of both mortality and gross morphological 
deformities (terata) in test solutions, compared to the controls. The 
test is useful for screening for teratogens because organisms are 
exposed during embryonic development.

METHOD 1002.0:

    Ceriodaphnia dubia Survival and Reproduction test. Ceriodaphnia 
neonates are exposed to a control water and at least five different 
concentrations of effluent, or to receiving water, in a static renewal 
system until 60% of control females have three broods of young, or a 
maximum of 8 days. Test results are based on survival and reproduction 
in test solutions, compared to the controls.

METHOD 1003.0:

    Algal (Selenastrum capricornutum) Growth Test. A Selenastrum 
population is exposed to a control water and to at least five different 
concentrations of effluent, or to receiving water, in a static system, 
for 96 hours. The test results are determined by the population 
responses in test solutions in terms of changes in cell density (cell 
counts per milliliter), biomass, chlorophyll content, or absorbance, 
compared to the controls.
    Toxicity Test Endpoints. The endpoints for the freshwater short-
term chronic toxicity tests with effluents and receiving waters are 
summarized as: (1) The NOEC, which is the highest percent effluent 
concentration at which no adverse effect on survival, growth, or 
reproduction is observed, and (2) the IC25 (Inhibition Concentration, 
25%), which is the effluent concentration at which growth or 
reproduction are reduced 25% from that of controls. Although both 
endpoints are permissible, EPA recommends the IC25 endpoint for 
regulatory use.
    The precision of the freshwater chronic toxicity tests is discussed 
in the respective methods sections in the methods manual (EPA/600/4-91/
002). NOECs from repetitive tests generally fall within one 
concentration interval of the median value, and when measured with the 
IC25, the precision is generally 

[[Page 53534]]
in the range of 30-60%. Precision can be improved by decreasing the 
concentration interval around the median value. This is accomplished by 
adding more concentration on either side of the median value.
    (b) Short-Term Chronic Toxicity Test Methods for Estuarine and 
Marine Organisms. The approved short-term chronic toxicity tests for 
estuarine and marine organisms are contained in the manual, Short-term 
Methods for Estimating the Chronic Toxicity of Effluents and Receiving 
Waters to Estuarine and Marine Organisms, Second Edition, July 1994 
(EPA/600/4-91/003). This manual describes six short-term (one-hour to 
nine-day) methods for estimating the chronic toxicity of effluents and 
receiving waters to five species: The sheepshead minnow, Cyprinodon 
variegatus; the inland silverside, Menidia beryllina; the mysid shrimp, 
Mysidopsis bahia; the sea urchin, Arbacia punctulata; and the red 
macroalga, Champia parvula.
    The marine chronic toxicity tests in today's rule do not apply to 
discharges into marine waters of the Pacific Ocean. Toxicity tests for 
such discharges will continue to be specified in NPDES permits on a 
case-by-case basis. EPA intends to propose standardized toxicity test 
methods based on the methods developed by the States and EPA 
laboratories on the Pacific Coast.
    Guidelines are included on laboratory safety, quality assurance, 
facilities and equipment, dilution water, effluent sampling methods and 
holding times and temperatures, data analysis, report preparation, and 
organism culturing and handling. Copies of computer programs for 
statistical analysis of the data referred to in the manual are 
available from EMSL-Cincinnati. The approved short-term chronic tests 
are:

METHOD 1004.0:

    Sheepshead Minnow (Cyprinodon variegatus) Larval Survival and 
Growth Test. Larvae (preferably less than 24 hours old) are exposed in 
a static renewal system to a control water and at least five 
concentrations of effluent, or to receiving water for seven days. Test 
results are determined on the survival and weight change of the larvae 
in test solutions, compared to the controls.

METHOD 1005.0:

    Sheepshead Minnow (Cyprinodon variegatus) Embryo-larval Survival 
and Teratogenicity Test. Sheepshead minnow embryos are exposed in a 
static renewal system to a control water and at least five different 
concentrations of effluent, or to receiving water, from shortly after 
fertilization of the eggs to hatch, and the larvae are exposed for an 
additional four days posthatch (total of nine days). Test results are 
determined based on the combined frequency of both mortality and gross 
morphological deformities (terata) in the test solutions, compared to 
the controls. The test is useful in screening for teratogens because 
organisms are exposed during embryonic development.

METHOD 1006.0:

    Inland silverside (Menidia beryllina), Larval Survival and Growth 
Test Larvae (preferably 7-11 days old) are exposed in a static renewal 
system to a control water and at least five concentrations of effluent, 
or to receiving water for seven days. Test results are determined on 
the survival and weight change of the larvae in the test solutions, 
compared to the controls.

METHOD 1007.0:

    Mysidopsis bahia Survival, Growth, and Fecundity Test. Seven-day 
old mysids are exposed in a static renewal system to a control water 
and at least five different concentrations of effluent, or to receiving 
water for seven days. Test results are determined on survival, growth, 
and egg production (fecundity) of the mysids in the test solutions, 
compared to the controls.

METHOD 1008.0:

    Arbacia punctulata Fertilization Test. Arbacia sperm are exposed 
one hour in a static system to control medium and at least five 
concentrations of effluent, or to receiving water. Eggs are then added 
to the sperm and both are exposed for an additional 20 minutes. The 
response is measured in terms of the percent fertilization of the eggs 
compared to the control.

METHOD 1009.0:

    Champia parvula Reproduction Test. Branches of male and female 
plants are placed together for 48 hours in a static system and exposed 
to a control medium and at least five concentrations of effluent, or in 
receiving water. The exposed plants are then transferred to control 
medium for a recovery period of 5-7 days. After the recovery period, 
the numbers of reproductive structures (cystocarps) that develop on the 
female plants as a result of fertilization in the test solutions are 
compared to the controls.
    Test Endpoints. The endpoints for the estuarine and marine short-
term chronic toxicity tests with effluents and receiving waters 
include: (1) The NOEC, which is the highest percent effluent 
concentration at which no adverse effect on survival, growth, or 
reproduction is observed, and (2) the IC25 (Inhibition Concentration, 
25%), which is the effluent concentration at which growth or 
reproduction are reduced 25% from that of controls. Although both 
endpoints are permissible, EPA recommends the IC25 endpoint for 
regulatory use.
    The precision of the chronic toxicity tests is discussed in the 
respective methods sections in the manual (EPA/600/4-91/003). NOECs 
from repetitive tests generally fall within one concentration interval 
of the median value. The precision of these test methods is also given 
in the Technical Support Document (second edition) that provides 
additional data points.

IV. Summary of Response to Comments for Aquatic Toxicity Tests

    This section of the preamble summarizes the changes to the three 
methods manuals and significant comments received. The rest of the 
comments are summarized in the Supplementary Information Document (SID) 
which is available in the Water Docket.

A. Summary of Changes

    One of the most commonly mentioned comments in the proposal was to 
have all three manuals formatted similarly, so that the documents would 
be easier to use. The three documents incorporated by reference in this 
rulemaking are now formatted in the same way, and as a result, are more 
``user friendly''.
    With this rule, several technical and editorial changes are made in 
the manual, Methods for Measuring the Acute Toxicity of Effluents and 
Receiving Waters to Freshwater and Marine Organisms, to respond to 
public comments on the Proposed Rule, December 4, 1989, and to make 
certain technical and policy language consistent with the revised 
freshwater and marine short-term chronic toxicity test manuals (EPA/
600/4-91/002, EPA/600/4-91/003). Most of the substantive method changes 
made pursuant to public comment were made in the acute toxicity manual. 
Changes to the chronic toxicity manuals were largely related to format 
and consistency between the manuals. Briefly the changes are explained 
below.
    Two paragraphs have been added to the introduction. The first 
paragraph cautions against making unauthorized changes in the methods, 
and the second paragraph makes a statement about experience needed by 
users of the methods. In Section 7, on the selection of dilution water 
for tests, ``ground water'' is added as an acceptable 

[[Page 53535]]
``natural'' water. In Section 8, on sample collection and handling, the 
description of sample ``holding time'' was expanded, but holding 
conditions and limits on sample holding time were not changed. In 
Section 9, on toxicity test procedures, an explanation was added on how 
an increase in pH during a toxicity test can be reduced or avoided by 
using a static renewal or flow-through approach. In Section 9, on 
toxicity test procedures, one footnote was added to each of two tables 
of test summary conditions, listing an additional species that could be 
used with the test conditions. These changes were made in response to 
comments on the proposed rule.

B. Effluent and Receiving Water Toxicity Tests with Fish and Aquatic 
Life

1. Test Variability
    Comment: Toxicity test results are too variable, and methods are 
not sufficiently well standardized or validated with round robin data 
to include in 40 CFR part 136.
    Response: EPA agrees that methods approved under part 136 should be 
validated scientifically. Further, EPA recognizes that an 
interlaboratory study (round robin) provides a useful and desirable 
means of validating an analytical method. However, EPA does not 
consider such a study to be a requirement for approval under Part 136 
for a variety of reasons. First, prior to each interlaboratory study 
conducted with aquatic toxicity tests methods, EPA conducted 
intralaboratory studies that demonstrated similar, satisfactory 
precision. Where the Agency does not have interlaboratory data for a 
species, adequate data on intralaboratory precision are available. 
Second, quality assurance and quality control procedures specified in 
the toxicity test methods manuals are designed to minimize any 
variability due to analyst error or stress in test cultures due to 
factors other than effluent toxicity. Finally, the toxicity test 
methods specify a procedure for a series of initial repetitive tests to 
ensure that laboratory results during any particular analysis establish 
a pattern of satisfactory performance and define that laboratory's 
intralaboratory variability.
    EPA does consider the precision of candidate methods in approving 
such methods under part 136. The essential criterion is that the 
precision of the methods fall within the approximate range of other 
Agency methods (including those in part 136), and that approved methods 
provide valid results. For some of the chemical-specific methods, e.g., 
for manganese, the variability at the low end of the measurement 
detection range exceeds that of the toxicity test methods. Compare 
Technical Support Document for Water Quality-based Toxics Control at 3, 
Table 1-3 (EPA/505/2-90-001). A large amount of intra- and inter-
laboratory precision data are available on the toxicity tests approved 
in today's rule, and representative data sets are included in the 
methods manuals. On the basis of these data, EPA is comfortable with 
the conclusion that whole effluent toxicity tests are no more variable 
than chemical analytical methods in Part 136 and, therefore, stands 
behind the conclusion that toxicity tests in NPDES permits provide 
reliable indicators of whole effluent toxicity.
2. Quality Assurance/Quality Control (QA/QC)
    Some commenters expressed the opinion that the Agency's QA 
requirements were excessively time-consuming and costly, whereas other 
commenters stated that the requirements were too lenient. See the SID 
for additional QA/QC information, such as the requirements for five 
initial toxicity tests, cleaning labware and apparatus, and food 
quality. The major comments on QA were as follows:
a. Existence of QA Guidelines for Toxicity Tests
    Comment: The proposed methods do not contain the necessary QA 
protocols.
    Response: EPA disagrees. Each of the toxicity test methods manuals 
incorporated by reference into Table IA, 40 CFR part 136, contains 
separate, detailed, QA/QC guidelines, and each analytical method within 
these manuals discusses all aspects of the tests which relate to QA/QC.
b. Reference Toxicant Tests
    Comment: The requirement for monthly chronic QA tests of the 
sensitivity of organisms cultured within the laboratory is excessive. 
Monthly acute tests, or monthly acute and quarterly chronic tests for 
such organisms should be sufficient.
    Response: EPA believes that the condition of organisms produced in 
``in house'' laboratory cultures can change rapidly, requiring monthly 
verification of test organism sensitivity with the appropriate acute 
and/or short-term chronic toxicity test(s), using reference toxicants. 
Without this assessment, changes in the cultures can lead to less 
precision in the tests. It is sufficient to use a single reference 
toxicant with one or all test species (e.g., sodium chloride, potassium 
chloride, sodium dodecyl sulfate, or other suitable substance). The 
tests can be limited to acute toxicity tests if the laboratory performs 
only acute tests with effluents and receiving waters. However, EPA does 
not agree that acute tests can be used instead of short-term chronic 
tests for the monthly verification of the sensitivity of test organisms 
to be used in short-term chronic tests with effluents and receiving 
waters.
    Comment: Where effluent and reference toxicant tests are performed 
concurrently with organisms from the same batch shipped to a 
laboratory, and only the reference toxicant test is invalid (e.g., for 
failure to meet acceptability criteria or control chart limits), the 
permittee should not be required to repeat both the effluent toxicity 
and reference toxicant tests.
    Response: EPA believes that the probability that an effluent 
toxicity test could be valid when the side-by-side reference toxicant 
test does not meet acceptability criteria is very slight. Under these 
circumstances, therefore, the results of both tests are rejected and 
the tests must be repeated.
    If the reference toxicant test meets the acceptability criteria but 
the results fall outside the control limits, the results of both the 
reference toxicant and effluent tests should be considered provisional 
and subject to careful review. Good laboratories that have developed 
very narrow control limits may be unfairly penalized if test results 
that fall outside the control limits are rejected. For this reason, the 
width of the control limits should be considered by the permitting 
authority in determining if the reference toxicant and effluent 
toxicity data should be rejected on the basis of the control chart 
limits.
    The requirement for side-by-side reference toxicant tests with 
shipped organisms could be waived if the test organism supplier 
provides reference toxicant and control charts data from monthly tests 
conducted with young from the same source cultures during the previous 
five-month period, using the same reference toxicants and same toxicity 
test conditions.
    Comment: EPA should provide guidance on the acceptable performance 
of each reference toxicant (e.g., as it has done with chemical QC 
samples).
    Response: EPA believes that the laboratory conducting the WET tests 
should derive response data by conducting a range-finding test prior to 
the definitive test. Accuracy of toxicity test results cannot be 
ascertained, only the precision of toxicity can be estimated, therefore 
it is not appropriate to provide such information. 

[[Page 53536]]

    Comment: EPA should provide reference toxicants and standard test 
organisms.
    Response: The Agency is currently divesting itself from the 
production and distribution of QC materials for chemical methods and 
transferring those tasks to the private sector under cooperative 
research and development agreements (CRADAs) authorized by the Federal 
Technology Transfer Act of 1986, (Pub.L. No. 99-502). However, 
biological QC materials, such as reference toxicants and reference 
Artemia cysts, are still available in limited quantity from the Quality 
Assurance Research Division, Environmental Monitoring Systems 
Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268. 
Further information can be obtained by writing to the laboratory or 
calling 513-569-7325.
    Adequate supplies of test organisms are currently available from 
the private sector, and the market place has and is expected to respond 
quickly to any increased demand for test organisms.
3. Sample Collection, Holding Time and Temperature
a. Sample Containers
    Comment: Glass sample containers should be used instead of plastic 
containers because there is less adsorption of toxics from the samples. 
However, plastic sample containers would be acceptable if the users are 
warned of this problem.
    Response: The use of plastic containers for collection and shipment 
of effluent samples is preferred over glass bottles, which are more 
easily broken during shipment. It must be recognized, however, that the 
loss of toxics from samples (and possible reduction in toxicity) by 
adsorption to plastic surfaces may be greater with plastic containers 
than with glass ones. Prolonged storage of samples in plastic 
containers before use, therefore, should be avoided to the extent 
possible.
b. Sample Holding Time and Temperature
    Comment: The sample holding time (36 hours) prior to the start of 
the toxicity test is too restrictive.
    Response: EPA believes that 36 hours provides sufficient time to 
deliver the samples to the performing laboratories in most cases. In 
the isolated cases where the permittee can document that this delivery 
time cannot be met, the permitting authority may allow an option for 
on-site testing, or a variance to extend the holding time. The request 
for a variance in sample delivery time (directed to the Regional 
Administrator under 40 CFR 136.4 and 40 CFR 136.5) must include 
supportive data which show that the toxicity of the effluent sample is 
not reduced (e.g., because of biodegradation, chemical transformation, 
volatilization and/or sorption of toxics on the sample container 
surfaces) by extending the holding time beyond 36 hours. In no case 
should more than 72 hours elapse between collection and first use of 
the sample.
    Comment: Current guidance on sample collection in the toxicity test 
manuals does not clearly indicate when sample holding time begins.
    Response: EPA agrees and provides the following clarification in 
the manual. Sample holding time begins when the last grab sample in a 
series is taken (e.g., when a series of four grab samples are taken 
over a 24 hours period), or when a 24 hours composite sampling period 
is completed.
    Comment: It is not possible to regularly maintain a sample 
temperature of 4  deg.C during sample shipment.
    Response: EPA agrees that the requirement to maintain sample 
temperature at 4  deg.C may be difficult to achieve. However, the 
temperature requirement is important to minimize possible loss of 
toxicity due to chemical transformations and microbial degradation 
during transit and holding. Sufficient ice should be placed with the 
samples in the shipping container to ensure that ice is still present 
when the samples arrive at the laboratory. However, even if ice is 
present when a sample arrives at the laboratory, the analyst should 
measure and record the temperature of the samples to confirm that the 4 
 deg.C temperature maximum has not been exceeded. In the isolated cases 
where the permittee or the analyst can document that the 4  deg.C 
shipping temperature cannot be met, the permittee can be given the 
option of on-site testing or can request a variance in sample shipping 
temperature. The request for a variance must include supportive data to 
demonstrate that the toxicity of the effluent samples is not reduced 
when the holding temperature is increased to the level proposed.
4. Toxicity Testing Species
a. Addition of the MICROTOXR Test System
    Comment: Many commenters requested the inclusion of and provided 
information on a toxicity test known as the MICROTOXR Luminescent 
Bacteria Toxicity Test using the organism, Photobacterium phosphoreum. 
Information supplied included performance characteristics of the method 
and its use. Commenters urged inclusion of the test because of its 
alleged simplicity, cost effectiveness, reproducibility, and widespread 
use. One commenter suggested use of the method for compliance testing, 
toxicity reduction evaluations, and pretreatment evaluations.
    Response: While EPA agrees that MICROTOXR is a relatively 
rapid and simple test system that can provide data useful for in-plant 
toxicity screening, today's rule does not include any test methods to 
measure the toxic effect of effluent on bacteria. Consistent with the 
public notice in the proposed rule and the test manuals incorporated by 
reference therein, today's final rule only includes methods that 
measure toxicity to representative species from certain phylogenetic 
groups: i.e., fish, invertebrates, and algae. Information available to 
the Agency does not, at this time, indicate that the MICROTOXR 
test system provides an acceptable, sensitive indicator of the toxic 
effects of effluents to the fish, invertebrates, or algae included in 
the test methods promulgated today.
    The Agency hastens to add, however, that today's rule does not 
restrict the use of the MICROTOXR test as an additional or 
supplemental test method for use in states with federally-approved 
NPDES programs. EPA also notes that tests such as MICROTOXR may 
provide the permittee the additional benefit of a diagnostic tool for 
the purposes of in-plant toxicity screening for the protection of 
biological (microbial) treatment processes. Under EPA regulations, when 
a permittee conducts any testing required by the permit using an 
analytical method approved in 40 CFR part 136, all test results must be 
reported (40 CFR 122.41(l)(4)(ii)). Thus, a diagnostic test not 
included in 40 CFR part 136 provides permittees with the opportunity 
for internal effluent evaluation undisclosed to the permitting 
authority. The Agency notes, however, that results of any biological 
testing of ``end-of-pipe'' discharge or receiving waters must be 
reported in subsequent permit applications.
b. Indigenous (Feral) Test Organisms
    Comment: The use of indigenous species from the receiving water 
should be allowed in effluent toxicity tests.
    Response: The use of feral (feral indicates wild) indigenous 
species from the receiving water is not allowed due to lack of control 
in the quality of the test organisms, including such factors as range 
in age, possible previous exposure to contaminants, disease, and injury 
during collection, all of which might 

[[Page 53537]]
significantly affect organism sensitivity to toxicants, and the 
precision and reproducibility of the test. However, the above 
discussion does not mean that EPA is adverse to persons developing 
credible toxicity methods based on other organisms, including methods 
based on organisms indigenous to specific surface waters. These 
toxicity methods would need to include QA/QC provisions that assure a 
proper level of precision and reproducibility, and would need to use 
test organisms cultured in a laboratory that are unaffected by 
environmental stresses. Such methods could be submitted for approval as 
an alternative test procedure (40 CFR 136.4 (a) and (d)).
c. Supplemental Species
    Comment: Some commenters noted that some State laws prohibit the 
import of non-indigenous species. One commenter noted that the list of 
recommended test species in the acute toxicity test manual (EPA/600/4-
90/027) did not include any test species indigenous to Pacific coastal 
waters. The commenter provided data from side-by-side testing 
(Homesimysis costata) suggesting that a west coast test species (that 
the commenter thought should be included) was at least as sensitive to 
toxicity as one of the test species recommended in the acute manual. 
The State of California expressed concern that test methods it had 
developed and has been including in NPDES permits would be displaced by 
today's rule.
    Response: The species selected by EPA for effluent toxicity tests 
in the NPDES program represent a ``performance standard'' or indicator 
of sensitivity to toxicity for a given phylogenetic category. 
Therefore, to use a species other than the recommended species, the 
permittee or the permitting authority should provide data from side-by-
side testing showing that the proposed substitute test species is at 
least as sensitive as the recommended test species for that 
phylogenetic category.
    Toxicity test methods will not require use of non-indigenous test 
organisms when State law prohibits import of such species. However, the 
toxicity test manuals provide instructions for the disposal of test 
organisms and, if these instructions are followed, the use of non-
indigenous organisms will not result in establishment of populations of 
these organisms in local waters that will threaten indigenous wildlife.
    Appendix B in the acute toxicity test manual (EPA/600/4-90/027F) 
contains a list of ``supplemental'' test species that may be 
appropriate for use in acute toxicity testing under certain test 
conditions. EPA accepts the use of Notropis leedsi (Bannerfish Shiner) 
in place of Pimephales promelas (Fathead Minnow), if the same test 
conditions are used, and the use of the mysid, Homesimysis costata, in 
place of Mysidopsis bahia, with the same test conditions except at a 
temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C, and a 
salinity of 32-34, instead of 5-30), where their 
use is required test organisms in discharge permits. However, other 
species on the list are not currently approved for use as recommended 
species.
    California is correct in its conclusion that the standardization of 
methods by today's rule will displace unapproved methods (for NPDES 
permits issued after today's rule). In response to this concern, EPA is 
restricting the applicability of today's rule. The marine chronic tests 
in today's rule do not apply to discharges into marine waters of the 
Pacific Ocean. EPA seeks to minimize disruption in the administration 
of NPDES permit programs in those States with Pacific coastal waters. 
EPA intends to propose approval of marine chronic methods applicable to 
colder, Pacific coast waters in the near future. Marine acute west 
coast WET methods are included in the acute testing manual.
5. Test Conditions
    See the SID for response to comments on the following: Dilution 
water, test temperature and pH, renewal of test solutions, age of test 
organisms, test duration, feeding before/during the tests, dilution 
factor, replication, dissolved oxygen and aeration, and the number of 
effluent concentrations used in tests.
6. Applicability of Tests
a. Criteria for Test Selection
    Comment: In initially preparing, and subsequently revising, the 
toxicity test manuals, EPA failed to establish criteria for toxicity 
test selection. The toxicity tests proposed by the Agency did not 
satisfy the criteria for determining adequacy of testing methods.
    Response: EPA believes the commenter refers to the criteria 
described in the EPA report to Congress entitled, ``Availability, 
Adequacy, and Comparability of Testing Procedures for the Analysis of 
Pollutants Established Under Section 304(h) of the Federal Water 
Pollution Control Act,'' EPA/600/9-87/030, September 1988. In that 
document, EPA compared biological analyses to chemical analyses for the 
purpose of assessing the adequacy of a given biological method. The 
document explained the attributes of biological tests that were 
significant for assessing adequacy: biological detection limits, 
precision, and applicability.
    In toxicity tests, the detection limit is determined by the 
``sensitivity'' of the test organisms. The sensitivity of organisms to 
pollutants is an intrinsic quality, which may vary greatly between 
species, but also varies somewhat among organisms within the same 
species, and is affected by the condition or ``health'' of the 
organisms. Because the sensitivity of the test organisms cannot be 
``calibrated'' before each toxicity test, the tests must include 
standards to ensure data integrity. The final rule promulgated today 
includes the use of standard ``reference'' toxicants to maintain that 
integrity.
    To assess the precision of biological tests, the EPA report 
indicated that the methods must account for inherent variability of 
response and natural variability of within-species sensitivity. The 
methods in the final rule account for that variability by use of 
replicate testing; the toxicity methods require that a series of 
controls be run concurrently with pollutant exposures. These methods 
also contain criteria for determining the acceptability of data from a 
toxicity test based on the performance of the control organisms.
    The final attribute for assessing the adequacy of biological 
methods, as discussed in the EPA report, was applicability. The key 
criterion identified for determining biological test applicability was 
whether special conditions in the laboratory or a unique laboratory 
location is required to perform the test. For a test method to be 
applicable, it must be adaptable to a wide variety of laboratories. 
Applicability of a biological test depends on the ease with which the 
test can be performed on a routine basis and the consistency of 
availability of test organisms. The methods in this rule use readily 
available test organisms and can be competently performed by 
laboratories following the QA/QC guidelines described in the manuals.
    EPA disagrees with the commenter's central proposition that to 
establish applicability, each method requires inter-laboratory 
validation. In validating each method, EPA considered intra-laboratory 
testing. For those tests for which EPA further relies on 
interlaboratory testing, comparable coefficients of variation 
(precision) were achieved. Based on the high degree of correlation 
between coefficients of variation between intralaboratory tests and 
interlaboratory tests, EPA is confident in its reliance on 

[[Page 53538]]
intralaboratory studies to establish the applicability of the test 
methods to a wide variety of laboratories.
b. Ceriodaphnia Test
    Comment: There are problems with the Ceriodaphnia dubia short-term 
chronic toxicity test as evidenced by the low rate of successful test 
initiation (61%) and test completion (56%) in the Battelle Columbus 
(1987) round robin.
    Response: The Ceriodaphnia dubia short-term chronic toxicity test 
method (especially the diet) has been significantly improved since the 
Battelle round robin, as evidenced by the higher rates of successful 
test initiation and completion in a round robin supervised by EPA 
Region 4 in 1989 (EPA/505/2-90-001). In this inter-laboratory study, 36 
(80%) of 45 tests were successfully completed. The endpoints (No 
Observed Effect Concentrations, or NOECs) of 35 of the 36 tests, fell 
on two adjacent concentrations. Also, an interlaboratory study of the 
Ceriodaphnia dubia 7-day chronic test conducted by the San Francisco 
Bay Regional Water Quality Control Board (Environ. Toxicol. Chem. 
10:143-145, 1991), resulted in a coefficient of variation of 29%, 
demonstrating good precision.
c. Test Validation in Receiving Waters
    Comment: The relationship between laboratory data on effluent 
toxicity and effects on aquatic life in receiving waters has not been 
established by the Agency.
    Response: Numerous freshwater and marine site studies have been 
made to determine this relationship (see the Technical Support 
Document, EPA/505/2-90-001, 1991). These studies comprise a large data 
base specifically collected to determine the validity of toxicity tests 
to predict receiving water community impacts. The results of these 
studies clearly show the direct relationship between laboratory data on 
effluent toxicity and its adverse effect on aquatic life in receiving 
water.
d. Stage of Development of Toxicity Test Methods
    Comment: EPA toxicity test methods are still in a developmental 
stage, and have not been properly peer reviewed.
    Response: The acute toxicity tests have been widely used in the 
public and private sector for the past two decades, and the short-term 
chronic tests have been in general use in the NPDES permit program for 
six to nine years. The toxicity test manuals were widely distributed to 
expert peer reviewers in academia, major industries and trade 
organizations, consulting firms, and government agencies prior to 
publication, and were subject to further review during the public 
comment period following issuance of the Proposed Rule. Codification of 
these methods was proposed December 4, 1989, because they were 
considered adequately standardized for use in the NPDES Program. 
Furthermore, these methods have been published in highly respected, 
peer reviewed journals.
e. Ability of Laboratories to Perform the Arbacia and Champia Tests
    Comment: Few laboratories have the capability to perform some of 
the short-term chronic toxicity tests, such as the Champia and Arbacia 
tests.
    Response: EPA agrees that the number of laboratories with the 
capability of conducting Champia and Arbacia tests is currently 
limited. However, as the requirements for use of these organisms in the 
NPDES permits program increases, EPA's past experience indicates that 
the resulting increase in market demand will result in an increase in 
the number of laboratories that are capable of performing these tests.

C. Statistical Analysis of Results of Toxicity Tests with Fish and 
Other Aquatic Life

    Twenty-four sets of comments were received on statistical methods 
for toxicity data analysis. Some of the comments and responses are 
discussed below and the rest are in the SID.
    Comment: The use of Coefficients of Variation (CVs) of point 
estimates, such as the LC50, and the range in NOEC's and/or LOEC's 
(Lowest Observed Effect Concentration) are an inappropriate measure of 
test precision. The use of the NOEC and LC50 endpoints for precision 
estimates is not consistent with the calculation of precision of 
chemical methods. Therefore comparison of toxicity test precision to 
chemical method precision is inappropriate.
    Response: In the case of toxicity tests, test precision is a 
measure of agreement of successive test results. Toxicity results are 
expressed in terms of a point estimate, such as the LC1 (Concentration 
at which 1% of the organisms die), LC50, IC25, or a NOEC-LOEC pair 
derived from hypothesis testing. The CV is a widely used and acceptable 
method of expressing variability (precision) of point estimates from 
toxicity tests, such as LC50's, and is comparable to the calculation of 
precision of chemical methods. However, NOEC's and LOEC's are not point 
estimates, and it is not possible to express the precision of these 
values in terms of a similar statistic. In this case, precision can 
only be described by listing the NOEC-LOEC interval for each test, and 
indicating the range in these values. For a more general discussion of 
statistical analysis using hypothesis testing versus point estimates, 
see page 11 of the ``Technical Support Document for Water Quality-based 
Toxics Control'', EPA/505/2-90-001, PB91-127415, March 1991.
    Comment: The choice of statistical methods is not justified in the 
guidance documents.
    Response: EPA recognizes that the statistical methods recommended 
in the toxicity test methods manuals are not the only possible methods 
of statistical analysis. In selecting the methods for the manuals, EPA 
statisticians evaluated and considered many other analyses. The methods 
finally selected were chosen, among other reasons, because there are: 
(1) Well tested and well documented; (2) applicable to most different 
toxicity test data sets for which they are recommended, but still 
powerful; (3) most easily understood by non-statisticians; and (4) 
amenable to use without a computer, if necessary.
    Comment: Statistical analysis of toxicity test results is very 
complicated and should require the review and evaluation of a qualified 
statistician.
    Response: The statistical analyses recommended in the three 
toxicity test manuals (acute, freshwater short-term chronic, and marine 
short-term chronic) cited in the proposed rule had been subjected to 
extensive peer review in the private and public sectors prior to their 
proposal. The reviewers included EPA statisticians, government contract 
statisticians, and statisticians from academia. EPA believes that this 
constitutes an objective peer review of the recommended statistical 
analyses by qualified statisticians. In addition, the methods have also 
been published in highly regarded peer reviewed journals. The manuals 
also provide detailed, stepwise guidance for the statistical analyses 
of individual test results.
    Comment: It is not always obvious that an effect level that is 
determined to be statistically significant is also biologically 
significant.
    Response: The implied question, concerning the ``biological 
significance'' of (threshold) ``statistically significant' occurrences 
of adverse biological effects observed in toxicity tests, is an 
implementation question, and is not addressed in this rulemaking. 
However, in a related area, the Agency's water quality criteria for 
fish and other aquatic life are based on ``safe concentrations'' of 
toxicants which are defined as the highest concentration of toxicant 
not showing a ``statistically significant'' occurrence of an adverse 
biological 

[[Page 53539]]
effect (NOEC) with the assumption that a ``statistically significant'' 
reduction in an important biological response will adversely affect the 
success of the organisms and, therefore, is a ``significant''biological 
effect.
    Comment: Only surviving adult females should be used for 
Ceriodaphnia reproduction analysis.
    Response: The exclusion of reproduction data from females that do 
not survive to the end of the test would bias the results in favor of 
the organisms that are more tolerant to pollution. Therefore, EPA 
believes that it is best to use the reproduction data from all the test 
organisms in the analysis, except for those from test concentrations 
that have significantly greater mortality than the test controls. Data 
from the latter are not included in the determination of the 
reproductive endpoint.
    Comment: More guidance is needed in selecting alternative 
statistical methods when replicate values are found to reflect wide 
variation in survival values.
    Response: The freshwater and marine short-term chronic toxicity 
test methods manuals contain detailed flowcharts on the recommended 
statistical analyses. It is not possible to provide guidelines to cover 
all contingencies of toxicity data analysis. Therefore these 
recommendations were intended to cover most types of data that would 
occur in toxicity testing. As stated in the manuals, EPA advises 
analysts to consult with a qualified statistician for cases that are 
not covered by the recommended analyses.
    Comment: The NOEC is not a meaningful endpoint and is too dependent 
upon the concentration intervals utilized in the test.
    Response: EPA recognizes that the NOEC is dependent upon the 
concentration intervals used in a test, but disagrees that it is not a 
meaningful endpoint. The NOEC is the most commonly used endpoint in 
chronic toxicity tests and, prior to the development of the Linear 
Interpolation (or Inhibition Concentration) Method, was the only 
endpoint available for determination of ``safe concentrations.'' The 
Agency's water quality criteria for fish and other aquatic life are 
based on ``safe concentrations'' of toxicants which are defined as the 
highest concentration of toxicant not causing a ``statistically 
significant'' difference in biological response (such as growth or 
reproduction). Use of the NOEC in effluent and receiving water toxicity 
tests is described in the Agency's ``Technical Support Document for 
Water Quality-based Toxics Control'', EPA/505/2-90-001, PB91-127415, 
March 1991.
    Comment: Statistical methods which require log or geometric 
dilution series should be discussed.
    Response: The use of graphical method to determine the LC50 is 
recommended by EPA (EPA/600/4-90/027F) only when the response is ``all 
or nothing,'' i.e., only two levels of response--zero mortality at 
lower test concentrations and 100% mortality at higher test 
concentrations. Results of this type occur in a high proportion (60% or 
more) of effluent toxicity tests. When such an all or nothing response 
occurs, the results are not amenable to statistical analysis. According 
to Finney, a leading authority on the analysis of acute toxicity data, 
a graphically-derived estimate of the LC50, which employs the known 
logarithmic relationship between toxicant concentration and mortality, 
is ``the only reasonable approach'' (Finney, D.J. 1985. Arch. Toxicol. 
56:215-218). However, the graphical method is unable to provide 
confidence limits for the endpoints. When partial mortalities occur at 
one or more test concentrations, EPA recommends the use of the Trimmed 
Spearman-Karber or Probit Analysis.
    Comment: Regression (point estimation) should be used as an 
interpretive tool for the data rather than exclusively using a ``mean'' 
system.
    Response: The selection of the statistical analysis (in the two 
short-term chronic manuals) is dependant upon the intended use of the 
data. For example, in the NPDES permitting program, the recommended 
statistical procedure is the point estimate, because confidence 
intervals can be placed around the point estimate.
    Comment: There must be an adequate concentration response or the 
test is of little value in calculation of a LC50 or EC50.
    Response: Data from toxicity tests frequently show an all or 
nothing response, and in these instances the appropriate statistical 
procedure to estimate the LC50 are the Graphical Method and/or the 
Trimmed Spearman Karber. The alternative LC50 statistical procedures do 
require that the data show a dose response above and below the LC50 
concentration.

D. Implementation and Miscellaneous Issues

    Approximately 23 comments were related to the application and 
implementation of EPA Policy on the Water Quality-Based Toxics Control 
Program and other issues which were not specifically applicable to the 
technical methods contained in this rulemaking. These comments are 
addressed in the SID which is part of the administrative record for 
this rulemaking.

VI. Regulatory Analyses

A. Unfunded Mandates Reform Act of 1995

    Under section 202 of the Unfunded Mandates Reform Act of 1995 
(``Unfunded Mandates Act''), signed into law on March 22, 1995, EPA 
must prepare a written statement to accompany rules where the estimated 
costs to State, local, or tribal governments, or to the private sector, 
will be $100 million or more in any one year. Under section 205, EPA 
must select the most cost-effective and least burdensome alternative 
that achieves the objective of such a rule and that is consistent with 
statutory requirements. Section 203 requires EPA to establish a plan 
for informing and advising any small governments that may be 
significantly and uniquely affected by the rule.
    EPA estimates that the costs to State, local, or tribal 
governments, or the private sector, from this rule will be less than 
$100 million. This rulemaking should have minimal impact, if any, on 
the current regulatory burden imposed on NPDES permittees because the 
rulemaking merely standardizes methods (that are currently contained in 
guidance) to determine compliance with whole effluent toxicity 
limitations required under existing regulations. EPA has determined 
that an unfunded mandates statement therefore is unnecessary. 
Similarly, the standardized methods in today's rule do not establish 
any regulatory requirements that might significantly or uniquely affect 
small governments; any such requirements would have been established 
previously in NPDES regulations providing for inclusion of whole 
effluent toxicity limitations.

B. Regulatory Flexibility Act

    Under the Regulatory Flexibility Act, 5 U.S.C. 601 et seq., EPA is 
required to determine whether a regulation will significantly affect a 
substantial number of small entities so as to require a regulatory 
analysis. The regulation requires no new reports beyond those now 
required. The analytical techniques approved here either can be handled 
by small facilities, or are widely available by contract at a 
reasonable price. Therefore, in accordance with 5 U.S.C. 605(b), I 
hereby certify that this rule will not have significant adverse 
economic 

[[Page 53540]]
impact on a substantial number of small facilities.

C. Paperwork Reduction Act

    This rule does not impose any additional information requirements 
on respondents, and consequently is not subject to the Paperwork 
Reduction Act, 44 U.S.C. 3501 et seq.

D. Executive Order 12866

    Under Executive Order 12866, EPA must judge whether a regulation is 
``major'' and therefore subject to the requirement of a ``Regulatory 
Impact Analysis.'' This regulation is not major for the following 
reasons:

    1. The rule only prescribes analytical methods and sample 
handling requirements that ensure a uniform measure of pollutants 
across all wastewater discharges within minimum acceptance criteria. 
The rule itself does not require that analyses actually be 
performed. Other existing rules require such analyses in certain 
circumstances. The purpose is to ensure that the quality of the 
environmental monitoring data meets certain minimum standards.
    2. The impact of this regulation will be far less than $100 
million. The regulation affects unit monitoring cost for the NPDES 
programs, e.g., effluent guidelines regulations and the NPDES 
implementation regulations, and the pretreatment programs. However, 
the rule does not itself impose those costs. The monitoring costs 
for other programs are considered in the rulemaking for each 
program.

    Under Executive Order 12866 The Office of Management and Budget 
waived review on October 26, 1994.
    The range in cost for the acute and chronic methods, on a per test 
basis, is approximately $200.00-$2800.00. Clustered at the low end of 
the cost range estimate are the acute 96 hour test methods, and at the 
higher end the short-term chronic test methods. The majority of testing 
laboratories charged between $200.00-$1500.00 per test. EPA believes 
that the overall range of cost per test, particularly at the high end, 
will decrease as a result of promulgation of the methods. This is 
because the number of approved tests will be limited to those in the 
rule, as opposed to the many variations of each test method now being 
conducted. Experience has shown that the cost of the tests has 
decreased over time as the testing laboratories have become more 
competent in performing the different test methods. EPA estimates that 
the overall cost will drop by 20% (ranging from $160.00-$2240.00 for 
all labs, and $160.00-$1200.00 for the majority of labs) as a result of 
promulgation of this rule.

VII. Materials Incorporated by Reference Into 40 CFR Part 136

    1. USEPA. 1993. Methods for Measuring the Acute Toxicity of 
Effluents to Freshwater and Marine Organisms. Fourth Edition, August 
1993. Environmental Monitoring Systems Laboratory, U.S. 
Environmental Protection Agency, Cincinnati, Ohio (EPA/600/4-90/
027F). Table 1A, Note 7.
    2. USEPA. 1994. Short-term Methods for Estimating the Chronic 
Toxicity of Effluents and Receiving Waters to Freshwater Organisms. 
Third Edition, July 1994. Environmental Monitoring Systems 
Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. 
(EPA/600/4-91/002). Table 1A, Note 9.
    3. USEPA. 1994. Short-term Methods for Estimating the Chronic 
Toxicity of Effluents and Receiving Waters to Marine and Estuarine 
Organisms. Second Edition, July 1994. Environmental Monitoring 
Systems Laboratory, U.S. Environmental Protection Agency, 
Cincinnati, Ohio. (EPA/600/4-91/003). Table 1A, Note 10.

VIII. Public Availability of Materials To Be Incorporated by Reference

    Copies of the documents incorporated by reference in today's 
rulemaking will be available to the general public from the following 
sources at no cost:
    National Center for Environmental Publications and Information 
(NCEPI): available 24 hours a day, 7 days a week; (513) 489-8190, or 
FAX (513) 489-8695, identifying the name of the document or the 
publication number listed in section VII of this preamble. Available 
formats: paper copies and 3\1/2\ inch or 5 inch discs.
    EPA Office of Water Resource Center: available 24 hours a day, 7 
days a week; (202) 260-7786. Contract staff will assist caller in 
identifying a document from document title, publication number, or a 
description of the subject matter. Available formats: paper copies and 
3\1/2\ inch or 5 inch discs.
    EPA Regional Office Libraries: EPA has 10 Regional offices around 
the country, each with a publically accessible library. Copies of these 
documents can be viewed and copied at these EPA Regional libraries. EPA 
Region I, JFK Federal Building, One Congress Street, Boston, MA 02203, 
(617) 565-3420; EPA Region 2, 290 Broadway, New York, NY 10007-1866, 
(212) 637-3000; EPA Region 3, 841 Chestnut Building, Philadelphia, PA 
19107, (215) 597-9800; EPA Region 4, 345 Courtland Street, NE., 
Atlanta, GA 30365, (404) 347-4727; EPA Region 5, 77 West Jackson Blvd., 
Chicago, IL 60604-3507, (312) 353-2000; EPA Region 6, First Interstate 
Bank Tower at Fountain Place, 1445 Ross Avenue, 12th Floor, Suite 1200, 
Dallas, TX 75202-2733, (214) 665-6444; EPA Region 7, 726 Minnesota 
Avenue, Kansas City, KS 66101, (913) 551-7000; EPA Region 8, 999 18th 
Street, Suite 500, Denver, CO 80202-2466, (303) 293-1603; EPA Region 9, 
75 Hawthorne Street, San Francisco, CA 94105, (415) 744-1305; EPA 
Region 10, 1200 Sixth Avenue, Seattle, WA 98101, (206) 553-1200.
    Internet, EPA operates a ``public access server,'' also known as 
``Earth 1,'' through which EPA will include all of the ways that copies 
of the test methods manuals are available. The Office of Water will put 
the directions about electronic retrieval of the test methods manuals 
on EPA's Internet ``homepage.'' By doing so, persons interested in 
electronic copies of the methods manuals may obtain copies either (1) 
retrieving the documents from EPA's file transfer protocol (FTP) site 
on the Internet at ftp.epa.gov or gopher.epa.gov (2) retrieving the 
documents by dial-in access at 919-558-0335, or (3) by requesting 
floppy disks from NCEPI, including requests through the Office of Water 
Resource Center. EPA would explain the limitations some users may 
encounter trying to print out diagrams, tables, charts and graphs, 
which would may require special ``read'' software. Later this year, the 
Office of Water will have its own Internet ``homepage'' which will 
include all Office of Water rules and information on how to obtain 
copies of all technical support documents.
    By the end of 1995, EPA will be a participant in the Government 
Information Locator Service (GILS) consistent with Office of Management 
and Budget requirements. GILS is a ``list of lists'' on the Internet, 
of all U.S. Government publications, describing the publication and how 
to get it. The Office of Water will describe the means of electronic 
access to the whole effluent toxicity test methods manuals through the 
GILS system.
    Public Libraries, A description of the whole effluent toxicity 
methods final rule and the test methods manuals has been placed in the 
combined catalogues of the Online Computer Library Center (OCLC) in 
Columbus, Ohio, available to all member libraries across the country 
(approximately 13,000). This summary will facilitate public access 
through interlibrary loans from the Regional EPA libraries. Through 
OCLC, EPA has placed the summary and access information in the Online 
Library System. Finally, EPA has provided the national association of 
public libraries with a summary of the whole effluent toxicity methods 
rule and the test methods manuals, as a way of emphasizing their 
availability through this means.

[[Page 53541]]

    Copies of these documents will also be available for viewing and 
copying at the State Libraries: Alabama Library Association, 400 S. 
Union Street, Suite 255, Montgomery, AL 36104; Alaska Library 
Association, PO Box 81084; Fairbanks, AL 99708-1084; Arizona State 
Library Association, 13832 32d. Street, Phoenix, AZ 85032; Arkansas 
Library Association, 1100 N. University, #109, Little Rock, AR 72204; 
California Library Association, 717 K. Street, Suite 300, Sacramento, 
CA 95814-3477; Colorado Library Association, 114 Pinecliffe Road, 
Pinecliffe, CO 80471; Connecticut Library Association, Box 1016, 
Hartford, CT 06360; Delaware Library Association, PO Box 816, 
Wilmington, DE 19903; District of Columbia Library Association, PO Box 
14177, Benjamin Franklin Station, Washington, DC 20044; Florida Library 
Association, 1133 W. Morse Blvd., Suite 201, Winter Park, Fl 32789-
3788; Georgia Library Association, Young Harris College, PO Box 39, 
Young Harris, GA 30582; Guam Library Association, PO Box 22515 GFM, 
Barrigada, GU 96921; Hawaii Library Association, PO Box 4441, Honolulu, 
HI 96814-4441; Idaho Library Association, Boise State University, 
Boise, ID 83725; Illinois Library Association, 33 W. Grand Avenue, 
#301, Chicago, IL 60610; Indiana Library Federation 6408 Carrollton 
Avenue, Indianapolis, IN 46220-1615; Iowa Library Association, 823 
Insurance Exchange Building, Des Moines, IA 50309; Kansas Library 
association, South Central Kansas Library System, 901 N. Main, 
Hutchinson, KS 67501-4401; Kentucky Library Association, 1501 Twilight 
Tr., Frankfort, KY 40601; Louisiana Library Association, PO Box 3058, 
Baton Rouge, LA 70821; Maine Library Association, Community Drive, 
Augusta, ME 04330; Maryland Library Association, 400 Cathedral Street, 
3d Floor, Baltimore, MD 21201; Massachusetts Library Association, 
Countryside Offices 707 Turnpike St., North Andover, MA 08145; Michigan 
Library Association, 1000 Long Blvd. Suite 1, Lansing, MI 48911; 
Minnesota Library Association, 1315 Lowrey Avenue, N. Minneapolis, MN 
55411-1398; Mississippi Library Association, PO Box 20488, Jackson, MS 
39209-1448; Missouri Library Association, 11306 Business 63 South, 
Suite B, Columbia, MO 65201; Montana Library Association, 507 Fifth 
Avenue, Helena, MT 59601-4359; Nebraska Library Association, 5302 S. 
75th Street, Ralston, NE 68127-3903; Nevada Library Association, Elko 
County Public Library, 720 Court Street, Elko, NV 89801; New Hampshire 
Library Association, Franklin Public Library, 310 Central Street, 
Franklin, NH 03235; New Jersey Library Association, 4 W. Lafayette, 
Trenton, NJ 08608; New Mexico Library Association, San Juan College 
Library, 4601 College Avenue, Farmington, NM 87401; New York Library 
Association, 252 Hudson Avenue, Albany, NY 12210; North Carolina 
Library Association, Southeastern Technical Asst. Center, 2013 Lejeune 
Blvd., Jacksonville, NC 28546-7027; North Dakota Library Association, 
University of North Dakota-Lake Region, 1800 N. College Drive, Devil's 
Lake, ND 58301; Ohio Library Council, 35 E. Gay Street, Columbus, OH 
43215; Oklahoma Library Association, 300 Hardy Drive, Edmond, OK 73013; 
Oregon Library Association, 1270 Chemeketa Street, NE, Salem, OR 97301; 
Pennsylvania Library Association, 1919 N. Front Street, Harrisburg, PA 
17110; Rhode Island Library Association, 300 Richmond Street, 
Providence, RI 02903; South Carolina Library Association, Rt 2, Box 
139F, Denmark, SC 29042; South Dakota Library Association, PO Box 673, 
Pierre, SD 57501; Tennessee Library Association, Memphis State 
University Library, Memphis, TN 30152; Texas Library Association, 3355 
Bee Cave Road, #401, Austin, TX 78746; Utah Library Association, 365 
Emory, Salt Lake City, UT 84101; Vermont Library Association, Box 803, 
Burlington, VT 05402-0803; St. Thomas/St. John Library Associationa, 
University of Virgin Islands, St. Thomas, VI 00802; St. Croix Library 
Association, PO Box 306164, Veteran's Drive Station, Charlotte Amalie, 
VI 00803; Virginia Library Association, 669 S. Washington Street, 
Alexandria, VA 22314-4109; Washington Library Association, Ft. 
Vancouver Regional Library, 1007 E. Mill Plain Blvd. Vancouver, WA 
98603-3504; West Virginia Library Association, West Virginia Library 
Community, Science and Culture Center, Charleston, WV 35305; Wisconsin 
Library Association, 4785 Hayes Road, Madison, WI 53704-2764; Wyoming 
Library Association, Sweetwater County Library, PO Box 550, Green 
River, WY 82935.
    A limited number of copies will be available from the EPA Regional 
offices, and the State NPDES permitting offices. Finally, after the 
first printing, hard copies will be available from the National 
Technical Information Service (NTIS) in Springfield, Virginia for 
$31.00, $31.00, and $45.00, respectively for ``Short-Term Methods for 
Estimating the Chronic Toxicity of Effluents and Receiving Water to 
Marine and Estuarine Organisms, Second Edition'' July 1994, EPA/600/4-
91/003, ``Short-Term Methods for Estimating the Chronic Toxicity of 
Effluents and Receiving Water to Freshwater Organisms, Third Edition'' 
July 1994, EPA/600/4-91/002, and ``Methods for Measuring the Acute 
Toxicity of Effluents and Receiving Waters to Freshwater and Marine 
Organisms, Fourth Edition'' August 1993, EPA/600/4-90/027F. (NTIS is an 
organization within the U.S. Department of Commerce.)
    EPA is also notifying the following groups of the availability of 
these documents: International Association of Environmental Testing 
Laboratories; American Society of Testing Materials; Society of 
Environmental Toxicology and Chemistry; American Chemical Society; 
Water Environment Federation; Association of Metropolitan Sewerage 
Agencies; Association of Analytical Chemists; and the Discharge 
Monitoring Requirement Quality Assurance Program.

IX. References

Federal Register: U.S. Environmental Protection Agency. Policy for 
the Development of Water Quality-Based Permit Limitations for Toxic 
Pollutants, 49 FR 9016; Mar. 9, 1984.
Anderson, S.L. and T.J. Norberg-King. 1991. Precision of ShortTerm 
Chronic Toxicity Tests in the Real World. Environmental Toxicology 
and Chemistry 10(2):143-145.
Finney, D.J. 1985. The Median Lethal Dose and its Estimation. Arch. 
Toxicol. 56:215-218.
U.S. Environmental Protection Agency. July 1994. Whole Effluent 
Toxicity (WET) Control Policy. EPA 833-B-94-002.
U.S. Environmental Protection Agency. 1991. Technical Support 
Document for Water Quality-Based Toxics Control, March 1991, EPA/
505/2-90/001; PB91-127415.
U.S. Environmental Protection Agency. September 1988. Report to 
Congress: Availability, Adequacy, and Comparability of Testing 
Procedures for the Analysis of Pollutants Established Under Section 
304(h) of the Federal Water Pollution Control Act. EPA/600/9-87/030.

List of Subjects in 40 CFR Part 136

    Environmental protection, Water pollution control, Incorporation by 
reference.

    Dated: October 3, 1995.
Carol M. Browner,
Administrator.

    For the reasons set out in the preamble, part 136 of title 40 of 
the Code of Federal Regulations is amended as follows: 

[[Page 53542]]


PART 136--[AMENDED]

    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, 
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. In Sec. 136.3(a), Table IA is revised to read as follows:


Sec. 136.3  Identification of test procedures.

* * * * *

                                                     Table IA.--List of Approved Biological Methods                                                     
--------------------------------------------------------------------------------------------------------------------------------------------------------
        Parameter and units                 Method \1\                    EPA            Standard methods, 18th Ed.      ASTM              USGS         
--------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria:                                                                                                                                               
    1. Coliform (fecal), number     Most Probable Number        p. 132 \3\              9221C E \4\                  ...........  ......................
     per 100 mL.                     (MPN), 5 tube.             p. 124 \3\              9222D \4\                                 B-0050-85 \5\         
                                    3 dilution, or Membrane                                                                                             
                                     filter (MF) \2\, single                                                                                            
                                     step.                                                                                                              
    2. Coliform (fecal) in          MPN, 5 tube, 3 dilution,    p. 132 \3\              9221C E \4\                  ...........  ......................
     presence of chlorine, number    or.                        p. 124 \3\              9222D \4\                                                       
     per 100 mL.                    MF, single step \6\.......                                                                                          
    3. Coliform (total), number     MPN, 5 tube, 3 dilution,    p. 114 \3\              9221B \4\                    ...........  ......................
     per 100 mL.                     or.                        p. 108 \3\              9222B \4\                                 B-0025-85 \5\         
                                    MF \2\ single step or two                                                                                           
                                     step.                                                                                                              
    4. Coliform (total), in         MPN, 5 tube, 3 dilution,    p. 114 \3\              9221B \4\                    ...........  ......................
     presence of chlorine, number    or.                        p. 111 \3\              9222(B+B.5c) \4\                                                
     per 100 mL.                    MF \2\ with enrichment....                                                                                          
    5. Fecal streptococci, number   MPN, 5 tube, 3 dilution...  p. 139 \3\              9230B \4\                    ...........  ......................
     per 100 mL.                    MF \2\, or................  p. 136 \3\              9230C \4\                                 B-0055-85 \5\         
                                    Plate count...............  p. 143 \3\                                                                              
Aquatic Toxicity:                                                                                                                                       
    6. Toxicity, acute, fresh       Daphnia, Ceriodaphnia,      Sec. 9 \7\              ...........................  ...........  ......................
     water organisms, LC50,          Fathead Minnow, Rainbow                                                                                            
     percent effluent.               Trout, Brook Trout, or                                                                                             
                                     Bannerfish Shiner                                                                                                  
                                     mortality.                                                                                                         
    7. Toxicity, acute, estuarine   Mysid, Sheepshead Minnow,   Sec. 9 \7\              ...........................  ...........  ......................
     and marine organisms, LC50,     or Menidia spp. mortality.                                                                                         
     percent effluent.                                                                                                                                  
    8. Toxicity, chronic, fresh     Fathead minnow larval       1000.0 \8\              ...........................  ...........  ......................
     water organisms, NOEC or        survival and growth.       1001.0 \8\              ...........................                                     
     IC25, percent effluent.        Fathead minnow embryo-                              ...........................                                     
                                     larval survival and        1002.0 \8\              ...........................                                     
                                     teratogenicity.            1003.0 \8\              ...........................                                     
                                    Ceriodaphnia survival and                                                                                           
                                     reproduction.                                                                                                      
                                    Selenastrum growth........                                                                                          
    9. Toxicity, chronic,           Sheepshead minnow larval    1004.0 \9\              ...........................  ...........  ......................
     estuarine and marine            survival and growth.       1005.0 \9\              ...........................                                     
     organisms, NOEC or IC25,       Sheepshead minnow embryo-                                                                                           
     percent effluent.               larval survival and        1006.0 \9\              ...........................                                     
                                     teratogenicity.            1007.0 \9\                                                                              
                                    Menidia beryllina larval    1008.0 \9\              ...........................                                     
                                     and growth.                1009.0 \9\              ...........................                                     
                                    Mysidopsis bahia survival,                                                                                          
                                     growth, and fecundity.                                                                                             
                                    Arbacia punctulata                                                                                                  
                                     fertilization.                                                                                                     
                                    Champia parvula                                                                                                     
                                     reproduction.                                                                                                      
--------------------------------------------------------------------------------------------------------------------------------------------------------
Notes to Table IA:                                                                                                                                      
\1\ The method must be specified when results are reported.                                                                                             
\2\ A 0.45 um membrane filter (MF) or other pore size certified by the manufacturer to fully retain organisms to be cultivated and to be free of        
  extractables which could interfere with their growth.                                                                                                 
\3\ USEPA. 1978. Microbiological Methods for Monitoring the Environment, Water, and Wastes. Environmental Monitoring and Support Laboratory, U.S.       
  Environmental Protection Agency, Cincinnati, Ohio. EPA/600/8-78/017.                                                                                  
\4\ APHA. 1992. Standard Methods for the Examination of Water and Wastewater. American Public Health Association. 18th Edition. Amer. Publ. Hlth.       
  Assoc., Washington, DC.                                                                                                                               
\5\ USGS. 1989. U.S. Geological Survey Techniques of Water-Resources Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and
  Analysis of Aquatic Biological and Microbiological Samples, U.S. Geological Survey, U.S. Department of Interior, Reston, Virginia.                    
\6\ Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Most Probable Number method will be required to 
  resolve any controversies.                                                                                                                            
\7\ USEPA. 1993. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. Fourth Edition. Environmental Monitoring     
  Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. August 1993, EPA/600/4-90/027F.                                           
\8\ USEPA. 1994. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Third Edition.       
  Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency USEPA. 1994, Cincinnati, Ohio (July 1994, EPA/600/4-91/002).        
\9\ Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. Second Edition.         
  Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio (July 1994, EPA/600/4-91/003). These methods do   
  not apply to marine waters of the Pacific Ocean.                                                                                                      

    3. Section 136.3(b) is amended by revising references (2), (6), and 
(11) and by adding references (34), (38), and (39) to read as follows:


Sec. 136.3  Identification of test procedures.

* * * * *


[[Page 53543]]

    (b) * * *
References, Sources, Costs, and Table Citations
* * * * *
    (2) USEPA. 1978. Microbiological Methods for Monitoring the 
Environment, Water, and Wastes. Environmental Monitoring and Support 
Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. 
EPA/600/8-78/017. Available from: National Technical Information 
Service, 5285 Port Royal Road, Springfield, Virginia 22161, Publ. No. 
PB-290329/AS. Cost: $36.95. Table IA, Note 3.
* * * * *
    (6) American Public Health Association. 1992. Standard Methods for 
the Examination of Water and Wastewater. 18th Edition. Amer. Publ. 
Hlth. Assoc., 1015 15th Street NW, Washington, DC 20005. Cost: $160.00. 
Table IA, Note 4.
* * * * *
    (11) USGS. 1989. U.S. Geological Survey Techniques of Water-
Resources 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 the 
Interior, Reston, Virginia. Available from: USGS Books and Open-File 
Reports Section, Federal Center, Box 25425, Denver, Colorado 80225. 
Cost: $18.00. Table IA, Note 5.
* * * * *
    (34) USEPA. 1993. Methods for Measuring the Acute Toxicity of 
Effluents to Freshwater and Marine Organisms. Fourth Edition, December 
1993. Environmental Monitoring Systems Laboratory, U.S. Environmental 
Protection Agency, Cincinnati, Ohio (EPA/600/4-90/027F). Available 
from: National Technical Information Service, 5285 Port Royal Road, 
Springfield, Virginia 22161, Publ. No. PB-91-167650. Cost: $31.00. 
Table IA, Note 17. See changes in the manual, listed in Part V of this 
rule.
* * * * *
    (38) USEPA. 1994. Short-term Methods for Estimating the Chronic 
Toxicity of Effluents and Receiving Waters to Freshwater Organisms. 
Third Edition. July 1994. Environmental Monitoring Systems Laboratory, 
U.S. Environmental Protection Agency, Cincinnati, Ohio. (EPA/600/4-91/
002). Available from: National Technical Information Service, 5285 Port 
Royal Road, Springfield, Virginia 22161, Publ. No. PB-92-139492. Cost: 
$31.00. Table IA, Note 8.
    (39) USEPA. 1994. Short-term Methods for Estimating the Chronic 
Toxicity of Effluents and Receiving Waters to Marine and Estuarine 
Organisms. Second Edition, July 1994. Environmental Monitoring Systems 
Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. 
EPA/600/4-91/003. Available from: National Technical Information 
Service, 5285 Port Royal Road, Springfield, Virginia 22161, Publ. No. 
PB-92-139484. Cost: $45.00. Table IA, Note 9.
    4. In Sec. 136.3(e), Table II is amended by revising the entry for 
``Table IA-Bacteria Tests:'' and adding an entry for ``Table IA-Aquatic 
Toxicity Tests:'' and by revising footnote 1 and adding footnote 16 to 
read as follows:

                    Table II. Required Containers, Preservation Techniques, and Holding Times                   
----------------------------------------------------------------------------------------------------------------
                                                                                            Maximum holding time
         Parameter No./name                Container \1\          Preservation \2\,\3\               \4\        
----------------------------------------------------------------------------------------------------------------
Table IA--Bacteria Tests:                                                                                       
    1-4 Coliform, fecal and total...  P,G                     Cool, 4C, 0.008% Na2S2O3 5..  6 hours.            
    5 Fecal streptococci............  P,G                     Cool, 4C, 0.008% Na2S2O3 5..  6 hours.            
Table IA--Aquatic Toxicity Tests:                                                                               
    6-10 Toxicity, acute and chronic  P,G                     Cool, 4C \16\...............  6 hours.            
                                                                                                                
*                  *                  *                  *                  *                  *                
                                                        *                                                       
----------------------------------------------------------------------------------------------------------------
\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 automatic sampler makes it         
  impossible to preserve each aliquot, then chemical samples may be preserved by maintaining at 4C until        
  compositing and sample splitting is completed.                                                                
\3\ When any sample is to be shipped by common carrier or sent through the United States Mails, it must comply  
  with the Department of Transportation Hazardous Materials Regulations (49 CFR Part 172). The person offering  
  such material for transportation is responsible for ensuring such compliance. For the preservation            
  requirements of Table II, the Office of Hazardous Materials, Transportation Bureau, Department of             
  Transportation, has determined that the Hazardous Materials Regulations do not apply to the following         
  materials: Hydrochloric Acid (HCl) in water solutions at concentrations of 0.04% by weight or less (pH about  
  1.96 or greater); Nitric Acid (HNO3) in water solutions of 0.15% by weight or less (pH about 1.62 or greater);
  Sulfuric Acid (H2SO4) in water solutions of 0.35% 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 in the table are the      
  maximum times that samples may be held before analyses and still be considered valid. Samples used for        
  toxicity tests are to be used for test initiation or for renewal of test solutions within 36 h of collection  
  as grab samples, or within 36 hours of the collection of the last sample of the composite. Samples for        
  bacteria or chemical analysis may be held for longer periods than specified in this table only if the         
  permittee or monitoring laboratory has data on file to show that 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
  Para. 136.3(e). Some samples may not be stable for the maximum time period given in the table. A permittee or 
  monitoring laboratory is obligated to hold the samples for a shorter time if knowledge exists to show that    
  this is necessary to maintain sample stability. See Para. 136.3(e) for details. The term ``analyze            
  immediately'' usually means within 15 minutes or less of sample collection.                                   
\5\ Should only be used in the presence of residual chlorine.                                                   
*                  *                  *                  *                  *                  *                
   *                                                                                                            
\16\ Sufficient ice should be placed with the samples in the shipping container to ensure that ice is still     
  present when the samples arrive at the laboratory. However, even if ice is present when the samples arrive, it
  is necessary to immediately measure the temperature of the samples and confirm that the 4C temperature maximum
  has not been exceeded. In the isolated cases where it can be documented that this holding temperature can not 
  be met, the permittee can be given the option of on-site testing or can request a variance. The request for a 
  variance should include supportive data which show that the toxicity of the effluent samples is not reduced   
  because of the increased holding temperature.                                                                 


[[Page 53544]]

[FR Doc. 95-25348 Filed 10-13-95; 8:45 am]
BILLING CODE 6560-50-P