[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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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
