[Federal Register Volume 67, Number 223 (Tuesday, November 19, 2002)]
[Rules and Regulations]
[Pages 69952-69972]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 02-29072]



[[Page 69951]]

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Part V





Environmental Protection Agency





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40 CFR Part 136



Guidelines Establishing Test Procedures for the Analysis of Pollutants; 
Whole Effluent Toxicity Test Methods; Final Rule

  Federal Register / Vol. 67, No. 223 / Tuesday, November 19, 2002 / 
Rules and Regulations  

[[Page 69952]]


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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 136

[FRL-7408-6]
RIN 2040-AD73


Guidelines Establishing Test Procedures for the Analysis of 
Pollutants; Whole Effluent Toxicity Test Methods; Final Rule

AGENCY: Environmental Protection Agency (EPA).

ACTION: Final rule.

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SUMMARY: In this final regulation, EPA ratifies approval of several 
test procedures for measuring the toxicity of effluents and receiving 
waters. The test procedures are commonly referred to as whole effluent 
toxicity or WET test methods. EPA also withdraws two WET test methods 
from the list of nationally-approved biological test procedures for the 
analysis of pollutants. This action also revises some of the WET test 
methods to improve performance and increase confidence in the 
reliability of the results. Today's action will satisfy settlement 
agreement obligations designed to resolve litigation over an earlier 
rulemaking that originally approved WET test methods.

DATES: This regulation is effective December 19, 2002. For judicial 
review purposes, this final rule is promulgated as of 1:00 p.m. Eastern 
Standard Time on December 3, 2002 in accordance with 40 CFR 23.7. The 
incorporation by reference of certain publications listed in this rule 
is approved by the Director of the Federal Register as of December 19, 
2002.

FOR FURTHER INFORMATION CONTACT: Marion Kelly; Engineering and Analysis 
Division (4303T); Office of Science and Technology; Office of Water, 
U.S. Environmental Protection Agency; Ariel Rios Building; 1200 
Pennsylvania Avenue, NW; Washington, DC 20460, or call (202) 566-1045, 
or E-mail at [email protected]. For technical information regarding 
method changes in today's rule, contact Debra L. Denton, USEPA Region 
9, c/o SWRCB, 1001 I Street, Sacramento, CA 95814, or call (916) 341-
5520, or E-mail [email protected].

SUPPLEMENTARY INFORMATION

I. General Information
    A. Potentially Regulated Entities
    B. How Can I Get Copies Of Related Information?
    1. Docket
    2. Electronic Access
II. Statutory Authority
III. Background
    A. Regulatory History
    B. Settlement Agreement
    C. Proposed Rule
IV. Summary of Final Rule
    A. Proposed WET Method Changes
    B. Additional Revisions to WET Test Methods
    C. Ratification and Withdrawal of Methods
    D. Amendment to 40 CFR 136.3, Table IA
V. Changes from the Proposed Rule
    A. Proposed WET Method Changes
    1. Blocking by Known Parentage
    2. pH Drift
    3. Nominal Error Rates
    4. Dilution Series
    5. Dilution Waters
    6. Pathogen Interference
    7. EDTA in the Selenastrum capricornutum Growth Test
    B. Additional Revisions to WET Test Methods
    1. Variability Criteria
    2. Minimum Number of Replicates
    3. Test Requirements/Recommendations
    4. Sample Collection and Holding Times
    5. Reference Toxicant Testing
    6. Sample Holding Temperature
    7. Biomass
    8. Total Residual Chlorine
    9. Ceriodaphnia dubia Survival and Reproduction Test Termination 
Criteria
    10. Additional Minor Corrections
    C. Ratification and Withdrawal of Methods
VI. Response to Major Comments
    A. Proposed WET Method Changes
    1. Cost
    2. Concentration-Response Relationships
    3. Confidence Intervals
    B. Additional Revisions to WET Test Methods
    1. Method Flexibility
    2. Test Acceptability Criteria
    3. Quality Assurance/Quality Control Requirements
    4. Statistical Methods
    C. Ratification and Withdrawal of Methods
    1. Validation of Performance Characteristics
    2. Interlaboratory Variability Study
    3. Variability
    4. Successful Test Completion Rate
    5. False Positive Rate
    6. Implementation
VII. Statutory and Executive Order Reviews
    A. Executive Order 12866: Regulatory Planning and Review
    B. Paperwork Reduction Act
    C. Regulatory Flexibility Act
    D. Unfunded Mandates Reform Act
    E. Executive Order 13132: Federalism
    F. Executive Order 13175: Consultation and Coordination with 
Indian Tribal Governments
    G. Executive Order 13045: Protection of Children from 
Environmental Health and Safety Risks
    H. Executive Order 13211: Actions Concerning Regulations that 
Significantly Affect Energy Supply, Distribution, or Use
    I. National Technology Transfer Advancement Act
    J. Congressional Review Act
VIII. References

I. General Information

A. Potentially Regulated Entities

    EPA Regions, as well as States, Territories, and Tribes authorized 
to implement the National Pollutant Discharge Elimination System 
(NPDES) program, issue permits that comply with the technology-based 
and water quality-based requirements of the Clean Water Act. In doing 
so, NPDES permitting authorities make a number of discretionary choices 
associated with permit writing, including the selection of pollutants 
to be measured and, in many cases, limits for those pollutants in 
permits. If EPA has ``approved'' (i.e., promulgated through rulemaking) 
standardized test procedures for a given pollutant, the NPDES 
permitting authority must specify one of the approved testing 
procedures or an EPA-approved alternate test procedure for the 
measurements required under the permit. In addition, when a State, 
Territory, or authorized Tribe provides certification of Federal 
licenses under Clean Water Act section 401, States, Territories and 
Tribes are directed to use the approved testing procedures. Categories 
and entities that may be regulated include:

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                                   Examples of potentially regulated
           Category                             entities
------------------------------------------------------------------------
Federal, State, Territorial,   Federal, State, Territorial, and Tribal
 and Indian Tribal              entities authorized to administer the
 Governments.                   NPDES permitting program; Federal,
                                State, Territorial, and Tribal entities
                                providing certification under Clean
                                Water Act section 401.
Municipalities...............  Municipal operators of NPDES facilities
                                required to monitor whole effluent
                                toxicity.
Industry.....................  Private operators of NPDES facilities
                                required to monitor whole effluent
                                toxicity.
------------------------------------------------------------------------

    This table is not intended to be exhaustive, but rather provides a 
guide for readers regarding entities likely to be regulated by this 
action. This table lists the types of entities that EPA is now aware 
could potentially be regulated by

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this action. Other types of entities not listed in the table could also 
be regulated. To determine whether your facility or organization is 
regulated by this action you should carefully examine 40 CFR 
122.41(j)(4), 122.44(i)(1)(iv), and 122.21. If you have questions 
regarding the applicability of this action to a particular entity, 
consult the first person listed in the preceding FOR FURTHER 
INFORMATION CONTACT section.

B. How Can I Get Copies of Related Information?

1. Docket
    EPA has established an official public docket for this action under 
Docket ID No. WET-X (Electronic Docket No. OW-2002-0024). The official 
public docket consists of the documents specifically referenced in this 
action, any public comments received, and other information related to 
this action. Although a part of the official docket, the public docket 
does not include Confidential Business Information (CBI) or other 
information whose disclosure is restricted by statute. The official 
public docket is the collection of materials that is available for 
public viewing at the Office of Water (OW) Docket, in the EPA Docket 
Center (EPA/DC), EPA West, Room B-102, 1301 Constitution Avenue NW., 
Washington, DC. The EPA Docket Center Public Reading Room is open from 
8:30 a.m. to 4:30 p.m. EST, Monday through Friday, excluding legal 
holidays. The telephone number for the Public Reading Room is (202) 
566-1744, and the telephone number for the OW Docket is (202) 566-2426.
2. Electronic Access
    You may access this Federal Register document electronically 
through the EPA Internet under the ``Federal Register'' listings at 
http://www.epa.gov/fedrgstr/.
    An electronic version of the public docket is available through 
EPA's electronic public docket and comment system, EPA Dockets. You may 
use EPA Dockets at http://www.epa.gov/edocket/ to view public comments, 
access the index listing of the contents of the official public docket, 
and to access those documents in the public docket that are available 
electronically. Although not all docket materials may be available 
electronically, you may still access any of the publicly available 
docket materials through the docket facility identified in Unit I.B.1. 
Once in the system, select ``search,'' then key in the appropriate 
docket identification number.

II. Statutory Authority

    EPA promulgates today's rule pursuant to the authority of sections 
301, 304(h), 402, and 501(a) of the Clean Water Act (``CWA'' or the 
``Act''), 33 U.S.C. 1311, 1314(h), 1342, 1361(a) (the ``Act''). Section 
101(a) of the Act sets forth the ``goal of restoring and maintaining 
the chemical, physical, and biological integrity of the nation's 
waters'' and prohibits ``the discharge of toxic pollutants in toxic 
amounts.'' Section 301 of the Act prohibits the discharge of any 
pollutant into navigable waters unless the discharge complies with a 
National Pollutant Discharge Elimination System (NPDES) permit, issued 
under section 402 of the Act. Section 304(h) of the Act requires the 
Administrator of the EPA to ``promulgate guidelines establishing test 
procedures for the analysis of pollutants that shall include the 
factors which must be provided in any certification pursuant to section 
401 of this Act or permit applications pursuant to section 402 of this 
Act.'' Section 501(a) of the Act authorizes the Administrator to 
``prescribe such regulations as are necessary to carry out his function 
under this Act.'' EPA publishes CWA analytical method regulations at 40 
CFR part 136. The Administrator also has made these test procedures 
applicable to monitoring and reporting of NPDES permits (40 CFR part 
122, Sec. Sec.  122.21, 122.41, 122.44, and 123.25), and implementation 
of the pretreatment standards issued under section 307 of the Act (40 
CFR part 403, Sec. Sec.  403.10 and 403.12).

III. Background

A. Regulatory History

    On October 16, 1995, EPA amended the ``Guidelines Establishing Test 
Procedures for the Analysis of Pollutants,'' 40 CFR part 136, to add a 
series of standardized toxicity test methods to the list of Agency 
approved methods for conducting required testing of aqueous samples 
under the CWA (60 FR 53529) (WET final rule). The WET final rule 
amended 40 CFR 136.3 (Tables IA and II) by adding acute toxicity 
methods and short-term methods for estimating chronic toxicity. These 
methods measure the toxicity of effluents and receiving waters to 
freshwater, marine, and estuarine organisms. Acute methods (USEPA, 
1993) generally use death of some percentage of the test organisms 
during 24 to 96 hour exposure durations as the measured effect of an 
effluent or receiving water. The short-term methods for estimating 
chronic toxicity (USEPA, 1994a; USEPA, 1994b) use longer durations of 
exposure (up to nine days) to ascertain the adverse effects of an 
effluent or receiving water on survival, growth, and/or reproduction of 
the organisms. The methods listed at 40 CFR part 136 for measuring 
aquatic toxicity are referred to collectively as ``WET test methods,'' 
methods specific to measuring acute toxicity are referred to as 
``acute'' test methods, and short-term methods for estimating chronic 
toxicity are referred to as ``chronic'' methods.
    EPA standardized the test procedures for conducting the approved 
acute and chronic WET test methods in the following three method 
manuals, which were incorporated by reference in the WET final rule: 
Methods for Measuring the Acute Toxicity of Effluents and Receiving 
Water to Freshwater and Marine Organisms, Fourth Edition, August 1993, 
EPA/600/4-90/027F (acute method manual); 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 
(freshwater chronic method manual); and 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 (marine chronic method manual). EPA explains in the Technical 
Support Document for Water Quality-Based Toxics Control (TSD) (USEPA, 
1991) that these WET test methods, along with chemical controls and 
bioassessments, are a component of EPA's integrated strategy for water 
quality-based toxics control. The TSD recommends that WET tests using 
the most sensitive of at least three test species from different phyla 
be used for monitoring the toxicity of effluents.
    Since the 1995 WET final rule, EPA has issued several rulemakings 
and guidance documents in fulfillment of settlement agreements to 
resolve judicial challenges to the WET final rule (see Settlement 
Agreement discussion in Section III.B). On February 2, 1999, EPA 
published technical corrections that incorporated into the WET final 
rule an errata document to correct minor errors and omissions, provide 
clarification, and establish consistency among the WET final rule and 
method manuals (64 FR 4975; February 2, 1999). On July 18, 2000, EPA 
announced the availability of a WET Variability Guidance Document (65 
FR 44528; July 18, 2000). On July 28, 2000, EPA published the 
availability of a WET Method Guidance Document (65 FR 46457; July 28, 
2000). On September 28, 2001, EPA proposed specific revisions to the 
WET test methods, and EPA proposed to ratify its previous

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approval of these methods (66 FR 49794; September 28, 2001) (see 
section III.C). Today, EPA takes final action on the September 2001 
proposal.

B. Settlement Agreement

    Following promulgation of the WET methods on October 16, 1995, 
several parties challenged the rulemaking (Edison Electric Institute v. 
EPA, No. 96-1062 (D.C. Cir.); Western Coalition of Arid States v. EPA, 
No. 96-1124 (D.C. Cir.); and Lone Star Steel Co. v. EPA, No. 96-1157 
(D.C. Cir.)). To resolve the litigation, EPA entered into settlement 
agreements with the various parties and agreed to publish a technical 
correction notice, publish a method guidance document and a variability 
guidance document, conduct an interlaboratory variability study, 
publish a peer-reviewed interlaboratory variability study report 
(including a table of coefficients of variation), address pathogen 
contamination, propose specific technical method changes, and propose 
to ratify or withdraw WET test methods evaluated in the interlaboratory 
variability study. Today's final action fulfills EPA's obligations 
under the settlement agreements.

C. Proposed Rule

    On September 28, 2001, EPA proposed modifications to the WET test 
methods (66 FR 49794). The proposal included updates to the methods, 
minor corrections and clarifications, and specific technical changes in 
response to stakeholder concerns. Specifically, EPA proposed technical 
changes to (1) require ``blocking'' by known parentage in the 
Ceriodaphnia dubia Survival and Reproduction Test; (2) specify 
procedures to control pH drift that may occur during testing; (3) 
incorporate review procedures for the evaluation of concentration-
response relationships; (4) clarify recommendations regarding nominal 
error rate assumptions; (5) clarify limitations in the generation of 
confidence intervals; (6) add guidance on dilution series selection; 
(7) clarify requirements regarding acceptable dilution waters; and (8) 
add procedures for determining and minimizing the adverse impact of 
pathogens in the Fathead Minnow Survival and Growth Test.
    EPA also solicited comment on other modifications to improve the 
performance of the methods, including the incorporation of variability 
criteria and increases in the minimum number of test replicates. EPA 
proposed to incorporate WET method changes into new editions of each of 
the WET method manuals (USEPA, 1993; USEPA, 1994a; USEPA, 1994b) and to 
update Table IA at 40 CFR part 136 to cite the new method manual 
editions.
    In the September 28, 2001 proposed rule, EPA also proposed to 
ratify 11 of the 12 WET methods evaluated in EPA's WET Interlaboratory 
Variability Study. EPA proposed to ratify the Ceriodaphnia dubia Acute 
Test; Fathead Minnow Acute Test; Sheepshead Minnow Acute Test; Inland 
Silverside Acute Test; Ceriodaphnia dubia Survival and Reproduction 
Test; Fathead Minnow Larval Survival and Growth Test; Selenastrum 
capricornutum Growth Test; Sheepshead Minnow Larval Survival and Growth 
Test; Inland Silverside Larval Survival and Growth Test; Mysidopsis 
bahia Survival, Growth, and Fecundity Test; and Champia parvula 
Reproduction Test. To support ratification of these methods, EPA 
presented the results of the WET Interlaboratory Variability Study 
(USEPA, 2001a; USEPA, 2001b), a national study of 12 WET methods 
involving 56 laboratories and over 700 samples. EPA proposed to 
withdraw Holmesimysis costata as an acceptable substitute species for 
use in the Mysidopsis bahia Acute Test method protocol. In its place, 
EPA proposed a new Holmesimysis costata Acute Test protocol.
    EPA invited public comment for 60 days and later extended the 
comment period for an additional 45 days (66 FR 58693; November 23, 
2001). EPA received 38 comment packages during the allotted comment 
period.

IV. Summary of Final Rule

A. Proposed WET Method Changes

    Today's action incorporates most of the method changes proposed on 
September 28, 2001 (66 FR 49794) with minor modifications to address 
public comments. For a summary of major changes from the proposed rule, 
including proposed actions not incorporated in today's rule, see 
Section V of this preamble. Method manual revisions promulgated in 
today's action include:
    [sbull] Minor corrections and clarifications,
    [sbull] Incorporation of updated method precision data,
    [sbull] Requirement for ``blocking'' by known parentage in the 
Ceriodaphnia dubia Survival and Reproduction Test,
    [sbull] Specification of procedures to control pH drift that may 
occur during testing,
    [sbull] Review procedures for the evaluation of concentration-
response relationships,
    [sbull] Clarification of limitations in the generation of 
confidence intervals,
    [sbull] Guidance on dilution series selection,
    [sbull] Clarification of requirements regarding acceptable dilution 
waters,
    [sbull] Procedures for determining and minimizing the adverse 
impact of pathogens in the Fathead Minnow Survival and Growth Test,
    [sbull] Requirement for the use of ethylenediaminetetraacetic acid 
(EDTA) in the Selenastrum capricornutum Growth Test.

B. Additional Revisions to WET Test Methods

    In addition to requesting comment on the specific modifications to 
WET test methods mentioned above, EPA solicited comment on any 
additional modifications that would improve the overall performance of 
the methods. Specifically, EPA solicited comment on application of 
variability criteria to test results, modification of test 
acceptability criteria, and increases in test replication requirements. 
In response to comments, today's final rule also incorporates the 
following additional modifications to WET test methods:
    [sbull] Requirement to meet specific variability criteria when 
NPDES permits require sublethal WET testing endpoints expressed using 
hypothesis testing,
    [sbull] Increases in the required minimum number of replicates for 
several tests,
    [sbull] Clarification of required and recommended test conditions 
for the purposes of reviewing WET test data submitted under NPDES 
permits,
    [sbull] Additional clarification of sample holding times,
    [sbull] Clarification of requirements for reference toxicant 
testing and additional guidance on evaluating reference toxicant test 
results,
    [sbull] Clarification of allowable sample holding temperatures,
    [sbull] Clarification of biomass as the measured endpoint in 
survival and growth tests,
    [sbull] Clarification of requirements for measuring total residual 
chlorine in WET samples,
    [sbull] Modification of the test termination criteria for the 
Ceriodaphnia dubia Survival and Reproduction Test to exclude the 
counting of fourth brood neonates,
    [sbull] Additional minor corrections identified by commenters.

C. Ratification and Withdrawal of Methods

    Based on the WET Interlaboratory Variability Study, peer review 
comments, and comments on the proposed rule, EPA is ratifying ten 
methods evaluated in the WET

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Interlaboratory Variability Study and withdrawing two methods. EPA is 
ratifying the Ceriodaphnia dubia Acute Test; Fathead Minnow Acute Test; 
Sheepshead Minnow Acute Test; Inland Silverside Acute Test; 
Ceriodaphnia dubia Survival and Reproduction Test; Fathead Minnow 
Larval Survival and Growth Test; Selenastrum capricornutum Growth Test; 
Sheepshead Minnow Larval Survival and Growth Test; Inland Silverside 
Larval Survival and Growth Test; and Mysidopsis bahia Survival, Growth, 
and Fecundity Test. In accordance with EPA's Report to Congress on the 
Availability, Adequacy, and Comparability of testing procedures (USEPA, 
1988), EPA has confirmed that the methods ratified today are repeatable 
and reproducible (i.e., exhibit adequate within-laboratory and between-
laboratory precision), available and applicable (i.e., adaptable to a 
wide variety of laboratories and use widely available organisms and 
supplies), and representative (i.e., predictive of receiving system 
impacts). See section VI.C.1 of this preamble.
    EPA's WET Interlaboratory Variability Study demonstrated that the 
methods ratified today generally have a high rate of successful 
completion, do not often produce false positive results, and exhibit 
precision comparable to chemical methods approved at 40 CFR part 136. 
Table 1 summarizes the performance characteristics for the ten WET test 
methods ratified today. In ratifying these WET test methods, EPA 
reaffirms the conclusion expressed in the 1995 WET final rule (60 FR 
53529; October 16, 1995), that these methods, including the 
modifications in today's rule, are applicable for use in NPDES permits.

                    Table 1.--Summary of Performance Characteristics for Ratified WET Methods
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                                                                  Successful                     Interlaboratory
                         Test method                           test completion   False positive  precision (%CV)
                                                                   rate (%)       rate \a\ (%)         \b\
----------------------------------------------------------------------------------------------------------------
Ceriodaphnia dubia Acute Test................................             95.2             0.00             29.0
Ceriodaphnia dubia Survival and Reproduction Test............             82.0             3.70             35.0
Fathead Minnow Acute Test....................................              100             0.00             20.0
Fathead Minnow Larval Survival and Growth Test...............             98.0             4.35             20.9
Selenastrum capricornutum Growth Test........................             63.6             0.00             34.3
Mysidopsis bahia Survival, Growth, and Fecundity Test........             97.7             0.00             41.3
Sheepshead Minnow Acute Test.................................              100             0.00             26.0
Sheepshead Minnow Larval Survival and Growth Test............              100             0.00             10.5
Inland Silverside Acute Test.................................             94.4             0.00             38.5
Inland Silverside Larval Survival and Growth Test............              100             0.00            43.8
----------------------------------------------------------------------------------------------------------------
\a\ False positive rates reported for each method represent the higher of false positive rates observed for
  hypothesis testing or point estimate endpoints.
\b\ Coefficients of variation (CVs) reported for each method represent the CV of LC50 values for acute test
  methods and IC25 values for chronic test methods. CVs reported are based on total interlaboratory variability
  (including within-laboratory and between-laboratory components of variability) and averaged across sample
  types.

    EPA is withdrawing the Holmesimysis costata Acute Test and the 
Champia parvula Reproduction Test methods from 40 CFR part 136. EPA was 
unable to obtain interlaboratory precision data for these methods in 
the WET Interlaboratory Variability Study due to laboratory 
unavailability. EPA was unable to contract with a minimum of six 
laboratories qualified and willing to conduct these test methods within 
the time frame of the Study. Due to this lack of interlaboratory 
precision data generated from the Study for these methods, several 
commenters recommended that these methods not be approved at 40 CFR 
part 136 for national use. In response, today's action removes the 
Holmesimysis costata Acute Test method (1995 version) and the Champia 
parvula Reproduction Test method from the list of test methods approved 
for nationwide use at 40 CFR part 136.
    By withdrawing these methods from 40 CFR part 136 for nationwide 
use, EPA does not reject their use on more limited bases. Today's 
withdrawal simply reflects that the Agency has not validated these 
methods for national use. EPA continues to support the use of these 
methods for applications other than for the determination of compliance 
with NPDES permit limits, as well for limited, localized, or regional 
use where the methods have been validated by other entities. In 
addition, EPA continues to support the use of the Holmesimysis costata 
Acute Test to measure toxicity to marine organisms of the Pacific 
Ocean. Because test procedures for measuring toxicity to estuarine and 
marine organisms of the Pacific Ocean are not listed at 40 CFR part 
136, permit writers may include (under 40 CFR 122.41(j)(4) and 
122.44(i)(1)(iv)) requirements for the use of test procedures that are 
not approved at part 136, such as the Holmesimysis costata Acute Test 
and other West Coast WET methods (USEPA, 1995b) on a permit-by-permit 
basis.

D. Amendment to 40 CFR 136.3, Table IA

    Today's rule amends 40 CFR 136.3 by removing the Champia parvula 
Reproduction Test method (Method 1009.0) from Table IA, modifying the 
reference to acute ``mysid'' tests in Table IA to include only 
Mysidopsis bahia (and not Holmesimysis costata), adding method numbers 
to acute tests, revising the parameter measured in marine tests to 
refer to organisms ``of the Atlantic Ocean and Gulf of Mexico,'' and 
modifying footnotes and references to cite the updated versions of the 
method manuals.

V. Changes From the Proposed Rule

A. Proposed WET Method Changes

    On September 28, 2001, EPA proposed technical method changes to 
improve the performance and clarity of WET test methods and to address 
specific stakeholder concerns. These provisions were presented and 
discussed in section III of the proposed rule preamble (66 FR 49794) 
and detailed in the document titled, Proposed Changes to Whole Effluent 
Toxicity Method Manuals (USEPA, 2001e). In today's action, EPA is 
withdrawing or revising some of the proposed revisions based on 
comments received on the proposed rule. These revisions are discussed 
below. Other comments that EPA addressed but did not result in changes 
from the proposal are discussed in section VI.

[[Page 69956]]

1. Blocking by Known Parentage
    EPA proposed specific method manual modifications that would 
require blocking by known parentage in the Ceriodaphnia dubia Survival 
and Reproduction Test method. Today, EPA is finalizing the proposed 
method changes with a minor modification to clarify that neonates from 
a single known parent may be used in the initiation of more than one 
test. This minor modification mitigates some commenters' concerns 
regarding the increased cost of blocking by known parentage. Blocking 
by known parentage requires the use of at least six neonates from each 
of at least ten separate parents. If more than six neonates from a 
given parent remain after allocating organisms to a test, those 
remaining neonates may be discarded, used as future culture organisms, 
or used in another test initiated on the same day (provided that the 
neonates meet age requirements).
2. pH Drift
    During the conduct of static or static-renewal WET tests, the pH in 
test containers may fluctuate or drift from the initial pH value. EPA 
proposed specific procedures that may be used to control this pH drift 
in chronic WET tests. Today, EPA is revising the specified procedures 
in response to stakeholder comments. Some commenters requested that EPA 
clarify the pH that should be maintained in pH-controlled tests. 
Today's action clarifies that, when the test objective is to determine 
the toxicity of an effluent in the receiving water, the target pH to 
maintain in a pH-controlled test is the pH of the receiving water 
measured at the edge of any mixing zone authorized in a permit. When 
the test objective is to determine the absolute toxicity of the 
effluent, the target pH to maintain in a pH-controlled test is the pH 
of the sample upon completion of collection. The revisions also clarify 
that in pH-controlled tests, the pH should be maintained within +/-0.2 
pH units of the target pH in freshwater chronic tests and within +/-0.3 
pH units for marine/estuarine chronic tests. EPA also added guidance on 
interpreting the results of parallel testing.
    The revisions also remove language from the proposed method manual 
changes that warned about effects from pH drift in the absence of pH-
dependent toxicants. To address the concern that the daily cycle of pH 
drift and renewal caused artifactual toxicity by ``shocking'' test 
organisms, EPA proposed language in the method manuals that warned of 
such potential interference from pH drift even when pH-dependent 
toxicants were not present. EPA specifically requested that commenters 
provide ``any data that show the value of proposed pH control measures 
in situations where ammonia or other pH-dependent toxicants are not 
present.'' EPA did not receive such data. EPA believes that pH drift 
alone is not a test interference if pH is within the organism's 
tolerance range. The degree of pH drift typically observed in effluent 
samples should generally only interfere with test results if the sample 
contains a compound with toxicity that is pH dependent and at a 
concentration that is near the toxicity threshold. Because EPA did not 
receive data to suggest otherwise, EPA is removing any reference to pH 
drift interference in the absence of pH-dependent toxicants.
    Many commenters recommended that EPA include the proposed pH 
control guidance for acute test methods as well as chronic methods 
because of the insufficiency of static renewal testing to control the 
pH drift and the impracticability and cost of flowthrough testing. In 
today's action, EPA has not provided additional techniques that involve 
modification of the sample to control pH drift in acute test methods, 
because EPA believes that the current acute methods provide adequate 
remedies for pH drift without modifying the sample. In acute tests, pH 
drift may be remedied by more frequent test renewals or use of 
flowthrough testing. While EPA agrees that flowthrough testing is more 
costly than static or static renewal testing, today's action does not 
impose any additional costs by requiring flowthrough testing. Today's 
action simply retains the options for pH control that are currently 
described in the acute method manual and does not add additional 
options.
3. Nominal Error Rates
    Today's action does not incorporate the proposed method manual 
changes regarding nominal error rates. The method manuals maintain the 
original statement recommending a nominal error rate of 0.05. EPA 
proposed changes to its recommendation regarding nominal error rate 
assumptions, specifically, the change from 0.05 to 0.01 under specific 
circumstances. EPA proposed changes to its recommended error rate 
assumptions based on the settlement agreement, which identified the 
circumstances under which EPA would change its recommendations 
regarding nominal error rate reductions. These specified circumstances 
do not necessarily represent cases where the risk of false positive 
results increase, but rather situations for which the petitioners 
sought specific relief.
    Commenters on the proposed rule commented that there was no 
scientific justification for reducing nominal error rate assumptions in 
only these circumstances and recommended reducing the nominal error 
rate in all circumstances. EPA agrees with the commenters that there is 
not a scientific justification for allowing reduced nominal error rates 
in these specific circumstances, but disagrees that nominal error rates 
should be reduced in all circumstances. Some commenters claimed that a 
reduced nominal error rate is needed to improve confidence in the test 
results. Reducing the nominal error rate, however, does not inherently 
improve confidence in test results. Because of the relationship between 
Type I and Type II statistical errors, reductions in nominal error 
rates improve confidence in results that identify toxicity, but reduce 
confidence in results that do not identify toxicity. This reduces the 
power of the test and the chance of identifying toxic discharges, 
thereby reducing environmental protection. In addition, the statistical 
test designs (i.e., test replication requirements) of WET methods and 
all supporting method validation data were based on a nominal error 
rate of 0.05. Because there is no scientific justification for 
recommending reductions in nominal error rates in the circumstances 
proposed and commenters did not provide such supporting rationale or 
data, EPA has not incorporated the proposed method manual 
recommendations regarding nominal error rates. The method manuals 
maintain the original recommendation to assume a nominal error rate of 
0.05.
4. Dilution Series
    EPA is finalizing the proposed guidance on the selection of 
dilution series in WET testing. In addition to the proposed guidance, 
EPA has made minor modifications in response to comments to further 
clarify that no one particular dilution series is required. Specific 
dilution series used in the WET method manuals are provided as examples 
and recommendations, not requirements.
5. Dilution Waters
    EPA is finalizing the proposed guidance on the selection of 
dilution waters in WET testing. In addition to the proposed guidance, 
EPA has made minor modifications in response to comments to further 
clarify that no single dilution water type is required for all tests. 
The method manuals now

[[Page 69957]]

clarify that receiving waters, synthetic waters, or synthetic waters 
adjusted to approximate receiving water characteristics may be used for 
dilution water, provided that the water meets the qualifications for an 
acceptable dilution water. EPA clarified in the method manuals that an 
acceptable dilution water is one which is appropriate for the 
objectives of the test; supports adequate performance of the test 
organisms with respect to survival, growth, reproduction, or other 
responses that may be measured in the test (i.e., consistently meets 
test acceptability criteria for control responses); is consistent in 
quality; and does not contain contaminants that could produce toxicity. 
EPA also provided clarification on the use of dual controls. When using 
dual controls, the dilution water control should be used for 
determining the acceptability of the test and for comparisons with the 
tested effluent. If test acceptability criteria (e.g., minimum 
survival, reproduction, or growth) are not met in the dilution water 
control, the test must be repeated on a newly collected sample. 
Comparisons between responses in the dilution water control and in the 
culture water control can be used to determine if the dilution water, 
which may be a receiving water, possesses ambient toxicity.
6. Pathogen Interference
    In today's action, EPA finalizes the proposed guidance on 
controlling pathogen interference in the Fathead Minnow Larval Survival 
and Growth Test with several modifications to address commenter 
concerns. Some commenters were concerned that the proposed guidance 
allowed the use of pathogen control techniques such as UV, 
chlorination, filtration, and antibiotics only after the recommended 
modified test design (fewer fish per cup) failed to control pathogen 
interference. Today's revisions clarify that EPA recommends pathogen 
control techniques that do not modify the sample, such as the modified 
test design technique, over ones that do. Upon approval by the 
regulatory authority, however, analysts also may use various sample 
sterilization techniques that modify the sample to control pathogen 
interference, provided that parallel testing of altered and unaltered 
samples further confirms the presence of pathogen interference and 
demonstrates successful pathogen control.
    The manuals also now provide further explanation regarding the 
purpose for and required extent of pathogen source determination. 
Commenters were concerned that EPA was requiring permittees to generate 
data that was irrelevant to correcting for pathogen test interference. 
This is not the case. Determining whether tests are adversely affected 
by pathogens in the effluent or pathogens in the receiving water used 
for test dilution is an important first step in selecting an 
appropriate pathogen control technique. If the source of interfering 
pathogens in the test is the receiving water used as the dilution 
water, then pathogen interference may be controlled by simply using an 
alternative dilution water. If the source of interfering pathogens in 
the test is the effluent, then pathogen control techniques are 
appropriate to control the interference. To further address the 
comments, EPA removed mention of pathogen source identification beyond 
determining whether the pathogen source was the effluent or dilution 
water. EPA also made several minor modifications in response to 
comments, including an acknowledgment that pathogen control techniques 
may not eliminate pathogens, but should minimize the adverse influence 
of pathogens so that test results are not confounded by mortality due 
to pathogens.
7. EDTA in the Selenastrum capricornutum Growth Test
    In the WET Interlaboratory Variability Study, EPA found that 
performance of the Selenastrum capricornutum Growth Test was much 
higher (lower interlaboratory variability and lower false positive 
rate) when the test was conducted with EDTA (ethylenediaminetetraacetic 
acid). Based on this finding, EPA proposed to recommend the use of EDTA 
in the Selenastrum capricornutum Growth Test. Several commenters 
expressed concern that EPA only recommended, rather than required, the 
use of EDTA. Commenters stated that this recommendation was not 
sufficient to ensure the acceptable performance of the method and 
encouraged EPA to require the use of EDTA. To address these comments, 
the Selenastrum capricornutum Growth Test now requires the addition of 
EDTA to nutrient stock solutions when conducting the Selenastrum 
capricornutum Growth Test and submitting data under NPDES permits. To 
address concerns that EDTA may interfere with (i.e., mask) the toxicity 
of metals, the method continues to caution that the addition of EDTA 
may cause the Selenastrum capricornutum Growth Test to underestimate 
the toxicity of metals. EPA cautions regulatory authorities to consider 
this possibility when selecting test methods for monitoring effluents 
that are suspected to contain metals. As recommended in EPA's Technical 
Support Document for Water Quality-Based Toxics Control (TSD) (USEPA, 
1991), the most sensitive of at least three test species from different 
phyla should be used for monitoring the toxicity of effluents.

B. Additional Revisions to WET Test Methods

1. Variability Criteria
    Today's action incorporates mandatory variability criteria for five 
chronic test methods. EPA recommends the use of point estimation 
techniques over hypothesis testing approaches for calculating endpoints 
for effluent toxicity tests under the NPDES Permitting Program. 
However, to reduce the within-test variability and to increase 
statistical sensitivity when test endpoints are expressed using 
hypothesis testing rather than the preferred point estimation 
techniques, variability criteria must be applied as a test review step 
when NPDES permits require sublethal hypothesis testing endpoints 
(i.e., no observed effect concentration (NOEC) or lowest observed 
effect concentration (LOEC)) and the effluent has been determined to 
have no toxicity at the permitted receiving water concentration These 
variability criteria must be applied for the following methods: Fathead 
Minnow Larval Survival and Growth Test; Ceriodaphnia dubia Survival and 
Reproduction Test; Selenastrum capricornutum Growth Test; Mysidopsis 
bahia Survival, Growth, and Fecundity Test; and Inland Silverside 
Larval Survival and Growth Test. Within-test variability, measured as 
the percent minimum significant difference (PMSD), must be calculated 
and compared to upper bounds established for test PMSDs. Under this new 
requirement, tests conducted under NPDES permits that fail to meet the 
variability criteria (i.e., PMSD upper bound) and show ``no toxicity'' 
at the permitted receiving water concentration (i.e., no significant 
difference from the control at the receiving water concentration or 
above) are considered invalid and must be repeated on a newly collected 
sample. Lower bounds on the PMSD are also applied, such that test 
concentrations shall not be considered toxic (i.e., significantly 
different from the control) if the relative difference from the control 
is less than the lower PMSD bound.
    In the proposed rule, EPA solicited comment on the required use of 
upper and lower PMSD bounds in the calculation of NOEC and LOEC values.

[[Page 69958]]

According to the proposed approach, any test treatment with a 
percentage difference from the control (i.e., [mean control response--
mean treatment response]/ mean control response * 100) that is greater 
than the upper PMSD bound would be considered as significantly 
different; and any test treatment with a percentage difference from the 
control that is less than the lower PMSD bound would not be considered 
as significantly different.
    EPA received comments on this proposed approach that expressed 
concern that variability criteria were used only to adjust NOEC and 
LOEC values and not to invalidate tests. Commenters argued that the 
proposed approach does not control variability unless tests failing to 
meet the variability criteria are invalidated. In response to these 
comments, EPA has modified the application of variability criteria in 
today's action. Rather than implementing variability criteria as a 
component of endpoint calculation, today's method modifications 
implement variability criteria (upper and lower PMSD bounds) as a test 
review step that is required when NPDES permits require sublethal WET 
testing endpoints expressed using hypothesis testing for the five test 
methods previously listed. Reviewed tests that fail to meet the 
variability criteria and do not detect toxicity at the receiving water 
concentration are invalid and must be repeated on a newly collected 
sample.
    EPA received comments both for and against implementation of 
variability criteria as test acceptability criteria. To balance these 
comments, the final rule implements the variability criteria as a 
required test review step when NPDES permits require sublethal WET 
testing endpoints expressed using hypothesis testing for the five test 
methods previously listed. As such, the variability criteria have the 
potential to invalidate highly variable tests. Invalidation, however, 
is contingent upon other data evaluation steps. For instance, tests 
that exceed the variability criteria are only invalidated when the test 
also fails to detect toxicity at the permitted receiving water 
concentration. The method manuals continue to restrict use of the term 
``test acceptability criteria'' to biological measurements in test 
controls (i.e., control survival, reproduction, and growth) that 
independently assess test acceptability. Unlike the variability 
criteria instituted today, the use of ``test acceptability criteria'' 
to invalidate tests are not contingent on any other data evaluation 
steps. For this reason, the term ``test acceptability criteria'' is not 
applicable to the variability criteria established in today's action.
    EPA received comments that recommended alternative measures for 
controlling within test variability, such as limits on the coefficient 
of variation (CV) for the control treatment. In developing variability 
criteria, EPA considered other measures of test precision, including 
the standard deviation and coefficients of variation for treatments and 
control, minimum significant difference (MSD), and the mean square for 
error from the analysis of variance of treatment effects. EPA considers 
the PMSD to be the measure that is most easily understood and that is 
most directly applied to determination of NOEC and LOEC values. The 
PMSD quantifies the smallest percentage difference between the control 
and a treatment (effluent dilution) that could be declared as 
statistically significant. It thus includes exactly that variability 
affecting determination of the NOEC and LOEC. The CV for the control or 
any one treatment, or selected treatments, represents only a portion of 
the variability affecting the NOEC and LOEC. Some State or Regional WET 
programs have requirements on the CV for the control and the treatment 
representing the receiving water concentration (RWC). Such requirements 
can provide finer control over the variability influencing a single 
comparison between the control and the RWC treatment. The PMSD upper 
bound provides control over the total within-test variability and is 
intended specifically for multi-concentration tests in which the NOEC 
or LOEC are determined by using hypothesis testing. Regulatory 
authorities may continue to use variability control strategies adopted 
within their jurisdiction, but when NPDES permits require sublethal WET 
testing endpoints expressed using hypothesis testing, the variability 
criteria required by today's action must be implemented as well. 
Requiring such variability criteria provides national consistency and 
control of WET test precision when hypothesis testing approaches are 
chosen. In today's action, EPA reiterates the recommendation of the 
method manuals and the TSD (USEPA, 1991) by stating that for the NPDES 
Permit Program, point estimation techniques are preferred over 
hypothesis testing approaches for calculating endpoints for effluent 
toxicity tests.
    EPA received comments that the upper and lower bounds established 
for PMSD variability criteria were arbitrary or unrepresentative. EPA 
established the proposed variability criteria as performance-based 
standards set at the 10th and 90th percentiles of PMSD values from 
EPA's evaluation of national reference toxicant test data (USEPA, 
2000c). In today's action, EPA has revised the variability criteria to 
reflect the 10th and 90th percentiles of PMSD values based on EPA's 
Interlaboratory Variability Study. The use of data from this study 
reflects not only tests performed on reference toxicants, but tests 
performed on effluents, receiving waters, and non-toxic ``blank'' 
samples as well. Data from this study also is representative of 
qualified laboratories that routinely conduct WET testing for 
permittees (see Section VI.C.2 of this preamble). In method 
development, EPA routinely uses such data from interlaboratory 
validation studies to set performance-based criteria.
    In September 2001, EPA proposed variability criteria for four 
methods. Some commenters recommended that EPA expand the variability 
criteria to other test methods and other test endpoints. EPA did not 
propose variability criteria for the Selenastrum capricornutum Growth 
Test and the Sheepshead Minnow Larval Survival and Growth Test because 
these methods showed lower within-test variability in EPA's evaluation 
of national reference toxicant test data (USEPA, 2000c). EPA's WET 
Interlaboratory Variability Study confirmed that the Sheepshead Minnow 
Larval Survival and Growth Test was less variable than the methods for 
which EPA proposed variability criteria, however, the Selenastrum 
capricornutum Growth Test showed comparable within-test variability to 
methods for which EPA proposed variability criteria. For this reason, 
EPA is today requiring variability criteria for the Selenastrum 
capricornutum Growth Test in addition to the four methods for which 
variability criteria were proposed.
    As previously stated in the method manuals (USEPA, 1993; USEPA, 
1994a; USEPA, 1994b) and EPA's Technical Support Document (USEPA, 
1991), EPA recommends the use of point estimation techniques over 
hypothesis testing approaches for calculating endpoints for effluent 
toxicity tests under the NPDES Permitting Program. EPA is instituting 
variability criteria to reduce within-test variability and to increase 
statistical sensitivity when test endpoints are expressed using 
hypothesis testing rather than the preferred point estimation 
techniques. For the five methods for which EPA is instituting 
variability criteria when test results are analyzed by hypothesis test 
methods, less than 90% of tests are able to detect

[[Page 69959]]

a 25% reduction in growth or reproduction (from the control treatment) 
as statistically significant using the hypothesis test. A 25% reduction 
in growth or reproduction is equivalent to the effect level measured 
using the preferred point estimation endpoint for chronic methods 
(i.e., the IC25). Instituting variability criteria for these five 
chronic methods will improve the overall statistical sensitivity when 
using hypothesis testing and allow hypothesis testing approaches to 
achieve a level of statistical sensitivity that is more comparable to 
the preferred point estimation endpoint (IC25).
    EPA is not requiring variability criteria for the Sheepshead Minnow 
Larval Survival and Growth Test, because the WET Interlaboratory 
Variability Study confirmed that this method is less variable than the 
five methods for which EPA is requiring variability criteria. In EPA's 
WET Interlaboratory Variability Study, all Sheepshead Minnow Larval 
Survival and Growth Tests were able to detect effects of 25% or less as 
statistically significant in hypothesis testing without instituting 
variability criteria. The 90th percentile PMSD for the Sheepshead 
Minnow Larval Survival and Growth Test was 17%, compared to 29%, 47%, 
30%, 37%, and 28% for the five methods for which EPA is requiring 
variability criteria. For the chronic methods that were not evaluated 
in the WET Interlaboratory Variability Study, EPA does not have 
sufficient data to support the implementation of mandatory variability 
criteria at this time.
    EPA is not requiring variability criteria for survival endpoints of 
acute methods because, in general, these methods are less variable than 
sublethal chronic test methods, and hypothesis testing approaches are 
able to achieve a level of statistical sensitivity similar to the 
preferred point estimation endpoint for acute methods and survival 
endpoints (i.e., the LC50). The preferred point estimation endpoint for 
the analysis of survival in acute methods is the LC50, which represents 
an effect level of 50% mortality. Over 90% of acute tests in the WET 
Interlaboratory Variability Study were able to detect effects of 50% 
mortality or less as statistically significant in hypothesis testing 
without instituting variability criteria. The 90th percentile of PMSD 
values in the WET Interlaboratory Variability Study was 39% for the 
Fathead Minnow Acute Test, 25% for the Ceriodaphnia dubia Acute Test, 
17% for the Sheepshead Minnow Acute Test, and 31% for the Inland 
Silverside Acute Test. Based on these measured PMSD values, well over 
90% of acute tests should be able to detect effects at the LC50 as 
statistically significant without instituting variability criteria.
    By requiring application of variability criteria today in five 
methods, EPA does not intend to discourage permitting authorities from 
applying variability criteria for other endpoints or methods, or from 
applying more stringent variability criteria for the five chronic 
methods subject to today's action. While EPA continues to recommend 
that permitting authorities apply variability criteria to additional 
methods as recommended in EPA guidance (USEPA, 2000c), today's rule 
does not require such variability criteria for additional methods or 
endpoints.
2. Minimum Number of Replicates
    EPA solicited comment on increasing the minimum number of 
replicates in certain WET tests from three to four. Commenters were 
supportive of this proposed change and stated that this change was 
needed to support the use of non-parametric hypothesis tests as 
outlined in the method manuals. In today's action, EPA is increasing 
the minimum number of replicates as proposed.
3. Test Requirements/Recommendations
    Several commenters on the proposed rule expressed concern that WET 
methods do not adequately differentiate between mandatory test 
conditions (i.e., those required using the words ``must'' or ``shall'') 
and discretionary test conditions (i.e., those recommended using the 
word ``should''). Commenters claimed that this situation causes 
difficulty in reviewing, validating, and certifying test results 
submitted under NPDES permits. To address this concern, EPA modified 
the WET methods to clearly distinguish between required and recommended 
test conditions for the purposes of reviewing WET test data submitted 
under NPDES permits. In today's action, EPA has modified the tables of 
test conditions and test acceptability criteria presented in the method 
manuals for each method, such that each test condition is identified as 
required or recommended. In addition, EPA has added to each method 
manual a section on test review. This section provides guidance on the 
review of sampling and handling procedures, test acceptability 
criteria, test conditions, statistical methods, concentration-response 
relationships, reference toxicant testing, and test variability. This 
section also establishes two new requirements for WET test review: 
mandatory review of concentration-response relationships and, for some 
methods, the mandatory variability criteria described earlier.
4. Sample Collection and Holding Times
    In today's action, EPA has further clarified the requirements for 
sample collection and sample holding times. EPA made these 
modifications in response to comments requesting additional 
clarification and additional flexibility. In today's action, EPA has 
not modified the default maximum 36 hour sample holding time (up to 72 
hours with regulatory authority approval), which must be met for first 
use of the sample, but EPA has provided additional clarification and 
additional flexibility for the use of samples for test renewals when 
the samples meet the initial sample holding times for first use. Sample 
holding times apply to ``first use of the sample,'' and samples may be 
used for renewal at 24, 48, and/or 72 hours after first use.
    The method manuals also now provide additional flexibility when 
shipment of renewal samples is delayed during an ongoing test. If 
shipping problems (e.g., unsuccessful Saturday delivery) are 
encountered with renewal samples after a test has been initiated, the 
permitting authority may allow the continued use of the most recently 
used sample for test renewal. EPA also clarified that sample collection 
on days one, three, and five is the recommended (not required) sample 
collection scheme. A minimum of three samples are required for seven-
day chronic tests, but variations in the sampling scheme (i.e., the 
days on which new samples are collected) also are allowed.
5. Reference Toxicant Testing
    Today's action clarifies the purpose and requirements of reference 
toxicant testing and the appropriate use of reference toxicant test 
results. Several commenters identified inconsistencies in the 
requirements for reference toxicant testing and recommended that EPA 
clarify the purpose of generating reference toxicant test data. In 
today's action, EPA clarifies that reference toxicant testing is used 
to (1) initially demonstrate acceptable laboratory performance, (2) 
assess the sensitivity and health of test organisms, and (3) document 
ongoing laboratory performance. EPA has made method manual 
modifications consistent with this stated purpose. Regardless of the 
source of test organisms (in-house cultures or purchased from external 
suppliers), the testing laboratory must perform at least one acceptable 
reference toxicant test per month for each type of toxicity test method 
conducted in that month. If a test

[[Page 69960]]

method is conducted only monthly, or less frequently, a reference 
toxicant test must be performed concurrently with each effluent 
toxicity test. This requirement will document ongoing laboratory 
performance and assess organism sensitivity and consistency when 
organisms are cultured in-house. When organisms are obtained from 
external suppliers, concurrent reference toxicant tests must be 
performed with each effluent sample, unless the test organism supplier 
provides control chart data from at least the last five months of 
reference toxicant testing. This requirement assesses organism 
sensitivity and health when organisms are obtained from external 
vendors. To initially demonstrate acceptable laboratory performance, 
the method manuals require a laboratory to obtain consistent, precise 
results with reference toxicants before it performs toxicity tests with 
effluents under NPDES permits.
    In today's action, EPA also clarifies the appropriate use of 
reference toxicant test results. Commenters recommended that EPA 
provide additional guidance on evaluating reference toxicant test 
results and using these results to validate toxicity tests on test 
samples of unknown toxicity. In response, EPA clarifies that reference 
toxicant test results should not be used as a de facto criterion for 
rejection of individual effluent or receiving water tests. Reference 
toxicant testing is used for evaluating the sensitivity and consistency 
of organisms over time and for documenting initial and ongoing 
laboratory performance. EPA clarified the steps to take when more than 
1 in 20 reference toxicant tests falls outside of control chart limits, 
or when a reference toxicant test result falls ``well'' outside of 
control limits. Under these circumstances, the laboratory should 
investigate sources of variability, take corrective actions to reduce 
identified sources of variability, and perform an additional reference 
toxicant test during the same month.
    In response to comments that reference toxicant testing only 
compares variability within a laboratory, EPA added guidance for 
evaluating test precision among laboratories and for limiting excessive 
variability in reference toxicant testing. EPA has recommended that 
laboratories compare the calculated coefficient of variation, also 
referred to as the CV (i.e., standard deviation/mean), of the IC25 or 
LC50 for the 20 most recent data points to the distribution of 
laboratory CVs reported nationally for reference toxicant testing 
(USEPA, 2000c). If the calculated CV exceeds the 75th percentile of CVs 
reported nationally for LC50s or IC25s, the laboratory should use the 
75th and 90th percentiles to calculate warning and control limits, 
respectively, and the laboratory should investigate options for 
reducing variability.
    Several commenters recommended standardizing reference toxicants 
and acceptance ranges for reference toxicant test results. Other 
comments opposed mandatory reference toxicants and required acceptance 
ranges claiming that insufficient guidance and data are available for 
instituting such requirements and that such requirements would impose 
additional costs on laboratories. In today's action, EPA is not 
requiring the use of specific reference toxicants or setting required 
acceptance ranges for reference toxicant testing. EPA agrees that 
requiring specific reference toxicants and acceptance ranges would 
increase laboratory costs. Many laboratories would be forced to develop 
initial and ongoing documentation of laboratory performance (e.g., 
reference toxicant control charts) using a new reference toxicant. For 
these laboratories, years of historic performance information using the 
original reference toxicant would be rendered useless. In addition, EPA 
believes that certain advantages gained by requiring reference toxicant 
acceptance ranges are already provided by method modifications 
instituted in today's action. For instance, today's action institutes 
variability criteria when NPDES permits require sublethal WET testing 
endpoints expressed using hypothesis testing. This method modification 
limits WET test variability, which would be one of the primary purposes 
of any standardized reference toxicant acceptance ranges.
6. Sample Holding Temperature
    Today's action clarifies the allowable sample holding temperatures 
for WET samples as 0[deg]-6[deg]C. EPA received comments that the 
Agency should establish acceptable ranges for the current sampling 
holding temperature of 4[deg]C. EPA has defined the acceptable range as 
0[deg]-6[deg]C based on current NELAC (National Environmental 
Laboratory Accreditation Conference) standards which state that, ``for 
samples with a specified storage temperature of 4[deg]C, storage at a 
temperature above the freezing point of water to 6[deg]C shall be 
acceptable'' (NELAC, 2001). EPA also clarifies that hand-delivered 
samples used on the day of collection do not need to be cooled to 
0[deg]-6[deg]C prior to test initiation.
7. Biomass
    Today's action clarifies that the sublethal endpoint used in 
survival and growth tests is based on the number of initial organisms 
exposed. Comments expressed concern that by calculating the chronic 
endpoint based on the number of initial organisms (rather than 
surviving organisms), the growth endpoint was in error and biased. EPA 
disagrees. In the 1995 WET final rule, EPA changed the test endpoint 
from a growth endpoint that was based on the number of surviving 
organisms, to a combined growth and survival endpoint that is based on 
the number of initial organisms. This does not represent an error in 
the endpoint calculation, but rather a change in the endpoint itself. 
EPA made this change: (1) to provide consistency with other methods 
(e.g., Ceriodaphnia dubia Survival and Reproduction Test) that 
incorporate survival along with sublethal effects, and (2) because the 
survival and growth endpoint is a more sensitive measure than the 
growth endpoint alone. While the 1995 WET final rule changed the test 
endpoint to a combined survival and growth endpoint, the method manuals 
continued to refer to the endpoint as a ``growth'' endpoint. Today's 
action clarifies that the endpoint is, in fact, a combined survival and 
growth endpoint that is more accurately termed biomass.
8. Total Residual Chlorine
    Today's action clarifies the requirements for measuring total 
residual chlorine in WET test samples. Several commenters stated that 
certain requirements for measuring total residual chlorine were 
unnecessary when the absence of the chemical has already been 
determined. In response to these comments, EPA has clarified that if 
total residual chlorine is not detected in effluent or dilution water 
at test initiation, it is unnecessary to measure total residual 
chlorine at test solution renewal or at test termination. If total 
residual chlorine is detected at test initiation, then measurement of 
total residual chlorine at test solution renewal and test termination 
would continue to be required. EPA also has clarified that the 
measurement of total residual chlorine is unnecessary in laboratory 
prepared synthetic dilution water.
    Commenters also recommended that EPA remove the requirement for the 
analysis of total residual chlorine immediately following sample 
collection. EPA has maintained this requirement in today's action, 
because information on chlorine at the site and

[[Page 69961]]

time of collection is important for evaluating the effectiveness of 
chlorination/dechlorination processes and comparing the results of WET 
testing with instream effects.
9. Ceriodaphnia dubia Survival and Reproduction Test Termination 
Criteria
    Commenters recommended various modifications to the test 
termination criteria in the Ceriodaphnia dubia Survival and 
Reproduction Test. Some commenters recommended a strict seven-day test, 
and others recommended that the test last no longer than seven days. 
Other commenters recommended that the test be terminated when 80% of 
control females produce three broods, rather than the current criteria 
of 60%. Still other commenters recommended that fourth brood neonates 
not be counted. To evaluate the recommended approaches to terminating 
Ceriodaphnia dubia Survival and Reproduction Tests, EPA analyzed test 
data from the WET Interlaboratory Variability Study using each of the 
recommended test termination criteria. EPA compared the recommended 
criteria to the current criteria by calculating within-test variability 
and successful test completion rates under each of the test termination 
scenarios. While some of the recommended test termination criteria 
(such as termination when 80% of control females produce three broods 
or a maximum of seven days) slightly improved the within-test 
variability of the method (from a median PMSD of 23.2% to 19.9%), these 
criteria caused significant reductions in successful test completion 
(from 83% successful completion to 66%). Only the recommendation to 
exclude fourth brood neonates resulted in a decrease in within-test 
variability without an offsetting decrease in the rate of successful 
test completion. Based on these results, EPA is modifying the 
Ceriodaphnia dubia Survival and Reproduction Test to specify that 
neonates from fourth broods are excluded from the number of neonates 
counted in the test. With the exception of excluding fourth brood 
neonates, EPA is maintaining the current test termination criteria. 
These criteria state that the test is terminated when 60% or more of 
the surviving control females have produced their third brood, or at 
the end or eight days, whichever occurs first. These criteria may be 
met at six, seven, or eight days.
10. Additional Minor Corrections
    Some commenters identified additional errors in the WET method 
manuals or the proposed changes that EPA was not aware of at the time 
of proposal. In today's action, EPA has made these additional 
corrections and minor clarifications.

C. Ratification and Withdrawal of Methods

    In the September 28, 2001 proposal, EPA proposed to ratify the 
following eleven test methods evaluated in the WET Interlaboratory 
Variability Study: Ceriodaphnia dubia Acute Test; Fathead Minnow Acute 
Test; Sheepshead Minnow Acute Test; Inland Silverside Acute Test; 
Ceriodaphnia dubia Survival and Reproduction Test; Fathead Minnow 
Larval Survival and Growth Test; Selenastrum capricornutum Growth Test; 
Sheepshead Minnow Larval Survival and Growth Test; Inland Silverside 
Larval Survival and Growth Test; Mysidopsis bahia Survival, Growth, and 
Fecundity Test; and Champia parvula Reproduction Test. EPA proposed to 
withdraw the Holmesimysis costata Acute Test and, in its place, 
proposed a revised version of the method. As explained previously, EPA 
is ratifying ten of these methods today based on the results of EPA's 
WET Interlaboratory Variability Study that demonstrate the adequacy, 
availability, and comparability of the methods (see Section IV.C). For 
these ten methods, EPA generated sufficient interlaboratory validation 
data, and those data justify ratification. EPA's WET Interlaboratory 
Study evaluated interlaboratory precision, successful test completion 
rates, and false positive rates of the WET methods from the testing of 
over 700 samples in 56 laboratories. For each method ratified in 
today's action, EPA obtained interlaboratory data on four sample 
matrices from at least seven laboratories to as many as 35 
laboratories.
    Several commenters expressed concern that EPA did not properly 
validate WET test methods, specifically, the Champia parvula 
Reproduction Test and the Holmesimysis costata Acute Test. EPA was 
unable to obtain interlaboratory precision data for these methods in 
the WET Interlaboratory Variability Study. Because these WET methods 
are not used widely in NPDES permits, EPA was unable to contract with a 
minimum of six laboratories qualified and willing to conduct these test 
methods within the time frame of the Study. In the proposed rule, EPA 
supported these methods with intralaboratory precision data and limited 
interlaboratory precision data (two trials of the Holmesimysis costata 
Acute Test in two laboratories), but commenters questioned the 
sufficiency of such data for validating methods for nationwide use, as 
well as the necessity to approve such methods for nationwide use.
    EPA has reviewed its proposal to ratify the Champia parvula 
Reproduction Test in light of comments received and has decided to 
withdraw the method from the list of nationally-approved test methods 
at 40 CFR part 136. At the current time, an insufficient number of 
laboratories nationwide have the capabilities to perform the method. As 
noted, EPA was thus unable to obtain a rigorous multi-laboratory 
performance data set to comprehensively evaluate this method. EPA had 
predicted that as the requirements for use of this organism in the 
NPDES permit program increased, the resulting increase in market demand 
would result in an increase in the number of laboratories capable of 
performing the test. However, the number of permits requiring the 
Champia parvula chronic test has remained low (DeGraeve et al., 1998), 
so few laboratories have invested in developing Champia parvula 
cultures or standard operating procedures for the method. While today's 
action removes the Champia parvula chronic test method from the 40 CFR 
part 136 listing, EPA retains the standardized method in the marine 
chronic method manual with an explanation that the method is not listed 
at 40 CFR part 136 for nationwide use. Accordingly, retention of the 
method in the method manual continues to enable standardization of the 
method for developmental and other non-regulatory purposes and may 
foster laboratories to maintain or even develop expertise in performing 
the method.
    EPA also has reviewed its proposal of the Holmesimysis costata 
Acute Test in light of comments received. As proposed, EPA now 
withdraws Holmesimysis costata as an acceptable species for use in the 
Mysidopsis bahia Acute Test method. EPA does not, however, promulgate 
the proposed Holmesimysis costata Acute Test method as a nationally-
approved method at 40 CFR part 136 at this time. Because the 
Holmesimysis costata Acute Test is used in only a small number of 
permits on the West Coast, EPA was unable to obtain sufficient 
interlaboratory data on this method during the time that the WET 
Interlaboratory Variability Study was conducted to support today's 
rulemaking. While today's action removes the Homesimysis costata Acute 
Test from the 40 CFR part 136 listing, EPA includes the proposed method 
in the method manual with an explanation

[[Page 69962]]

that the method has not yet been approved at 40 CFR part 136 for 
nationwide use.
    Three commenters, including the California State Water Resources 
Control Board, supported ratification of the Holmesimysis costata Acute 
Test method. The California State Water Resources Control Board added 
that ratification of this method was ``particularly important, as it is 
the only method employing a marine species that is indigenous to the 
Pacific coast.'' The California State Water Resources Control Board has 
been proactive in developing, testing, validating, and implementing WET 
test methods specific to West Coast species (USEPA, 1995b), and EPA 
does not intend to frustrate that effort by today's action. For this 
reason, EPA is specifying in Table IA of 40 CFR part 136 that the 
marine acute and marine chronic test methods ratified in today's 
rulemaking measure toxicity to estuarine and marine organisms ``of the 
Atlantic Ocean and Gulf of Mexico.'' By defining the parameter measured 
by promulgated marine methods as toxicity to organisms ``of the 
Atlantic Ocean and Gulf of Mexico,'' today's action does not displace 
West Coast methods that have been approved for use in States such as 
California. Because test procedures for measuring toxicity to estuarine 
and marine organisms of the Pacific Ocean are not listed at 40 CFR part 
136, permit writers may include (under 40 CFR 122.41(j)(4) and 
122.44(i)(1)(iv)) requirements for the use of test procedures that are 
not approved at part 136, such as West Coast WET methods (USEPA, 1995b) 
on a permit-by-permit basis. Furthermore, this rule does not preclude 
permit writers addressing marine or estuarine waters of the Pacific 
Ocean from requiring, on a permit-by-permit basis, any method 
designated as approved for ``estuarine and marine organisms of the 
Atlantic Ocean and Gulf of Mexico,'' where such method is suitable for 
the specific application.

VI. Response to Major Comments

    EPA encouraged public participation in this rulemaking and 
requested comments on the proposed revision and ratification of WET 
methods. EPA also requested data supporting comments, if available. 
Thirty-eight stakeholders provided comments on the proposal. 
Stakeholders included eight laboratories, eight regulatory authorities, 
11 industries/industry groups, nine publicly-owned treatment works 
(POTWs), and two environmental consulting companies.
    This section summarizes major comments received on the proposed 
rule that were not previously addressed in Section V and provides a 
summary of EPA's responses. The complete comment summary and response 
document can be found in the public record for this final rule.

A. Proposed WET Method Changes

    EPA received comments on each of the proposed method changes, and 
those comments that prompted modifications to the proposed method 
changes are discussed in pection V of this preamble. Other substantial 
comments on proposed method changes follow.
1. Cost
    Several commenters expressed concern that proposed method 
modifications will increase test costs. Of the WET method modifications 
instituted in today's action, only four are additional mandatory 
changes that have the potential to increase test costs. These four 
modifications include: (1) The requirement for blocking by known 
parentage in the Ceriodaphnia dubia Survival and Reproduction Test; (2) 
the requirement to review test results for concentration-response 
relationships; (3) the incorporation of mandatory variability criteria 
for certain test methods when NPDES permits require sublethal WET 
testing endpoints expressed using hypothesis testing; and (4) the 
increase in the minimum number of replicates for the Fathead Minnow 
Larval Survival and Growth Test, Selenastrum capricornutum Growth Test, 
Sheepshead Minnow Larval Survival and Growth Test, Inland Silverside 
Larval Survival and Growth Test, and Sea Urchin Fertilization Test. EPA 
believes that the overall cost increases due to these changes will be 
minor and that the potential benefits of these modifications outweigh 
the incremental costs. EPA has estimated that the total cost of these 
modifications for all permittees will be less than five million dollars 
per year nationwide for all tests (Table 2 and USEPA, 2002). EPA 
believes that these costs also would be alleviated by a potential 
reduction in costs for retesting and additional investigations (e.g., 
toxicity identification evaluations). The modifications should result 
in improved test performance and increased confidence in the 
reliability of testing results.

 Table 2.--Estimated Total Cost Resulting From WET Method Modifications
              Required by Today's Action (From USEPA, 2002)
------------------------------------------------------------------------
                        Modification                         Cost ($/yr)
------------------------------------------------------------------------
Blocking-by-parentage......................................     $352,592
Concentration-response relationship........................       98,069
Increased replicates.......................................      886,634
Variability criteria.......................................    2,595,873
                                                            ------------
    Total..................................................    3,933,168
------------------------------------------------------------------------

2. Concentration-Response Relationships
    Today, EPA is finalizing proposed method modifications to require 
the review of concentration-response relationships for all multi-
concentration tests. Under this requirement, the concentration-response 
relationship generated for each multi-concentration test must be 
reviewed to ensure that calculated test results are interpreted 
appropriately. In conjunction with this requirement, EPA has provided 
recommended guidance for concentration-response relationship review 
(USEPA, 2000a).
    Several commenters expressed concern that the proposed method 
modifications require that the concentration-response relationship be 
reviewed but does not require that a concentration-response 
relationship be established before determining that toxicity is 
present. Commenters recommended that EPA require the establishment of a 
``valid'' concentration-response relationship prior to determining 
toxicity. Though within the scope of the proposed rule, EPA does not 
consider such a requirement appropriate for several reasons. First, WET 
methods and the WET testing program rely on the measurement of specific 
test endpoints (NOECs, LC50s, IC25s) for determining toxicity, not on 
achievement of specified concentration-response patterns. Second, the 
concentration-response guidance is a component of test review that 
ensures that test endpoints, which are used to determine toxicity, are 
calculated and interpreted appropriately. Second, concentration-
response relationships are empirical; and a single definition for a 
``valid'' concentration-response relationship is not appropriate. A 
range of toxicants may produce an infinite range of different shaped 
responses. In addition, a single response pattern may be due to several 
different reasons, some indicating toxicity, and some not. For example, 
the presence of pathogens,

[[Page 69963]]

considered an adverse effect confounding WET tests, may produce the 
same concentration-response pattern as a true toxicant. For this 
reason, EPA designed the guidance as a step-by-step review process that 
investigates the causes for non-ideal concentration-response patterns 
and provides for proper interpretation of test endpoints. Third, WET 
testing has inherent characteristics that may limit the ability to 
achieve ideal concentration-response relationships. For instance, WET 
testing is constrained to 100% effluent sample as the highest test 
concentration. This sometimes inhibits the ability to establish an 
ideal concentration-response relationship that extends gradually from 
no effect at one concentration to complete effect at some higher 
concentration. Traditional toxicology on pure substances, from which 
the concentration-response relationship concept is borrowed, is not 
similarly constrained. Test concentrations can be increased or lowered 
until an ideal response is generated. The typical WET test design of 
five concentrations and a control also may limit the ability to 
generate ideal concentration-response relationships. The location or 
spacing of these five concentrations may miss the gradual transition 
from no effect to complete effects. In traditional toxicology using 
pure substances, tests can be rerun with altered or additional test 
concentrations of the same compound, but in WET testing each individual 
sample and test is unique and cannot be exactly duplicated due to the 
complex and dynamic nature of the test samples over time. Non-ideal 
concentration-response relationships will occasionally be encountered 
in WET testing, and the goal of concentration-response relationship 
review is to properly interpret these non-ideal patterns.
    Fourth, the concentration-response relationship guidance has been 
shown to be very effective at reducing false positives. For instance, 
in the WET Interlaboratory Variability Study, the use of the 
concentration-response relationship guidance reduced false positive 
incidences from above 14% to below 5% for some methods (USEPA, 2001a).
3. Confidence Intervals
    EPA is finalizing the proposed method modifications that provide 
guidance when confidence intervals are not generated. This guidance 
clarifies that confidence intervals may not be generated by EPA 
software when test data do not meet specific assumptions required by 
the statistical methods, when point estimates are outside of the test 
concentration range, or when specific limitations imposed by the 
software are encountered. EPA also provides guidance for proceeding 
under each circumstance. Some commenters stressed the importance of 
obtaining confidence intervals in all circumstances and recommended 
that EPA use confidence intervals in assessing the reliability of 
results and determining compliance. EPA believes that the failure to 
generate confidence intervals should not adversely affect WET test 
result reporting because confidence intervals surrounding point 
estimates are not currently reported in the Permit Compliance System 
(the national database tracking compliance with NPDES permits) or used 
in compliance determinations. Compliance with permit requirements is 
based on the point estimate itself and not confidence intervals 
surrounding the estimate. This approach is no different in WET testing 
than in chemical testing, where compliance is also based on the 
analytical result itself. EPA demonstrated in the WET Interlaboratory 
Variability Study that the WET methods provide adequate precision and 
adequate protection from false positives. Therefore, EPA is not 
altering the compliance determination approach to include the use of 
confidence intervals.

B. Additional Revisions to WET Test Methods

    In addition to receiving comment on proposed method modifications, 
EPA received comments recommending additional method modifications. 
Those recommendations that EPA incorporated in today's action and those 
comments that prompted additional modifications are discussed in 
section V of this preamble. Other substantial comments on additional 
method changes are discussed below.
1. Method Flexibility
    EPA received comments that requested additional requirements be 
added to WET test methods, as well as comments that WET test methods 
are overly restrictive and would benefit from additional flexibility. 
As with all promulgated methods, EPA has attempted to balance these two 
opposing objectives. EPA has prescribed certain method elements when 
necessary to ensure the reliability of results, and allowed flexibility 
in other method elements so that the performance of analytical methods 
can be optimized. As noted in section V.B.3, EPA reevaluated the use of 
mandatory and discretionary terms in the WET test methods to ensure 
that the terms are included in the manuals as intended.
    EPA received comments that WET test methods do not adequately 
distinguish between required and recommended procedures. In response, 
EPA modified the tables of test conditions and test acceptability 
criteria presented in the method manuals for each method, such that 
each item is identified as required or recommended. In addition, EPA 
added to each method manual a section on test review. This section 
provides direction on the review of sampling and handling procedures, 
test acceptability criteria, test conditions, statistical methods, 
concentration-response relationships, reference toxicant testing, and 
test variability.
    EPA believes that these method modifications clarify the 
requirements for acceptable WET test results submitted under NPDES 
permits. However, EPA acknowledges that these method modifications will 
not solve all commenters concerns regarding inconsistencies in WET test 
review and acceptance. In the WET test methods, EPA established the 
minimum requirements for acceptable WET tests. In some cases, NPDES 
permits incorporate recommendations from the WET test method manuals as 
requirements in the permit (on a permit-by-permit basis). Authorized 
States retain the authority to establish more stringent requirements or 
to require additional procedures, test conditions, or QC elements. 
Thus, WET requirements ultimately reflected as NPDES permit 
requirements may continue to differ among States.
2. Test Acceptability Criteria
    In the proposed rule, EPA solicited comments on increasing the test 
acceptability criteria for mean control reproduction in the 
Ceriodaphnia dubia Survival and Reproduction Test and mean control 
weight in the Fathead Minnow Larval Survival and Growth Test. EPA also 
requested that commenters submit supporting data. EPA received comments 
both in favor of and opposed to increasing test acceptability criteria 
for these methods, but these comments were not accompanied by 
supporting data. Because EPA does not currently possess and did not 
receive data indicating that such changes would improve the performance 
of the methods, EPA is not modifying the survival, growth and 
reproduction test acceptability criteria for these methods in today's 
action.
    EPA also received comments recommending the Agency establish 
requirements for additional test acceptability criteria, such as limits 
on

[[Page 69964]]

control variability. Today's action does establish mandatory 
variability criteria when NPDES permits require sublethal WET testing 
endpoints expressed using hypothesis testing. EPA has incorporated 
these variability criteria as a required test review step for five 
methods rather than as test acceptability criteria, meaning that, 
depending on the reviewed result, retesting may be necessary. EPA 
continues to use the term ``test acceptability criteria'' only to refer 
to the evaluation of biological measurements in test controls (i.e., 
control survival, reproduction, and growth).
3. Quality Assurance/Quality Control Requirements
    Some commenters expressed concern that WET test methods do not 
contain adequate quality assurance/quality control (QA/QC) 
requirements. Each of the toxicity test method manuals contains 
separate, detailed, QA/QC guidelines, and each analytical method within 
these manuals discusses all aspects of the tests which are related to 
QA/QC. Section 4 of each method manual provides QA/QC requirements and 
guidance for facilities, equipment, and test chambers; test organisms; 
culturing and test dilution water; effluent and receiving water 
sampling and handling; test conditions; food quality; test 
acceptability criteria; calibration and standardization; replication 
and test sensitivity; demonstrating acceptable laboratory performance; 
documenting ongoing laboratory performance; and record keeping. The 
primary QA/QC requirements of WET test methods, as contained in section 
4 of the method manuals, remain the requirements for acceptable 
biological performance (survival, reproduction, and growth) in test 
controls and the requirement for the routine analysis of reference 
toxicants. In today's action, however, EPA added additional QA/QC 
requirements including the required review of concentration-response 
relationships and mandatory variability criteria when NPDES permits 
require sublethal WET testing endpoints expressed using hypothesis 
testing. EPA believes that the QA/QC requirements of WET tests will 
adequately ensure that results are reliable and of known and acceptable 
quality.
4. Statistical Methods
    Several commenters recommended that EPA approve and use alternative 
statistical methods (such as percent effect approaches and Generalized 
Linear Models). EPA has not included such alternative statistical 
methods in today's modifications to WET test methods. EPA believes that 
the statistical methods currently recommended in the WET methods are 
appropriate, and acknowledges that these recommended statistical 
methods are not the only appropriate techniques. The method manuals 
state that, ``the statistical methods recommended in this manual are 
not the only possible methods of statistical analysis.'' The 
recommended statistical methods described in the method manuals were 
selected because they are ``(1) applicable to most of the different 
toxicity test data sets for which they are recommended, (2) powerful 
statistical tests, (3) hopefully ``easily'' understood by 
nonstatisticians, and (4) amenable to use without a computer, if 
necessary'' (see subsection 9.4.1.2 of USEPA, 1994a).
    Several commenters also expressed concern over bias introduced by 
the smoothing technique that is used in the recommended Inhibition 
Concentration Procedure (ICp). EPA has acknowledged in the method 
manuals and in method guidance (USEPA, 2000a) that the smoothing 
process may result in an upward adjustment in the control mean. EPA has 
provided guidance on concentration-response relationship review that 
corrects anomalous results that may arise from this smoothing procedure 
(USEPA, 2000a). This guidance warns that results from point estimation 
techniques should be interpreted carefully when the response pattern 
includes stimulation at low concentrations and no significant effect at 
higher concentrations. Under these conditions, the smoothing process 
could result in anomalous results, so EPA guidance recommends 
evaluating the ICp calculation without smoothing in these cases. If the 
percent effect at the receiving water concentration (RWC) is less than 
25% when calculated without smoothing, and the response at the RWC is 
not statistically significantly different from the control response, 
then a calculated IC25 of less than the RWC should be noted as 
anomalous and the effluent determined to be non-toxic at the RWC.

C. Ratification and Withdrawal of Methods

1. Validation of Performance Characteristics
    Several commenters stated that EPA did not properly validate WET 
test methods because it did not evaluate essential performance 
characteristics. Commenters referenced EPA's Report to Congress on the 
Availability, Adequacy, and Comparability of Testing Procedures (USEPA, 
1988) and stated that EPA failed to validate the following performance 
characteristics required by this report: accuracy, precision, dynamic 
range, detection limits, interferences, ruggedness (applicability), 
reporting, and representativeness/method comparability. EPA disagrees 
with this assertion and maintains that the WET test methods ratified in 
today's action were adequately validated according to all of the 
applicable criteria identified in the 1988 Report to Congress.
    The list of performance characteristics cited by the commenters is 
provided in the 1988 Report to Congress within the context of chemical 
methods, and several of these characteristics are not applicable to 
biological test methods such as the WET methods that EPA is ratifying 
today. The 1988 Report to Congress specifically notes that not all such 
criteria apply to biological testing. The Report explains that the 
generation of scientifically accurate and valid biological measurements 
for environmental pollutants requires approximately the same criteria 
for assessing the adequacy of a method as previously described for 
chemical analyses, however, there are several differences which are 
important. Detection limits and dynamic range are specifically listed 
as characteristics that ``are not usually appropriate concepts for all 
biological measurements unless instrumentation is required.'' Because 
some performance characteristics listed in the 1988 Report to Congress 
for chemical methods are not applicable to biological test methods, EPA 
did not (and, in fact, could not) evaluate those inapplicable 
performance characteristics for WET test method validation.
    In ratifying the previously approved WET test methods, EPA applied 
the availability, adequacy, and comparability criteria identified in 
the Report as relevant to biological measurements. The WET test methods 
ratified today are ``available'' because EPA has identified a 
sufficient number of laboratories that can conduct the test and culture 
the test organisms. The ratified WET test methods are ``adequate'' 
because the multi-laboratory tests (as well as aggregation of single 
laboratory tests) demonstrate high degrees of precision; the tests are 
reproducible. In addition, the manuals identify interferences and ways 
to control interference. Finally, the test acceptability criteria for 
control performance and requirements for reference toxicant testing 
provide sufficient standards to ensure data integrity, absent the 
``calibration'' procedures available with non-living analytical 
instrumentation.

[[Page 69965]]

    The Report specifically identified detection limits and dynamic 
range as performance characteristics that are usually not applicable to 
biological measurements, and the 1988 conclusions remain true today. In 
addition, accuracy is a performance characteristic that is not 
completely applicable to WET testing. Accuracy as a performance 
characteristic of a measurement system describes the closeness of 
measured results to a known result. Chemical methods generally measure 
some surrogate property (e.g., absorption of light at a particular 
wavelength) of an analyte (e.g., copper) to determine the concentration 
of that analyte. To confirm that the surrogate measure accurately 
represents the true concentration of the analyte, the pure analyte can 
be weighed, diluted to a known concentration, and measured using the 
analytical procedure under study. This procedure cannot be conducted 
for whole effluent toxicity. Toxicity cannot be purified, weighed, or 
diluted to a known concentration of ``toxicity.'' Toxicity is only 
defined by its effects on organisms, and it is these effects that are 
directly measured in the toxicity test. Because toxicity is inherently 
defined by the measurement system (a ``method-defined analyte''), and 
toxicity cannot be independently measured apart from a toxicity test, 
accuracy as a performance characteristic is not completely applicable. 
The inapplicability of the accuracy performance characteristic does not 
mean that WET tests are not accurate or that permittees are incapable 
of certifying the accuracy of WET test results reported on discharge 
monitoring reports. It means simply that the procedures commonly used 
in analytical testing to measure the performance characteristic that is 
termed ``accuracy'' cannot be applied to WET test methods.
    Notwithstanding the previous explanation, one component of accuracy 
can be described for WET tests. The American Society for Testing and 
Materials (ASTM) defines accuracy as ``a measure of the degree of 
conformity of a single test result generated by a specific procedure to 
the assumed or accepted true value and includes both precision and 
bias'' (ASTM, 1998; emphasis added). Bias is defined as ``the 
persistent positive or negative deviation of the average value of a 
test method from the assumed or accepted true value'' (ASTM, 1998). 
Precision is defined as ``the degree of agreement of repeated 
measurements of the same property, expressed in terms of dispersion of 
test results about the arithmetical mean result obtained by repetitive 
testing of a homogeneous sample under specified conditions' (ASTM, 
1998). Like ASTM, the 1988 Report to Congress (USEPA, 1988) also 
explains that accuracy includes both bias and precision. As explained 
previously, EPA conducted an Interlaboratory Variability Study of the 
ratified methods in order to, among other things, generate a quantified 
estimate of the precision for each method studied. WET tests are 
therefore amenable to the precision portion of accuracy. It is the bias 
portion of accuracy that is not applicable to WET test methods and 
cannot be described for WET as it is described for chemical analytes.
    The additional performance characteristics listed in the 1988 
Report to Congress, namely precision, interferences, ruggedness 
(applicability), reporting, and representativeness, are applicable to 
biological test methods, and EPA evaluated and considered these 
characteristics in ratifying the WET test methods. To establish the 
precision of the methods, EPA conducted an Interlaboratory Variability 
Study for each of the WET methods ratified today. From the Study, EPA 
established single-laboratory and multi-laboratory precision estimates 
for multiple sample matrices for each method (USEPA, 2001a; USEPA, 
2001b). EPA also conducted a study of within laboratory precision 
measured when testing reference toxicants (USEPA, 2000c). In today's 
action, EPA is modifying the WET method manuals to include this new and 
updated single-laboratory and multi-laboratory precision data for each 
method. Precision data from the WET Interlaboratory Variability Study 
confirmed that the WET test methods provided adequate precision (CVs 
ranged from 10.5 to 43.8%). The measured precision ranges for the 
ratified toxicity tests demonstrate the tests are comparable to (no 
more variable than) chemical analytical methods approved at 40 CFR part 
136. Finally, the precision had improved since the time the methods 
were promulgated in 1995, thus confirming EPA's conclusions that 
precision would improve with time, i.e., as analysts developed more 
expertise the methods would be ``validated by use.''
    In addition to precision, EPA evaluated and considered the 
performance characteristic of interferences. Each WET test method 
contains a section describing possible test interferences. In today's 
action, EPA has expanded that section to address two additional 
interference concerns that were raised by stakeholders by including 
guidance for controlling test interference that could be due to pH 
drift in the test and interference caused by pathogens.
    EPA also evaluated and considered the performance characteristic of 
ruggedness or applicability. The methods ratified today use materials 
that are widely available and organisms that can be easily cultured in 
the laboratory. By conducting a national interlaboratory study of these 
methods, EPA also confirmed that the methods are adaptable to a wide 
variety of laboratories and that the methods generate reproducible 
results in those laboratories. In the WET Interlaboratory Variability 
Study, EPA documented successful test completion rates of 63.6% to 100% 
for WET methods. EPA anticipates that method modifications instituted 
today will improve the successful test completion rate for methods at 
the bottom of this range, such as the Selenastrum capricornutum Growth 
Test. Today, EPA is requiring the use of EDTA in this test. As 
laboratories gain experience in performing the test with EDTA, EPA 
anticipates that successful test completion rates will improve. See 
section VI.C.4 of this preamble.
    EPA also considered the aspect of result reporting in its 
development and validation of WET test methods. Each method manual 
contains a section devoted to test review and reporting. In today's 
action, EPA has supplemented this section by providing guidance on the 
review of sampling and handling, test acceptability criteria, test 
conditions, statistical methods, concentration-response relationships, 
reference toxicant testing, and test variability. In addition, EPA 
clarified the required and recommended test conditions when submitting 
data under NPDES permits.
    EPA documented and considered the representativeness or 
comparability of WET methods. Prior to approving the WET test methods 
in the 1995 WET final rule, EPA conducted several studies that 
demonstrated the ability of WET tests to predict impacts of effluents 
on the biological integrity of receiving waters (USEPA, 1991). In a 
1995 workshop of nationally recognized WET experts (the Society of 
Environmental Toxicology and Chemistry's Pellston Workshop), including 
those from academia, government, and the regulated community (e.g., 
POTWs and industry), the experts concluded that ``WET testing is an 
effective tool for predicting receiving system impacts when appropriate 
considerations of

[[Page 69966]]

exposure are considered'' (Waller et al., 1996). The workgroup also 
agreed that ``further laboratory-to-field validation is not essential 
for the continued use of WET testing'' (Waller et al., 1996).
2. Interlaboratory Variability Study
    Several commenters expressed concern that EPA used data from the 
Interlaboratory Variability Study that was of poor quality and would 
have been discarded in a regulatory context. In conducting the WET 
Interlaboratory Variability Study, EPA's objective was to validate the 
WET methods as promulgated. EPA was not attempting to validate the 
diversity of testing requirements that may be implemented in various 
States. State regulatory authorities retain the discretion to enhance 
the requirements of a method for implementation in their State as well 
as to require procedures that EPA otherwise recommends. In the WET 
Interlaboratory Variability Study, EPA appropriately evaluated data 
according to the promulgated methods and ASTM guidance for measuring 
interlaboratory method precision. EPA accurately invalidated tests 
according to test acceptability criteria specified in each method. EPA 
acknowledges that the promulgated methods allow flexibility in the 
review of test conditions. The method manuals state that departures in 
specified test condition ranges do not necessarily invalidate test 
results. In today's action EPA modified the methods to better clarify 
this allowable flexibility. For the purposes of reviewing data 
submitted under NPDES permits, the manuals now clearly distinguish 
between requirements of the method and recommended test condition 
ranges.
    Several commenters expressed concern that EPA did not use the 
results of reference toxicant tests from the WET Interlaboratory 
Variability Study to qualify or disqualify data. EPA agrees. EPA used 
reference toxicant tests in the manner in which they are described in 
the method manuals. Failure of reference toxicant tests do not 
necessarily invalidate a test. In today's action, EPA has incorporated 
method modifications to clarify reference toxicant testing requirements 
and the appropriate use of reference toxicant test data. EPA has 
clarified that reference toxicant test results should not be used as a 
de facto criterion for rejection of individual effluent or receiving 
water tests, but rather, reference toxicant testing is used for 
evaluating the health and sensitivity of organisms over time and for 
documenting initial and ongoing laboratory performance.
    Several commenters expressed concern that too few data points were 
used to estimate method performance in the WET Interlaboratory 
Variability Study. In accordance with ASTM guidance on determining 
interlaboratory method precision, EPA set a data quality objective of a 
minimum of six complete and useable data sets for each WET test method 
evaluated in the Study. To meet this data quality objective, EPA 
endeavored to sponsor a minimum of nine laboratories per method. For 
all of the methods that EPA is ratifying today, seven or more 
laboratories participated in interlaboratory testing. For several 
individual sample matrices and test method combinations that were 
tested (blank sample analyzed using the Selenastrum capricornutum 
Growth Test, receiving water sample analyzed using the Selenastrum 
capricornutum Growth Test without EDTA, and the receiving water sample 
analyzed using the Inland Silverside Acute Test), fewer than six 
useable data sets were obtained. EPA did not, however, establish 
precision criteria in today's rule based on results from a single 
sample matrix. EPA tested four sample matrices (blank, reference 
toxicant, effluent, and receiving water) with each test method, and 
precision estimates were based on the combined results of reference 
toxicant, effluent and receiving water testing. Because multiple sample 
matrices were used to generate precision estimates, more than six 
useable data sets were used for each method. In fact, at least 17 data 
sets were used to establish precision estimates for each method.
    Several commenters also expressed concern that the selection of 
laboratories for the WET Interlaboratory Variability Study was biased. 
EPA disagrees. EPA believes that the laboratories that participated in 
the WET Interlaboratory Variability Study were representative of the 
laboratory community that commonly conducts WET testing for permittees. 
From the outset, EPA and the regulated community wanted to ensure that 
participants in the Study were representative. Industry trade groups, 
such as AMSA (Association of Metropolitan Sewerage Agencies), surveyed 
their member permittees to identify the laboratories that provide their 
routine WET testing services. AMSA requested that members sponsor those 
laboratories' participation in the Study. Of the 55 participant 
laboratories involved in the Study, 44 (or 80%) were specifically 
recommended by AMSA with commitments from AMSA members to sponsor such 
laboratories' participation in the Study. Thirty-seven of these 
laboratories were ultimately sponsored by AMSA members to analyze 
samples using one or more methods. The remaining seven laboratories had 
commitments of sponsorship from AMSA members, but were ultimately 
sponsored by EPA in the Study because their bids were among the nine 
lowest. The high percentage (80%) of laboratories in the Study that 
were sponsored by permittees for participation demonstrates that the 
laboratories involved in the Study are representative of those that 
commonly conduct WET testing for permittees.
    Several commenters expressed concern that a majority of 
laboratories did not detect toxicity in the reference toxicant sample 
type distributed for the Ceriodaphnia dubia Survival and Reproduction 
Test method. Prior to interlaboratory testing in the WET 
Interlaboratory Variability Study, referee laboratories conducted 
preliminary testing to determine the appropriate composition of samples 
to prepare for the Study. This preliminary testing was important for 
ensuring that test samples prepared for the Study produced results 
within the test concentration range. Despite these preliminary testing 
efforts, the spiking level selected for the reference toxicant sample 
type in the Ceriodaphnia dubia Survival and Reproduction Test method 
was insufficient to produce the targeted level of effect. The spiking 
concentration of KCl for this sample was selected to achieve an IC25 of 
approximately 50% sample based on preliminary testing, but the spiked 
sample missed this targeted effect level. The prepared sample was only 
slightly toxic and could not be detected as toxic in 67% of tests. 
Depending on the sensitivity of test organisms at individual 
laboratories, some laboratories identified the sample as toxic, while 
other laboratories did not. Similarly, marginally toxic effluents may 
exhibit intermittent toxicity in routine monitoring. In such cases, 
permittees and regulatory authorities should consult EPA guidance that 
addresses marginal and intermittent toxicity (USEPA, 1991; USEPA, 
2000c; USEPA, 2001f).
    The reference toxicant sample used in the Study also was prepared 
as an ampule that was reconstituted at each participant laboratory. 
This reconstitution process also likely produced minor variations (from 
laboratory to laboratory) in the final sample composition that 
influenced whether toxicity was detected. While the concentration of 
potassium ions was not measured in each final reconstituted

[[Page 69967]]

sample, conductivity was measured and can be used as an approximate 
surrogate measure. In samples that showed toxicity, the average 
conductivity was 873 [mu]mhos, and in samples that did not show 
toxicity, the average conductivity was 797 [mu]mhos. The differences in 
conductivity between tests that indicated toxicity and tests that did 
not were statistically significantly different (at the alpha = 0.05 
level). This finding indicates that those samples which were less 
diluted in the reconstitution process, were also more likely to be 
toxic.
    Several commenters also expressed concern over the way EPA handled 
outlier data points in the WET Interlaboratory Variability Study. EPA 
believes that outliers were treated according to standard practice and 
according to ASTM standards for measuring method precision. EPA 
identified outliers using ASTM's h and k statistics, and discarded 
outliers only when a probable cause for the outlier was identified. In 
all, only eight tests in the entire study of 698 tests were excluded 
based on outlier analysis.
3. Variability
    Several commenters stated that the variability of the WET methods 
(measured in terms of CV) is too high for use in NPDES permits. 
Commenters also recommended that specific steps be taken to account for 
variability in the permit limit derivation and compliance determination 
process. EPA believes that the WET Interlaboratory Variability Study 
accurately estimated the precision of WET test methods, and that this 
precision is adequate for regulatory use of the WET methods. The 
precision measured for the WET test methods is comparable to that of 
chemical methods. While EPA agrees with commenters that WET test 
methods cannot be compared in all aspects to chemical methods, the 
comparison of interlaboratory precision values does demonstrate that 
WET test methods are no more variable than other methods approved at 40 
CFR part 136 and used for regulatory compliance purposes.
    In a recent peer-reviewed guidance document (USEPA, 2000c), EPA 
thoroughly evaluated the issue of WET test method variability and 
accounting for such variability in NPDES applications. The document 
concluded that ``comparisons of WET method precision with method 
precision for analytes commonly limited in NPDES permits clearly 
demonstrate that the variability of the promulgated WET methods is 
within the range of variability experienced in other types of [required 
regulatory] analyses.'' The analytical variability of WET test methods 
is accounted for appropriately in the development of permit limits 
derived according to EPA's Technical Support Document (TSD) (USEPA, 
1991). The TSD approach accounts for both effluent variability and 
method variability. The TSD statistical approach to determination of 
reasonable potential and permit limit derivation considers combined 
effluent and analytical variability through the CV of measured effluent 
values. Because the determination of effluent variability is based on 
empirical measurements, the variability estimated for effluent 
measurements includes the variability of pollutant levels, sampling 
variability, and a smaller component owed to method variability.
    EPA does not recommend additional approaches or factors to account 
for variability, because the TSD approach appropriately accounts for 
method variability in the permit derivation process. In the guidance 
document, EPA evaluated additional approaches to account for 
variability in the permit derivation process and concluded that such 
approaches would not ensure adequate protection of water quality. The 
TSD approach was designed to provide a reasonable degree of protection 
for water quality as well as from effluent and analytical variability. 
Alternative approaches would undermine these objectives.
    Some commenters expressed specific concern that the Selenastrum 
capricornutum Growth Test method was too variable. EPA believes that 
the variability of the Selenastrum capricornutum Growth Test method, as 
measured in the WET Interlaboratory Variability Study (USEPA, 2001a) 
and variability guidance document (USEPA, 2000c), is acceptable for the 
intended regulatory use of the methods. EPA observed in the WET 
Interlaboratory Variability Study that the variability of the 
Selenastrum capricornutum Growth Test method was lower when the method 
was conducted with the addition of EDTA. In today's action, EPA is 
removing the option to conduct the test without the addition of EDTA 
when data is submitted under NPDES permits. EPA believes that this 
modification will improve the overall performance of the test method. 
False positive rates decreased from 33.3% to 0.00% and interlaboratory 
variability decreased from 58.5% to 34.3% when EDTA was added. EPA 
cautions, however, that the required addition of EDTA may make the 
Selenastrum capricornutum Growth Test less sensitive, thus less useful, 
for measuring the toxicity of some test samples, specifically, samples 
that contain toxic levels of metals.
4. Successful Test Completion Rate
    Some commenters stated that EPA incorrectly calculated successful 
test completion rates in the WET Interlaboratory Variability Study by 
failing to invalidate tests that did not meet specific test condition 
ranges. As previously discussed (see section VI.C.2 of this preamble), 
EPA accurately invalidated tests according to the test acceptability 
criteria specific to each method, and successful test completion rates 
were based on meeting these criteria. EPA acknowledges that the 
promulgated methods allow flexibility in the review of test conditions. 
The method manuals state that departures in specified test condition 
ranges do not necessarily invalidate test results. In today's action 
EPA has modified the methods to better clarify this allowable 
flexibility. For the purposes of reviewing data submitted under NPDES 
permits, the manuals now clearly distinguish between requirements of 
the method and recommended test condition ranges.
    Several commenters stated that the successful test completion rate 
measured for the Ceriodaphnia dubia Survival and Reproduction Test 
method was unacceptable and indicates a lack of ruggedness. EPA 
believes that the successful test completion rate observed for the 
Ceriodaphnia dubia Survival and Reproduction Test method in the WET 
Interlaboratory Variability Study was artificially suppressed by very 
poor performance in a small subset of laboratories. Only ten of the 34 
participant laboratories performed invalid tests, but eight of these 
laboratories performed invalid tests on 50% or more of the samples 
tested. The low rate of successful test completion in these eight 
laboratories may have been influenced by the Study's strict testing 
schedule, which required that each test be conducted on a given day and 
that all tests be conducted within a 15-day time period. When invalid 
tests conducted in a given laboratory were likely due to marginal or 
poor health of the test organism cultures, then it was logical that the 
laboratory would fail a high percentage of tests during the Study 
because culture health was unlikely to fully recover within 15 days. 
EPA believes that measuring an individual laboratory's rate of 
successful test completion over a 15-day period may not be 
representative of that laboratory's overall successful test completion 
rate. For instance, several laboratories had successful test completion 
rates of 0% during the WET

[[Page 69968]]

Interlaboratory Variability Study. Obviously, this result is not 
indicative of the laboratory's overall successful test completion rate. 
If so, the laboratory would not be in business or would not have been 
able to prequalify for participation in the Study. EPA believes that 
successful test completion rates for this method are higher in routine 
use because testing laboratories are allowed flexibility in the timing 
of sample collection and can avoid initiating tests during periods of 
marginal to poor culture health.
    Some commenters expressed concern that the successful test 
completion rate for the Selenastrum capricornutum Growth Test method 
was too low. In today's action, EPA is removing the option to conduct 
the test without the addition of EDTA. EPA believes that this 
modification will improve successful test completion rates for the 
method as laboratories consistently culture and test with EDTA. The 
successful test completion rate of 63.6% (when conducted with EDTA) was 
in part due to laboratory inexperience in using both the with and 
without-EDTA techniques. For example, two laboratories that cultured 
organisms without EDTA and generally conducted tests without EDTA 
showed poor successful test completion rates (failing eight of eight 
tests) when EDTA was used. These laboratories failed all eight tests 
conducted with EDTA and passed all but one test (seven of eight) 
without EDTA. Commenters point out that laboratories were prequalified 
for participation in the WET Interlaboratory Variability Study, but 
this prequalification required only experience with the method, not 
experience with both the with and without-EDTA procedures of the 
method. Some laboratories cultured organisms and typically conducted 
tests with EDTA, and other laboratories cultured organisms and 
typically conducted tests without EDTA.
5. False Positive Rate
    Several comments stated that EPA underestimated the false positive 
rates measured in the WET Interlaboratory Variability Study and that 
the measured rates are unacceptably high for regulatory use. In the 
context of WET methods, the false positive rate is the rate at which 
tests conducted on non-toxic dilution waters indicate the presence of 
toxicity (i.e., NOEC, LC50, or IC25 test endpoints are <100% effluent). 
EPA disagrees with comments that stated that false positive rates for 
WET test methods are unacceptably high. EPA's WET Interlaboratory 
Variability Study conclusively showed that measured false positive 
rates were below the theoretical rate of 5% estimated for the methods. 
Measured false positive rates were 3.7% for the Ceriodaphnia dubia 
Survival and Reproduction Test method, 4.35% for the Fathead Minnow 
Larval Survival and Growth Test method, and 0% for all other methods 
evaluated in the WET Interlaboratory Variability Study (with the 
exception of the Selenastrum capricornutum Growth Test conducted 
without EDTA, which EPA is removing as an option in today's action). A 
total of 150 valid WET tests were conducted on blank samples in the 
Study. Of these, only two tests (1.3%) resulted in a false positive 
result.
    The WET Interlaboratory Variability Study conclusively demonstrated 
that the false positive rate of WET methods is at or below the level 
expected for the methods. While this rate is low (below 5%), false 
positives do occur. EPA accounts for this possibility in the compliance 
and enforcement guidance. EPA policy states that ``EPA does not 
recommend that the initial response to a single exceedance of a WET 
limit, causing no known harm, be a formal enforcement action with a 
civil penalty'' (USEPA, 1995a). EPA policy suggests additional testing 
is an appropriate initial response to a single WET limit exceedance.
    Several commenters expressed concern that WET tests do not have 
method detection limits as contained in chemical methods to protect 
from reporting false positive results. As previously discussed (see 
section VI.C.1 of this preamble), method detection limit concepts are 
not applicable to WET test methods and have not been applied 
historically to toxicity testing methods developed by EPA or by 
voluntary consensus standards bodies.
    EPA established the method detection limit (MDL) concept 
specifically for chemical methods, where results generally consist of a 
single measurement of the pollutant of interest by an analytical 
instrument. The MDL concept uses information about the variability of 
the measurement system to determine a response level at which the 
measurement can be reliably distinguished from background ``noise,'' 
thus providing protection from false positive results. In WET testing, 
the final result is not based on a single measurement, but is the 
product of a series of replicated measurements on a range of effluent 
concentrations. The additional measurements, controls, replication, and 
statistical approaches included in the WET test method ``measurement 
system'' ensure that measured responses can be reliably distinguished 
from background noise.
    While results from chemical methods may rely on a single instrument 
measurement, each WET test is designed as an experiment. WET tests 
contain at least six treatments, each replicated from four to ten 
times. Measurements are made on each replicate of each treatment, so 
that results reflect average responses and the variability of those 
responses can be estimated. Each test also includes a control 
treatment, which is also replicated. This control treatment provides a 
measure of the background response and the ``noise'' or variability 
associated with that response.
    The control response is then compared to the response in effluent 
treatments using statistical methods to test the hypothesis that 
treatments containing effluent are not significantly different from the 
control treatment. If this hypothesis is rejected (considering the 
measured background or control responses, the treatment responses, and 
the variability associated with those responses), then the effluent is 
considered toxic. Hypothesis testing techniques provide protection from 
false positive results by specifically setting the Type I error rate 
allowed in rejecting the null hypothesis. Point estimation techniques 
use regression analysis to determine the effluent concentration that 
produces a specified level of response (e.g., the IC25 endpoint 
specifies a 25% difference between control and effluent treatment 
response in order for the effluent to be determined as toxic). In this 
case, false positive protection is inherently provided by the level of 
response required for generation of the selected endpoint. EPA believes 
that the test design employed in WET testing (including controls, 
replication, and hypothesis testing or point estimation) provides 
adequate protection from false positives.
6. Implementation
    Some commenters commented on issues specifically related to the 
implementation of WET permits, such as reasonable potential 
determinations, independent applicability of WET limits, discharge 
monitoring report certifications, and use of WET methods in NPDES 
permits. Many such comments are beyond the scope of this rulemaking. In 
the proposed rulemaking, EPA invited comments ``only on the conduct of 
WET test methods and not on the implementation of WET control 
strategies through NPDES permits.'' EPA recognizes that NPDES 
permittees have continuing

[[Page 69969]]

concerns about implementation of WET requirements in NPDES permits. In 
a `WHEREAS' clause to the Settlement Agreement described previously, 
EPA acknowledged that the provisions of the Settlement Agreement, which 
focused primarily on test methodology and, to a lesser extent, 
interpretation of test results, did not address all of the litigants' 
concerns regarding applicability of WET testing requirements to 
particular waterbodies (with specific reference to intermittent or 
effluent dependent waterbodies located in the Arid West) and did not 
address many of the litigants' concerns regarding regulatory 
implementation of WET control programs (e.g., toxicity identification 
evaluation requirements, toxicity reduction evaluation requirements, 
compliance determinations, and trigger thresholds). In addition, the 
Settlement Agreement also acknowledged that the 1995 rule, which 
incorporated the WET test methods in dispute, did not specify means to 
adjust for the frequency, duration, or magnitude of instream exposure 
conditions, and that such decisions are to be made by the regulatory 
authority in the context of water quality standard setting and/or NPDES 
permitting decisions. EPA continues to acknowledge these continuing 
concerns and will continue to address implementation concerns as they 
arise in concrete circumstances or through guidance, as appropriate.

VII. Statutory and Executive Order Reviews

A. Executive Order 12866: Regulatory Planning and Review

    Under Executive Order 12866 (58 FR 51735; October 4, 1993), the 
Agency must determine whether the regulatory action is ``significant'' 
and therefore subject to Office of Management and Budget (OMB) review 
and the requirements of the Executive Order. The Executive Order 
defines ``significant regulatory action'' as one that is likely to 
result in a rule that may:
    (1) Have an annual effect on the economy of $100 million or more, 
or adversely affect in a material way the economy, a sector of the 
economy, productivity, competition, jobs, the environment, public 
health or safety, or State, local, or Tribal governments or 
communities;
    (2) Create a serious inconsistency or otherwise interfere with an 
action taken or planned by another agency;
    (3) Materially alter the budgetary impact of entitlements, grants, 
user fees, or loan programs or the rights and obligations of recipients 
thereof; or
    (4) Raise novel legal or policy issues arising out of legal 
mandates, the President's priorities, or the principles set forth in 
the Executive Order.
    It has been determined that this rule is not a ``significant 
regulatory action'' under the terms of Executive Order 12866 and is 
therefore not subject to OMB review.

B. Paperwork Reduction Act

    This action does not impose an information collection burden under 
the provisions of the Paperwork Reduction Act, 44 U.S.C. 3501 et seq. 
This rule revises and ratifies test methods that are currently approved 
for use in NPDES permits and does not impose any additional information 
collection requirements.
    Burden means the total time, effort, or financial resources 
expended by persons to generate, maintain, retain, or disclose or 
provide information to or for a Federal agency. This includes the time 
needed to review instructions; develop, acquire, install, and utilize 
technology and systems for the purposes of collecting, validating, and 
verifying information, processing and maintaining information, and 
disclosing and providing information; adjust the existing ways to 
comply with any previously applicable instructions and requirements; 
train personnel to be able to respond to a collection of information; 
search data sources; complete and review the collection of information; 
and transmit or otherwise disclose the information.
    An Agency may not conduct or sponsor, and a person is not required 
to respond to a collection of information unless it displays a 
currently valid OMB control number. The OMB control numbers for EPA's 
regulations are listed in 40 CFR part 9 and 48 CFR chapter 15.

C. Regulatory Flexibility Act

    The Regulatory Flexibility Act (RFA) generally requires an agency 
to prepare a regulatory flexibility analysis of any rule subject to 
notice and comment rulemaking requirements under the Administrative 
Procedure Act or any other statute unless the agency certifies that the 
rule will not have a significant economic impact on a substantial 
number of small entities. Small entities include small businesses, 
small organizations, and small governmental jurisdictions.
    For purposes of assessing the impacts of today's rule on small 
entities, small entity is defined as: (1) A small business as defined 
by the U.S. Small Business Administration definitions at 13 CFR 
121.201; (2) a small governmental jurisdiction that is a government of 
a city, county, town, school district or special district with a 
population of less that 50,000; and (3) a small organization that is 
any not-for-profit enterprise which is independently owned and operated 
and is not dominant in its field.
    After considering the economic impacts of today's final rule on 
small entities, I certify that this action will not have a significant 
economic impact on a substantial number of small entities. Today's rule 
revises and ratifies EPA WET test methods currently approved for use at 
40 CFR part 136. Overall, the costs of these revisions are minimal. 
While some of the revisions may increase costs (e.g., quality control 
requirements), EPA believes that these costs will be alleviated by a 
potential reduction in retesting and additional investigations (e.g., 
accelerated testing, toxicity identification evaluations, or toxicity 
reduction evaluations) by the permittee that may result from improved 
test performance and increased confidence in the reliability of testing 
results. Many of the laboratories that conduct WET testing are already 
implementing the additional requirements, further minimizing any 
potential cost increases. EPA estimates that the average incremental 
cost per permit per year for today's method revisions is $276. Because 
monitoring frequency is typically less frequent for small entities than 
large entities, EPA expects the average incremental cost per permit per 
year to be even less than $276 for small entities. Using a cost of $276 
and average revenue information for small governmental jurisdictions 
and businesses, EPA estimates that the incremental costs for these 
method revisions are less than 0.1 percent of revenue for small 
entities.

D. Unfunded Mandates Reform Act

    Title II of the Unfunded Mandates Reform Act of 1995 (UMRA), Public 
Law 104-4, establishes requirements for Federal agencies to assess the 
effects of their regulatory actions on State, Tribal, and local 
governments and the private sector. Under section 202 of the UMRA, EPA 
generally must prepare a written statement, including a cost-benefit 
analysis, for proposed and final rules with ``Federal mandates'' that 
may result in expenditures to State, Tribal, and local governments, in 
the aggregate, or to the private sector, of $100 million or more in any 
one year. Before promulgating an EPA rule for which a written statement 
is needed, section 205 of the UMRA generally requires EPA to identify 
and consider a reasonable number of regulatory alternatives and adopt 
the least costly, most cost-

[[Page 69970]]

effective or least burdensome alternative that achieves the objectives 
of the rule. The provisions of section 205 do not apply when they are 
inconsistent with applicable law. Moreover, section 205 allows EPA to 
adopt an alternative other than the least costly, most cost-effective 
or least burdensome alternative if the Administrator publishes with the 
final rule an explanation of why that alternative was not adopted.
    Before EPA establishes any regulatory requirements that may 
significantly or uniquely affect small governments, including Tribal 
governments, it must have developed under section 203 of the UMRA a 
small government agency plan. The plan must provide for the 
notification of potentially affected small governments, enabling 
officials of affected small governments to have meaningful and timely 
input in the development of EPA regulatory proposals with significant 
Federal intergovernmental mandates, and informing, educating, and 
advising small governments on compliance with the regulatory 
requirements.
    EPA has determined that today's rule does not contain a Federal 
mandate that may result in expenditures of $100 million or more for 
State, Tribal, and local governments, in the aggregate, or the private 
sector in any one year. This rule promulgates revisions to WET test 
methods that are currently approved for use in NPDES permits and 
certification of Federal licenses under the CWA. The revisions are 
minor and the cost to implement them is minimal. Thus, today's rule is 
not subject to sections 202 and 205 of the UMRA. For the same reasons, 
EPA has also determined that this rule contains no regulatory 
requirements that might significantly or uniquely affect small 
governments. Thus, today's rule also is not subject to the requirements 
of section 203 of the UMRA.

E. Executive Order 13132: Federalism

    Executive Order 13132, entitled ``Federalism'' (64 FR 43255; August 
10, 1999), requires EPA to develop an accountable process to ensure 
``meaningful and timely input by State and local officials in the 
development of regulatory policies that have federalism implications.'' 
``Policies that have federalism implications'' is defined in the 
Executive Order to include regulations that have ``substantial direct 
effects on the States, on the relationship between the national 
government and the States, or on the distribution of power and 
responsibilities among the various levels of government.''
    This final rule does not have federalism implications. It will not 
have substantial direct effects on the States, on the relationship 
between the national government and the States, or on the distribution 
of power and responsibilities among the various levels of government, 
as specified in Executive Order 13132. Today's rule promulgates 
revisions to WET test methods that are currently approved for use in 
NPDES permits and certification of Federal licenses under the CWA. The 
revisions are minor and the cost to implement them is minimal. Thus, 
Executive Order 13132 does not apply to this rule.

F. Executive Order 13175: Consultation and Coordination with Indian 
Tribal Governments

    Executive Order 13175, entitled ``Consultation and Coordination 
with Indian Tribal Governments'' (65 FR 67249; November 9, 2000), 
requires EPA to develop an accountable process to ensure ``meaningful 
and timely input by Tribal officials in the development of regulatory 
policies that have Tribal implications.'' ``Policies that have Tribal 
implications'' is defined in the Executive Order to include regulations 
that have ``substantial direct effects on one or more Indian Tribes, on 
the relationship between the Federal government and the Indian Tribes 
or on the distribution of power and responsibilities between the 
Federal government and Indian Tribes.''
    This final rule does not have Tribal implications. It will not have 
substantial direct effects on Tribal governments, on the relationship 
between the Federal government and Indian Tribes, or on the 
distribution of power and responsibilities between the Federal 
government and Indian Tribes, as specified in Executive Order 13175. 
Today's rule promulgates revisions to WET test methods that are 
currently approved for use in NPDES permits and certification of 
Federal licenses under the CWA. The revisions are minor and the cost to 
implement them is minimal. Thus, Executive Order 13175 does not apply 
to this rule.

G. Executive Order 13045: Protection of Children From Environmental 
Health and Safety Risks

    Executive Order 13045 (62 FR 19885; April 23, 1997) applies to any 
rule that: (1) is determined to be ``economically significant'' as 
defined under Executive Order 12866, and (2) concerns an environmental 
health or safety risk that EPA has reason to believe may have a 
disproportionate effect on children. If the regulatory action meets 
both criteria, the Agency must evaluate the environmental health or 
safety effects of the planned rule on children, and explain why the 
planned regulation is preferable to other potentially effective and 
reasonably feasible alternatives considered by the Agency. This rule is 
not subject to the Executive Order because it is neither ``economically 
significant'' as defined in Executive Order 12866, nor does it concern 
an environmental health or safety risk that EPA has reason to believe 
may have a disproportionate effect on children.

H. Executive Order 13211: Actions Concerning Regulations That 
Significantly Affect Energy Supply, Distribution, or Use

    This rule is not subject to Executive Order 13211, ``Actions 
Concerning Regulations That Significantly Affect Energy Supply, 
Distribution, or Use'' (66 FR 28355; May 22, 2001) because it is not a 
significant regulatory action under Executive Order 12866.

I. National Technology Transfer and Advancement Act

    Section 12(d) of the National Technology Transfer and Advancement 
Act of 1995, (``NTTAA''), Public Law 104-113, section 12(d) (15 U.S.C. 
272 note), directs EPA to use voluntary consensus standards in its 
regulatory activities unless to do so would be inconsistent with 
applicable law or otherwise impractical. Voluntary consensus standards 
are technical standards (e.g., material specifications, test methods, 
sampling procedures, business practices) that are developed or adopted 
by voluntary consensus standards bodies (VCSBs). The NTTAA directs EPA 
to provide Congress, through the Office of Management and Budget (OMB), 
explanations when the Agency decides not to use available and 
applicable voluntary consensus standards.
    This rulemaking would revise existing EPA WET test methods. For the 
methods that EPA is revising, the Agency did not conduct a search to 
identify potentially applicable voluntary consensus standards, because 
the revisions EPA is promulgating today would merely incorporate more 
specificity and detail into currently approved EPA test methods. EPA 
did, however, consult available voluntary consensus standards, such as 
ASTM standards, for guidance in conducting the Interlaboratory 
Variability Study and in defining certain performance characteristics 
of the methods.

J. Congressional Review Act

    The Congressional Review Act, 5 U.S.C. 801 et seq., as added by the 
Small Business Regulatory Enforcement

[[Page 69971]]

Fairness Act of 1996 (SBREFA), generally provides that before a rule 
may take effect, the agency promulgating the rule must submit a rule 
report, which includes a copy of the rule, to each House of the 
Congress and to the Comptroller General of the United States. EPA will 
submit a report containing this rule and other required information to 
the U.S. Senate, the U.S. House of Representatives, and the Comptroller 
General of the United States prior to publication of the rule in the 
Federal Register. A major rule cannot take effect until 60 days after 
it is published in the Federal Register. This action is not a ``major 
rule'' as defined by 5 U.S.C. 804(2). This rule will be effective on 
December 19, 2002.

VIII. References

    American Society for Testing and Materials. 1998. Standard 
Practice for Determination of Precision and Bias of Applicable Test 
Methods of Committee D-19 on Water. D 2777-98. American Society for 
Testing and Materials, Philadelphia, PA.
    DeGraeve, G.M., G.J. Smith, W.H. Clement, D.O. McIntyre, and T. 
Forgette. 1998. WET Testing Program: Evaluation of Practices and 
Implementation. Project 94-HHE-1. Water Environment Research 
Foundation, Alexandria, VA.
    Edison Electric Institute et al. v. EPA, Settlement Agreement, 
July 24, 1998. U.S. Court of Appeals, D.C. Circuit, No. 96-1062.
    Lone Star Steel v. EPA, Settlement Agreement, March 4, 1998. 
U.S. Court of Appeals, D.C. Circuit, No. 96-1157.
    National Environmental Laboratory Accreditation Conference, 
2001. Quality Systems. National Environmental Laboratory 
Accreditation Conference.
    U.S. Environmental Protection Agency. 1988. Availability, 
Adequacy, and Comparability of Testing Procedures for the Analysis 
of Pollutants Established Under Section 304(h) of the Federal Water 
Pollution Control Act; Report to Congress. EPA/600/9-87/030. U.S. 
Environmental Protection Agency, Environmental Monitoring Systems 
Laboratory, Cincinnati, OH.
    U.S. Environmental Protection Agency. 1991. Technical Support 
Document for Water Quality-Based Toxics Control. EPA/505/2-90/001. 
U.S. Environmental Protection Agency, Office of Water Enforcement 
and Permits, and Office of Water Regulations and Standards, 
Washington, DC.
    U.S. Environmental Protection Agency. 1993. Methods for 
Measuring the Acute Toxicity of Effluents and Receiving Waters to 
Freshwater and Marine Organisms, 4th ed. EPA/600/4-90/027F. U.S. 
Environmental Protection Agency, Environmental Monitoring Systems 
Laboratory, Cincinnati, OH.
    U.S. Environmental Protection Agency. 1994a. Short-term Methods 
for Estimating the Chronic Toxicity of Effluents and Receiving 
Waters to Freshwater Organisms, 3rd ed. EPA/600/4-91/002. U.S. 
Environmental Protection Agency, Environmental Monitoring Systems 
Laboratory, Cincinnati, OH.
    U.S. Environmental Protection Agency. 1994b. Short-term Methods 
for Estimating the Chronic Toxicity of Effluents and Receiving 
Waters to Marine and Estuarine Organisms, 2nd ed. EPA/600/4-91/003. 
U.S. Environmental Protection Agency, Environmental Monitoring 
Systems Laboratory, Cincinnati, OH.
    U.S. Environmental Protection Agency. 1995a. National Policy 
Regarding Whole Effluent Toxicity Enforcement, July 6, 1995, 
memorandum from Brian Maas, U.S. Environmental Protection Agency, 
Office of Enforcement and Compliance Assurance, Washington DC.
    U.S. Environmental Protection Agency. 1995b. Short-term Methods 
for Estimating the Chronic Toxicity of Effluents and Receiving 
Waters to West Coast Marine and Estuarine Organisms, 1st ed. EPA/
600/R-95/136. U.S. Environmental Protection Agency, National 
Exposure Research Laboratory, Cincinnati, OH.
    U.S. Environmental Protection Agency. 1995c. Whole effluent 
toxicity: guidelines establishing test procedures for the analysis 
of pollutants, final rule. 60 FR 53529-53563.
    U.S. Environmental Protection Agency. 1999. Whole effluent 
toxicity: guidelines establishing test procedures for the analysis 
of pollutants, whole effluent toxicity tests; final rule, technical 
correction. 64 FR 4975-4991.
    U.S. Environmental Protection Agency. 2000a. Method Guidance and 
Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR 
Part 136). EPA/821/B-00/004. U.S. Environmental Protection Agency, 
Office of Water, Washington, DC.
    U.S. Environmental Protection Agency. 2000b. Notice of 
availability, ``Understanding and accounting for method variability 
in WET applications under the NPDES program.'' 65 FR 44528-44529.
    U.S. Environmental Protection Agency. 2000c. Understanding and 
Accounting for Method Variability in Whole Effluent Toxicity 
Applications Under the National Pollutant Discharge Elimination 
System Program. EPA/833/R-00/003. U.S. Environmental Protection 
Agency, Office of Wastewater Management, Washington, DC.
    U.S. Environmental Protection Agency. 2000d. Whole effluent 
toxicity test methods guidance; notice of availability. 65 FR 46457-
46458.
    U.S. Environmental Protection Agency. 2001a. Final Report: 
Interlaboratory Variability Study of EPA Short-term Chronic and 
Acute Whole Effluent Toxicity Test Methods, Vol. 1. EPA /821/B-01/
004. U.S. Environmental Protection Agency, Office of Water, 
Washington, DC.
    U.S. Environmental Protection Agency. 2001b. Final Report: 
Interlaboratory Variability Study of EPA Short-term Chronic and 
Acute Whole Effluent Toxicity Test Methods, Vol. 2: Appendix. EPA/
821/B-01-005. U.S. Environmental Protection Agency, Office of Water, 
Washington, DC.
    U.S. Environmental Protection Agency. 2001c. Guidelines 
establishing test procedures for the analysis of pollutants; whole 
effluent toxicity test methods; proposed rule. 66 FR 49794-49816.
    U.S. Environmental Protection Agency. 2001d. Guidelines 
establishing test procedures for the analysis of pollutants; whole 
effluent toxicity test methods; extension of comment period. 66 FR 
58693-58694.
    U.S. Environmental Protection Agency. 2001e. Proposed Changes to 
Whole Effluent Toxicity Method Manuals. EPA/821/B-01/002. U.S. 
Environmental Protection Agency, Office of Water, Washington, DC.
    U.S. Environmental Protection Agency. 2001f. Clarifications 
Regarding Toxicity Reduction and Identification Evaluations in the 
National Pollutant Discharge Elimination System Program. U.S. 
Environmental Protection Agency, Office of Water, Washington, DC.
    U.S. Environmental Protection Agency. 2002. Cost Estimate for 
Required Method Modifications in EPA's WET Final Rule, October 16, 
2002, memorandum from Marion Kelly, U.S. Environmental Protection 
Agency, Office of Water, Office of Science and Technology, 
Washington, DC.
    Waller, T.W., L.P. Ammann, W.J. Birge, K.L. Dickson, P.B. Dorn, 
N.E. LeBlanc, D.I. Mount, B.R. Parkhurst, H.R. Preston, S.C. 
Schimmel, A. Spacie, and G.B. Thursby. 1996. Discussion synopsis, 
predicting instream effects from WET tests. Chapter 9 In: Whole 
Effluent Toxicity Testing: An Evaluation of Methods and Prediction 
of Receiving System Impacts. D.R. Grothe, K.L. Dickson, and D.K. 
Reed-Judkins, eds., SETAC Press, Pensacola, FL, pp. 271-286.

List of Subjects at 40 CFR Part 136

    Environmental protection, Incorporation by reference, Reporting and 
recordkeeping requirements, Water pollution control.

    Dated: November 8, 2002.
Christine Todd Whitman,
Administrator.

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

PART 136--GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANALYSIS 
OF POLLUTANTS

    1. The authority citation for part 136 continues to read as 
follows:

    Authority: Secs. 301, 304(h), 307, and 501(a), Pub. L. 95-217, 
91 Stat. 1566, et seq. (33 U.S.C. 1251, et seq.) (The Federal Water 
Pollution Control Act Amendments of 1972 as amended by the Clean 
Water Act of 1977).


    2. Section 136.3 is amended:
    a. In Table IA of paragraph (a) by revising entries 6 to 9.
    b. In paragraph (b) by revising references (34), (38), and (39).
    c. In paragraph (b) by removing and reserving reference (42).
    The revisions read as follows:

[[Page 69972]]

Sec.  136.3  Identification of test procedures.

    (a) * * *

                                                     Table IA.--List of Approved Biological Methods
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                                Standard methods
       Parameter and units                           Method \1\                      EPA      18th, 19th, 20th Ed.         ASTM               USGS
--------------------------------------------------------------------------------------------------------------------------------------------------------
 
                                                                      * * * * * * *
Aquatic Toxicity:
    6. Toxicity, acute, fresh      Ceriodaphnia dubia acute.....................        \7\
     water organisms, LC50,        Daphnia pulex and Daphnia magna acute........     2002.0
     percent effluent.             Fathead minnow, Pimephales promelas, and             \7\
                                    Bannerfin shiner, Cyprinella leedsi, acute.      2021.0
                                                                                        \7\
                                                                                     2001.0
                                   Rainbow trout, Oncorhynchus mykiss, and brook        \7\
                                    trout, Salvelinus fontinalis, acute.             2019.0
    7. Toxicity, acute, estuarine  Mysid, Mysidopsis, bahia, acute..............        \7\
     and marine organisms of the   Sheepshead minnow, Cyprinodon variegatus,         2007.0
     Atlantic Ocean and Gulf of     acute.                                              \7\
     Mexico, LC50, percent         Silverside, Menidia beryllina, Menidia            2004.0
     effluent.                      menidia, and Menidia peninsulae, acute.             \7\
                                                                                     2006.0
    8. Toxicity, chronic, fresh    Fathead minnow, Pimephales promelas, larval          \8\
     water organisms, NOEC or       survival and growth.                             1000.0
     IC25, percent effluent.       Fathead minnow, Pimephales promelas, embryo-   .........
                                    larval survival and teratogenicity.                 \8\
                                                                                     1001.0
                                   Daphnia, Ceriodaphnia dubia, survival and            \8\
                                    reproduction.                                    1002.0
                                   Green alga, Selenastrum capricornutum, growth        \8\
                                                                                     1003.0
    9. Toxicity, chronic,          Sheepshead minnow, Cyprinodon variegatus,            \9\
     estuarine and marine           larval survival and growth.                      1004.0
     organisms of the Atlantic     Sheepshead minnow, Cyprinodon variegatus,      .........
     Ocean and Gulf of Mexico,      embryo-larval survival and teratogenicty.           \9\
     NOEC or IC25, percent         Inland silverside, Menidia beryllina, larval      1005.0
     effluent.                      survival and growth.                          .........
                                   Mysid, Mysidopsis bahia, survival, growth,           \9\
                                    and fecundity.                                   1006.0
                                                                                  .........
                                                                                        \9\
                                                                                     1007.0
                                   Sea urchin, Arbacia punctulata, fertilization        \9\
                                                                                     1008.0
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ The method must be specified when results are reported.
* * * * * * *
\7\ USEPA. October 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition.
  U.S. Environmental Protection Agency, Office of Water, Washington, D.C. EPA 821-R-02-012.
\8\ USEPA. October 2002. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth
  Edition. U.S. Environmental Protection Agency, Office of Water, Washington, D.C. EPA 821-R-02-013.
\9\ USEPA. October 2002. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms.
  Third Edition. U.S. Environmental Protection Agency, Office of Water, Washington, D.C. EPA 821-R-02-014.
* * * * * * *

* * * * *
    (b) * * *

References, Sources, Costs, and Table Citations

* * * * *
    (34) USEPA. October 2002. Methods for Measuring the Acute Toxicity 
of Effluents and Receiving Waters to Freshwater and Marine Organisms. 
Fifth Edition. U.S. Environmental Protection Agency, Office of Water, 
Washington, D.C. EPA 821-R-02-012. Available from: National Technical 
Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, 
Publ. No. PB2002-108488. Table IA, Note 7.
* * * * *
    (38) USEPA. October 2002. Short-Term Methods for Estimating the 
Chronic Toxicity of Effluents and Receiving Waters to Freshwater 
Organisms. Fourth Edition. U.S. Environmental Protection Agency, Office 
of Water, Washington, D.C. EPA 821-R-02-013. Available from: National 
Technical Information Service, 5285 Port Royal Road, Springfield, 
Virginia 22161, Publ. No. PB2002-108489. Table IA, Note 8.
    (39) USEPA. October 2002. Short-Term Methods for Estimating the 
Chronic Toxicity of Effluents and Receiving Waters to Marine and 
Estuarine Organisms. Third Edition. U.S. Environmental Protection 
Agency, Office of Water, Washington, D.C. EPA 821-R-02-014. Available 
from: National Technical Information Service, 5285 Port Royal Road, 
Springfield, Virginia 22161, Publ. No. PB2002-108490. Table IA, Note 9.
* * * * *
    (42) [RESERVED]
* * * * *
[FR Doc. 02-29072 Filed 11-18-02; 8:45 am]
BILLING CODE 6560-50-P