[Federal Register Volume 66, Number 189 (Friday, September 28, 2001)]
[Proposed Rules]
[Pages 49794-49816]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 01-24374]
[[Page 49793]]
-----------------------------------------------------------------------
Part VI
Environmental Protection Agency
-----------------------------------------------------------------------
40 CFR Part 136
Guidelines Establishing Test Procedures for the Analysis of Pollutants;
Whole Effluent Toxicity Test Methods; Proposed Rule
Federal Register / Vol. 66, No. 189 / Friday, September 28, 2001 /
Proposed Rules
[[Page 49794]]
-----------------------------------------------------------------------
ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 136
[FRL-7069-7]
Guidelines Establishing Test Procedures for the Analysis of
Pollutants; Whole Effluent Toxicity Test Methods
AGENCY: Environmental Protection Agency (EPA).
ACTION: Proposed rule.
-----------------------------------------------------------------------
SUMMARY: Today, EPA proposes to ratify its approval of several analytic
test procedures measuring ``whole effluent toxicity,'' which the Agency
standardized in an earlier rulemaking. Today's proposal also would
modify some of those test procedures. EPA is proposing today's notice
to satisfy obligations in a settlement agreement designed to resolve
litigation over that earlier rulemaking. The proposed changes are
intended to improve the performance of whole effluent toxicity (WET)
tests, and thus increase confidence in the reliability of the results
obtained using the test procedures.
DATES: Comments on this proposal must be postmarked, delivered by hand,
or electronically mailed on or before November 27, 2001. Comments
provided electronically will be considered timely if they are submitted
electronically by 11:59 p.m. Eastern Standard Time (EST) on November
27, 2001.
ADDRESSES: Send written or electronic comments on the proposed rule to
``Whole Effluent Toxicity (WET) Test Method Changes'' Comment Clerk
(WETEU-IX); Water Docket (4101); Environmental Protection Agency; Ariel
Rios Building; 1200 Pennsylvania Avenue, NW; Washington, DC--P 20460.
EPA requests that commenters submit copies of any references cited in
comments. Commenters also are requested to submit an original and three
copies of their written comments and enclosures. Commenters that want
receipt of their comments acknowledged should include a self-addressed,
stamped envelope. All written comments must be postmarked or delivered
by hand. No facsimiles (faxes) will be accepted. Hand deliveries should
be delivered to EPA's Water Docket at 401 M Street, SW, Room EB57,
Washington, D.C. 20460.
Comments may be submitted electronically to: [email protected].
Electronic comments must be submitted as a Word Perfect 5/6/7/8 file or
an ASCII file, avoiding the use of special characters and any form of
encryption. Comments and data also will be accepted on disks in
WordPerfect 5/6/7/8 or ASCII file format. Electronic comments on this
proposed rule may be filed online at any Federal Depository Library.
All electronic comments must be identified by docket number (WET-IX).
Electronic comments will be transferred into a paper version for the
official record. EPA will attempt to clarify electronic comments if
there is an apparent error in transmission.
The record for this rulemaking has been established under docket
number WET-IX. A copy of the supporting documents cited in this
proposal is available for review at EPA's Water Docket, East Tower
Basement (Room EB 57), 401 M Street, SW, Washington, DC 20460. For
access to docket materials, call (202) 260-3027 on Monday through
Friday, excluding Federal holidays, between 9:00 a.m. and 3:30 p.m. EST
to schedule an appointment.
This Federal Register document has been placed on the Internet for
public review and downloading at the following location: http://www.epa.gov/fedrgstr/. The final report of EPA's WET Interlaboratory
Variability Study, Volumes 1 and 2 (USEPA, 2001a; USEPA, 2001b) and the
document titled, Proposed Changes to Whole Effluent Toxicity Method
Manuals (USEPA, 2001d), which is referenced in today's rule and
provides details of proposed changes, also are available on the
Internet at http://www.epa.gov/waterscience/WET.
FOR FURTHER INFORMATION CONTACT: For regulatory information regarding
this proposal, contact Marion Kelly, Engineering and Analysis Division
(4303), Office of Science and Technology, Office of Water, U.S.
Environmental Protection Agency, 1200 Pennsylvania Avenue, NW,
Washington, DC 20460 (e-mail: [email protected]) or call (202) 260-
7117. For technical information regarding method changes proposed in
today's rule, contact Teresa J. Norberg-King, National Health and
Environmental Effects Research Laboratory, Mid-Continent Ecology
Division, Office of Research and Development, U.S. Environmental
Protection Agency, 6201 Congdon Boulevard, Duluth, MN 55804 (e-mail:
[email protected]) or call (218) 529-5163.
SUPPLEMENTARY INFORMATION:
Potentially Regulated Entities
EPA Regions, as well as State, 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, the
NPDES permitting authority, including authorized States, Territories,
and Tribes, make a number of discretionary choices associated with
permit writing, including the selection of pollutants to be measured
and, in many cases, limited in permits. If EPA has ``approved'' (i.e.,
promulgated through rulemaking) standardized testing procedures for a
given pollutant, the NPDES permitting authority must specify one of the
approved test procedures or an approved alternate test procedure for
the measurements required under the permit. In addition, when a States,
Territory, or authorized Tribe provides certification of Federal
licenses under CWA section 401, measurements required by such
certifications must be made using the approved testing procedures.
Categories and entities that may be regulated include:
------------------------------------------------------------------------
Examples of potentially
Category affected/regulated entities
------------------------------------------------------------------------
States, Territorial, and Indian Tribal States, Territories, and Tribes
Governments. authorized to administer the
NPDES permitting program;
States, Territories, and
Tribes that certify Federal
licenses.
------------------------------------------------------------------------
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 this action. Other types of entities
not listed in the table also could be regulated. If you have questions
regarding the applicability of this action to a particular entity,
consult the persons listed in the preceding FOR FURTHER INFORMATION
CONTACT section.
[[Page 49795]]
Outline of Preamble
I. Statutory Authority
II. Regulatory Background
III. Explanation of Today's Action
A. Introduction
B. Proposed Method Changes
1. Updates
a. Incorporation of Previous Addenda and Errata
b. Update of Method Precision Data
2. Minor Corrections and Clarifications
3. Specific Stakeholder Concerns
a. Blocking by Known Parentage
b. pH Drift
c. Concentration-Response Relationships
d. Nominal Error Rates
e. Confidence Intervals
f. Dilution Series
g. Dilution Waters
h. Pathogen Interference
C. Ratification or Withdrawal of Methods
1. WET Variability Study
2. Ceriodaphnia dubia Acute Test, Ceriodaphnia dubia Survival
and Reproduction Test, Fathead Minnow Acute Test, Fathead Minnow
Larval Survival and Growth Test, Sheepshead Minnow Acute Test,
Sheepshead Minnow Larval Survival and Growth Test, and Inland
Silverside Acute Test
3. Inland Silverside Larval Survival and Growth Test
4. Champia parvula Reproduction Test
5. Mysidopsis bahia Survival, Growth, and Fecundity Test
6. Selenastrum capricornutum Growth Test
7. Holmesimysis costata Acute Test
IV. Regulatory Requirements
A. Executive Order 12866--Regulatory Planning and Review
B. Unfunded Mandates Reform Act
C. Regulatory Flexibility Act (RFA), as Amended by the Small
Business Regulatory Enforcement Fairness Act of 1996 (SBREFA), 5
U.S.C. 601 et seq.
D. Paperwork Reduction Act
E. National Technology Transfer and Advancement Act
F. Executive Order 13045--Protection of Children From
Environmental Health Risks and Safety Risks
G. Executive Order 13175--Consultation and Coordination With
Indian Tribal Governments
H. Executive Order 13132--Federalism
I. Executive Order 13211--Energy Effects
J. Plain Language Directive
V. Request for Comments and Available Data
A. pH Drift
B. Percent Minimum Significant Difference
C. Other Method Modifications
VI. References
I. Statutory Authority
Today's proposal is pursuant to the authority of sections 101(a),
301, 304(h), 402, and 501(a) of the Clean Water Act (CWA), 33 U.S.C.
1251(a), 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.''
II. Regulatory Background
Standardized analytical procedures for monitoring and reporting
required in NPDES permits (40 CFR part 122, Secs. 122.21, 122.41,
122.44, and 123.25), and in the implementation of the pretreatment
standards issued under section 307 of the Act (40 CFR part 403,
Secs. 403.10 and 402.12) appear at 40 CFR part 136. There may be
discharges that require limitations for certain parameters using test
procedures not yet approved under 40 CFR part 136. Under 40 CFR
122.41(j)(4) and 122.44(i)(1)(iv) permit writers may include, through
permit proceedings, parameters requiring the use of test procedures
that are not approved part 136 methods. EPA also may include such
parameters in accordance with the provisions prescribed at 40 CFR
401.13, ``Test Procedures for Measurements.'' Permits may include, for
example, effluent limitations for WET using standardized testing
procedures other than those published at 40 CFR part 136 that are
approved for nationwide use. In such cases, use of the particular test
species and test protocols would remain subject to challenge on a case-
by-case basis in permit proceedings (except, for example, if an
authorized State conducted rulemaking to standardize a particular
testing procedure applicable within the State).
In 1995, EPA amended the ``Guidelines Establishing Test Procedures
for the Analysis of Pollutants,'' 40 CFR part 136, to add a series of
standardized whole effluent toxicity (WET) test methods to the list of
Agency approved methods for CWA data gathering and compliance
monitoring programs (60 FR 53529; October 16, 1995) (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, 1993b) generally use death 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. For
this rulemaking notice, the short-term methods for estimating chronic
toxicity will be referred to as chronic methods for ease of notation.
Standardized test procedures for conducting the approved acute and
chronic WET tests are provided 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).
After promulgation of the WET methods, a variety of parties filed
suit challenging the EPA rulemaking (Edison Electric Institute v. EPA,
No. 96-1062 (D.C. Cir.); Western Coalition of Arid States v. EPA, No.
96-1124; Lone Star Steel Co. v. EPA, No. 96-1157 (D.C. Cir.)). To
resolve that litigation, EPA entered into settlement agreements with
the various parties. EPA proposes actions today to fulfill obligations
under some of those settlement agreements.
In February 1999, EPA published a technical corrections notice 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). Further background on the WET test methods and these
technical documents are included in the Federal Register notices cited
above (60 FR 53529 and 64 FR 4975).
[[Page 49796]]
III. Explanation of Today's Action
A. Introduction
Today's proposal would make a number of revisions to the currently
approved WET test methods. See section III.B. Also in today's action,
EPA presents final results of an interlaboratory variability study of
WET test methods and, based on these results, proposes to ratify 11 of
the 12 methods evaluated in the study (see section III.C). Today's
proposal requests public comment on the inclusion of additional
technical changes to the approved WET test methods and on EPA's
proposal to ratify 11 of 12 WET test methods.
Although today's action fulfills portions of settlement agreements
resolving litigation over the 1995 WET test method rulemaking, EPA
acknowledges that some stakeholders still have significant concerns
related to implementation of WET control strategies through NPDES
permits. By today's proposal, EPA intends to focus only on analytic
testing methodologies to measure WET, not on WET implementation
generally.
Since the 1995 WET final rule, EPA and authorized States have taken
additional actions to improve and enhance implementation of WET control
strategies. EPA, for example, has published additional guidance on the
conduct of a toxicity identification evaluation (TIE) and a toxicity
reduction evaluation (TRE), as well as guidance on the circumstances
that trigger such evaluations (USEPA, 1999c; USEPA, 2001g).
Other questions have arisen about the significance of EPA action to
standardize WET testing procedures through rulemaking. For example,
some stakeholders question whether, by promulgating WET test methods,
EPA has published recommended water quality criteria (pursuant to CWA
section 304(a)) for ``toxicity.'' To respond and clarify, EPA's
promulgation of WET test procedures are not water quality criteria
recommendations under section 304(a). When States develop and implement
water quality standards, including narrative water quality criteria,
States should translate those criteria into measurable expressions of
toxicity. The test methods themselves are not per se translators of the
narrative criterion: ``no toxics in toxic amounts.'' The test methods
are merely the measurement tools according to which such criteria may
be translated.
Today's proposed revisions include changes to the three method
manuals (USEPA, 1993b; USEPA, 1994a; USEPA, 1994b) incorporated by
reference in the WET rule (60 FR 53529; October 16, 1995) and amend the
``Guidelines Establishing Test Procedures for the Analysis of
Pollutants'' (40 CFR part 136) to reference the updated editions of the
method manuals. Modifications to the method manuals are intended to
update the methods, provide additional minor corrections and
clarifications, and address specific stakeholder concerns (see Section
III.B). EPA proposes to update the methods (1) by incorporating
previous method addenda and errata and (2) by revising method precision
statements to reflect results from recent EPA studies (USEPA, 2000d;
USEPA, 2001a). In addition to corrections identified in previous method
addenda and errata, EPA proposes to correct other minor technical
errors and omissions. EPA also seeks comment on an additional
modification to WET test methods that would require the application of
upper and lower bounds on the percent minimum significant difference
(PMSD) calculated in WET tests (see section V.B).
EPA also proposes method revisions in response to specific
stakeholder concerns. Specifically, these revisions include: requiring
``blocking'' by known parentage in the Ceriodaphnia dubia Survival and
Reproduction Test; adding procedures to control pH drift that may occur
during testing; incorporating review procedures for the evaluation of
concentration-response relationships; clarifying allowable nominal
error rate adjustments; clarifying limitations in the generation of
confidence intervals; adding guidance on dilution series selection;
clarifying dilution water acceptability; and adding procedures for
determining and minimizing the impact of pathogens in the Fathead
Minnow Survival and Growth Test. These are summarized below in section
III.B and detailed in the document titled, Proposed Changes to Whole
Effluent Toxicity Method Manuals (USEPA, 2001d). Proposal of these
revisions partially fulfills the requirements of two settlement
agreements between stakeholders and EPA (Edison Electric Institute, et
al. v. EPA, No. 96-1062 & consolidated case (D.C. Cir.), Settlement
Agreement, July 24, 1998; Lone Star Steel v. EPA, No. 96-1157 (D.C.
Cir.), Settlement Agreement, March 4, 1998).
EPA requests public comment on the proposed changes to the WET test
methods and on the proposal to ratify the WET test methods (see section
V). When EPA takes final action on today's proposal, the Agency intends
to incorporate the modifications proposed today into the text of new
editions of each of the WET method manuals.
B. Proposed Method Changes
Today, EPA proposes to revise each of the WET method manuals
(USEPA, 1993b; USEPA, 1994a; USEPA, 1994b). Proposed method changes
include: (1) updates to the methods, (2) minor corrections and
clarifications, and (3) modifications to address specific stakeholder
concerns. These method changes are described in Sections 1 through 3
below and are detailed in the document titled, Proposed Changes to
Whole Effluent Toxicity Method Manuals (USEPA, 2001d), which is
included in the docket supporting today's rule and is available online
at http://www.epa.gov/waterscience/WET.
1. Updates
a. Incorporation of Previous Addenda and Errata
Subsequent to promulgating the WET final rule in 1995, EPA issued
several documents to correct and amend that rule and its supporting
documentation. Specifically, in February 1999, EPA published a final
rule that incorporated into the WET rule an errata document (USEPA,
1999a) to correct minor errors and omissions in the WET method manuals
(64 FR 4975; February 2, 1999). In addition, a 1996 addenda document
(USEPA, 1996a) revised the 1993 acute method manual (USEPA, 1993b).
Today, EPA proposes to incorporate the changes noted in the errata and
the addenda documents into the text of the appropriate method manuals
by issuing revised editions of each of the three method manuals. EPA
plans to issue the revised editions when it takes final action on this
proposal. The incorporation of the errata and addenda into the method
manual text would not further alter the methods. This action would
simply assist users of the method manuals by incorporating all previous
corrections into updated editions.
b. Update of Method Precision Data
Since publishing the WET method manuals, EPA has conducted two
large-scale studies of WET test method precision. During 1999 and 2000,
EPA conducted an interlaboratory variability study (the WET Variability
Study) of 12 of the 17 WET test methods promulgated at 40 CFR part 136.
This study generated data from more than 700 blind samples tested in 55
laboratories. EPA published interlaboratory precision results from the
WET Variability Study in 2000 (USEPA, 2000b; USEPA, 2000c) and
submitted the study results for expert
[[Page 49797]]
peer review in 2001 (USEPA, 2001c). Following expert peer review, EPA
published a final study report (USEPA, 2001a; USEPA, 2001b).
In addition to the WET Variability Study, EPA conducted a study of
intralaboratory WET test precision based on routine laboratory
reference toxicant test data. EPA compiled a database of more than
1,800 reference toxicant tests conducted for 23 different methods
between 1988 and 1999 in 75 laboratories. EPA used this database to
quantify estimates of precision for each of the WET methods. EPA
published this precision data and additional guidance on reducing
method variability in a guidance document titled, Understanding and
Accounting for Method Variability in Whole Effluent Toxicity
Applications Under the National Pollutant Discharge Elimination System
Program (USEPA, 2000d) (the Variability Guidance Document).
In today's action, EPA proposes to modify the WET method manuals by
updating statements and inserting tables regarding the multi-laboratory
(interlaboratory) and single-laboratory (intralaboratory) precision of
the methods using data from the WET Variability Study and the
Variability Guidance Document. Results from these two studies represent
the most current and complete data available on intralaboratory and
interlaboratory precision of WET test methods. The proposed changes
would modify the chronic method manuals (USEPA, 1994a; USEPA, 1994b) by
revising subsections on precision and accuracy for several test
methods. The proposed changes also would modify Section 4 (Quality
Assurance) of each of the method manuals (USEPA, 1993b; USEPA, 1994a;
USEPA, 1994b) to update statements on test method variability and
precision. The specifics of the proposed method manual changes related
to updating precision statements are detailed in the document titled,
Proposed Changes to Whole Effluent Toxicity Method Manuals (USEPA,
2001d).
2. Minor Corrections and Clarifications
In addition to the incorporation of changes identified in the 1999
errata (USEPA, 1999a) and the acute manual addenda (USEPA, 1996a), EPA
proposes to correct additional minor errors and omissions in the WET
method manuals. All of the minor corrections and clarifications
identified to date are detailed in the document titled, Proposed
Changes to Whole Effluent Toxicity Method Manuals (USEPA, 2001d). This
list may not be exhaustive, and EPA proposes to correct additional
minor errors and omissions that become apparent during the correcting
or revising of sections of the WET method manuals.
3. Specific Stakeholder Concerns
Today, EPA also proposes to modify the WET method manuals to
address specific stakeholder concerns. The proposed modifications are
summarized in Sections a through h below and are detailed in the
document titled, Proposed Changes to Whole Effluent Toxicity Method
Manuals (USEPA, 2001d), which is included in the docket supporting
today's rule and is available online at http://www.epa.gov/waterscience/WET. Proposal of these revisions partially fulfills the
requirements of two settlement agreements between stakeholders and EPA
(Edison Electric Institute, et al. v. EPA, Settlement Agreement, July
24, 1998; Lone Star Steel v. EPA, Settlement Agreement, March 4, 1998).
a. Blocking by Known Parentage
EPA proposes to amend the Ceriodaphnia dubia Survival and
Reproduction Test (section 13 of USEPA, 1994a) to require that test
organisms be allocated using ``blocking by known parentage.'' Blocking
by known parentage is a block randomization technique for allocating
test organisms among test chambers such that offspring from a single
female are distributed evenly among the test treatments (one per
treatment). In this arrangement, a block consists of the set of six
test chambers (one for each test treatment) containing organisms
derived from a single female parent.
Currently, the promulgated method describes a blocking by known
parentage procedure for use in test setup, but the method does not
require the use of this procedure. Today's proposal would require the
use of blocking by known parentage by using compulsory terms such as
``must'' and ``shall.'' The procedure described for test setup in the
current promulgated method would be retained as an example of how
blocking by known parentage may be accomplished.
In association with a blocking by known parentage requirement,
today's proposal also would add guidance on the treatment of males that
may occur in tests. The proposed changes would require exclusion of an
entire block from reproduction analysis (i.e., calculation of the no
observed effect concentration for reproduction and the 25% inhibition
concentration for reproduction) when 50% or more of the surviving
organisms in that block are identified as males. If less than 50% of
surviving organisms in a block are identified as males, only those
males would be excluded from the reproduction analysis. The proposed
changes also would stipulate that a test is invalid if fewer than eight
replicates remain in the control after excluding individual males and
necessary blocks (i.e., those having 50% or more of surviving organisms
identified as males). The specifics of all proposed method manual
changes related to blocking by known parentage are detailed in the
document titled, Proposed Changes to Whole Effluent Toxicity Method
Manuals (USEPA, 2001d).
Blocking by known parentage provides at least two benefits to the
performance of the Ceriodaphnia dubia Survival and Reproduction Test
(USEPA, 2001e). First, this technique of test organism allocation
ensures that any ``brood effect'' is evenly distributed among the test
treatments. Brood effects include differences in organism fecundity or
sensitivity that may be attributed to the health or genetics of the
parent organism. Blocking by known parentage minimizes any potential
bias that may be caused by one test treatment receiving an inordinate
number of underperforming (or overperforming) young from the same
parent organism. In an analysis of 389 tests from EPA's reference
toxicant test database (USEPA, 2000d) and 102 tests from EPA's WET
Variability Study (USEPA, 2001a), 9% and 25% of tests, respectively,
showed statistically significant (alpha = 0.05) block effects on the
reproduction endpoint (USEPA, 2001e). This means that, for these tests,
the number of offspring produced by test organisms was significantly
affected by the parental source of those test organisms. The blocking
by known parentage technique distributes this effect evenly across the
test treatments to ensure that observed differences in reproduction
between treatments are due to the effect of the treatment and not the
parental source of test organisms.
A second benefit of blocking by known parentage would be that it
provides a means of minimizing the impact of male production on test
performance. In healthy cultures, Ceriodaphnia dubia generally
reproduce parthenogenetically to produce cloned females for use in
testing. Under conditions of environmental stress, however, cladocerans
(such as Ceriodaphnia dubia and Daphnia magna) are known to produce
males (Pennak, 1989), which can negatively affect the performance of
toxicity tests designed to measure reproductive
[[Page 49798]]
effects (Haynes et al., 1989). When using blocking by known parentage,
males produced by a given brood female are contained within a single
block of the test rather than randomly scattered throughout the test.
If a large number of males are produced from a given brood female, the
associated block may be removed from the analysis of reproduction,
thereby minimizing the effect of those males on the test. Blocking by
known parentage also allows the source of males to be identified, so
that potential problems with culture health can be more easily
isolated.
b. 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. This
pH drift may be upward or downward depending upon test conditions and
sample characteristics. For instance, the addition of food substances
such as algae may cause a decrease in pH, while the loss of carbon
dioxide (CO2) from supersaturated effluent samples may cause
an increase in pH. A change in pH during testing means that an effluent
sample might be tested for toxicity at a different pH than the effluent
sample pH at the point of discharge. Under certain circumstances, this
pH drift could influence sample toxicity and be considered a test
interference. For this reason, EPA is proposing to provide guidance in
the chronic method manuals (USEPA, 1994a; USEPA, 1994b) on how to
identify if pH drift is a test interference and how to control test pH
if artifactual toxicity due to pH drift is confirmed.
For most tests, the range of pH drift is small, is well within the
organisms' tolerance range, and does not interfere with the analysis of
whole effluent toxicity. In EPA's WET Variability Study (USEPA, 2001a),
daily pH drift in blank samples averaged only +0.1 units (with a range
of -0.3 to +0.8 among 35 tests) in the Ceriodaphnia dubia Survival and
Reproduction Test and -0.1 units (with a range of -1.4 to +0.7 among 25
tests) in the Fathead Minnow Larval Survival and Growth Test. For
effluent samples (municipal wastewater spiked with KCl) analyzed in
EPA's WET Variability Study, pH drift in the 100% sample increased
slightly for the Ceriodaphnia dubia Survival and Reproduction Test,
averaging +0.3 units (with a range of -0.2 to +1.1 among 28 tests). For
the Fathead Minnow Larval Survival and Growth Test, daily pH drift in
effluent samples averaged -0.1 units (with a range of -0.6 to +0.4
among 28 tests), the same degree of drift observed in blank samples.
Ninety percent of Ceriodaphnia dubia Survival and Reproduction Tests
(126 tests) experienced absolute pH drift (up or down) of less than 0.7
units, and 90% of Fathead Minnow Larval Survival and Growth Tests (105
tests) experienced absolute pH drift of less than 0.5 units.
While pH drift was relatively mild for most samples analyzed in the
WET Variability Study (USEPA, 2001a), other effluent samples may
routinely exhibit a greater degree of pH drift. For example, municipal
wastewater from Publicly-Owned Treatment Works (POTW) is typically
discharged at a pH of 7.2-7.4, but the pH may equilibrate after contact
with air and stabilize at 8.0-8.5 (USEPA, 1992). In a 1998 survey of
433 POTWs, 39% of respondents indicated that upward drift of effluent
sample pH had been observed during acute or chronic WET testing
(DeGraeve et al., 1998). Upward pH drift in POTW effluent is generally
caused by dissipation of CO2 from the sample. Biological
treatment often produces an effluent that is supersaturated with
CO2. As dissolved CO2 in the supersaturated
sample equilibrates with the atmospheric CO2 concentration,
CO2 is lost from the sample. Because dissolved
CO2 acts as a weak acid, pH increases as CO2 is
lost. In cases where pH drift is due to the effluent characteristics,
the degree of drift will be greatest in the 100% effluent concentration
and will decrease with decreasing test concentrations.
EPA does not consider pH drift alone to be an interference in WET
testing if pH is within the organism's tolerance range (typically pH 6
to 9). Belanger and Cherry (1990) showed that Ceriodaphnia dubia
survival and reproduction did not differ significantly in receiving
water tests conducted at pH values ranging from 6 to 9. 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. Compounds with pH-dependent toxicity are those with
chemical characteristics that allow sufficient differences in
dissociation, solubility, or speciation to occur within a
physiologically tolerable pH range of 6 to 9 (Schubauer-Berigan et al.,
1993). Examples of such compounds include ammonia, metals, hydrogen
sulfide, cyanide, and ionizable organics. Ammonia, for instance, is
very common in effluent samples, and its toxicity changes sharply
within the typical effluent pH range of 7 to 8.5. As pH increases and
the temperature is held relatively constant, the percent of total
ammonia in the un-ionized form increases (USEPA, 1994a; Emerson et al.,
1975). Because the un-ionized form of ammonia (NH3) is significantly
more toxic than the ionized form (NH4+), toxicity
increases as pH increases. For metals, toxicity may increase or
decrease with increasing pH. Lead and copper were found to be more
acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel and zinc
were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). pH-dependent
toxicity is likely to be affected by temperature, dissolved oxygen,
CO2 concentrations, and total dissolved solids (USEPA,
1992). When pH-dependent compounds are present at concentrations near
the threshold for toxicity, pH drift during WET testing may produce
artifactual toxicity, or toxicity that would not have been observed if
the initial test pH had been maintained.
In addition to the issue of pH drift affecting toxicity in the
presence of pH-dependent compounds, stakeholders have raised concerns
about daily pH drift and sample renewal cycles producing toxicity even
in the absence of pH-dependent compounds. The circumstance of concern
would be in static-renewal tests, where the pH may change between the
time test organisms are placed into the test solutions and the time at
which the test solution is renewed. At renewal, the pH of test
solutions may be quickly returned to the initial sample pH. For chronic
tests that require daily renewal, a daily cycle of pH drift and renewal
may be established. Stakeholders expressed concern that, if the
difference in pH between the test solution and the renewal solution is
great, these adjustments in pH at renewal may cause shock to the test
organisms. Because the control treatment does not always experience the
same pH drift as effluent treatments, any shock resulting from daily
renewal would be experienced only in effluent treatments and
artifactual toxicity could result. In a 1998 settlement agreement with
these stakeholders (Edison Electric Institute, et al. v. EPA,
Settlement Agreement, July 24, 1998), EPA agreed to propose changes to
the WET methods that would provide methodological solutions for
controlling pH drift.
Currently, the WET method manuals (USEPA, 1993b; USEPA, 1994a;
USEPA, 1994b) provide guidance for effluent samples that arrive (i.e.,
at the testing laboratory prior to testing) with a pH outside of the
6.0 to 9.0 range. This range represents the general organism tolerance
range, so pH values outside of this range may produce toxic effects due
to pH alone. For samples that arrive
[[Page 49799]]
with a pH outside of this range, the current method manuals require
adjustment of the sample to pH 7 for freshwater testing or pH 8 for
marine testing. The method manuals also suggest brief aeration of
samples prior to use if dissolved oxygen levels are not at or near
saturation. Aeration provides the benefit of bringing other dissolved
gases (e.g., CO2) into equilibrium with the atmosphere and
stabilizing pH, but use of aeration should be minimized to reduce the
loss of volatile chemicals.
In 1996, EPA issued additional guidance on ammonia and pH control
in chronic testing (USEPA, 1996b). This guidance recognized that the
analyst has flexibility to control artifactual toxicity caused by pH
drift in chronic tests provided that the analyst verifies that the
source of toxicity is, in fact, artifactual. To verify that the
toxicity is artifactual, EPA recommended parallel testing using one
test with an adjusted pH and one test without an adjusted pH. If
toxicity is removed or reduced when pH is adjusted, the source of
toxicity could be artifactual and pH could be controlled in the testing
of the effluent. This guidance acknowledged that pH could be controlled
during testing with procedures that do not significantly alter the
nature of the sample.
Today, EPA proposes to modify the chronic method manuals (USEPA,
1994a; USEPA, 1994b) to incorporate procedures for controlling pH drift
in static-renewal tests when sample toxicity is confirmed to be
artifactual and caused by pH drift. EPA proposes adding guidance that
is consistent with the 1996 USEPA guidance on pH and ammonia control in
chronic testing (USEPA, 1996b), and extending this guidance to include
situations where artifactual toxicity is caused by pH drift in the
absence of ammonia.
The proposed method changes would require that, prior to the use of
pH control techniques, the analyst must confirm that observed toxicity
is artifactual and caused by pH drift. Evidence of artifactual toxicity
would be demonstrated by conducting parallel tests: one with controlled
pH and one with uncontrolled pH. Several such parallel tests conducted
on a given effluent may be required by the regulatory authority to
verify that the toxicity observed in that effluent is artifactual and
caused by pH drift (as opposed to variability in effluent samples).
Following this determination, the regulatory authority may allow pH
control in subsequent chronic toxicity testing of the effluent. The
proposed method changes would specify the use of acid/base addition
and/or a CO2-controlled atmosphere technique for adjusting
and controlling pH in chronic tests.
The CO2-controlled atmosphere technique that is proposed
for pH control in chronic tests is conducted using enclosed test
chambers with CO2 injected into the headspace above the test
solution (USEPA, 1991a; USEPA, 1992; USEPA, 1996c; Mount and Mount,
1992). An enriched-CO2 environment increases the dissolution
of CO2 into the sample, which acts as a weak acid to prevent
pH increases. This technique uses the natural carbonate buffering
system to control pH and requires minimal alteration of the sample.
This technique is one method recommended for adjusting pH in toxicity
identification evaluations (TIEs) (USEPA, 1991a; USEPA, 1992; USEPA,
1996c).
In acute testing, the proposed method changes would recommend the
use of static-renewal testing or flow-through testing when artifactual
toxicity due to pH drift is suspected. The use of static-renewal
testing may reduce the degree of pH drift (compared to static non-
renewal tests), and flow-through testing should eliminate pH drift that
could occur due to static testing conditions. In flow-through testing,
new sample is continually added to the test chambers, so drift from the
initial sample pH should not occur. Flow-through testing also
eliminates any potential for organism shock from pH drift and renewal
cycles, because test renewal is continuous. Because flow-through
testing provides an available option for reducing pH drift in acute
tests without modifying the sample, EPA does not propose additional
techniques (such as acid/base addition and/or CO2-controlled
atmosphere techniques that are proposed for chronic test methods) for
pH control in acute test methods.
The specifics of all proposed method manual changes related to pH
drift are detailed in the document titled, Proposed Changes to Whole
Effluent Toxicity Method Manuals (USEPA, 2001d). The proposed changes
related to pH drift will affect all methods in the freshwater chronic
method manual (USEPA, 1994a), except for the Selenastrum capricornutum
Growth Test; and all methods in the marine chronic method manual
(USEPA, 1994b), except for the Arbacia punctulata Fertilization Test
and the Champia parvula Reproduction Test. The Selenastrum, Arbacia,
and Champia tests do not require test solution renewal, so daily pH
fluctuations should not be a concern. Proposed changes to the acute
method manual (USEPA, 1993b) would simply recommend the use of static-
renewal testing or flow-through testing when artifactual toxicity due
to pH drift is suspected. EPA invites comments on how pH drift would
and should be addressed in WET testing (see Section V.A).
c. Concentration-Response Relationships
The concentration-response relationship established between the
concentration of a toxicant and the magnitude of the response is a
fundamental principle of toxicology. This principle assumes that there
is a causal relationship between the dose of a toxicant (or
concentration for toxicants in solution) and a measured response. A
response may be any measurable biochemical or biological parameter that
is correlated with exposure to the toxicant. The classical
concentration-response relationship is depicted as a sigmoidal-shaped
curve with detrimental responses increasing as the concentration of the
toxicant increases. Not all concentration-response relationships,
however, are represented by the classical sigmoidal-shaped curve. A
corollary of the concentration-response concept is that every toxicant
should exhibit a concentration-response relationship, given that the
appropriate response is measured and given that the concentration range
evaluated is appropriate. Use of this concept can be helpful in
determining whether an effluent sample causes toxicity and in
identifying anomalous test results.
In July 2000, EPA published guidance on evaluating concentration-
response relationships to assist in determining the validity of WET
test results (USEPA, 2000a). This document explained the concentration-
response concept and provided review steps for 10 different
concentration-response patterns that may be encountered in WET test
data. Based on the results of the review, the guidance anticipates one
of three determinations: (1) that calculated effect concentrations are
reliable and should be reported; (2) that calculated effect
concentrations are anomalous and should be explained; or (3) that the
test was inconclusive and should be repeated with a newly collected
sample.
In today's action, EPA proposes to require the review of
concentration-response relationships generated for all multi-
concentration WET tests reported under the NPDES program. EPA proposes
to modify section 10 of the two chronic method manuals (USEPA, 1994a;
USEPA, 1994b) and section 12 of the acute method manual (USEPA, 1993b)
to incorporate this required test review procedure. The modified
sections would explain the
[[Page 49800]]
concentration-response concept, require the review of concentration-
response relationships, and reference EPA guidance (USEPA, 2000a)
describing various forms of concentration-response relationships and
review procedures. Use of the concentration-response review procedures
(USEPA, 2000a) would ensure that a valid concentration-response
relationship is demonstrated prior to the determination of toxicity.
EPA intends to maintain the review procedures described in the guidance
document (USEPA, 2000a) as ``guidance'' because these procedures may be
revised as new information on the review of concentration-response
relationships (including additional forms of concentration-response
relationships) becomes available.
To demonstrate the effectiveness of the proposed concentration-
response review steps, EPA used the guidance on concentration-response
relationships (USEPA, 2000a) in the review and reporting of results
from EPA's WET Variability Study (USEPA, 2001a). In this study, 635
valid tests (i.e., those that met test acceptability criteria) were
reviewed according to the proposed concentration-response evaluation
procedures. Based on these review procedures, the calculated effect
concentrations in 14 tests were determined to be anomalous, and the
effect concentrations calculated in 9 tests were determined to be
inconclusive. Eight of the 23 test results that were considered
anomalous or inconclusive had erroneously indicated toxicity in blank
samples. These results would have been reported as false positives if
the concentration-response review procedures had not been used. This
study indicates that the proposed concentration-response review
procedures are effective in reducing the incidence of false positives
in WET testing. The use of these review procedures reduced the rate of
reported false positives in the WET Variability Study from 11.1% to
3.7% for the Ceriodaphnia dubia Survival and Reproduction Test; from
12.5% to 4.35% for the Fathead Minnow Larval Survival and Growth Test;
from 14.3% to 0% for the Mysidopsis bahia Survival, Growth, and
Fecundity Test; and from 14.3% to 0% for the Inland Silverside Larval
Survival and Growth Test.
In addition to requiring the review of concentration-response
relationships, EPA proposes to modify section 12 of the acute method
manual (USEPA, 1993b) and section 10 of the two chronic method manuals
(USEPA, 1994a; USEPA, 1994b) to consolidate other important test review
components that are described elsewhere in the method manuals. These
revised sections, titled ``Report Preparation and Test Review,'' would
describe the review of sample collection and handling conditions, test
acceptability criteria, test conditions, statistical methods,
concentration-response relationships, reference toxicant testing, and
test variability. The specifics of the proposed method manual changes
related to concentration-response relationship evaluation and other
test review components are detailed in the document titled, Proposed
Changes to Whole Effluent Toxicity Method Manuals (USEPA, 2001d).
The quality of WET Variability Study data (USEPA, 2001a; USEPA,
2001b) used to make decisions for this rulemaking is of primary
importance to the Agency and to stakeholders. These data and the test
review and acceptance criteria used in the WET Variability Study are
detailed in a final study report contained in the record for this
rulemaking (USEPA, 2001a). Some stakeholders believe that EPA
improperly applied different standards in accepting or rejecting data
generated in the WET Variability Study and departed from the stated
objectives of the study design. EPA is proposing test review procedures
consistent with the test reviews that EPA conducted on data developed
in the WET Variability Study (though EPA notes that the objectives of
the study differ from those associated with compliance monitoring). EPA
proposes modifications to standardize the minimum elements of WET test
review. While some of these test review components provide specific
criteria for the acceptance or rejection of test results (e.g., the
method test acceptability criteria), others (e.g., review of test
conditions, reference toxicant testing, and concentration-response
relationships) must be reviewed within the context of the test
objective. Also, State and/or regional regulatory authorities may
require additional test review components and criteria to further
standardize the reporting and review of WET test data. EPA requests
comment on the acceptance, interpretation, and use of the WET
Variability Study data and on the proposed section of the method
manuals titled, ``Report Preparation and Test Review''.
d. Nominal Error Rates
WET test results (i.e., effect concentrations) may be determined by
point estimation or hypothesis testing techniques (USEPA, 1994a; USEPA,
1994b). Hypothesis testing techniques compare responses in the control
treatment with responses in other treatments to test the ``null
hypothesis'' that there is no statistically significant difference
between the treatments (i.e., that the effluent is not toxic). To
determine when a difference between treatments is large enough to be
statistically significant, the statistician or analyst must select a
nominal error rate. The nominal error rate, or alpha level, is an
intended upper bound on the probability of incorrectly concluding that
the treatments are different when, in fact, they are not (a Type I
statistical error). The larger the alpha level, the greater the
probability of incorrectly rejecting the null hypothesis (i.e.,
determining that the effluent is toxic when, in fact, it is not). For
all WET tests, EPA recommends using an alpha level of 0.05, which
corresponds to a 5% probability of making a Type I error.
In response to stakeholder concerns that an alpha level of 0.05
does not adequately protect against Type I errors (Moore et al., 2000;
Edison Electric Institute, et al. v. EPA, Settlement Agreement, July
24, 1998), EPA published guidance on nominal error rate selection
(USEPA, 2000a). This guidance clarifies that the alpha level may be
reduced to 0.01 in specific circumstances. These circumstances include
instances when sublethal endpoints from Ceriodaphnia dubia or fathead
minnow tests are reported under NPDES permit requirements, or when WET
permit limits (based on any WET method) are derived without allowing
for receiving water dilution. Even under these circumstances, however,
the alpha level may be reduced only in tests that meet a fixed
criterion for test sensitivity because reductions in the alpha level
also reduce statistical power. Specifically, the percent minimum
significant difference (PMSD) calculated for the test using an alpha
level of 0.01 should be less than or equal to criteria set forth in the
guidance document (USEPA, 2000a). The document also provides guidance
on determining the need for additional test replication to meet PMSD
criteria and guidance on the decision process for reducing the nominal
error rate in hypothesis testing.
In today's action, EPA proposes to modify the chronic WET method
manuals (USEPA, 1994a; USEPA, 1994b) to clarify the circumstances under
which the recommended alpha level may be reduced. The proposed change
would modify subsection 9.4.6 (Recommended Alpha Levels) of the two
chronic method manuals (USEPA, 1994a; USEPA, 1994b). This subsection
would maintain the current recommendation that an alpha level of 0.05
be used for hypothesis testing. In
[[Page 49801]]
addition, the subsection would identify the specific circumstances
where the alpha level used for hypothesis testing could appropriately
be reduced from 0.05 to 0.01. The subsection would describe these
circumstances and reference the published guidance (USEPA, 2000a) for
information on determining adequate test sensitivity and determining
the appropriateness of reductions in the alpha level. The specifics of
the proposed method manual changes related to nominal error rates are
detailed in the document titled, Proposed Changes to Whole Effluent
Toxicity Method Manuals (USEPA, 2001d).
e. Confidence Intervals
Point estimation techniques described in the WET method manuals are
used to generate effect concentrations and associated 95% confidence
intervals (USEPA, 1993b; USEPA, 1994a; USEPA, 1994b). Software used to
conduct these statistical procedures occasionally do not provide the
associated confidence intervals. This situation may arise when test
data do not conform with specific assumptions required by the
statistical methods, when point estimates are outside of the test
concentration range, and when specific limitations imposed by the
software are encountered. In July 2000, EPA published guidance on the
specific circumstances under which confidence intervals are not
generated or are not suitable (USEPA, 2000a).
In today's action, EPA proposes to modify the WET method manuals to
clarify the circumstances under which confidence intervals are not
generated by point estimation techniques and to reference the published
guidance on this issue (USEPA, 2000a). The proposed change would modify
subsection 9.3.2 (Point Estimation Techniques) of the two chronic
method manuals (USEPA, 1994a; USEPA, 1994b) and subsection 11.2
(Determination of the LC50 from Definitive, Multi-Effluent-
Concentration Acute Toxicity Tests) of the acute method manual (USEPA,
1993b). The specifics of the proposed method manual changes related to
confidence intervals are detailed in the document titled, Proposed
Changes to Whole Effluent Toxicity Method Manuals (USEPA, 2001d).
f. Dilution Series
In multi-concentration (definitive) WET tests, organism effects are
measured in a range of effluent concentrations. The dilution series
selected for the test defines the concentrations of effluent tested.
The WET methods recommend preparing test concentrations using a
dilution factor of greater than or equal to 0.5 and provide an example
dilution series of 100%, 50%, 25%, 12.5%, and 6.25% effluent. While
this particular dilution series is commonly used in WET testing, test
concentrations for each test should be selected independently based on
the objective of the study, the expected range of toxicity, the
receiving water concentration (or instream waste concentration), and
any available historical testing information on the effluent. The
dilution series should be selected to optimize the precision of
calculated effect concentrations and assist in establishing
concentration-response relationships. In July 2000, EPA published
guidance on selecting appropriate dilution series for WET testing
(USEPA, 2000a).
In today's action, EPA proposes to modify the WET method manuals to
reference the published guidance on selecting dilution series (USEPA,
2000a) and to clarify that dilution series should be selected
independently for each test based on the objective of the study, the
expected range of toxicity, the receiving water concentration (or
instream waste concentration), and any available historical testing
information on the effluent. The proposed change would modify
subsection 8.10 (Multi-concentration [Definitive] Effluent Toxicity
Tests) of the two chronic method manuals (USEPA, 1994a; USEPA, 1994b)
and subsection 9.3 (Multi-concentration [Definitive] Effluent Toxicity
Tests) of the acute method manual (USEPA, 1993b). The specifics of the
proposed method manual changes related to dilution series selection are
detailed in the document titled, Proposed Changes to Whole Effluent
Toxicity Method Manuals (USEPA, 2001d).
g. Dilution Waters
Test concentrations in definitive WET tests are prepared by
diluting the effluent sample with an appropriate dilution water. The
WET methods allow the use of natural receiving waters or synthetically
prepared waters for dilution. Because the choice of dilution water can
affect WET test results (Cooney et al., 1992; Belanger et al., 1989;
DeLisle and Roberts, 1988), selecting an appropriate dilution water is
important. To assist in this process, EPA published guidance on
dilution water selection (USEPA, 2000a) that clarifies what EPA
considers to be an acceptable dilution water. An acceptable dilution
water is one that 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. The guidance also provides
recommendations on how to select an appropriate dilution water based on
the objectives of the test, the condition and quality of ambient
receiving water, in-stream dilution potential, and recommendations or
requirements from local regulatory authorities. Lastly, the guidance
explains the use of dual controls when dilution water differs from
organism culture water.
In today's action, EPA proposes to modify the WET method manuals by
clarifying the definition of acceptable dilution waters and referencing
the published guidance (USEPA, 2000a) for more information on selecting
appropriate dilution waters. The proposed change would modify
subsection 7.1 (Types of Dilution Water) of each of the method manuals
(USEPA, 1993b; USEPA, 1994a; USEPA, 1994b). The specifics of the
proposed method manual changes related to dilution waters are detailed
in the document titled, Proposed Changes to Whole Effluent Toxicity
Method Manuals (USEPA, 2001d).
h. Pathogen Interference
WET testing is designed to measure the aggregate toxicity of an
aqueous test sample. The presence of pathogens and/or parasites in the
test sample, however, may confound this measurement of toxicity by
causing sporadic mortality among test organisms. Today, EPA proposes to
modify the Fathead Minnow (Pimephales promelas) Larval Survival and
Growth Test to provide guidance on the adverse effects of pathogens
and/or parasites on test performance (i.e., pathogen and/or parasite
test interference). EPA proposes procedures to control pathogen and/or
parasite effects without compromising the capacity of the test to
measure the toxicity of the test sample. The proposed method
modifications are summarized below and detailed in the document titled,
Proposed Changes to Whole Effluent Toxicity Method Manuals (USEPA,
2001d).
Pathogens that interfere with the test may come from the receiving
water used for test dilutions, from the effluent, or from the receiving
water that is used as intake water. Most receiving waters contain all
the common fish pathogens, but these fish pathogens do not cause a
problem in the stream. At times, however, the test conditions during
[[Page 49802]]
WET tests (e.g., 24 hour durations between sample renewals, beakers
used for seven days without change, or uneaten brine shrimp) may
promote bacterial growth. Some opportunistic bacteria take advantage of
these conditions and flourish or ``bloom.'' The bacteria that bloom may
be harmless or they may be fish pathogens. Blooms may even differ
between replicates. In some cases, the presence of uncontrolled
pathogen and/or parasite effects in the WET test may suggest the
selection of a different test species.
Stakeholders have identified particular concerns with the adverse
effect of pathogens on the performance of the Fathead Minnow Larval
Survival and Growth Test. A typical indication that pathogen
interference has occurred in a WET test is when test organisms exhibit
``sporadic mortality.'' This sporadic mortality phenomenon is
characterized by an unexpected concentration-response relationship
(i.e., effects that do not increase with increasing effluent
concentration) and fathead minnow survival that varies greatly among
replicates and among effluent dilutions. The observed sporadic
mortality among replicates tends to occur in receiving water controls
and in lower effluent concentrations (or occasionally in the full-
strength effluent samples) on day three or day four of the Fathead
Minnow Larval Survival and Growth Test. EPA does not have evidence of
such sporadic mortality occurring in concurrently conducted chronic
tests using the cladoceran, Ceriodaphnia dubia, or concurrent acute
tests with the fathead minnow, C. dubia, or other acute test species.
When sporadic mortality is observed, often a fungal growth occurs
directly on the fish, especially in the gill area. This growth
interferes with measuring toxicity in the WET test. Biological test
interference due to this type of fungal growth may occur during the
toxicity test when effluents and water samples tested are derived from
the receiving water (i.e., their source is a receiving water intake) or
when the receiving water is used as the diluent. The fungal growth has
been attributed to Saprolegnia sp. (Downey et al., 2000) which may be a
secondary infection following infection from a known fish pathogen.
Microbiological evaluations on receiving waters, the fish, and their
food indicated the ubiquitous nature of pathogenic organisms (e.g.,
Flexibacter spp., Aeromonas hydrophila). Eradicating these types of
organisms from the test through the decontamination of the fish and
their food has not been practical (Geis et al., 2000a).
Data from the WET test must be reviewed carefully to ascertain if
pathogens are suspected. The key indicators that pathogen interference
has occurred are the presence of an unexpected concentration-response
relationship (i.e., effects that do not increase with increasing
effluent concentration), and organism survival that varies greatly
among replicates and among effluent dilutions. The analyst should
evaluate the test data to determine a cause for any unexpected
concentration-response pattern and subsequently to determine the
validity of calculated results (USEPA, 2000a). Normal, reversed, or
bimodal concentration-response relationships are not considered
indicators of test interference by pathogenic bacteria (USEPA, 2000a).
The analyst also should evaluate the responses at each test
concentration for unusually high mortality and/or for unevenness of
mortalities among replicates. If the within-treatment coefficient of
variation (CVs) for survival in an effluent treatment is greater than
40% and relatively low for control replicates in standard synthetic
water, pathogen interference should be considered. Following data
evaluations, additional testing would be required to ascertain that
sporadic mortality observed in the WET test is due to interference by
pathogenic bacteria. Parallel tests should be conducted using
reconstituted water and receiving water as diluents with the effluent.
Before modifying any test procedures that will allow the analyst to
account for pathogen interference, all available options within the
flexibility of the method should be exhausted. Samples should be
filtered through a 2-4 mm mesh opening (as described in Subsection
8.8.2 of the freshwater chronic method manual (USEPA, 1994a)) to remove
indigenous organisms. Tests should be conducted using separate
glassware, pipettes, and siphons for each concentration to minimize
cross contaminating replicates of all treatments. The analyst also must
keep laboratory equipment clean and dry when not in use. Use of
reconstituted laboratory waters instead of receiving waters may
eliminate the interference, and the use of reconstituted water would be
preferable to invalid tests. However, for those instances when
receiving water is required as the diluent or when the effluent and the
subsequent dilutions exhibit the interference, EPA recommends modifying
the test design to prevent the spread of the pathogen among the test
chambers during the test.
Once pathogenic test interference has been confirmed by additional
testing, the proposed modifications to the Fathead Minnow Larval
Survival and Growth Test would recommend use of an altered test design
to minimize the effects of the pathogenic interference. The use of
fewer fish per test chamber and new test chambers daily has been the
most effective technique for controlling the effects of pathogenic
bacteria in the Fathead Minnow Larval Survival and Growth Test. Use of
small plastic 30-ml cups containing two fish per cup showed the
greatest improvement to the test method, removing the pathogenic effect
91% of the time (Geis et al., 2000a). For instance, use of 20 ml of
test solution in a 1 ounce plastic cup and two fish per beaker
significantly reduced the sporadic mortality not attributed to the
effluent toxicity. The total number of fish tested is not reduced
(i.e., 40 per treatment), and the fish are combined at the end of the
test into the typical number of replicates so that data analysis
following the test method manuals is unchanged.
When parallel testing has confirmed pathogen interference and the
modifications to the test design for the number of fish per chamber
does not reduce the pathogen interference, the regulatory authority may
allow modifications of the effluent samples to remove or inactivate the
pathogens. The analyst should apply TIE filtration steps (USEPA, 1991a;
USEPA, 1992) in combination with various sterilization techniques
listed below to ascertain and control adverse influences on tests
caused by pathogens in the intake or receiving waters used for
dilution. For some samples, one or more techniques such as irradiation
with ultraviolet light, pasteurization, filtration (0.2 m
pore size), and addition of antibiotics has been shown to improve
survival and reduce variability among replicates effectively (SETAC,
1999). EPA cautions that some treatment methods that might control
pathogens in the test, (e.g., ultraviolet light treatment or the
addition of antibiotics (Downey et al., 2000)) may also improperly
reduce or increase the toxicity of the sample. Filtration also may
remove some toxicity in the sample as shown in toxicity identification
evaluations (USEPA, 1991a; 1992; 1993a). The use of ultrafiltration on
an effluent sample containing particulate matter to which process-
induced metals have adsorbed may improperly remove a significant source
of process-related toxicity. Also, chlorination and dechlorination may
be
[[Page 49803]]
a treatment option where pathogenic bacteria are suspected as the sole
source of toxicity in the ambient intake waters. However, when the
analyst prepares samples using techniques of chlorination and/or
dechlorination, potential exists for oxidation and reduction of other
compounds (USEPA, 1991a; 1992). All toxicity tests conducted on
modified samples (e.g., sterilized) must include an additional blank
preparation (control) consisting of similarly treated reconsituted
laboratory water (USEPA, 1991a; 1992).
Procedures to control the adverse influences of pathogens must not
be used to reduce process-related sources of toxicity. With effluents
and ambient waters, the pathogen(s) may mask the presence of a chemical
that is, by itself, toxic. It is also possible that the pathogen
infection is induced by some predisposing factor in the receiving water
and would not occur without that factor. The need to evaluate both
intake water and effluent samples to determine the cause of the
pathogen or the source of pathogens is essential before applying any
pathogen/parasite control technology and cannot be overemphasized. The
analyst must evaluate whether the intake water is contributing the
interference observed in the toxicity test of the final effluent.
The method modifications proposed today provide techniques to
assess and control the effects of pathogens in the Fathead Minnow
Larval Survival and Growth Test. Today's proposal does not address,
however, the determination as to the conditions under which this
control is appropriate for purposes of NPDES permit compliance. By
today's proposal, EPA does not concede that the discharge of toxic
biological agents to waters of the US is appropriate or authorized but
merely that pathogens in test samples may confound measurement of whole
effluent toxicity.
C. Ratification or Withdrawal of Methods
In a 1998 settlement agreement with Edison Electric Institute et
al. (Edison Electric Institute, et al. v. EPA, No. 96-1062 &
consolidated case (D.C. Cir.), Settlement Agreement, July 24, 1998),
EPA agreed to conduct an interlaboratory variability study of 12 of the
17 approved WET test methods (the WET Variability Study). The 12
methods evaluated in the study (Table 1) represent a combination of
acute and chronic test methods; freshwater and marine test methods; and
invertebrate, fish, and algal species. EPA conducted the WET
Variability Study in 1999 through 2000, and published preliminary
results from the study in October 2000 (USEPA, 2000b; USEPA, 2000c). In
2001, EPA submitted the preliminary results of the study for expert
peer review (USEPA, 2001c). The peer review comments and EPA's response
to those comments are included in the record established for this
rulemaking (see Addresses section of this rule). Based on peer review
comments, EPA revised the preliminary study report to produce a final
study report. In conjunction with today's action, EPA is publishing a
final study report (USEPA, 2001a; USEPA, 2001b) that presents the final
results of EPA's WET Variability Study. These results are discussed in
section III.C.1 below.
The settlement agreement (Edison Electric Institute, et al. v. EPA,
Settlement Agreement, July 24, 1998) also required that EPA propose to
ratify or withdraw each of the 12 WET test methods evaluated in the WET
Variability Study. Based on the results of the WET Variability Study,
consideration of peer review comments, and an overall evaluation of the
WET program, EPA proposes to ratify 11 of the methods evaluated in the
WET Variability Study. EPA proposes to ratify nine of these methods, in
an amended form, as described in Section III.B of this rule. EPA
proposes to ratify two other methods (the Selenastrum capricornutum
Growth Test and the Mysidopsis bahia Survival, Growth and Fecundity
Test) with additional modifications (i.e., in addition those described
in Section III.B of this rule) to improve the performance of the
methods. EPA proposes to withdraw and propose a new Holmesimysis
costata Acute Test method. The Holmesimysis costata Acute Test method
was promulgated and tested in the WET Variability Study using acute
test procedures designed for the Mysidopsis bahia Acute Test (except at
a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C; and a
salinity of 32-34, instead of 5-30). Results of the
WET Variability Study revealed that acute test procedures designed for
Mysidopsis bahia were insufficient for successful test conduct using
Holmesimysis costata. For this reason, EPA proposes to withdraw
Holmesimysis costata as an acceptable species for use in the Mysidopsis
bahia Acute Test method and to propose it as an acute toxicity test
method designed specifically for Holmesimysis costata. Sections 2-7
below discuss the proposed ratification and/or withdrawal of each
method evaluated in the WET Variability Study.
Table 1.--Whole effluent toxicity test methods included in EPA's WET
Variability Study
------------------------------------------------------------------------
Test method
Test method Common test method name No. a
------------------------------------------------------------------------
Cladoceran, Ceriodaphnia dubia, Ceriodaphnia-- dubia ...........
Acute Test. Acute Test.
Cladoceran, Ceriodaphnia dubia, Ceriodaphnia dubia 1002.0
Survival and Reproduction Test. Survival and
Reproduction Test.
Fathead Minnow, Pimephales Fathead Minnow Acute ...........
promelas, Acute Test. Test.
Fathead Minnow, Pimephales Fathead Minnow Larval 1000.0
promelas, Larval Survival and Survival and Growth
Growth Test. Test.
Green Alga, Selenastrum Selenastrum 1003.0
capricornutum, Growth Test. capricornutum Growth
Test.
Mysid, Mysidopsis bahia, Mysidopsis bahia 1007.0
Survival, Growth, and Fecundity Survival, Growth, and
Test. Fecundity Test.
Sheepshead Minnow, Cyprinodon Sheepshead Minnow Acute ...........
variegatus, Acute Test. Test.
Sheepshead Minnow, Cyprinodon Sheepshead Minnow Larval 1004.0
variegatus, Larval Survival and Survival and Growth
Growth Test. Test.
Inland Silverside, Menidia Inland Silverside Acute ...........
beryllina, Acute Test. Test.
Inland Silverside, Menidia Inland Silverside Larval 1006.0
beryllina, Larval Survival and Survival and Growth
Growth Test. Test.
Red Macroalga, Champia parvula, Champia parvula 1009.0
Reproduction Test b. Reproduction Test.
Mysid, Holmesimysis costata, Holmesimysis costata ...........
Acute Test b c. Acute Test.
------------------------------------------------------------------------
a Test method numbers were not designated for acute test methods in
USEPA, 1993b.
b Due to insufficient laboratory support, interlaboratory data were not
obtained for this method.
c The EPA-approved acute test with Holmesimysis costata was performed
using the test conditions for the Mysidopsis bahia Acute Test method
(except at a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C;
and a salinity of 32-34, instead of 5-30).
[[Page 49804]]
In ratifying WET test methods, EPA reaffirms the conclusion
expressed in the 1995 WET final rule (60 FR 53529; October 16, 1995),
that these methods are applicable for use in NPDES permits. In the 1995
WET final rule, this conclusion was based on the well-established use
of the methods, the existence of extensive guidance on quality
assurance and routine quality control activities, and validation data
from a number of studies conducted by EPA, State programs, and
universities. Since promulgation of the methods, this basis for
approval has been strengthened by more widespread use of the methods,
additional guidance on quality assurance and quality control issues
(USEPA, 2000a; USEPA, 2000d), and the WET Variability Study to confirm
method performance data from original validation studies (USEPA, 2001a;
USEPA, 2001b).
1. WET Variability Study
EPA designed the WET Variability Study to characterize
interlaboratory variability, the rate of successful test completion,
and the rate of ``false positive'' incidence (i.e., the measurement of
toxicity in non-toxic blank samples) for the 12 test methods listed in
Table 1. For two of these methods (the Champia parvula Reproduction
Test and the Holmesimysis costata Acute Test), EPA was unable to obtain
interlaboratory data due to laboratory unavailability (i.e., 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). Intralaboratory data were obtained for the Champia parvula
Reproduction Test, but no valid intralaboratory or interlaboratory data
were obtained for the Holmesimysis costata acute test. For each of the
remaining 10 methods, 7 to 35 laboratories participated in multi-
laboratory testing of 3 or 4 ``blind'' test samples. Laboratories
received some combination of the following test sample types: reagent
water (or ``blank''); reference toxicant; municipal or industrial
effluent; and receiving water. Participant laboratories were required
to analyze each blind test sample according to the promulgated WET test
method manuals and specific instructions in participant laboratory
standard operating procedures developed for the study (appendix B,
USEPA, 2001b). In total, the study generated interlaboratory precision
data from testing more than 700 blind samples among 55 participant
laboratories. EPA had not previously conducted a study of this
magnitude with these objectives in this time frame.
The results of the WET Variability Study (Table 2) supported the
conclusions of the 1995 WET final rule and confirmed the acceptability
of the WET test methods for use in NPDES permits, except as noted below
in sections 2 through 7. The analysis of successful test completion
rates revealed that most WET test methods could be consistently and
reliably performed by qualified testing laboratories. For the purposes
of the study, EPA defined successful test completion rates to be the
percentage of initiated and properly terminated tests that met the test
acceptability criteria as specified in the WET method manuals.
Successful test completion rates were above 90% for 8 of the 10 methods
evaluated during interlaboratory testing. Only the Ceriodaphnia dubia
Survival and Reproduction Test method (see section 2 below) and the
Selenastrum capricornutum Growth Test method (see section 5 below)
produced successful test completion rates less than 90%.
The analysis of false positive rates revealed that the WET test
methodologies, including applicable guidance on reviewing WET test
results (USEPA, 2000a), effectively control the incidence of falsely
identifying toxicity in non-toxic ``blank'' samples. False positive
rates were defined as the percentage of valid tests conducted on blank
samples that indicated toxicity by producing LC50 (median lethal
concentration), NOEC (no observed effect concentration), or IC25 (25%
inhibition concentration) values of less than 100% sample. False
positive results were reported for three test methods, and the rates of
false positives were below the theoretical false positive rate of 5%
(based on the recommended 0.05 alpha level for hypothesis testing) for
all but the Selenastrum capricornutum Growth Test conducted without
EDTA.
The analysis of interlaboratory precision data revealed that the
WET test methods are sufficiently precise for use in NPDES permits.
Interlaboratory coefficients of variation (CVs) calculated in the WET
Variability Study ranged from 10.5% to 58.5% (Table 2). This observed
range of interlaboratory variability is consistent with the range of
variability reported for chemical methods approved at 40 CFR part 136
(USEPA, 1991b). For chemical methods measuring metals at the low end of
the detection range, interlaboratory CVs range from 18% to 129%, with a
median CV of 45%. Interlaboratory CVs for chemical methods for organic
analyses range from greater than 12% to 91%, and interlaboratory CVs
for nonmetal inorganic analyses range from 4.6% to 70%.
Table 2.--Summary of Test Results From EPA's WET Variability Study
------------------------------------------------------------------------
Successful
test False Interlaboratory
Test method completion positive precision (%
rate (%) ratea (%) CV) b
------------------------------------------------------------------------
Ceriodaphnia dubia Acute Test 95.2 0.00 29.0
Ceriodaphnia dubia Survival 82.0 3.70 35.0
and Reproduction Test.......
Fathead Minnow Acute Test.... 100 0.00 20.0
Fathead Minnow Larval 98.0 4.35 20.9
Survival and Growth Test....
Selenastrum capricornutum 63.6 0.00 34.3
Growth Test (with EDTA) c...
Selenastrum capricornutum 65.9 33.3 58.5
Growth Test (without EDTA) c
Mysidopsis bahia Survival, d 97.7 0.00 41.3
Growth, and Fecundity Test..
Sheepshead Minnow Acute Test. 100 0.00 26.0
Sheepshead Minnow Larval 100 0.00 10.5
Survival and Growth Test....
Inland Silverside Acute Test. 94.4 0.00 38.5
Inland Silverside Larval 100 0.00 43.8
Survival and Growth Test....
Champia parvula Reproduction ND ND f ND
Test e......................
Holmesimysis costata Acutee.. ND ND ND
------------------------------------------------------------------------
a False positive rates reported for each method represent the higher of
false positive rates observed for hypothesis testing or point estimate
endpoints.
[[Page 49805]]
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.
c The Selenastrum capricornutum Growth Test method was conducted with
and without ethylenediaminetetraacetic acid (EDTA) as a component of
the nutrients added to test and control treatments. Due to improved
test performance with the addition of EDTA, EPA is proposing to
recommend the addition of EDTA in the Selenastrum capricornutum Growth
Test.
d Successful test completion for the optional fecundity endpoint was
50%.
e ND = not determined. Due to insufficient laboratory support,
interlaboratory data were not obtained for the Champia parvula
Reproduction Test method and the Holmesimysis costata Acute Test
method.
f While interlaboratory test data were not obtained for the Champia
parvula Reproduction Test method, intralaboratory data was obtained
from the referee laboratory. Intralaboratory CVs were 27.6%, 49.7%,
and 50.0% for reference toxicant, receiving water, and effluent sample
types, respectively.
2. Ceriodaphnia dubia Acute Test, Ceriodaphnia dubia Survival and
Reproduction Test, Fathead Minnow Acute Test, Fathead Minnow Larval
Survival and Growth Test, Sheepshead Minnow Acute Test, Sheepshead
Minnow Larval Survival and Growth Test, and Inland Silverside Acute
Test
Today, EPA proposes to ratify its previous rulemaking standardizing
the following WET test methods: Ceriodaphnia dubia Acute Test,
Ceriodaphnia dubia Survival and Reproduction Test, Fathead Minnow Acute
Test, Fathead Minnow Larval Survival and Growth Test, Sheepshead Minnow
Acute Test, Sheepshead Minnow Larval Survival and Growth Test, and the
Inland Silverside Acute Test. At the time of method promulgation,
interlaboratory precision data were available for each of these test
methods. Based on these precision data, EPA concluded that toxicity
tests are no more variable than chemical analytical methods in 40 CFR
part 136, and that toxicity tests provide reliable indicators of whole
effluent toxicity. At that time, EPA also anticipated that laboratory
performance would improve with use of the methods over time. Results
from the WET Variability Study not only confirmed the level of
precision previously cited for these methods, but indicated that the
methods currently exhibit even lower variability than estimated at the
time of method promulgation (60 FR 53529; October 16, 1995). Such data
also confirm EPA's assumptions regarding the likely improvement in
laboratory performance over time. The average of interlaboratory CVs
reported (in the WET method manuals and/or the Technical Support
Document for Water Quality-based Toxics Control (USEPA, 1991b)) for
each method at the time of promulgation ranged from 34% to 44.2% (Table
3). Interlaboratory CVs reported for these methods in the WET
Variability Study ranged from 10.5% to 38.5%. For each method,
interlaboratory variability measured in the WET Variability Study was
lower than that cited at the time of promulgation (Table 3).
Interlaboratory CVs measured in the WET Variability Study were 4% to
34% lower than average values cited in the method manuals for the same
methods. On average, interlaboratory variability measured in the WET
Variability Study was 15% lower than originally reported at the time of
method promulgation. These results strongly confirm EPA's conclusions
that these methods provide sufficient precision for use in NPDES
permits.
Table 3.--Comparison of Interlaboratory Method Precision at the Time of Method Promulgation and Measured in
EPA's WET Variability Study
----------------------------------------------------------------------------------------------------------------
Interlaboratory
precision Updated
estimates (%CV) interlaboratory
Method at time of precision Improved precision?
method estimates (%CV)
promulgation \b\
----------------------------------------------------\a\---------------------------------------------------------
Ceriodaphnia dubia Acute Test............... 44.2 29.0 Yes
Ceriodaphnia dubia Survival and Reproduction 42 35.0 Yes
Test.
Fathead Minnow Acute Test................... 35 20.0 Yes
Fathead Minnow Larval Survival and Growth 34 20.9 Yes
Test.
Selenastrum capricornutum Growth Test....... \c\ NR \d\ 34.3 NA \e\
Mysidopsis bahia Survival, Growth, and \c\ NR 41.3 NA \e\
Fecundity Test.
Sheepshead Minnow Acute Test................ 42 26.0 Yes
Sheepshead Minnow Larval Survival and Growth 44.2 10.5 Yes
Test.
Inland Silverside Acute Test................ 42.2 38.5 Yes
Inland Silverside Larval Survival and Growth \c\ NR 43.8 NA \e\
Test.
Champia parvula Reproduction Test........... \c\ NR \c\ NR NA \f\
Holmesimysis costata Acute Test............. \c\ NR \c\ NR NA \f\
----------------------------------------------------------------------------------------------------------------
\a\ Precision estimates represent an average of all interlaboratory CVs reported for a given method in the WET
method manuals (USEPA, 1993b; USEPA, 1994a; USEPA, 1994b) and/or the Technical Support Document for Water
Quality-based Toxics Control (USEPA, 1991b). The number of significant figures displayed differs because these
data are obtained from various sources, which reported results to either two or three significant figures.
\b\ Precision estimates were obtained from EPA's WET Variability Study conducted in 1999-2000 (USEPA, 2001a).
\c\ NR = None reported.
\d\ Precision estimates for the Selenastrum capricornutum Growth Test method are based on conduct of the test
with Ethylenediaminetetraacetic acid (EDTA) as a component of the nutrients added to test and control
treatments.
\e\ NA = not applicable. Improved precision could not be determined because estimates of interlaboratory
precision were not reported at the time of method promulgation.
\f\ NA = not applicable. Improved precision could not be determined because estimates of interlaboratory
precision were not reported at the time of method promulgation or determined in the WET Variability Study.
[[Page 49806]]
Other test performance characteristics measured in the WET
Variability Study also confirmed EPA's conclusions that these methods
are applicable for use in NDPES permits. False positive rates for these
methods were below the theoretical false positive rate of 5% (based on
the recommended 0.05 alpha level for hypothesis testing), indicating
that the methods do not routinely indicate toxicity in non-toxic
samples. Successful test completion rates for these methods were also
at acceptable levels (82.0% to 100%), with 6 of these 7 methods
exhibiting successful test completion rates above 90%. While the 82.0%
successful test completion rate for the Ceriodaphnia dubia Survival and
Reproduction Test method was lower than for most other methods
evaluated in the WET Variability Study, this rate is consistent with
successful test completion rate information available for this method
at the time of promulgation. The 82.0% successful test completion rate
observed in the WET Variability Study is consistent with the 80% rate
reported for this method in a 1989 interlaboratory study (USEPA, 1991b)
and represents tremendous improvement from a 1987 interlaboratory study
that reported a successful test completion rate of 56% (DeGraeve et
al., 1992).
The overall successful test completion rate observed for the
Ceriodaphnia dubia Survival and Reproduction Test method in the WET
Variability Study was also suppressed by poor performance in a subset
of laboratories. Only 10 of the 34 participant laboratories performed
invalid tests, but 8 of these laboratories performed invalid tests on
50% or more of the samples tested. The low rate of successful test
completion in these 8 laboratories may have been influenced by the
study's strict testing schedule, which required each test to be
conducted on a given day and all tests to be conducted within a 15-day
time period. When invalid tests conducted in a given laboratory were
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 this study because culture health was unlikely to fully recover
within 15 days. EPA believes that successful test completion rates for
this method improve when testing laboratories are allowed flexibility
in the timing of sample collection and can avoid initiating tests
during periods of marginal to poor culture health.
3. Inland Silverside Larval Survival and Growth Test
EPA proposes to ratify the Inland Silverside Larval Survival and
Growth Test method. Similarly to the methods listed in section 2 above,
the Inland Silverside Larval Survival and Growth Test method exhibited
acceptable successful test completion rates and false positive rates
(Table 2). No false positives were observed for the method in the WET
Variability Study, and the successful test completion rate was 100%.
Unlike the methods listed in section 2 above, however, EPA cannot
compare interlaboratory precision data cited at the time of method
promulgation and data reported from the WET Variability Study because
EPA did not rely on interlaboratory precision data for this method at
the time of promulgation (Table 3). Instead, EPA relied on
intralaboratory data for the method. The Agency's previous experience
with method variability evaluations supported EPA's assumption that,
though WET tests typically have lower CVs (higher precision) in
intralaboratory studies than in interlaboratory studies, acceptable
ranges of precision demonstrated in intralaboratory studies tend to
subsequently be confirmed by interlaboratory studies.
In the WET Variability Study, an interlaboratory CV of 43.8% was
reported for the Inland Silverside Larval Survival and Growth Test
method. While interlaboratory variability for this method is higher
than for other methods reported in the study, it is within the range of
interlaboratory CVs (34% to 44.2%) cited for other WET methods at the
time of promulgation (Table 3). It is also within the range of
interlaboratory CVs reported for chemical methods approved at 40 CFR
part 136 (USEPA, 1991b). Therefore, EPA reaffirms the conclusions that
this method is no more variable than chemical analytical methods
approved at 40 CFR part 136 and that this method is applicable for use
in NPDES permits (60 FR 53529; October 16, 1995).
4. Champia parvula Reproduction Test
In the WET Variability Study, insufficient participant laboratory
support was available to conduct interlaboratory testing of the Champia
parvula Reproduction Test method within the time frame of the study. In
addition to the referee laboratory, only one laboratory submitted the
necessary quality control information to prequalify for participation
in the interlaboratory study of this method. Due to insufficient
laboratory support and failure to meet the study's data quality
objective of a minimum of six laboratories, EPA canceled
interlaboratory testing of the Champia parvula Reproduction Test
method. Though interlaboratory testing was canceled, the referee
laboratory conducted single-laboratory testing of the Champia parvula
Reproduction Test method. In the 1995 WET rule, EPA addressed the issue
of limited laboratory availability for conduct of the Champia parvula
Reproduction Test method. EPA 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 this test. However, the
number of permits requiring the Champia parvula Reproduction Test
method has remained low (DeGraeve et al., 1998), so few laboratories
have invested in developing Champia parvula cultures or standard
operating procedures for conduct of the method.
EPA believes that the limited use of the Champia parvula
Reproduction Test method does not reduce the value of the test method.
The Champia parvula Reproduction Test represents the only approved test
method for a marine plant species. Maintaining an approved test method
for this functional group (marine/plant/chronic test) is important for
proper implementation of the WET program. The Technical Support
Document for Water Quality-Based Toxics Control (USEPA, 1991b)
recommends the use of at least three marine species representing three
different phyla (e.g., a fish, an invertebrate, and a plant) for
testing the toxicity of effluents discharged to estuarine and marine
environments.
The limited use of the Champia parvula Reproduction Test method
also does not affect the performance of the test method in laboratories
that are qualified to conduct the test. While the WET Variability Study
did not provide interlaboratory precision data for the Champia parvula
Reproduction Test method, referee laboratory data confirmed the
estimates of intralaboratory precision cited at the time of method
promulgation (USEPA, 1994b). Intralaboratory CVs cited in the method
manual for Champia parvula Reproduction Tests conducted using copper
sulfate and sodium dodecyl sulfate averaged 63%. In preliminary testing
for the WET Variability Study, the referee laboratory achieved an
intralaboratory CV of 27.6% for 3 reference toxicant tests using copper
sulfate, and an intralaboratory CV of 49.7% for 4 tests of spiked
receiving water. Only one pair of replicate
[[Page 49807]]
effluent samples was tested using the Champia parvula Reproduction Test
method. Tests of these duplicate effluent samples yielded a CV of
50.0%. All other testing of the effluent sample type was conducted on
samples from different sampling dates, so additional precision
measurements were not obtained for this sample type. In addition to
intralaboratory test data from the WET Variability Study, EPA's
Variability Guidance Document (USEPA, 2000d) reported an
intralaboratory CV of 59% for the Champia parvula Reproduction Test
based on 23 reference toxicant tests conducted in 2 laboratories.
Intralaboratory data from both the WET Variability Study and the
Variability Guidance Document support the intralaboratory precision
data previously cited in the method manual (USEPA, 1994b) for the
Champia parvula Reproduction Test method. Based on the confirmation of
intralaboratory precision data cited at the time of method
promulgation, EPA proposes to ratify the Champia parvula Reproduction
Test method.
5. Mysidopsis bahia Survival, Growth, and Fecundity Test
The Mysidopsis bahia Survival, Growth, and Fecundity Test uses
three test endpoints to evaluate toxicity: survival, growth, and
fecundity (or reproduction). The survival and growth endpoints are
required endpoints and specific test acceptability criteria for these
endpoints must be met (80% survival and mean weight of 0.20 mg in the
control treatment) to produce a valid test. The fecundity endpoint is
optional and may be used if the test acceptability criterion for
fecundity (egg production by 50% or more of control females) is met.
Failure to meet the test acceptability criterion for fecundity does not
invalidate a test but means that the fecundity endpoint may not be used
in calculating test results. In the WET Variability Study, 97.7% of
tests met the required test acceptability criteria for survival and
growth, but only 50% of tests met the test acceptability criterion for
fecundity. While failure to generate fecundity data does not invalidate
a test, it may affect the sensitivity of the measurement. Researchers
have shown that the fecundity endpoint is often the most sensitive
endpoint and that the test most effectively estimates the chronic
toxicity of effluents when all three endpoints are used (Lussier et
al., 1999).
EPA proposes to ratify the Mysidopsis bahia Survival, Growth, and
Fecundity Test method with an additional modification to improve the
performance of the method. EPA proposes to add guidance to improve the
success of obtaining fecundity data. The specifics of the proposed
method manual changes to implement this modification are detailed in
the document titled, Proposed Changes to Whole Effluent Toxicity Method
Manuals (USEPA, 2001d). The additional guidance would recommend
optimizing temperature, feeding, and organism densities during the
seven-day pre-test holding period and during the testing period. These
factors are critical to the success of the fecundity endpoint, because
they control the rate of mysid development and maturation. While these
factors are typically controlled during the testing period, equal
attention should be paid to these factors during the pre-test holding
period to ensure maximum mysid development. Lussier et al. (1999) found
that by increasing holding temperature and test temperature from
26 deg.C 1 deg.C to 26 deg.C-27 deg.C and maintaining
holding densities at 10 organisms/L, the percentage of tests
meeting the test acceptability criteria for fecundity increased from
60% to 97%.
With the exception of the low successful test completion rate for
the fecundity endpoint, other test method performance measures
evaluated in the WET Variability Study for the Mysidopsis bahia
Survival, Growth, and Fecundity Test were acceptable. No false
positives were observed for the method, the successful test completion
rate was 97.7% for the survival and growth endpoints, and
interlaboratory variability (%CV) was 41.3% for the growth IC25
endpoint (Table 2). No interlaboratory precision data were reported for
the Mysidopsis bahia Survival, Growth, and Fecundity Test method at the
time of method promulgation; therefore interlaboratory precision data
from the WET Variability Study could not be compared to previously
cited values for this method (Table 3). While interlaboratory
variability for this method is higher than for most other methods
reported in the study, it is within the range of interlaboratory CVs
(34% to 44.2%) cited for other WET methods at the time of promulgation
(Table 3). It is also within the range of interlaboratory CVs reported
for chemical methods approved at 40 CFR part 136 (USEPA, 1991b).
Therefore, EPA reaffirms the conclusions that this method is applicable
for use in NPDES permits (60 FR 53529; October 16, 1995).
6. Selenastrum capricornutum Growth Test
In the WET Variability Study, the Selenastrum capricornutum Growth
Test method was conducted with and without the addition of
ethylenediaminetetraacetic acid (EDTA). In the approved Selenastrum
capricornutum Growth Test method, EDTA is an optional component of the
nutrient mixture that is added to test and control treatments. While
algal growth is enhanced by the addition of EDTA, the method recommends
excluding EDTA from the nutrient mixture when testing samples that may
contain metals. EDTA is a chelating agent that effectively binds
metals, thereby potentially reducing the toxic effect of metals present
in the analyzed sample. Because the presence of metals in WET samples
is often unknown at the time of testing, laboratories often conduct the
Selenastrum capricornutum Growth Test method without the addition of
EDTA.
Results from the WET Variability Study revealed that Selenastrum
capricornutum Growth Test method performance was substantially better
when EDTA was added to the nutrient mixture than when it was excluded.
No false positives were observed when EDTA was used, but 2 of the 6
blank samples (33.3%) analyzed without EDTA produced false positive
results (USEPA, 2001a). Interlaboratory variability of the Selenastrum
capricornutum Growth Test method was also much lower with EDTA (34.3%)
than without EDTA (58.5%). When conducted with EDTA, the Selenastrum
capricornutum Growth Test method exhibited interlaboratory precision
similar to other chronic methods evaluated in the WET Variability
Study. No interlaboratory precision data were reported for the
Selenastrum capricornutum Growth Test method at the time of method
promulgation, so interlaboratory precision data from the WET
Variability Study could not be compared to previously cited values for
this method. When compared to interlaboratory precision cited for other
WET test methods at the time of promulgation, the Selenastrum
capricornutum Growth Test method (conducted with EDTA) was well within
the range (Table 3).
The successful test completion rate of the Selenastrum
capricornutum Growth Test method was low for tests conducted with and
without EDTA (63.6% and 65.9%, respectively), however, the low
successful test completion rates were in part due to laboratory
inexperience in using both the with and without-EDTA techniques. Two
laboratories that cultured organisms without EDTA and generally
conducted tests without EDTA showed poor successful test completion
rates
[[Page 49808]]
(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) without EDTA. When these two laboratories were removed from the
analysis, the successful test completion rate for tests conducted with
EDTA increased to 77.8%.
Based on WET Variability Study results, EPA proposes to ratify the
Selenastrum capricornutum Growth Test method with a modification to
recommend the addition of EDTA to the nutrient mixture added to control
and test treatments. The specifics of the proposed method manual
changes to implement this modification are detailed in the document
titled, Proposed Changes to Whole Effluent Toxicity Method Manuals
(USEPA, 2001d). This method modification will improve overall test
method performance by reducing false positives and increasing
interlaboratory precision. EPA also believes that recommending the use
of EDTA will improve successful test completion rates for the method as
laboratories consistently culture and test with EDTA. In addition to
improving test method performance, the method modification to recommend
the use of EDTA is consistent with other established Selenastrum
capricornutum toxicity testing protocols. Both ASTM (1992) and
Environment Canada (1992) methods for toxicity testing using
Selenastrum capricornutum recommend the use of EDTA.
EPA recognizes that the proposed modification to the Selenastrum
capricornutum Growth Test method may cause the method to underestimate
the toxicity of metals. EPA believes, however, that this modification
is necessary to ensure adequate performance of the Selenastrum
capricornutum Growth Test method. EPA also believes that under
appropriate implementation of the WET program, this modification will
not significantly reduce environmental protection. The Technical
Support Document for Water Quality-based Toxics Control (USEPA, 1991b)
recommends that permitting decisions be based on testing using a
minimum of three species representing three different phyla (e.g., a
fish, invertebrate, and plant). This recommendation is based on the
recognition that species differ in their sensitivity to toxicants. By
using a battery of species to test the toxicity of an effluent,
permitting decisions can be made to protect the most sensitive species
tested. Using this approach, the addition of EDTA in the Selenastrum
capricornutum Growth Test method would affect environmental protection
only when Selenastrum capricornutum is determined to be the most
sensitive species and when the effluent contains metals whose toxicity
is reduced by the addition of EDTA. This situation should be
infrequent, and result in only minor decreases in test sensitivity.
Geis et al. (2000b) showed that Ceriodaphnia dubia was more sensitive
than Selenastrum capricornutum to three of five metals tested (copper,
nickel, and cadmium), and Selenastrum capricornutum was only slightly
more sensitive than Ceriodaphnia dubia to zinc and lead.
7. Holmesimysis costata Acute Test
Holmesimysis costata is a Pacific coast mysid species that was
elevated from the supplemental species list in the previous acute
method manual and added to the list of approved acute toxicity test
species at the time of the WET final rule (60 FR 53529; October 16,
1995). This species was added in response to comments that the
recommended test species in the acute method manual did not include any
invertebrate species indigenous to Pacific coastal waters. One
commenter also submitted data showing that Holmesimysis costata was at
least as sensitive to toxicants as the recommended acute toxicity test
species. Based on these comments, the acute method manual was modified
to add a footnote listing Holmesimysis costata as an acceptable species
for use with the Mysidopsis bahia Acute Test procedures. The footnote
to the table of test conditions for the Mysidopsis bahia Acute Test
states that ``Holmesimysis costata can be used with the test conditions
in this table, except at a temperature of 12 deg.C, instead of 20 deg.C
or 25 deg.C, and a salinity of 32-34, instead of
5-30, where it is the required test organism in
discharge permits.'' Because the acute method manual was incorporated
by reference in the final rule, the incorporation of this footnote
established Holmesimysis costata as an approved acute toxicity test
species. The WET final rule (60 FR 53529; October 16, 1995) clarified
this by stating that ``EPA accepts the use of * * * Holmesimysis
costata in place of Mysidopsis bahia, with the same test conditions
(except at a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C,
and a salinity of 32-34, instead of 5-
30).''
EPA decided to evaluate the Holmesimysis costata Acute Test method
in the WET Variability Study according to the protocol as the method
was promulgated, i.e., using the test conditions for Mysidopsis bahia
(except at a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C,
and a salinity of 32 to 34, instead of 5
to 30). Sufficient participant laboratory support, however,
was not available to conduct interlaboratory testing of the
Holmesimysis costata Acute Test method within the time frame of the
study. In addition to the referee laboratory, only two laboratories
submitted the necessary quality control information to prequalify for
participation in the interlaboratory study of this method. This method
is required only in NPDES permits issued in California, so few
laboratories currently conduct this test routinely. Due to insufficient
laboratory support and failure to meet the study's data quality
objective of a minimum of six laboratories, EPA canceled
interlaboratory testing of the Holmesimysis costata Acute Test method.
Though interlaboratory testing was canceled, the referee laboratory did
attempt to conduct single-laboratory testing of the Holmesimysis
costata Acute Test.
During the WET Variability Study, the referee laboratory initiated
five Holmesimysis costata acute tests. The referee laboratory did not
initiate additional tests due to difficulties in obtaining test
organisms. Juvenile Holmesimysis costata used for testing are generally
obtained from field-collected gravid females. The referee laboratory
was unable to collect sufficient numbers of gravid females during most
of the time frame for the WET Variability Study (September 1999 through
April 2000). Of the five tests that were initiated, none successfully
met test acceptability criteria and required test conditions. Three
tests failed to meet test acceptability criteria for control survival,
and two tests failed to meet requirements for the age of test organisms
(all within 24 hours). These test failures demonstrated the inadequacy
of Mysidopsis bahia Acute Test procedures for use in conducting acute
tests with Holmesimysis costata. EPA has since concluded that modified
test procedures are needed for successful conduct of the Holmesimysis
costata Acute Test. These modifications include more detailed organism
collection and holding procedures, specific dilution water
requirements, revised temperature requirements, and less restrictive
test organism age requirements.
Today, EPA proposes to withdraw Holmesimysis costata as an
acceptable species for use in the Mysidopsis bahia Acute Test method
and proposes a separate Holmesimysis costata Acute Test method. This
proposal would add
[[Page 49809]]
to the acute method manual a table of test conditions specific to
Holmesimysis costata and information in Appendix A.3 on the morphology,
taxonomy, collection, holding, culturing, feeding, and testing of
Holmesimysis costata. The specifics of the proposed Holmesimysis
costata Acute Test method and the method manual changes necessary to
implement the addition of this method are detailed in the document
titled, Proposed Changes to Whole Effluent Toxicity Method Manuals
(USEPA, 2001d).
The proposed Holmesimysis costata Acute Test method is based on
method development data from the California Water Resources Control
Board's Marine Bioassay Project (State Water Resources Control Board,
1990) and from peer-reviewed literature (Martin et al., 1989; Hunt et
al., 1997). These data show that given the appropriate test procedures
and test conditions, the Holmesimysis costata Acute Test can produce
reliable and sensitive toxicity results with adequate precision.
Single-laboratory testing of zinc with the Holmesimysis costata Acute
Test method yielded intralaboratory precision (CVs) of 19% and 23% in
48-h and 96-h acute tests, respectively. Multi-laboratory testing of
zinc with the Holmesimysis costata Acute Test method yielded
interlaboratory precision (CVs) of 24% and 1% in 2 separate trials.
In addition to the proposed Holmesimysis costata Acute Test method,
EPA requests comment on the applicability of similar methods published
by voluntary consensus standard bodies. A mysid toxicity test method
with specific test procedures for Holmesimysis costata is published in
Standard Methods for the Examination of Water and Wastewater (APHA et
al., 1998), and a West Coast mysid toxicity test method is published by
the American Society for Testing and Materials (ASTM, 1993). EPA does
not believe that these methods from voluntary consensus standard bodies
provide the detailed requirements necessary for routine use in
compliance monitoring, so EPA is proposing a new Holmesimysis costata
Acute Test method for inclusion in EPA's acute method manual (USEPA,
1993b). EPA invites comment, however, on whether to approve the other
organizations' testing procedures, including comment on their use for
compliance monitoring.
IV. Regulatory Requirements
A. Executive Order 12866--Regulatory Planning and Review
Under Executive Order 12866 (58 FR 51735 (October 4, 1993)), the
Agency must determine whether a 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.''
Pursuant to the terms of Executive Order 12866, it has been
determined that this rule is not a ``significant regulatory action.''
Therefore, this action is not subject to OMB review.
B. 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, local, and tribal
governments and the private sector. Under section 202 of the UMRA, EPA
generally must prepare a written statement, including a cost-benefit
analysis, for proposed and final rules with ``Federal mandates'' that
may result in expenditures to State, local, and tribal governments, in
the aggregate, or to the private sector, of $100 million or more in any
one year. Before promulgating an EPA rule for which a written statement
is needed, section 205 of the UMRA generally requires EPA to identify
and consider a reasonable number of regulatory alternatives and adopt
the least costly, most cost-effective or least burdensome alternative
that achieves the objectives of the rule. The provisions of section 205
do not apply when they are inconsistent with applicable law. Moreover,
section 205 allows EPA to adopt an alternative other than the least
costly, most cost-effective or least burdensome alternative if the
Administrator publishes with the final rule an explanation of why that
alternative was not adopted. Before EPA establishes any regulatory
requirements that may significantly or uniquely affect small
governments, including tribal governments, it must have developed,
under section 203 of the UMRA, a small government agency plan. The plan
must provide for the notification of potentially affected small
governments, enabling officials of affected small governments to have
meaningful and timely input in the development of EPA regulatory
proposals with significant Federal intergovernmental mandates, and
informing, educating, and advising small governments on compliance with
the regulatory requirements.
EPA has determined that today's proposed rule does not contain a
Federal mandate that may result in expenditures of $100 million or more
for State, local, and tribal governments, in the aggregate, or the
private sector in any one year. Today's rule proposes 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 the requirements of sections 202 and 205 of the UMRA.
EPA has determined that this rule contains no regulatory
requirements that might significantly or uniquely affect small
governments. It would not significantly affect them because any
incremental costs incurred are minimal, and it would not uniquely
affect them because it would affect entities of all sizes required to
test for whole effluent toxicity by a regulatory authority the same.
Further, whole effluent toxicity monitoring by small entities is
generally expected to be less frequent than such monitoring by larger
entities. Therefore, today's rule is not subject to the requirements of
section 203 of UMRA.
C. Regulatory Flexibility Act (RFA), as Amended by the Small Business
Regulatory Enforcement Fairness Act of 1996 (SBREFA), 5 U.S.C. 601 et
seq.
The 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 stature 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
[[Page 49810]]
than 50,000; and (3) a small organization that is any not-for-profit
enterprise which is independently owned and operated and is not
dominant in its field.
After considering the economic impacts of today's proposed rule on
small entities, I certify that this action will not have a significant
economic impact on a substantial number of small entities. Today's rule
proposes 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.
The proposed revisions are intended to improve the performance of WET
tests, and thus increase confidence in the reliability of the results
obtained using the test methods. EPA estimates that any incremental
costs associated with the proposed revisions would be alleviated by a
potential reduction in retesting that may result from improved test
performance and increased confidence in the reliability of testing
results. We continue to be interested in the potential impacts of the
proposed rule on small entities and welcome comments on issues related
to such impacts.
D. 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.
It does not contain any information, collection, reporting, or record
keeping requirements.
Burden means the total time, effort, or financial resources
expended by persons to generate, maintain, retain, or disclose or
provide information to or for a Federal agency. This includes the time
needed to review instructions; develop, acquire, install, and utilize
technology and systems for the 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.
E. 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., materials specifications, test methods,
sampling procedures, and business practices) that are developed or
adopted by voluntary consensus standards bodies. The NTTAA directs EPA
to provide Congress, through OMB, explanations when the Agency decides
not to use available and applicable voluntary consensus standards.
Today's action would revise existing EPA WET test methods and add a
new Holmesimysis costata Acute Test method. For the methods that EPA is
proposing to revise, the Agency did not conduct a search to identify
potentially applicable voluntary consensus standards, because the
revisions EPA proposes today would merely incorporate more specificity
and detail into already approved EPA test methods. EPA invites comment,
however, on the extent to which voluntary consensus standard
organizations' methods would be consistent with the EPA methods for
which revisions are proposed today. For the new Holmesimysis costata
Acute Test method, the Agency reviewed applicable voluntary consensus
standards and identified two mysid methods (ASTM, 1993; APHA et al.,
1998) that provide specific test procedures for use with Holmesimysis
costata. While EPA requests comment on the applicability of these
voluntary consensus standards, the Agency does not believe that these
methods would provide the additional detailed requirements EPA proposes
today. For this reason, EPA proposes a new EPA Holmesimysis costata
Acute Test method. EPA welcomes comments on this aspect of the proposed
rulemaking and, specifically, invites the public to identify
potentially-applicable voluntary consensus standards and to explain why
such standards should be used in this regulation.
F. Executive Order 13045--Protection of Children From Environmental
Health Risks 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 not 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.
G. 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 6, 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 proposed rule does not have tribal implications. It will not
have substantial direct effects on tribal governments, on the
relationship between the Federal government and Indian tribes, or on
the distribution of power and responsibilities between the Federal
government and Indian tribes, as defined in Executive Order 13175.
Today's proposed rule would revise 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. In
the spirit of Executive Order 13175, and consistent with EPA policy to
promote communications between EPA and tribal governments, EPA
specifically solicits comment on this proposed rule from tribal
officials.
H. Executive Order 13132--Federalism
Executive Order 13132, entitled ``Federalism'' (64 FR 43255; August
10, 1999), requires EPA to develop an
[[Page 49811]]
accountable process to ensure ``meaningful and timely input by State
and local officials in the development of regulatory policies that have
federalism implications.'' ``Policies that have federalism
implications'' is defined in the Executive Order to include regulations
that have ``substantial direct effects on the States, on the
relationship between the national government and the States, or on the
distribution of power and responsibilities among the various levels of
government.''
This proposed rule does not have federalism implications. It will
not have substantial direct effects on the States, on the relationship
between the national government and the States, or on the distribution
of power and responsibilities among the various levels of government,
as specified in Executive Order 13132. Today's rule proposes 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. In the sprit of
Executive Order 13132, and consistent with EPA policy to promote
communications between EPA and State and local governments, EPA
specifically solicits comment on this proposed rule from State and
local officials.
I. Executive Order 13211--Energy Effects
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.
J. Plain Language Directive
Executive Order 12866 requires each agency to write all rules in
plain language. We invite your comments on how to make this proposed
rule easier to understand. For example, have we organized the material
to suit your needs? Are the requirements in the rule clearly stated?
Does the rule contain technical language or jargon that isn't clear?
Would a different format (grouping and order of sections, use of
headings, paragraphing) make the rule easier to understand? Would more
(but shorter) sections be better? Could we improve clarity by adding
tables, lists, or diagrams? What else could we do to make the rule
easier to understand?
V. Request for Comments and Available Data
EPA requests public comments on this proposed rule. EPA invites
comment on the technical merit, applicability, and implementation of
the specific WET test method changes included in this proposal. EPA
also invites comments on the ratification of the methods listed. EPA
encourages commenters to provide copies of supporting data and/or
references cited in comments.
EPA recognizes that stakeholders continue to have concerns over a
variety of issues related to implementation of whole effluent toxicity
controls through NPDES permits. Today's notice, however, invites
comments only on the conduct of WET test methods and not on the
implementation of WET control strategies through NPDES permits. EPA is
interested in comments on the extent to which some aspect(s) of the
technical components of the method revisions proposed today may affect
implementation of WET control strategies. For example, today's notice
solicits comments related to the proposed application of percent
minimum significant difference (PMSD) approaches to evaluate the
precision of WET test results (see Section B below). Application of the
PMSD approach is intended to control the within-test variability in WET
methods. Nationwide, however, NPDES agencies have implemented other
concepts, such as limits on CVs to control for within-test variability
rather than the PMSD concepts about which EPA solicits comment today.
It is not EPA's objective to create conflict with the current
implementation of WET control strategies that do not presently apply
the PMSD concepts, but instead to enhance ongoing implementation
efforts by providing an additional review step for WET test results to
promote WET test precision. To the extent that application of the PMSD
concepts could result in conflicts with the current and ongoing WET
implementation, EPA invites comments on how to ameliorate any such
adverse effects on WET implementation.
A. pH Drift
In particular, EPA requests comments and available data to support
or refute test method changes related to pH drift (see Section
III.B.3.b). EPA requests 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 specifically requests
chronic toxicity data from parallel controlled-pH and uncontrolled-pH
tests on well-treated municipal or industrial effluents. Such data
should include raw toxicity test data sheets, ammonia measurements on
tested samples, and daily initial and final pH measurements on each
test treatment. EPA also requests data from multiple tests conducted on
a given effluent over time to demonstrate a trend of artifactual
toxicity due to pH drift in that effluent. Data sets should include
full strength effluent, as well as a range of effluent concentrations
and a dilution water control. Electronic as well as hard copy formats
of raw test data and statistical analysis are encouraged. Though EPA
continues to search for and may yet develop data supporting the need
for procedures to control pH drift in the absence of ammonia or other
pH-dependent toxicants, if sufficient data are not available at the
time of final action on today's proposal, EPA may not incorporate
changes to the methods beyond the 1996 guidance in the final rule.
B. Percent Minimum Significant Difference
The percent minimum significant difference (PMSD) is a measure of
within-test variability and test sensitivity. The PMSD for a given WET
test can be defined as the smallest percentage difference between the
control and a treatment (an effluent dilution) that could be declared
as statistically significant. As test variability increases, the
ability of a test to detect small toxic effects diminishes and the test
becomes a less sensitive measure of toxicity. Appendix C of the WET
method manuals (USEPA, 1994a; USEPA, 1994b) describes the calculation
of the minimum significant difference (MSD). The PMSD is simply the MSD
expressed as a percentage of the control response (i.e., PMSD = MSD/
control mean * 100).
In June 2000, EPA published guidance on WET test variability that
recommended placing upper and lower bounds on the PMSD to control
variability and ensure a specified range of test sensitivity (USEPA,
2000d). This guidance derived lower and upper bounds as the 10th and
90th percentiles, respectively, of PMSDs from a large number of
reference toxicant tests. Based on this guidance, tests for which the
PMSD exceeds an upper bound would be conducted again (with a newly
collected sample), if the test leads to a decision that there is no
significant toxicity at the concentration identified in the permit as a
limit (``Instream Waste Concentration'' (IWC) or ``Receiving Water
Concentration''). This
[[Page 49812]]
guidance also applies lower PMSD bounds for the purpose of determining
the no observed effect concentration (NOEC). The purpose of the lower
PMSD bound is to avoid declaring as ``significant'' toxic effects that
are smaller than those that can generally and routinely be detected by
the method as currently conducted by qualified laboratories.
Application of a lower bound does not imply that EPA has knowledge
that, or considers that, percent differences smaller than the lower
bound represent non-toxic effects. The lower bound PMSD is used here
not as a threshold for toxicity but as a measure of method precision.
Today, EPA seeks comment on proposing to require the application of
the upper and lower PMSD bounds for sublethal endpoints in the (1)
Ceriodaphnia dubia Survival and Reproduction Test; (2) Fathead Minnow
Larval Survival and Growth Test; (3) Mysidopsis bahia Survival, Growth,
and Fecundity Test; and (4) Inland Silverside Larval Survival and
Growth Test. The proposed requirement would apply to the determination
of the NOEC and LOEC (lowest observed effect concentration) for
sublethal endpoints in multi-concentration tests. In the proposed
application, the upper and lower PMSD bounds would be used to determine
when a treatment differs significantly from the control treatment. 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. 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. The specifics of
method manual changes proposed to institute the required application of
PMSD bounds are detailed in the document titled, Proposed Changes to
Whole Effluent Toxicity Method Manuals (USEPA, 2001d). The PMSD
procedures about which EPA invites comment today would not preclude
application of the current recommended guidance (USEPA 2000d) on PMSD
bounds because today's proposed procedures are less restrictive than
the guidance recommendation. EPA will consider using additional sources
of data for developing lower and upper bounds for PMSD, including, but
not limited to, data from EPA's WET Variability Study (USEPA, 2001a).
EPA considered the appropriateness of requiring PMSD bounds for the
growth endpoints of the Sheepshead Minnow Larval Survival and Growth
Test and the Selenastrum capricornutum Growth Test. At this time, EPA
does not believe that requiring PMSD bounds for these test methods
would be appropriate because: (a) These methods appear to achieve
smaller PMSDs than the other chronic methods (USEPA 2000d), and (b) the
PMSD bounds for these methods (USEPA 2000d) would be based upon fewer
laboratories and tests (albeit a substantial number) than the PMSD
bounds for the methods for which EPA invites comment today. EPA also
considered the appropriateness of PMSD bounds for the survival
endpoints of test methods for chronic toxicity, and test methods for
acute toxicity. At this time, EPA does not believe that imposing PMSD
bounds for the survival endpoints would be necessary because precision
for survival endpoints appears to be, in most cases, better than
precision for sublethal endpoints (USEPA 2000d). EPA seeks comment on
the appropriateness of imposing PMSD bounds for four test methods and
for sublethal endpoints.
EPA considered other measures of test precision, including the
standard deviations and coefficients of variation for treatments and
control, MSD, and the mean square for error from the analysis of
variance of treatment effects (USEPA 1994a, 1994b). EPA considers the
PMSD to be the measure that would be most easily understood and that
could be 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 for selected treatments, represents only a
portion of the variability affecting the NOEC, LOEC, and point
estimates. Some State or Regional WET programs have requirements on the
CV for the control and the treatment representing the IWC
concentration. Such requirements can provide finer control over the
variability influencing a comparison, especially a direct comparison
between the control and the IWC treatment. The PMSD upper bound
provides control over the average variability and would be used here
specifically for multi-concentration tests in which the NOEC or LOEC
are determined by using the MSD. EPA seeks comment on (1) the need for
increased within-test precision, (2) the merits and drawbacks of
applying PMSD bounds as described above, and (3) additional or
alternative applications of PMSD bounds to control test precision.
Alternative applications of PMSD bounds could include quality control
requirements for laboratories to track PMSD values over time (e.g.,
control charts for PMSD performance in reference toxicant and/or
effluent tests); a requirement to demonstrate recent, ongoing precision
(PMSD less than an upper bound) in multiple tests before starting an
effluent test; and/or use of PMSD bounds as a component of test review.
EPA also requests that commenters submit data (hard copy and electronic
format) to support their comments or recommendations regarding the
application of PMSDs.
C. Other Method Modifications
In addition to the method modifications proposed today, EPA seeks
comment and recommendations on other method modifications that would
improve the performance of the WET test methods. Specifically, EPA
requests comment and recommendations on (1) increasing the test
acceptability criteria for mean control reproduction (number of young
per surviving female) in the Ceriodaphnia dubia Survival and
Reproduction Test; (2) increasing the test acceptability criteria for
mean control weight (mean weight per original) in the Fathead Minnow
Larval Survival and Growth Test; (3) increasing the number of replicate
chambers per concentration from a minimum of three to a minimum of four
in the Fathead Minnow Larval Survival and Growth Test Method,
Sheepshead Minnow Larval Survival and Growth Test Method, the Inland
Silverside Larval Survival and Growth Test Method, and the Sea Urchin
Fertilization Test Method; and (4) increasing the minimum number of
replicates in the Ceriodaphnia dubia Survival and Reproduction Test
Method. Modifications to the minimum number of replicates would be made
to improve the precision of the test methods. EPA intends to evaluate
these and other options for improving WET test method performance using
existing data (from the WET Variability Study and the Variability
Guidance Document) and data submitted to EPA in response to this
request. EPA requests comments and recommendations on any additional
quality control measures that would increase test precision or the
overall quality of data generated. Comments should be supported by data
(hard copy and electronic format) and other technical information
whenever possible. Comments that contain
[[Page 49813]]
suggestions that are not supported by submitted data will be
considered, but will be given less weight than those supported by data.
EPA also requests that commenters submit information on estimated
increases in testing costs that may be associated with any recommended
method modification.
Lastly, EPA requests comment on the document titled, Study Report
and Recommended Standard Operating Procedure (SOP) for Shipping Large
Volume Samples at Less Than 4 deg.C (USEPA, 2001f), which is included
in the record for this rulemaking (see Addresses section of this rule
for more information on obtaining copies of referenced materials). This
report presents data to support a recommended SOP for meeting sample
temperature requirements (less than 4 deg.C) during shipping of WET
samples.
VI. References
American Public Health Association (APHA), American Water Works
Association (AWWA), and Water Environment Federation (WEF). 1998.
Standard Methods for the Examination of Water and Wastewater, 20th ed.
American Public Health Association, Washington, DC.
American Society for Testing and Materials. 1992. Standard guide for
conducting static 96-h toxicity tests with microalgae. E 1218-90. In
Annual Book of ASTM Standards, Vol. 11.04. American Society for Testing
and Materials, Philadelphia, PA, pp 874-885.
American Society for Testing and Materials. 1993. Standard guide for
conducting static and flow-through acute toxicity tests with mysids
from the West Coast of the United States. E 1463-92. In Annual Book of
ASTM Standards, Vol. 11.04. American Society for Testing and Materials,
Philadelphia, PA, pp 1278-1299.
Belanger, S.E., J.L. Farris, and D.S. Cherry. 1989. Effects of diet,
water hardness, and population source on acute and chronic copper
toxicity to Ceriodaphnia dubia. Arch. Environ. Contam. Toxicol. 18:
601-611.
Belanger, S.E. and D.S. Cherry. 1990. Interaction effects of pH
acclimation, pH, and heavy metals on acute and chronic toxicity to
Ceriodaphnia dubia (Cladocera). J. Crust. Biol. 10(2): 225-235.
Cooney, J.D., G.M. DeGraeve, E.L. Moore, B.J. Lenoble, T.L. Pollock,
and G.J. Smith. 1992. Effects of environmental and experimental design
factors on culturing and toxicity testing of Ceriodaphnia dubia.
Environ. Toxicol. Chem. 11: 839-850.
DeGraeve, G.M., J.D. Cooney, B.H. Marsh, T.L. Pollock, and N.G.
Reichenback. 1992. Variability in the performance of the 7-d
Ceriodaphnia dubia survival and reproduction test: an intra- and
interlaboratory study. Environ. Toxicol. Chem. 11: 851-866.
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.
DeLisle, P.F. and M.H. Roberts. 1988. The effect of salinity on cadmium
toxicity to the estuarine mysid Mysidopsis bahia: role of chemical
speciation. Aquat. Toxicol. 12(4): 357-370.
Downey, P.J., K. Fleming, R. Guinn, N. Chapman, P. Varner, and J.D.
Cooney. 2000. Sporadic mortality in chronic toxicity tests using
Pimephales promelas (Rafinesque): cases of characterization and
control. Environ. Toxicol. Chem. 19(1): 248-255.
Edison Electric Institute et al. v. EPA, Settlement Agreement, July 24,
1998. U.S. Court of Appeals, D.C. Circuit, No. 96-1062.
Emerson, K., R.C. Russo, R.E. Lund, and R.V. Thurston. 1975. Aqueous
ammonia equilibrium calculations; effect of pH and temperature. J. Fish
Res. Bd. Can. 32(12): 2380-2383.
Environment Canada. 1992. Biological test method: growth inhibition
test using the freshwater alga Selenastrum capricornutum. Report EPS 1/
RM/25. Environment Canada, Ottawa, ON.
Geis, S., K. Fleming, A. Mager, and K. Schappe. 2000a. Investigation of
the pathogenic effect in whole effluent toxicity (WET) chronic fathead
minnow tests. SETAC Abstract Book, 21st Annual Meeting, 12-16 November,
2000.
Geis, S.W., K.L. Fleming, E.T. Korthals, G. Searle, L. Reynolds, and
D.A. Karner. 2000b. Modifications to the algal growth inhibition test
for use as a regulatory assay. Environ. Toxicol. Chem. 19(1): 36-41.
Haynes, G.J., A.J. Stewart, and B.C. Harvey. 1989. Gender-dependent
problems in toxicity tests with Ceriodaphnia dubia. Bull. Environ.
Contam. Toxicol. 43(2): 271-279.
Hunt, J.W., B.S. Anderson, S.L. Turpen, A.R. Coulon, M. Martin, and F.
Palmer. 1997. Precision and sensitivity of a seven-day growth and
survival toxicity test using the west coast mysid crustacean,
Holmesimysis costata. Environ. Toxicol. Chem. 16(4): 824-834.
Lone Star Steel v. EPA, Settlement Agreement, March 4, 1998. U.S. Court
of Appeals, D.C. Circuit, No. 96-1157.
Lussier, S.M., A. Kuhn, and R. Comeleo. 1999. An evaluation of the
seven-day toxicity test with Americamysis bahia (formerly Mysidopsis
bahia). Environ. Toxicol. Chem. 18(12): 2888-2893.
Martin, M., J.W. Hunt, and B.S. Anderson. 1989. Experimental evaluation
of the mysid Holmesimysis costata as a test organism for effluent
toxicity testing. Environ. Toxicol. Chem. 8: 001-010.
Moore, T.F., S.P. Canton, and M. Grimes. 2000. Investigating the
Incidence of Type I Errors for Chronic Whole Effluent Toxicity Testing
Using Ceriodaphnia dubia. Environ. Toxicol. Chem. 19: 118-122.
Mount, D.R. and D.I. Mount. 1992. A simple method of pH control for
static and static renewal aquatic toxicity tests. Environ. Toxicol.
Chem. 11: 609-614.
Pennak, R.W. 1989. Fresh-water Invertebrates of the United States:
Protozoa to Mollusca, 3rd ed. John Wiley & Sons, New York.
Schubauer-Berigan, M.K., J.R. Dierkes, P.D. Monson, and G.T. Ankley.
1993. pH-dependent toxicity of Cd, Cu, Ni, Pb, and Zn to Ceriodaphnia
dubia, Pimephales promelas, Hyalella azteca, and Lumbriculus
variegatus. Environ. Toxicol. Chem. 12: 1261-1266.
Society of Environmental Toxicology and Chemistry (SETAC). 1999.
Potential pathogenic interference in short-term chronic WET tests using
fathead minnows. 7pp. http://www.setac.org/wetFAQs.html.
State Water Resources Control Board. 1990. Procedures Manual for
Conducting Toxicity Tests Developed by the Marine Bioassay Project.
Report 90-10WQ. California Environmental Protection Agency, Sacramento,
CA.
U.S. Environmental Protection Agency. 1991a. Methods for Aquatic
Toxicity Identification Evaluations: Phase I Toxicity Characterization
Procedures, 2nd ed. EPA/600/6-91/003. U.S. Environmental Protection
Agency, Office of Research and Development, Environmental Research
Laboratory, Duluth, MN.
U.S. Environmental Protection Agency. 1991b. Technical Support
[[Page 49814]]
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,
D.C.
U.S. Environmental Protection Agency. 1992. Toxicity Identification
Evaluation: Characterization of Chronically Toxic Effluents, Phase I.
EPA/600/6-91/005F. U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Research Laboratory, Duluth,
MN.
U.S. Environmental Protection Agency. 1993a. Methods for Aquatic
Toxicity Identification Evaluations: Phase II Toxicity Identification
Procedures for Acutely and Chronically Toxic Samples. EPA-600/R-92/080.
U.S. Environmental Protection Agency, Office of Research and
Development, Environmental Research Laboratory, Duluth, MN.
U.S. Environmental Protection Agency. 1993b. 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. 1995. Whole effluent toxicity:
guidelines establishing test procedures for the analysis of pollutants,
final rule. Fed. Reg. 60: 53529-53563.
U.S. Environmental Protection Agency. 1996a. Addenda for Acute Manual.
In U.S. Environmental Protection Agency, 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. 1996b. Clarifications Regarding
Flexibility in 40 CFR Part 136 Whole Effluent Toxicity (WET) Test
Methods, April 10, 1996, memorandum from Tudor Davies, U.S.
Environmental Protection Agency, Office of Science and Technology,
Washington D.C.
U.S. Environmental Protection Agency. 1996c. Marine Toxicity
Identification Evaluation (TIE): Phase I Guidance Document. EPA/600/R-
96/054. U.S. Environmental Protection Agency, National Health and
Environmental Effects Research Laboratory, Narragansett, RI.
U.S. Environmental Protection Agency. 1999a. Errata for Effluent and
Receiving Water Toxicity Test Manuals: Acute Toxicity of Effluents and
Receiving Waters to Freshwater and Marine Organisms; Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters
to Freshwater Organisms; and Short-term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters to Marine and
Estuarine Organisms. January 1999. EPA/600/R-98/182. U.S. Environmental
Protection Agency, Office of Research and Development, Duluth, MN.
U.S. Environmental Protection Agency. 1999b. Whole effluent toxicity:
guidelines establishing test procedures for the analysis of pollutants,
whole effluent toxicity tests; final rule, technical correction. FR 64:
4975-4991.
U.S. Environmental Protection Agency. 1999c. Toxicity Reduction
Evaluation Guidance for Municipal Wastewater Treatment Plants. EPA/833/
B-99/002. U.S. Environmental Protection Agency, Office of Water,
Washington, D.C.
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, D.C.
U.S. Environmental Protection Agency. 2000b. Preliminary Report:
Interlaboratory Variability Study of EPA Short-term Chronic and Acute
Whole Effluent Toxicity Test Methods, Vol. 1. EPA/821/R-00/028A. U.S.
Environmental Protection Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2000c. Preliminary Report:
Interlaboratory Variability Study of EPA Short-term Chronic and Acute
Whole Effluent Toxicity Test Methods, Vol. 2: Appendix. EPA/821/R-00/
028B. U.S. Environmental Protection Agency, Office of Water,
Washington, D.C.
U.S. Environmental Protection Agency. 2000d. 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, D.C.
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, D.C.
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,
D.C.
U.S. Environmental Protection Agency. 2001c. Summary Report: Peer
Review of ``Preliminary Report: Interlaboratory Variability Study of
EPA Short-term Chronic and Acute Whole Effluent Toxicity Test Methods''
(WET Study Report). U.S. Environmental Protection Agency, Office of
Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001d. Proposed Changes to Whole
Effluent Toxicity Method Manuals. EPA/821/B-01/002. U.S. Environmental
Protection Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001e. Report on the Analysis of
Block Effects. U.S. Environmental Protection Agency, Office of Water,
Washington, D.C.
U.S. Environmental Protection Agency. 2001f. Study Report and
Recommended Standard Operating Procedure (SOP) for Shipping Large
Volume Samples at Less Than 4 deg.C. U.S. Environmental Protection
Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001g. Clarifications Regarding
Toxicity Reduction and Identification Evaluations in the National
Pollutant Discharge Elimination System Program. U.S. Environmental
Protection Agency, Office of Water, Washington, D.C.
[[Page 49815]]
List of Subjects in 40 CFR Part 136
Environmental protection, Reporting and recordkeeping requirements,
Water pollution control.
Dated: September 24, 2001.
Christine Todd Whitman,
Administrator.
For the reasons set out in the preamble, title 40, chapter I of the
Code of Federal Regulations, is proposed to be amended as follows:
PART 136--GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANALYSIS
OF POLLUTANTS
1. The authority citation for Part 136 continues to read as
follows:
Authority: Secs. 301, 304(h), 307, and 501(a), Pub. L. 95-217,
91 Stat. 1566, et seq. (33 U.S.C. 1251, et seq.) (The Federal Water
Pollution Control Act Amendments of 1972 as amended by the Clean
Water Act of 1977).
2. Section 136.3 is amended:
a. In Table IA paragraph (a) by revising entries 6 to 9.
b. In paragraph (a) by revising footnotes 7-9 to Table IA.
c. In paragraph (b) by revising references (34), (38), and (39). d.
In paragraph (b) by removing and reserving reference (42).
Sec. 136.3 Identification of test procedures.
(a) * * *
Table IA.--List of Approved Biological Methods
--------------------------------------------------------------------------------------------------------------------------------------------------------
Standard
Parameter and units Method \1\ EPA methods 18th, ASTM AOAC USGS Other
19th, 20th Ed.
--------------------------------------------------------------------------------------------------------------------------------------------------------
* * * * * * *
Aquatic Toxicity:
6. Toxicity, acute, fresh Daphnia, Sec. 9 \7\
water organisms, LC50, Ceriodaphnia,
percent effluent.. Fathead Minnow,
Rainbow Trout,
Brook Trout, or
Bannerfin Shiner
mortality.
7. Toxicity, acute, Mysidopsis bahia, Sec. 9 \7\
estuarine and marine Holmesimysis
organisms, LC50, percent costata,
effluent.. Sheepshead
Minnow, or
Menidia spp.
mortality.
8. Toxicity, chronic, fresh Fathead minnow 1000.0 \8\
water organisms, NOEC or larval survival
IC25, percent effluent.. and growth.
Fathead minnow 1001.0 \8\
embryo-larval
survival and
teratogenicity.
Ceriodaphnia 1002.0 \8\
survival and
reproduction.
Selenastrum growth 1003.0 \8\
9. Toxicity, chronic, Sheepshead minnow 1004.0 \9\
estuarine and marine larval survival
organisms, NOEC or IC25, and growth.
percent effluent..
Sheepshead minnow 1005.0 \9\
embryo-larval
survival and
teratogenicity.
Menidia beryllina 1006.0 \9\
larval survival
and growth.
Mysidopsis bahia 1007.0 \9\
survival, growth,
a fecundity.
Arbacia punctulata 1008.0 \9\
fertilization.
Champia parvula 1009.0 \9\
reproduction.
* * * * * * *
--------------------------------------------------------------------------------------------------------------------------------------------------------
Notes to Table IA:
[[Page 49816]]
\1\ The method must be specified when results are reported.
\7\ USEPA. [Date: To be completed at final rule]. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. Fifth
Edition. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number: To be completed at final
rule].
\8\ USEPA. [Date: To be completed at final rule]. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisms. Fourth Edition. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number: To be
completed at final rule].
\9\ USEPA [Date: to be completed at final rule]. 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, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number:
To be completed at final rule]. These methods do not apply to marine waters of the Pacific Ocean.
* * * * *
(b) * * *
References, Sources, Costs, and Table Citations:
* * * * *
(34) USEPA. [Date: To be completed at final rule]. Methods for
Measuring the Acute Toxicity of Effluents and Receiving Water to
Freshwater and Marine Organisms. Fifth Edition. [Date: To be completed
at final rule]. U.S. Environmental Protection Agency, Environmental
Monitoring Systems Laboratory, Cincinnati, Ohio [EPA number: To be
completed at final rule]. Available from: National Technical
Information Service, 5285 Port Royal Road, Springfield, Virginia 22161,
Publ. No. [Publication number: To be completed at final rule]. Cost:
$[Cost: To be completed at final rule]. Table IA, Note 7.
* * * * *
(38) USEPA. [Date: To be completed at final rule]. Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Water to Freshwater Organisms. Fourth Edition. [Date: To be completed
at final rule]. U.S. Environmental Protection Agency, Environmental
Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number: To be
completed at final rule]. Available from: National Technical
Information Service, 5285 Port Royal Road, Springfield, Virginia 22161,
Publ. No. [Publication number: To be completed at final rule]. Cost:
$[Cost: To be completed at final rule]. Table IA, Note 8.
(39) USEPA. [Date: To be completed at final rule]. Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Water to Marine and Estuarine Organisms. Third Edition. [Date: To be
completed at final rule]. U.S. Environmental Protection Agency,
Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA
number: To be completed at final rule]. Available from: National
Technical Information Service, 5285 Port Royal Road, Springfield,
Virginia 22161, Publ. No. [Publication number: To be completed at final
rule]. Cost: $[Cost: To be completed at final rule]. Table IA, Note 9.
* * * * *
(42) [Reserved]
* * * * *
[FR Doc. 01-24374 Filed 9-27-01; 8:45 am]
BILLING CODE 6560-50-P