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ENVIRONMENTAL PROTECTION AGENCY
Western Area Power Administration
CFR Citation: 40 CFR Part 136
FRL ID: [FRL-7069-7]
NOTICE: Part VI
DOCUMENT ACTION: Proposed rule.
SUBJECT CATEGORY: Guidelines Establishing Test Procedures for the Analysis of Pollutants; Whole Effluent Toxicity Test Methods
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.
DOCUMENT 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.
SUMMARY: Environmental Protection Agency,
SUPPLEMENTAL 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 technologybased and water
qualitybased 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.
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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. ConcentrationResponse 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 12866Regulatory 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 13045Protection of Children From Environmental Health Risks and Safety Risks
G. Executive Order 13175Consultation and Coordination With Indian Tribal Governments
H. Executive Order 13132Federalism
I. Executive Order 13211Energy 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 bycase 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 shortterm 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 shortterm 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 shortterm 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/ 490/027F (acute method manual); ShortTerm Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms, Third Edition, July 1994, EPA/600/491/002 (freshwater chronic method manual); and ShortTerm Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Marine and Estuarine Organisms, Second Edition, July 1994, EPA/600/491/003 (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. 961062 (D.C. Cir.); Western Coalition of Arid States v. EPA, No. 961124; Lone Star Steel Co. v. EPA, No. 961157 (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).
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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 concentrationresponse 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. 961062 & consolidated case (D.C. Cir.), Settlement Agreement, July 24, 1998; Lone Star Steel v. EPA, No. 961157 (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
largescale 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
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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 multilaboratory (interlaboratory) and singlelaboratory (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
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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 staticrenewal 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 (CO
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 PubliclyOwned Treatment Works (POTW) is typically
discharged at a pH of 7.27.4, but the pH may equilibrate after contact
with air and stabilize at 8.08.5 (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 CO
CO
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 pHdependent 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 (SchubauerBerigan 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 unionized form increases (USEPA, 1994a; Emerson et al.,
1975). Because the unionized form of ammonia (NH3) is significantly
more toxic than the ionized form (NH
In addition to the issue of pH drift affecting toxicity in the presence of pHdependent compounds, stakeholders have raised concerns about daily pH drift and sample renewal cycles producing toxicity even in the absence of pHdependent compounds. The circumstance of concern would be in staticrenewal 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
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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., CO
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 staticrenewal 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 CO
The CO
In acute testing, the proposed method changes would recommend the
use of staticrenewal testing or flowthrough testing when artifactual
toxicity due to pH drift is suspected. The use of staticrenewal
testing may reduce the degree of pH drift (compared to static non
renewal tests), and flowthrough testing should eliminate pH drift that
could occur due to static testing conditions. In flowthrough testing,
new sample is continually added to the test chambers, so drift from the
initial sample pH should not occur. Flowthrough testing also
eliminates any potential for organism shock from pH drift and renewal
cycles, because test renewal is continuous. Because flowthrough
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 CO
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 flowthrough 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. ConcentrationResponse Relationships
The concentrationresponse 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 concentrationresponse relationship is depicted as a sigmoidalshaped curve with detrimental responses increasing as the concentration of the toxicant increases. Not all concentrationresponse relationships, however, are represented by the classical sigmoidalshaped curve. A corollary of the concentrationresponse concept is that every toxicant should exhibit a concentrationresponse 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 concentrationresponse 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
concentrationresponse 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
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concentrationresponse concept, require the review of concentration
response relationships, and reference EPA guidance (USEPA, 2000a)
describing various forms of concentrationresponse relationships and
review procedures. Use of the concentrationresponse review procedures
(USEPA, 2000a) would ensure that a valid concentrationresponse
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 concentrationresponse
relationships (including additional forms of concentrationresponse relationships) becomes available.
To demonstrate the effectiveness of the proposed concentration response review steps, EPA used the guidance on concentrationresponse 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 concentrationresponse 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 concentrationresponse review procedures had not been used. This study indicates that the proposed concentrationresponse 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 concentrationresponse 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, concentrationresponse relationships, reference toxicant testing, and test variability. The specifics of the proposed method manual changes related to concentrationresponse 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 concentrationresponse 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, MultiEffluent
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 multiconcentration (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 concentrationresponse 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 (Multiconcentration [Definitive] Effluent Toxicity Tests) of the two chronic method manuals (USEPA, 1994a; USEPA, 1994b) and subsection 9.3 (Multiconcentration [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, instream 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 concentrationresponse 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 concentrationresponse 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 concentrationresponse pattern and subsequently to determine the validity of calculated results (USEPA, 2000a). Normal, reversed, or bimodal concentrationresponse 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 withintreatment 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 24 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 30ml 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
[[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 processrelated 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. 961062 & 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 conju
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 (email: kelly.marion@epa.gov) or call (202) 260 7117. For technical information regarding method changes proposed in today's rule, contact Teresa J. NorbergKing, National Health and Environmental Effects Research Laboratory, MidContinent Ecology Division, Office of Research and Development, U.S. Environmental Protection Agency, 6201 Congdon Boulevard, Duluth, MN 55804 (email: norbergking.teresa@epa.gov) or call (218) 5295163.
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