United States Office of Water EPA 821-B-01-002
Environmental Protection (4303) September 2001
Agency
A ppA Proposed Changes to Whole
Effluent Toxicity Method Manuals
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DISCLAIMER
The Engineering and Analysis Division of the Office of Science and Technology (OST), has
reviewed and approved this report for publication. The OST directed, managed, and reviewed
the work of DynCorp in preparing this report. Neither the U.S. Government nor any of its
employees, contractors, subcontractors (DynCorp), or their employees make any warranty,
expressed or implied, or assumes any legal liability or responsibility for any third party's use of
or the results of such use of any information, apparatus, product, or process discussed in this
report, or represents that its use by such party would not infringe on privately owned rights.
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TABLE OF CONTENTS
I. Introduction 1
II. Minor Corrections and Clarifications 4
A. Acute Method Manual 4
1. Replace Subsection 5.1.4.1 on page 21 of the acute method manual with the
following 4
2. Replace Subsection 8.5.4 on page 43 of the acute method manual with the following.
4
3. Insert the following references into the Cited References section on page 121 of the
acute method manual 4
B. Freshwater Chronic Method Manual 5
1. Replace Subsection 5.4.2.1 on page 23 of the freshwater chronic method manual with
the following 5
2. Replace Subsection 6.1.4 on page 26 of the freshwater chronic method manual with
the following 5
3. Replace Subsections 6.5.7 and 6.5.8 on page 29 of the freshwater chronic method
manual with the following 6
4. Replace Subsection 8.5.4 on page 38 of the freshwater chronic method manual with
the following 6
5. Replace Subsection 8.8.2 on page 39 of the freshwater chronic method manual with
the following 6
6. Replace item 23 in Table 1 (continued) on page 80 of the freshwater chronic method
manual with the following 7
7. Replace Table 14 on page 100 of the freshwater chronic method manual with the
following 7
8. Replace Subsections 13.13.3.6.3 and 13.13.3.6.4 on page 182 of the freshwater
chronic method manual with the following 7
9. Replace Subsection 14.6.16.1 on page 199 of the freshwater chronic method manual
with the following 8
10. Replace item 19 in Table 3 (continued) on page 211 of the freshwater chronic
method manual with the following 8
11. Insert the following references into the Cited References section on page 229 of the
freshwater chronic method manual 8
C. Marine Chronic Method Manual 9
1. Replace Subsection 5.4.2.1 on page 25 of the marine chronic method manual with the
following 9
2. Replace Subsection 6.1.4 on page 27 of the marine chronic method manual with the
following 9
3. Replace Subsections 6.5.7 and 6.5.8 on page 30 of the marine chronic method manual
with the following 9
4. Replace Subsection 8.5.4 on page 40 of the marine chronic method manual with the
following 10
5. Replace Subsection 8.8.3 on page 41 of the marine chronic method manual with the
following 10
in
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6. Replace Subsection 11.13.3.5.1 on page 102 of the marine chronic method manual
with the following 10
7. Replace Subsections 11.13.3.6.3 and 11.13.3.6.4 on page 106 of the marine chronic
method manual with the following 10
8. Replace Subsection 12.5.2 on page 125 of the marine chronic method manual with the
following 11
9. Replace Subsection 12.5.3 on page 125 of the marine chronic method manual with the
following 11
10. Replace Subsection 12.6.10.1 on page 127 of the marine chronic method manual
with the following 11
11. Replace Subsection 12.6.10.2 on page 127 of the marine chronic method manual
with the following 11
12. Replace Note to Figure 1 on page 128 of the marine chronic method manual with the
following 11
13. Replace Subsection 12.10.7.1 on page 141 of the marine chronic method manual
with the following 12
14. Replace Subsection 13.10.9.4 on page 186 of the marine chronic method manual
with the following 12
15. Replace Table 12 on page 205 of the marine chronic method manual with the
following 12
16. Replace Table 13 on page 205 of the marine chronic method manual with the
following 13
17. Replace Subsection 13.13.3.5.2 on page 207 of the marine chronic method manual
with the following 13
18. Replace Table 14 on page 207 of the marine chronic method manual with the
following 13
19. Replace Subsection 13.13.3.5.5 and Table 15 on page 208 of the marine chronic
method manual with the following 14
20. Replace Subsections 13.13.3.6.3 and 13.13.3.6.4 on page 209 of the marine chronic
method manual with the following 14
21. Replace Subsection 14.5.3 on page 223 of the marine chronic method manual with
the following 15
22. Replace Subsection 14.6.2 on page 225 of the marine chronic method manual with
the following 15
23. Replace Subsection 14.6.13, Test Organisms, on page 230 of the marine chronic
method manual with the following 15
24. Replace Subsection 14.6.13.2.1 on page 233 of the marine chronic method manual
with the following 15
25. Replace Subsection 14.10.8.2.1 on page 239 of the marine chronic method manual
with the following 15
26. Replace Subsection 14.12.1 on page 243 of the marine chronic method manual with
the following 15
27. Replace Subsection 14.13.2.4 on page 252 of the marine chronic method manual
with the following 16
28. Replace Subsection 14.13.2.6.6 on page 257 of the marine chronic method manual
with the following 16
29. Replace Subsection 14.13.2.6.7 on page 258 of the marine chronic method manual
with the following 16
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30. Replace Subsections 14.13.3.6.3 and 14.13.3.6.4 on page 269 of the marine chronic
method manual with the following 16
31. Replace Table 21 on page 281 of the marine chronic method manual with the
following 17
32. Replace Subsection 14.14.1.1.1 on page 294 of the marine chronic method manual
with the following 18
33. Replace Subsection 15.5.2 on page 301 of the marine chronic method manual with
the following 18
34. Replace Subsections 15.6.16.3.8 to 15.6.17.7 on page 308 of the marine chronic
method manual with the following 18
35. Replace Table 3 (continued) on page 318 of the marine chronic method manual with
the following 19
36. Replace Table 3 on page 358 of the marine chronic method manual with the
following 20
37. Replace item 18 in Table 3 (continued) on page 359 of the marine chronic method
manual with the following 21
38. Insert the following references into the Cited References section on page 380 of the
marine chronic method manual 21
III. Precision 23
A. Acute Method Manual 23
1. Replace Subsection 4.13.4 on page 12 of the acute method manual with the following.
23
2. Insert the following table after Table 5 on page 17 of the acute method manual. . . 23
B. Freshwater Chronic Method Manual 25
1. Replace Subsection 4.14.3 on page 14 of the freshwater chronic method manual with
the following 25
2. Insert the following table after Table 1 on page 15 of the freshwater chronic method
manual 25
3. Insert the following into Subsection 11.14.1, Precision, on page 108 of the freshwater
chronic method manual 26
4. Insert the following into Subsection 11.14.1.1, Single-Laboratory Precision, on page
108 of the freshwater chronic method manual 26
5. Insert the following into Subsection 11.14.1.2, Multilaboratory Precision, on page 111
of the freshwater chronic method manual 26
6. Replace the footnotes to Table 20 on page 112 of the freshwater chronic method
manual with the following 27
7. Replace the footnotes to Table 21 on page 113 of the freshwater chronic method
manual with the following 27
8. Insert the following tables after Table 21 on page 113 of the freshwater chronic
method manual 27
9. Insert the following into Subsection 13.14.1, Precision, on page 189 of the freshwater
chronic method manual 29
10. Insert the following into Subsection 13.14.1.1, Single-Laboratory Precision, on page
189 of the freshwater chronic method manual 29
11. Insert the following into Subsection 13.14.1.2, Multilaboratory Precision, on page
192 of the freshwater chronic method manual 29
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12. Insert the following tables after Table 19 on page 195 of the freshwater chronic
method manual 30
13. Insert the following into Subsection 14.14.1, Precision, on page 225 of the
freshwater chronic method manual 31
14. Insert the following into Subsection 14.14.1.1, Single-Laboratory Precision, on page
225 of the freshwater chronic method manual 32
15. Replace Subsection 14.14.1.2.1 on page 225 of the freshwater chronic method
manual with the following 32
16. Insert the following tables after Table 12 on page 228 of the freshwater chronic
method manual 33
C. Marine Chronic Method Manual 34
1. Replace Subsection 4.14.3 on page 17 of the marine chronic method manual with the
following 34
2. Insert the following table after Table 1 on page 18 of the marine chronic method
manual 35
3. Insert the following into Subsection 11.14.1, Precision, on page 116 of the marine
chronic method manual 35
4. Insert the following into Subsection 11.14.1.1, Single-Laboratory Precision, on page
116 of the marine chronic method manual 35
5. Insert the following into Subsection 11.14.1.2, Multilaboratory Precision, on page 116
of the marine chronic method manual 36
6. Insert the following tables after Table 27 on page 123 of the marine chronic method
manual 36
7. Insert the following into Subsection 13.14.1, Precision, on page 216 of the marine
chronic method manual 37
8. Insert the following into Subsection 13.14.1.1, Single-Laboratory Precision, on page
216 of the marine chronic method manual 38
9. Replace Subsection 13.14.1.2.1 on page 216 of the marine chronic method manual
with the following 38
10. Insert the following tables after Table 22 on page 221 of the marine chronic method
manual 39
11. Insert the following into Subsection 14.14.1, Precision, on page 294 of the marine
chronic method manual 40
12. Insert the following into Subsection 14.14.1.1, Single-Laboratory Precision, on page
294 of the marine chronic method manual 40
13. Replace Subsection 14.14.1.2.1 on page 294 of the marine chronic method manual
with the following 40
14. Insert the following tables after Table 33 on page 299 of the marine chronic method
manual 41
15. Insert the following into Subsection 16.14.1.1, Single-Laboratory Precision, on page
373 of the marine chronic method manual 43
IV. Blocking by Known Parentage 44
A. Freshwater Chronic Method Manual 44
1. Replace Figure 1 on page 156 of the freshwater chronic method manual with the
following 44
2. Replace Subsection 13.10.2, Start of the Test, on page 160 of the freshwater chronic
method manual with the following 44
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3. Insert the following into Subsection 13.10.9, Termination of the Test, on page 165 of
the freshwater chronic method manual 46
4. Replace item 10 in Table 3 on page 168 of the freshwater chronic method manual
with the following 46
5. Insert the following into Subsection 13.13.1, General, on page 170 of the freshwater
chronic method manual 46
V. pH Drift 47
A. Acute Method Manual 47
1. Replace Subsection 9.5.9 on page 49 of the acute method manual with the following.
47
B. Freshwater Chronic Method Manual 47
1. Insert the following into Subsection 11.3, Interferences, on page 58 of the freshwater
chronic method manual 47
2. Insert the following into Subsection 12.3, Interferences, on page 114 of the freshwater
chronic method manual 48
3. Insert the following into Subsection 13.3, Interferences, on page 144 of the freshwater
chronic method manual 50
C. Marine Chronic Method Manual 51
1. Insert the following into Subsection 11.3, Interferences, on page 61 of the marine
chronic method manual 51
2. Insert the following into Subsection 12.3, Interferences, on page 124 of the marine
chronic method manual 53
3. Insert the following into Subsection 13.3, Interferences, on page 163 of the marine
chronic method manual 54
4. Insert the following into Subsection 14.3, Interferences, on page 222 of the marine
chronic method manual 56
VI. Concentration-response Relationships 58
A. Acute Method Manual 58
1. Replace Section 12, Report Preparation, on page 119 of the acute method manual with
the following 58
B. Freshwater Chronic Method Manual 63
1. Replace Section 10, Report Preparation, on page 55 of the freshwater chronic method
manual with the following 63
C. Marine Chronic Method Manual 68
1. Replace Section 10, Report Preparation, on page 58 of the marine chronic method
manual with the following 68
VII. Nominal Error Rates 73
A. Freshwater Chronic Method Manual 73
1. Insert the following into Subsection 9.4.6, Recommended Alpha Levels, on page 49
of the freshwater chronic method manual 73
B. Marine Chronic Method Manual 73
1. Insert the following into Subsection 9.4.6, Recommended Alpha Levels, on page 52
of the marine chronic method manual 73
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VIII. Confidence Intervals 74
A. Acute Method Manual 74
1. Insert the following into Subsection 11.2, Determination of the LC50 from Definitive,
Multi-effluent-concentration Acute Toxicity Tests, on page 76 of the acute
method manual 74
B. Freshwater Chronic Method Manual 74
1. Insert the following into Subsection 9.3.2, Point Estimation Techniques, on page 47
of the freshwater chronic method manual 74
C. Marine Chronic Method Manual 74
1. Insert the following into Subsection 9.3.2, Point Estimation Techniques, on page 50
of the marine chronic method manual 74
IX. Dilution Series 75
A. Acute Method Manual 75
1. Insert the following into Subsection 9.3, Multi-concentration (Definitive) Effluent
Toxicity Tests, on page 47 of the acute method manual 75
B. Freshwater Chronic Method Manual 75
1. Insert the following into Subsection 8.10, Multi-concentration (Definitive) Effluent
Toxicity Tests, on page 42 of the freshwater chronic method manual 75
C. Marine Chronic Method Manual 75
1. Insert the following into Subsection 8.10, Multi-concentration (Definitive) Effluent
Toxicity Tests, on page 45 of the marine chronic method manual 75
X. Dilution Waters 76
A. Acute Method Manual 76
1. Insert the following into Subsection 7.1, Types of Dilution Water, on page 33 of the
acute method manual 76
B. Freshwater Chronic Method Manual 76
1. Insert the following into Subsection 7.1, Types of Dilution Water, on page 31 of the
freshwater chronic method manual 76
C. Marine Chronic Method Manual 76
1. Insert the following into Subsection 7.1, Types of Dilution Water, on page 32 of the
marine chronic method manual 76
XI. Pathogen Interference 77
A. Freshwater Chronic Method Manual 77
1. Replace Subsection 11.3.4 on page 59 of the freshwater chronic method manual with
the following 77
XII. Selenastrum capricornutum Growth Test Method 80
A. Freshwater Chronic Method 80
1. Replace Subsection 14.6.16.2 on page 199 of the freshwater chronic method manual
with the following 80
2. Replace Subsection 14.12, Acceptability of Test Results, on page 209 of the
freshwater chronic method manual with the following 80
3. Replace Table 3 (continued) on page 211 of the freshwater chronic method manual
with the following 81
Vlll
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XIII. Mysidopsis bahia Survival, Growth, and Fecundity Test Method 82
A. Marine Chronic Method 82
1. Insert the following into Subsection 14.6.13.2, Test Organisms, on page 233 of the
marine chronic manual 82
XIV. Holmesimysis costata Acute Test Method 83
A. Acute Method Manual 83
1. Replace Subsections 6.1.2 and 6.1.3 on page 27 of the acute method manual with the
following 83
2. Insert the following footnote on page 27 of the acute method manual 83
3. Replace Subsection 9.17, Summary of Test Conditions for the Principal Test
Organisms, on page 56 of the acute method manual with the following 83
4. Replace Table 15 on page 65 of the acute method manual with the following 84
5. Insert the following table after Table 17 on page 70 of the acute method manual. . 85
6. Replace the text of Appendix A. 3 on page 172 of the acute method manual with the
following 87
7. Insert the following references into the Selected References section of Appendix A.3
on page 184 of the acute method manual 96
8. Replace footnote 2 in Appendix B on page 266 of the acute method manual with the
following 97
XV. Percent Minimum Significant Difference (PMSD) 98
A. Freshwater Chronic Method Manual 98
1. Insert the following into Subsection 11.13.1, General, on page 81 of the freshwater
chronic method manual 98
2. Insert the following into Subsection 11.13.3.7, Dunnett's Procedure, on page 102 of
the freshwater chronic method manual 98
3. Insert the following into Subsection 13.13.1, General, on page 170 of the freshwater
chronic method manual 99
4. Insert the following into Subsection 13.13.3.7, Dunnett's Procedure, on page 182 of
the freshwater chronic method manual 99
B. Marine Chronic Method Manual 100
1. Insert the following into Subsection 13.13.1, General, on page 186 of the marine
chronic method manual 100
2. Insert the following into Subsection 13.13.3.7, Dunnett's Procedure, on page 210 of
the marine chronic method manual 100
3. Insert the following into Subsection 14.13.1, General, on page 243 of the marine
chronic method manual 101
4. Insert the following into Subsection 14.13.3.7, Dunnett's Procedure, on page 269 of
the marine chronic method manual 101
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I. Introduction
In 1995, the U.S. Environmental Protection Agency (EPA) published a final rule standardizing 17 whole
effluent toxicity (WET) test methods for use in National Pollutant Discharge Elimination System
(NPDES) monitoring [60 FR 53529; October 16, 1995]. These WET test methods measure the toxicity
of effluents and receiving waters to freshwater, marine, and estuarine organisms. The approved methods
include acute toxicity methods and short-term methods for estimating chronic toxicity. Acute methods
generally use death of the test organisms during 24-96 hour exposure durations as the measured effect of
an effluent or receiving water. The short-term methods for estimating chronic toxicity 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. In this document, 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 WET test methods are published in the following three test
method manuals (the WET method manuals):
• U.S. Environmental Protection Agency. 1993. Methods for Measuring the Acute Toxicity of
Effluents and Receiving Waters to Freshwater and Marine Organisms, 4th ed. EPA/600/4-
90/027F. U.S. Environmental Protection Agency, Environmental Monitoring Systems
Laboratory, Cincinnati, OH. (the acute method manual)
• 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. (the freshwater chronic method manual)
• 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. (the marine chronic method manual)
Since the publishing of these method manuals, EPA has corrected minor errors and omissions by
providing the following errata and addenda documents.
• U.S. Environmental Protection Agency. 1996. 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. 1999. 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.
EPA anticipates proposing to revise each of the WET method manuals to incorporate the above errata
and addenda, update the methods, provide additional minor corrections and clarifications, and address
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specific stakeholder concerns. When EPA takes final action on this proposal, the Agency intends to
incorporate the proposed modifications into the text of new editions of each of the WET method
manuals. This document details the proposed changes to the WET method manuals in redline and
strikeout text formatting. The proposed changes and the method manual sections affected are
summarized in Table 1.
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Table 1. Summary of Proposed WET Method Manual Changes.
Chapter
II. Minor Corrections
and Clarifications
III. Precision
IV. Blocking by
Known Parentage
V. pH Drift
VI. Concentration-
response Relationships
VII. Nominal Error
Rates
VIII. Confidence
Intervals
IX. Dilution Series
X. Dilution Water
XL Pathogen
Interference
XII. Selenastmm
capricornutum Growth
Test Method
XIII. Mysidopsis bahia
Survival, Growth, and
Fecundity Test Method
XIV. Holmesimysis
cost at a Acute Test
Method
XV Percent Minimum
Significant Difference
(PMSD)
Summary of Change
correct minor technical errors and omissions and
add references cited in the proposed changes
update method precision statements (including
tables of CVs) with data from recent EPA studies
require blocking by known parentage in the
Ceriodaphnia dubia Survival and Reproduction
Test
add procedures for control of pH drift during
testing
incorporate required review procedures for
evaluating concentration-response relationships
clarify allowable nominal error rate adjustments
clarify limitations in the generation of confidence
intervals
add guidance on dilution series selection
clarify dilution water acceptability
add guidance on controlling pathogen
interference in the Fathead Minnow Larval
Survival and Growth Test
recommend use of EDTA
add guidance to improve success of the fecundity
endpoint
add acute method for Holmesimysis costata
including new table of test conditions and
supplementary information in Appendix A. 3
add required application of upper and lower
PMSD bounds for the Ceriodaphnia dubia
Survival and Reproduction Test; Fathead
Minnow Larval Survival and Growth Test;
Mysidopsis bahia Survival, Growth, and
Fecundity Test; and Inland Silverside Larval
Survival and Growth Test
Method Manual Sections Affected
Acute
Manual
Section 5,
8, &
References
Section 4
Section 9
Section 12
Section 1 1
Section 9
Section 7
Section 6,
9, App.
A.3,andB
Freshwater
Chronic
Manual
Section 5, 6,
8, 11, 13,
14, &
References
Section 4,
11, 13, 14
Section 13
Section 11,
12, 13
Section 10
Section 9
Section 9
Section 8
Section 7
Section 1 1
Section 14
Section 1 1
and 13
Marine
Chronic
Manual
Section 5, 6,
8, 11, 12, 13,
14, 15, 16, &
References
Section 4, 11,
13, 14, 16
Section 11,
12, 13, 14
Section 10
Section 9
Section 9
Section 8
Section 7
Section 14
Section 13
and 14
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II. Minor Corrections and Clarifications
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Replace Subsection 5.1.4.1 on page 21 of the acute method manual with the following.
5.1.4.1 A dcionizing system good quality, laboratory grade deionized water, providing a resistance of 18
megaohm-cm, laboratory grade water should must be provided with available in the laboratory and in
sufficient capacity quantity for laboratory needs. Deionized water may be obtained from MILLIPORE®
Milli-Q®, MILLIPORE QPAK™2 or equivalent system. If large quantities of high quality deionized
water are needed, it may be advisable to supply the laboratory grade water deionizer with preconditioned
water from a CULLIGEAN®, CONTINENTAL®, or equivalent mixed-bed water treatment system.
2. Replace Subsection 8.5.4 on page 43 of the acute method manual with the following.
8.5.4 Sample holding time begins when the last grab sample in a series is taken (i.e., when a series of
four grab samples are taken over a 24-h period), or when a 24-h composite sampling period is completed.
If the data from the samples are to be acceptable for use in the NPDES Program, the lapsed time (holding
time) from sample collection to first use of the each grab or composite sample in test initiation must not
exceed 36 h. EPA believes that 36 h is adequate time to deliver the sample to the laboratories performing
the test in most cases. In the isolated cases, where the permittee can document that this delivery time
cannot be met, the permitting authority can allow an option for on-site testing or a variance for an
extension of shipped sample holding time. The request for a variance in sample holding time, directed to
the USEPA Regional Administrator under 40 CFR 136.3(e), must include supportive data which show
that the toxicity of the effluent sample is not reduced (e.g., because of volatilization and/or sorption of
toxics on the sample container surfaces) by extending the holding time beyond more than 36 h.
However, in no case should more than 72 h elapse between collection and first use of the sample. In
static-renewal tests, the original each grab or composite sample may also be used to prepare test solutions
for renewal at 24 h, 48 h, and/or 72 h after test initiation 24 h and/or 48 h after first use, if stored at 4°C,
with minimum head space, as described in Subsection 8.5. Guidance for determining the persistence of
the sample is provided in Subsection 8.7.
3. Insert the following references into the Cited References section on page 121 of the acute
method manual.
Casarett, L.J. and J. Doull. 1975. Toxicology: the basic science of poisons. Macmillan Publishing Co.,
New York.
Martin, M., J.W. Hunt, B.S. Anderson, S.L. Turpen, and F.H. Palmer. 1989. Experimental evaluation of
the mysid Holmesimysis costata as a test organism for effluent toxicity testing. Environ. Toxicol.
Chem. 8: 1003-1012.
Price, W.W., R.W. Heard, and L. Stuck. 1994. Observations on the genus Mysidopsis Sars, 1864 with
the designation of a new genus, Americamysis, and the descriptions of Americamysis alleni and
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Americamysis stucki (Peracarida: Mysidacea: Mysidae), from the Gulf of Mexico. Proc. Biol.
Soc. Wash. 107: 680-698.
USEPA. 2000a. Method guidance and recommendations for whole effluent toxicity (WET) testing (40
CFR Part 136). Office of Water, U.S. Environmental Protection Agency, Washington, B.C.
20460. EPA/82l/B-00/004.
USEPA. 2000b. Understanding and accounting for method variability in whole effluent toxicity
applications under the national pollutant discharge elimination system program. Office of
Wastewater Management, U.S. Environmental Protection Agency, Washington, B.C. 20460.
EPA/833/R-00/003.
USEPA. 200 la. Final report: interlaboratory variability study of EPA short-term chronic and acute
whole effluent toxicity test methods, Vol. 1. Office of Water, U.S. Environmental Protection
Agency, Washington, B.C. 20460. EPA/821/B-01/004.
USEPA. 200 Ib. Final report: interlaboratory variability study of EPA short-term chronic and acute
whole effluent toxicity test methods, Vol. 2: Appendix. Office of Water, U.S. Environmental
Protection Agency, Washington, B.C. 20460. EPA/821/B-01/004.
B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Replace Subsection 5.4.2.1 on page 23 of the freshwater chronic method manual with the
following.
5.4.2.1 A good quality, laboratory grade deionized water, providing a resistance of 18 megaohm-cm,
laboratory grade water, should must be available in the laboratory and in sufficient capacity quantity for
laboratory needs. Beionized water may be obtained from MILLIPORE® Milli-Q®, MILLIPORE
QPAK™2 or equivalent system. If large quantities of high quality deionized water are needed, it may be
advisable to supply the laboratory grade water deionizer with preconditioned water from a Culligan®,
Continental®, or equivalent mixed-bed water treatment system.
2. Replace Subsection 6.1.4 on page 26 of the freshwater chronic method manual with the
following.
6.1.4 Some states have developed culturing and testing methods for indigenous species that may be as
sensitive, or more sensitive, than the species recommended in SubSsection 6.1.3. However, USEPA
allows the use of indigenous species only where state regulations require their use or prohibit importation
of the recommended species in SubScction 6.2.6 Subsection 6.1.3. Where state regulations prohibit
importation of non-native fishes or use of the recommended test species, permission must be requested
from the appropriate state agency prior to their use.
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3. Replace Subsections 6.5.7 and 6.5.8 on page 29 of the freshwater chronic method manual with
the following.
0.3. / 1'isii siiouid DC oDserved C3.rcru.iiy ettcii dciy tor SI^HS or Qisc3.sc, stress, pnysiccii dcttrici^e, 3.110.
iriortciirty. uecid 3110. 3.011011113.1 specimens snouid DC removed cis soon cis ODserved, it is HOT uncommon to
Hcive some nsii ^3~iu /oj mortciirty during me nrst H-O n in ci Holding tctnK Decciuse or individuals uicix re ruse
to reed on 3.1x111013.1 rood ctnd die or stcirvciiion.1'isii in uie Holding tctnKS snouid ^enercHiy De red cis in uie
cultures (^see curturin^ meuiods in uie respective meuiodsj.
6.5.&7 A daily record of feeding, behavioral observations, and mortality should be maintained.
4. Replace Subsection 8.5.4 on page 38 of the freshwater chronic method manual with the
following.
8.5.4 Sample holding time begins when the last grab sample in a series is taken (i.e., when a series of
four grab samples are taken over a 24-h period), or when a 24-h composite sampling period is completed.
If the data from the samples are to be acceptable for use in the NPDES Program, the lapsed time (holding
time) from sample collection to first use of the each grab or composite sample in test initiation must not
exceed 36 h. EPA believes that 36 h is adequate time to deliver the sample to the laboratories performing
the test in most cases. In the isolated cases, where the permittee can document that this delivery time
cannot be met, the permitting authority can allow an option for on-site testing or a variance for an
extension of shipped sample holding time. The request for a variance in sample holding time, directed to
the USEPA Regional Administrator under 40 CFR 136.3(e), must include supportive data which show
that the toxicity of the effluent sample is not reduced (e.g., because of volatilization and/or sorption of
toxics on the sample container surfaces) by extending the holding time beyond more than 36 h.
However, in no case should more than 72 h elapse between collection and first use of the sample. In
static-renewal tests, the original each grab or composite sample may also be used to prepare test solutions
for renewal at 24 h and/or 48 hr after test initiation first use, if stored at 4°C, with minimum head space,
as described in SubS-section 8.5. Guidance for determining the persistence of the sample is provided in
SubSsection 8.7.
5. Replace Subsection 8.8.2 on page 39 of the freshwater chronic method manual with the
following.
8.8.2 With the daphnids, Ceriodaphnia dubia, and fathead minnow, Pimephalespromelas, tests, effluent
and receiving waters should rrmst be filtered through a 60-|im plankton net to remove indigenous
organisms that may attack or be confused with test organisms (see the daphnid, Ceriodaphnia dubia test
for more details). Receiving waters used in green alga, Selenastrum capricornutum, toxicity tests must
be filtered through a 0.45-|im pore diameter filter before use. It may be necessary to first coarse-filter the
dilution and/or waste water through a nylon sieve having 2- to 4-mm mesh openings holes to remove
debris and/or break up large floating or suspended solids. Because filtration may increase the dissolved
oxygen (DO) in the effluent, the DO should be checked both before and after filtering. Low dissolved
oxygen concentrations will indicate a potential problem in performing the test. Caution: filtration may
remove some toxicity.
-------
6. Replace item 23 in Table 1 (continued) on page 80 of the freshwater chronic method manual
with the following.
23. Sampling requirements: For on-site tests, samples collected daily, and used
within 24 h of the time they are removed from the
sampling device; For off-site tests, a minimum of three
samples collected on days one, three and five with a
maximum holding time of 36 h before first use (see
Section 8, Effluent and Receiving Water Sampling,
Sample Handling, and Sample Preparation for Toxicity
Tests, and Subsection 8.5.4)
7. Replace Table 14 on page 100 of the freshwater chronic method manual with the following.
TABLE 14. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
i 34 X(n-I+1) - X®
1
2
3
4
5
6
7
8
9
10
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
0.352
0.303
6^3*0.181
0.106
0.105
0.065
0.049
0.031
0.012
0.008
X(20).X(D
X(19) . X(2)
X(18) . X(3)
X(17) . X(4)
X(16) . X(5)
X(15).X(6)
X(14).X(7)
X(13).X(8)
X(12).X(9)
Xdi).X(io)
8. Replace Subsections 13.13.3.6.3 and 13.13.3.6.4 on page 182 of the freshwater chronic method
manual with the following.
13.13.3.6.3 Bartlett's statistic is therefore:
p
B = [(45)ln(31.8)-9sln(S'/2)]/1.04
= [45(3-6- 3.46) -9
11. 15/1.04
10.72
-------
13.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees of freedom, when the
variances are in fact the same. Therefore, the appropriate critical value for this test, at a significance
level of 0.01 with four degrees of freedom, is 13.3. Since B = +£-1-10.7 is less than the critical value of
13.3, conclude that the variances are not different.
9. Replace Subsection 14.6.16.1 on page 199 of the freshwater chronic method manual with the
following.
14.6.16.1 Selenastrum capricornutum, a unicellular coccoid green alga is the test organism. The genus
and species name of this organism was formally changed to Pseudokirchneriella subcapitata (Hindak,
1990), however, the method manual will continue to refer to Selenastrum capricornutum to maintain
consistency with previous versions of the method.
10. Replace item 19 in Table 3 (continued) on page 211 of the freshwater chronic method manual
with the following.
19. Sampling requirements: For on-site tests, one sample collected at test initiation, and used
within 24 h of the time it is removed from the sampling device.
For off-site tests, holding time must not exceed 36 h before first
use (see Section 8, Effluent and Receiving Water Sampling,
Sample Handling, and Sample Preparation for Toxicity Tests,
and Subsection 8.5.4)
11. Insert the following references into the Cited References section on page 229 of the freshwater
chronic method manual.
Casarett, L.J. and J. Doull. 1975. Toxicology: the basic science of poisons. Macmillan Publishing Co.,
New York.
Downey, P.J.,K. Fleming, R. Guinn, N. Chapman, P. Varner, J. Cooney. 2000. Sporadic mortality in
chronic toxicity tests using Pimephales promelas (Rafmesque): cases of characterization and
control. Environ. Toxicol. Chem. 19(1): 248-255.
Geis, S., K. Fleming, A. Mager, and K. Schappe. 2000. Investigation of the pathogenic effect in whole
effluent toxicity (WET) chronic fathead minnow tests. SETAC Abstract Book, 21st Annual
Meeting, 12-16 November, 2000.
Hindak, F. 1990. Biologicke prace (Slovenskej Akademie Vied). 36: 209.
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.
USEPA. 2000a. Method guidance and recommendations for whole effluent toxicity (WET) testing (40
CFR Part 136). Office of Water, U.S. Environmental Protection Agency, Washington, D.C.
20460. EPA/82l/B-00/004.
USEPA. 2000b. Understanding and accounting for method variability in whole effluent toxicity
applications under the national pollutant discharge elimination system program. Office of
-------
Wastewater Management, U.S. Environmental Protection Agency, Washington, B.C. 20460.
EPA/833/R-00/003.
USEPA. 200 la. Final report: interlaboratory variability study of EPA short-term chronic and acute
whole effluent toxicity test methods, Vol. 1. Office of Water, U.S. Environmental Protection
Agency, Washington, D.C. 20460. EPA/821/B-01/004.
USEPA. 200 Ib. Final report: interlaboratory variability study of EPA short-term chronic and acute
whole effluent toxicity test methods, Vol. 2: Appendix. Office of Water, U.S. Environmental
Protection Agency, Washington, D.C. 20460. EPA/821/B-01/004.
C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Replace Subsection 5.4.2.1 on page 25 of the marine chronic method manual with the following.
5.4.2.1 A good quality, laboratory grade deionized water, providing a resistance of 18 megaohm-cm,
laboratory grade water, should must be available in the laboratory and with- in sufficient capacity quantity
for laboratory needs. Deionized water may be obtained from MILLIPORE® Milli-Q®, MILLIPORE
QPAK™2 or equivalent system. If large quantities of high quality deionized water are needed, it may be
advisable to supply the laboratory grade water deionizer with preconditioned water from a Culligean®,
Continental®, or equivalent mixed-bed water treatment system.
2. Replace Subsection 6.1.4 on page 27 of the marine chronic method manual with the following.
6.1.4 Some states have developed culturing and testing methods for indigenous species that may be as
sensitive or more sensitive, than the species recommended in Subsection 6.1.3. However, USEPA allows
the use of indigenous species only where state regulations require their use or prohibit importation of the
species in Section 6, Facilities, Equipment, and Supplies, Subsection 6.1.3. Where state regulations
prohibit importation of non-native fishes or use of the recommended test species, permission must be
requested from the appropriate state agency prior to their use.
3. Replace Subsections 6.5.7 and 6.5.8 on page 30 of the marine chronic method manual with the
following.
0.3. / 1'isii siiouiQ DC oDscrvcQ CcirGruii^ Gticn cicty tor SI^HS or Qisc3.sc, stress, pnysictii Qtirriti^G, ctnci
rriorTciirt^. UGcici tiiiQ ctDnorrricii specimens snouiQ DC removed cis soon cis ODscrvcQ. it is HOT uncommon to
Helve sonic iisii ^3~iu /o) mortciirty during TUG nrst H-O n in ci noicun^ tcinrc DGCciusG or murviciuciis uicit re ruse
to ICCQ on cirtiricicii rood cHici QIC or stcirvcition.1'isn in TUG noicnn^ TCUXKS snouiQ ^enGrcHiy DC ICQ cis 111 TUG
cultures (^SGG culturing incuiocis in TUG respective inctnocisj.
6.S.&7 A daily record of feeding, behavioral observations, and mortality should be maintained.
-------
4. Replace Subsection 8.5.4 on page 40 of the marine chronic method manual with the following.
8.5.4 Sample holding time begins when the last grab sample in a series is taken (i.e., when a series of
four grab samples are taken over a 24-h period), or when a 24-h composite sampling period is completed.
If the data from the samples are to be acceptable for use in the NPDES Program, the lapsed time (holding
time) from sample collection to first use of the each grab or composite sample in test initiation must not
exceed 36 h. EPA believes that 36 h is adequate time to deliver the sample to the laboratories performing
the test in most cases. In the isolated cases, where the permittee can document that this delivery time
cannot be met, the permitting authority can allow an option for on-site testing or a variance for an
extension of shipped sample holding time. The request for a variance in sample holding time, directed to
the USEPA Regional Administrator under 40 CFR 136.3(e), must include supportive data which show
that the toxicity of the effluent sample is not reduced (e.g., because of volatilization and/or sorption of
toxics on the sample container surfaces) by extending the holding time beyond more than 36 h.
However, in no case should more than 72 h elapse between collection and first use of the sample. In
static-renewal tests, the original each grab or composite sample may also be used to prepare test solutions
for renewal at 24 h and/or 48 h after test initiation first use, if stored at 4°C, with minimum head space, as
described in Paragraph Subsection 8.5. Guidance for determining the persistence of the sample is
provided in Subsection 8.7.
5. Replace Subsection 8.8.3 on page 41 of the marine chronic method manual with the following.
8.8.3 It may be necessary to first coarse-filter samples through a NYLON® sieve having 2 to 4 mm mesh
openings to remove debris and/or break up large floating or suspended solids. If samples contain
indigenous organisms that may attack or be confused with the test organisms, the samples must should be
filtered through a sieve with 60-|im mesh openings. Since filtering may increase the dissolved oxygen
(DO) in an effluent, the DO should be checked both before and after determined prior to filtering. Low
dissolved oxygen concentrations will indicate a potential problem in performing the test. Caution:
filtration may remove some toxicity.
6. Replace Subsection 11.13.3.5.1 on page 102 of the marine chronic method manual with the
following.
11.13.3.5.1 The first step of the test for normality is to center the observations by subtracting the mean
of all the observations within a concentration from each observation in that concentration. The centered
observations are summarized in Table 14.
7. Replace Subsections 11.13.3.6.3 and 11.13.3.6.4 on page 106 of the marine chronic method
manual with the following.
11.13.3.6.3 Bartlett's statistic is therefore:
p
0.0187)-
7=1
= [12(-4-290 3.979) - 3(-*fr96018.876)]/1.139
10
-------
= 53968.882/1.139
= 4-7377.798
11.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees of freedom, when the
variances are in fact the same. Therefore, the appropriate critical value for this test, at a significance
level of 0.01 with three degrees of freedom, is 1 1.345. Since B = 4.737 7.798 is less than the critical
value of 1 1 .345, conclude that the variances are not different.
8. Replace Subsection 12.5.2 on page 125 of the marine chronic method manual with the following.
12.5.2 Sheepshead minnow culture unit ~ see Subsection 6rt3 12.6.12 below. To perform toxicity tests
on-site or in the laboratory, sufficient numbers of newly fertilized eggs must be available, preferably
from an in-house sheepshead minnow culture unit. If necessary, embryos can be obtained from outside
sources if shipped in well oxygenated water in insulated containers.
9. Replace Subsection 12.5.3 on page 125 of the marine chronic method manual with the following.
12.5.3 Brine shrimp, Artemia, culture unit ~ for feeding sheepshead minnow larvae in the continuous
culture unit (see Subsection 6rt2 12.6. 1 1 below).
10. Replace Subsection 12.6.10.1 on page 127 of the marine chronic method manual with the
following.
12.6.10.1 Saline test and dilution water ~ The salinity of the test water must be in the range of 5 to 32%o.
The salinity should vary no more than ±2%o-t among chambers on a given day. If effluent and receiving
water tests are conducted concurrently, the salinities of the water should be similar.
11. Replace Subsection 12.6.10.2 on page 127 of the marine chronic method manual with the
following.
12.6.10.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and
estuarine systems contain little or no measurable salts. Exposure of sheepshead minnow embryos to
these effluents will require adjustments in the salinity of the test solutions. It is important to maintain a
constant salinity across all treatments, if In addition, it may be desirable to match the test salinity with
that of the receiving water. Two methods are available to adjust salinities ~ a hypersaline brine derived
from natural seawater or artificial sea salts.
12. Replace Note to Figure 1 on page 128 of the marine chronic method manual with the following.
Note: Final endpoint for this test is total mortality (combined total number of dead embryos, dead larvae,
and deformed larvae) (see Subsection 12.10.& 9 and 12.13).
11
-------
13. Replace Subsection 12.10.7.1 on page 141 of the marine chronic method manual with the
following.
12.10.7.1 Since feeding is not required, test chambers are not cleaned daily unless accumulation of
particulate matter at the bottom of the tank causes a problem.
14. Replace Subsection 13.10.9.4 on page 186 of the marine chronic method manual with the
following.
13.10.9.4 Immediately prior to drying, the preserved larvae are in distilled water. The rinsed larvae from
each test chamber are transferred, using forceps, to a tared weighing pans and dried at 60 °C for 24 h, or at
105°C for a minimum of 6 h. Immediately upon removal from the drying oven, the weighing pans are
placed in a desiccator to cool and to prevent the adsorption of moisture from the air until weighed.
Weigh all weighing pans containing the dried larvae to 0.01 mg, subtract the tare weight to determine dry
weight of larvae in each replicate. Record (Figure 4) the weights. Divide the dry weight by the number
of original larvae per replicate to determine the average dry weight, and record (Figures 4 and 5) on the
data sheets. For the controls, also calculate the mean weight per surviving fish in the test chamber to
evaluate if weights met test acceptability criteria (see Subsection 13.11). Complete the summary data
sheet (Figure 5) after calculating the average measurements and statistically analyzing the dry weights
and percent survival for the entire test. Average weights should be expressed to the nearest 0.001 mg.
15. Replace Table 12 on page 205 of the marine chronic method manual with the following.
TABLE 12. INLAND SILVERSIDE, MENIDIA BERYLLINA^ GROWTH DATA
Effluent Concentration %
Replicate Control 6.25 12.5 25.0 50.0 100.0
A
B
C
0.751
0.849
0.907
0.737
0.922
0.927
0.722
0.285
0.718
0.196
0.312
1-079 0.079 -
-
-
0.836 0^590.862 0.575 0.196
0.0062 0.01300.01170.0631 0.0136 -
1 2345
12
-------
16. Replace Table 13 on page 205 of the marine chronic method manual with the following.
TABLE 13. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Effluent Concentration (%)
Replicate
A
B
C
Control
-0.085
0.013
0.071
6.25 12.5
r\ i A n r\ i ^ c r\ r\r\ r\ i /i ^7
U. 14- / -U. 1ZJ U.UU U. 1H- /
=0^900.060 0^6 -0.290
0^43-0.065 -erWT1 0.143
17. Replace Subsection 13.13.3.5.2 on page 207 of the marine chronic method manual with the
following.
13.13.3.5.2 Calculate the denominator, D, of the test statistic:
D= X
Where:
For this set of data,
X; = the ith centered observation
X= the overall mean of the centered observations
n = the total number of centered observations.
n = 9
X=
J_(-0.002) = 0.000
18. Replace Table 14 on page 207 of the marine chronic method manual with the following.
TABLE 14. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
.A. 1
-0.290 6
=6r«? -0.125 7
-0.085 8
*»
0.071 0.065
ed^^ o.o7i
0.143
13
-------
4 e^ee o.ois 9 0.147
5 0^3 0.060
19. Replace Subsection 13.13.3.5.5 and Table 15 on page 208 of the marine chronic method manual
with the following.
13.13.3.5.5 Compute the test statistic, W, as follows:
W= 1[E a^-^
D i=\
The differences X(lw+1) - X® are listed in Table 15. For this set of data:
W = 1 (0.3997 0.3800)2 = 0^64 0.89
0.16570.162
TABLE 15. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
a,
0.5888
0.3244
0.1976
0.0947
x(n-i+1) - x®
0 437 X® - X1-1-1
0^60 0.268 X(8) - X(2)
0^- 0.156 X(7)-X(3)
r\ r\n i n n co v(6) v(4)
U.U / 1 U.UJZ A - A
20. Replace Subsections 13.13.3.6.3 and 13.13.3.6.4 on page 209 of the marine chronic method
manual with the following.
13.13.3.6.3 Bartlett's statistic is therefore:
p
5 = [(6)ln(e^74 0.027) - 2^ln(S? ) ] / ir2$ 1.222
7=1
= [6(-3.5972 -3.612)-2(ln(0.0062) I ln(0.0130) I ln(0.0631) -12.290)]/i^5 1.222
= [-26.583 - (-24.378)] 2.909/1:25 1.222
= 2^362.38
14
-------
13.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees of freedom, when the
variances are in fact the same. Therefore, the appropriate critical value for this test, at a significance
level of 0.01 with 2 degrees of freedom, is 9.210. Since B = 2.23G 2.38 is less than the critical value of
9.210, conclude that the variances are not different.
21. Replace Subsection 14.5.3 on page 223 of the marine chronic method manual with the
following.
14.5.3 Mysid, Mysldopsls bahia, culture unit ~ see Subsection 6 14.6.13 below. This test requires a
minimum of 240 7-day old (juvenile) mysids. It is preferable to obtain the test organisms from an in-
house culture unit. If it is not feasible to culture mysids in-house, juveniles can be obtained from other
sources, if shipped in well oxygenated saline water in insulated containers.
22. Replace Subsection 14.6.2 on page 225 of the marine chronic method manual with the
following.
14.6.2 Data sheets (one set per test) ~ for data recording (Figures 14, 15, and 16 2, 7, and 8).
23. Replace Subsection 14.6.13, Test Organisms, on page 230 of the marine chronic method
manual with the following.
14.6.13 TEST ORGANISMS, Mysidopsis bahia (see Rodgers etal, 1986 and USEPA, 1993a for
information on mysid ecology). The genus name of this organism was formally changed to
Americamysis (Price et al, 1994), however, the method manual will continue to refer to Mysidopsis
bahia to maintain consistency with previous versions of the method.
24. Replace Subsection 14.6.13.2.1 on page 233 of the marine chronic method manual with the
following.
14.6.13.2.1 The test is begun with 7-day-old juveniles. To have the test animals available and
acclimated to test conditions at the start of the test, they gravid females must be obtained from the stock
culture eight days in advance of the test. Whenever possible, brood stock should be obtained from
cultures having similar salinity, temperature, light regime, etc., as are to be used in the toxicity test.
25. Replace Subsection 14.10.8.2.1 on page 239 of the marine chronic method manual with the
following.
14.10.8.2.1 The number of live mysids are counted and recorded each day when the test solutions are
renewed (Figure 2 7). Dead animals and excess food should be removed with a pipette before test
solutions are renewed.
26. Replace Subsection 14.12.1 on page 243 of the marine chronic method manual with the
following.
14.12.1 The minimum requirements for an acceptable test are 80% survival and an average weight of at
least 0.20 mg/surviving mysid in the controls. If fecundity in the controls is adequate (egg production by
50% of females), fecundity should be used as a criterion of effect in addition to survival and growth.
15
-------
27. Replace Subsection 14.13.2.4 on page 252 of the marine chronic method manual with the
following.
14.13.2.4 Probit Analysis (Finney, 1971; see Appendix €r H) is used to estimate the concentration that
causes a specified percent decrease in survival from the control. In this analysis, the total mortality data
from all test replicates at a given concentration are combined. If the data do not fit the Probit model, the
Spearman-Karber method, the Trimmed Spearman-Karber method, or the Graphical method may be used
(see Appendices HI-K).
28. Replace Subsection 14.13.2.6.6 on page 257 of the marine chronic method manual with the
following.
14.13.2.6.6 Compute the test statistic, W, as follows:
W= - [E a.(^-/+1) -
D i=i
The differences x(n"1+1) - X(l) are listed in Table ? 8. For this data in this example:
W = 1 (1.0475)2 = 0.9167
1.197
29. Replace Subsection 14.13.2.6.7 on page 258 of the marine chronic method manual with the
following.
14.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.2.6.5 6 with
the critical value found in Table 6, Appendix B. If the computed W is less than the critical value,
conclude that the data are not normally distributed. For this set of data, the critical value at a significance
level of 0.01 and n = 40 observations is 0.919. Since W = 0.9167 is less than the critical value, conclude
that the data are not normally distributed.
30. Replace Subsections 14.13.3.6.3 and 14.13.3.6.4 on page 269 of the marine chronic method
manual with the following.
14.13.3.6.3 Bartlett's statistic is therefore:
p
B = [(28)ln(0.00162) - 7^1n(Sf ) ] / 1.06
7 = 1
= [28(-6.4315 -6.427) - 7(-25.9357 -25.9329)]/1.06
= [-180.082 -179.973 - (-181.5499 -181.530)]/1.06
= +3851.469
16
-------
14.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees of freedom, when the
variances are in fact the same. Therefore, the appropriate critical value for this test, at a significance
level of 0.01 with three degrees of freedom, is 9.210 11.34. Since B = 1.385 1.469 is less than the critical
value of 9.210, conclude that the variances are not different.
31. Replace Table 21 on page 281 of the marine chronic method manual with the following.
TABLE 21. MYSID, MYSIDOPSIS BAHIA, FECUNDITY DATA: PERCENT FEMALES WITH
EGGS
Replicate
Control
Test Concentration (ppb)
50.0 100.0
210.0
RAW
ARC SINE
TRANS-
FORMED1
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
MeanOQ
s2,
i
1.00
1.00
0.67
1.00
1.00
0.80
1.00
1.00
1.57
1.57
0.96
.57
.57
.12
.57
.57
1.44
0.064
1
0.50
0.33
0.67
-
0.40
0.50
0.25
0.33
0.78
0.61
0.96
-
0.68
0.78
0.52
0.61
0.71
0.021
2
0.33
0.50
0.00
0.50
0.67
0.00
0.25
-
0.61
0.78
0.00
0.78
0.96
0.00
0.52
-
0.52
0.147
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
-
-
-
-
-
-
-
-
4
1 Since the denominator of the proportion of females with eggs varies with the number of females
occurring in that replicate, the adjustment of the arc sine square root transformation for 0% and 100% is
not used for this data.
17
-------
32. Replace Subsection 14.14.1.1.1 on page 294 of the marine chronic method manual with the
following.
14.14.1.1.1 Data on the single -laboratory precision of the mysid survival, growth, and fecundity using
copper (Cu) sulfate and sodium dodecyl sulfate (SDS) in natural seawater and in artificial seawater
(GP2) are shown in Tables 29-33. Survival NOEC/LOEC pairs showed good precision, and were the
ScirriG in rour or TUG six tests \vrtn v^u ctiici oUo. vjrovvTii emu. iccuiicirt^' vvcrc ^Giicrciiiy HOT ttcccptciDiG
cndpoints in cither sets of tests. In Tables 29-30 the coefficient of variation for the IC25, ranges from
18.0 to 35.0 and the IC50, ranges from 5.8 to 47.8, indicating acceptable test precision. Data in Tables
31-33 show no detectable differences between tests conducted in natural or artificial seawaters.
33. Replace Subsection 15.5.2 on page 301 of the marine chronic method manual with the
following.
15.5.2 Laboratory sea urchins, Arbacia punctulata, culture unit ~ See Subsection 6rF? 15.6.19, culturing
methods below and Section 4, Quality Assurance. To test effluent or receiving water toxicity, sufficient
eggs and sperm must be available.
34. Replace Subsections 15.6.16.3.8 to 15.6.17.7 on page 308 of the marine chronic method manual
with the following.
15.6.-Kr 18.3.8 Table 1 illustrates the preparation of test solutions at 30%o if they are made by combining
effluent (0%0), deionized water and HSB (100%0), or FORTY FATHOMS® sea salts.
15.6.+6 18.4 Artificial sea salts: FORTY FATHOMS® brand sea salts (Marine Enterprises, Inc., 8755
Mylander Lane, Baltimore, MD 21204; 301-321-1 189) have been used successfully at the EMSL-
Cincinnati, for long-term (6-12 months) maintenance of stock cultures of sexually mature sea urchins and
to perform the sea urchin fertilization test. GP2 seawater formulation (Table 2) has also been used
successfully at ERL-Narragansett, RI.
15.6.-Kr 18.4. 1 Synthetic sea salts are packaged in plastic bags and mixed with deionized water or
equivalent. The instructions on the package of sea salts should be followed carefully, and the salts
should be mixed in a separate container ~ not in the culture tank. The deionized water used in hydration
should be in the temperature range of 21-26°C. Seawater made from artifical sea salts is conditioned
(Spotte, 1973; Spotte, etal., 1984; Bower, 1983).
15.6.-Kr 18.4.2 The GP2 reagent grade chemicals (Table 4-2) should be mixed with deionized (DI) water
or its equivalent in a container other than the culture or testing tanks. The deionized water used for
hydration should be between 21-26°C. The artificial seawater must be conditioned (aerated) for 24 h
before use as the testing medium. If the solution is to be autoclaved, sodium bicarbonate is added after
the solution has cooled. A stock solution of sodium bicarbonate is made up by dissolving 33.6 g
NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this stock solution for each liter of the GP2
artifical seawater.
15.6.i? 19 TEST ORGANISMS, SEA URCHINS, ARBACIA PUNCTULATA
15.6.-H? 19.1 Adult sea urchins, Arbacia punctulata, can be obtained from commercial suppliers. After
acquisition, the animals are sexed by briefly stimulating them with current from a 12 V transformer.
18
-------
Electrical stimulation causes the immediate release of masses of gametes that are readily identifiable by
color ~ the eggs are red, and the sperm are white.
15.6.-H? 19.2 The sexes are separated and maintained in 20-L, aerated fiberglass tanks, each holding
about 20 adults. The tanks are supplied continuously (approximately 5 L/min) with filtered natural
seawater, or salt water prepared from commercial sea salts is recirculated. The animals are checked daily
and any obviously unhealthy animals are discarded.
15.6.-H? 19.3 The culture unit should be maintained at 15 ± 3°C, with a water temperature control device.
15.6.-H? 19.4 The food consists of kelp, Laminaria sp., gathered from known uncontaminated zones or
obtained from commercial supply houses whose kelp comes from known uncontaminated areas, or
romaine lettuce. Fresh food is introduced into the tanks at approximately one week intervals. Decaying
food is removed as necessary. Ample supplies of food should always be available to the sea urchins.
15.6.-H? 19.5 Natural or artificial seawater with a salinity of 30%o is used to maintain the adult animals,
for all washing and dilution steps, and as the control water in the tests (see Subsection 15.6.-KH8).
15.6.-H? 19.6 Adult male and female animals used in field studies are transported in separate or
partitioned insulated boxes or coolers packed with wet kelp or paper toweling. Upon arrival at the field
site, aquaria (or a single partitioned aquarium) are filled with control water, loosely covered with a
styrofoam sheet and allowed to equilibrate to 15°C before animals are added. Healthy animals will attach
to the kelp or aquarium within hours.
15.6.-H? 19.7 To successfully maintain about 25 adult animals for 7 days at a field site, a
screen-partitioned, 40-L glass aquarium using aerated, recirculating, clean saline water (30%o) and a
gravel bed filtration system, is housed within a water bath, such as FORTY FATHOMS® or equivalent
(15°C). The inner aquarium is used to avoid contact of animals and water bath with cooling coils.
35. Replace Table 3 (continued) on page 318 of the marine chronic method manual with the
following.
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT AND
RECEIVING WATERS (CONTINUED)
12. Effluent Test concentrations: Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water or minimum of
5 and a control
13. Test d Dilution factor: Effluents: >0.5
Receiving waters: None or > 0.5
14. Test duration: 1 h and 20 min
15. Endpoint: Fertilization of sea urchin eggs
19
-------
16. Test acceptability criteria:
17. Sampling requirements:
18. Sample volume required:
70% - 90% egg fertilization in controls
For on-site tests, Oone sample collected at test initiation,
and preferably used within 24 h of the time it is removed
from the sampling device. For off-site tests, holding
time must not exceed 36 h before first use (see Section
8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests,
Subsection 8.5.4)
1 L per test
36. Replace Table 3 on page 358 of the marine chronic method manual with the following.
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE RED MACRO ALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST WITH
EFFLUENTS AND RECEIVING WATERS
1. Test type:
2. Salinity:
3. Temperature:
TT 1 liotopcnocl:
5:—Light intensity:
6 4. Light source quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. No. organisms
per test chamber:
10. No. replicate
per concentration:
11. No. organisms per
Static, non-renewal
30%0 (± 2%0 of the selected test salinity)
23 ± 1°C
10 ll llSlYt, o ll QcirKllGSS
75 nE/m*/3 (500 ft c)
Cool-white fluorescent lights
75 |iE/m2/s (500 ft-c)
16 h light, 8 h darkness
200 mL polystyrene cups, or 250 mL Erlenmeyer flasks
100 mL (minimum)
5 female branch tips and 1 male plant
4 (minimum of 3)
20
-------
concentrations: 24 (minimum of 18)
12. Dilution water: 30%o salinity natural seawater, or a combination of 50% of
30%o salinity natural seawater and 50% of 30%o salinity GP2
artificial seawater (see Section 7, Dilution Water)
13. Test concentrations: Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water or minimum of 5
and a control
14. T-estti- Dilution factor: Effluents: >0.5
Receiving waters: None or >0.5
37. Replace item 18 in Table 3 (continued) on page 359 of the marine chronic method manual with
the following.
18. Sampling requirements: For on-site tests, Oone sample collected at test initiation,
and preferably used within 24 h of the time it is removed
from the sampling device. For off-site tests, holding
time must not exceed 36 h before first use (see Section
8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests,
Subsection 8.5.4)
38. Insert the following references into the Cited References section on page 380 of the marine
chronic method manual.
Casarett, L.J. and J. Doull. 1975. Toxicology: the basic science of poisons. Macmillan Publishing Co.,
New York.
Lussier, S.M., A. Kuhn, and R. Comeleo. 1999. An evaluation of the seven-day toxicity test with
Americamysis bahia (formerlyMysidopsis bahia). Environ. Toxicol. Chem. 18(12): 2888-2893.
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.
Price, W.W., R.W. Heard, and L. Stuck. 1994. Observations on the genus Mysidopsis Sars, 1864 with
the designation of a new genus, Americamysis, and the descriptions of Americamysis alleni and
Americamysis stucki (Peracarida: Mysidacea: Mysidae), from the Gulf of Mexico. Proc. Biol.
Soc. Wash. 107: 680-698.
USEPA. 1996. Marine toxicity identification evaluation (TIE): Phase I guidance document. R.M.
Burgess, K.T. Ho, G.E. Morrison, G. Chapman, and D.L. Denton (eds.). National Health and
Environmental Effects Research Laboratory, U.S. Environmental Protection Agency,
Narragansett, RI. EPA/600/R-96/054.
21
-------
USEPA. 2000a. Method guidance and recommendations for whole effluent toxicity (WET) testing (40
CFR Part 136). Office of Water, U.S. Environmental Protection Agency, Washington, B.C.
20460. EPA/82l/B-00/004.
USEPA. 2000b. Understanding and accounting for method variability in whole effluent toxicity
applications under the national pollutant discharge elimination system program. Office of
Wastewater Management, U.S. Environmental Protection Agency, Washington, B.C. 20460.
EPA/833/R-00/003.
USEPA. 200 la. Final report: interlaboratory variability study of EPA short-term chronic and acute
whole effluent toxicity test methods, Vol. 1. Office of Water, U.S. Environmental Protection
Agency, Washington, B.C. 20460. EPA/821/B-01/004.
USEPA. 200 Ib. Final report: interlaboratory variability study of EPA short-term chronic and acute
whole effluent toxicity test methods, Vol. 2: Appendix. Office of Water, U.S. Environmental
Protection Agency, Washington, B.C. 20460. EPA/821/B-01/004.
22
-------
III. Precision
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Replace Subsection 4.13.4 on page 12 of the acute method manual with the following.
4.13.4 Inter-laboratory precision of acute toxicity tests from 253 reference toxicant tests with seven
species, listed in Tables 2, 3, 4, and 5 (expressed as CV% for LCSOs), ranged from 11% to 167%. Table
6 shows interlaboratory precision data from a study of acute toxicity test methods using reference
toxicant, effluent, and receiving water sample types (USEPA, 2001a; USEPA, 2001b). Averaged across
sample types, total interlaboratory precision (expressed as CV% for LCSOs) ranged from 13% to 38.5%
for the acute methods.
2. Insert the following table after Table 5 on page 17 of the acute method manual.
TABLE 6. NATIONAL INTERLABORATORY STUDY OF ACUTE TOXICITY TEST
PRECISION, 2000: PRECISION OF LC50 POINT ESTIMATES FOR REFERENCE
TOXICANT, EFFLUENT, AND RECEIVING WATER SAMPLE TYPES1
CV (%)2
Method Sample Type
Within-lab3 Between-lab4
Pimephales promelas KC1 7.62 19.7
Municipal effluent 10.3 19.2
Receiving water
Average 8.96 19.4
Ceriodaphnia dubia KC1 14.6 15.2
Municipal effluent 9.68 32.8
Receiving water
Average 12.1 24.0
Total5
21.1
21.8
17.2
20.0
21.1
34.2
31.8
29.0
23
-------
Cyprinodon variegatus KC1
Municipal effluent
Receiving water
26.0
19.4
32.5
Average
26.0
Menidia beryllina
CuSO46
Industrial effluent
Receiving water
9.91
49.7
50.7
26.3
Average
9.91
49.7
38.5
Holmesimysis costata1
Zn (48 h test)
Zn (96 h test)
Zn (interlaboratory trial 1)
Zn (interlaboratory trial 2)
19
23
24
1
Average
21
13
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
CVs were calculated based on the within-laboratory component of variability, the between-
laboratory component of variability, and total interlaboratory variability (including both within-
laboratory and between-laboratory components). For the receiving water sample type, within-
laboratory and between-laboratory components of variability could not be calculated since the
study design did not provide within-laboratory replication for this sample type. The study design
also did not provide within-laboratory replication for the Cyprinodon variegatus Acute Method.
The within-laboratory (intralaboratory) component of variability for duplicate samples tested at
the same time in the same laboratory.
The between-laboratory component of variability for duplicate samples tested at different
laboratories.
The total interlaboratory variability, including within-laboratory and between-laboratory
components of variability. The total interlaboratory variability is synonymous with
interlaboratory variability reported from other studies where individual variability components
are not separated.
Precision estimates were not calculated for the reference toxicant sample type since the majority
of results for this sample type were outside of the test concentration range (ie., >100).
24
-------
Holmesimysis costata Acute Test data were from Martin et al. (1989). Zn was tested in two
intralaboratory trials and in two interlaboratory trials. Data from this study was only reported to
two significant figures.
B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Replace Subsection 4.14.3 on page 14 of the freshwater chronic method manual with the
following.
4.14.3 Interlaboratory precision data from a 1991 study of chronic toxicity tests with two species using
the reference toxicants potassium chloride and copper sulfate are shown in Table 1. Table 2 shows
interlaboratory precision data from a study of three chronic toxicity test methods using effluent, receiving
water, and reference toxicant sample types (USEPA, 200la; USEPA, 200Ib). The effluent sample was a
municipal wastewater spiked with KC1, the receiving water sample was a river water spiked with KC1,
and the reference toxicant sample consisted of moderately-hard synthetic freshwater spiked with KC1.
Additional precision data for each of the tests described in this manual are presented in the sections
describing the individual test methods.
2. Insert the following table after Table 1 on page 15 of the freshwater chronic method manual.
TABLE 2. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 2000: PRECISION OF RESPONSES USING EFFLUENT, RECEIVING
WATER, AND REFERENCE TOXICANT SAMPLE TYPES1.
Organism Endpoint Number of Tests2 CV (%)3
Pimephalespromelas Growth, IC25 73 20.9
Ceriodaphnia dubia Reproduction, IC25 34 35.0
Selenastrum capricornutum
(with EDTA)
Selenastrum capricornutum
(without EDTA)
Growth, IC25
Growth, IC50
Growth, IC25
Growth, IC50
21
22
21
22
34.3
32.2
58.5
58.5
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
25
-------
2 Represents the number of valid tests (i.e., those that met test acceptability criteria) that were used
in the analysis of precision. Invalid tests were not used.
3 CVs based on total interlaboratory variability (including both within-laboratory and between-
laboratory components of variability) and averaged across sample types. IC25s or ICSOs were
pooled for all laboratories to calculate the CV for each sample type. The resulting CVs were
then averaged across sample types.
3. Insert the following into Subsection 11.14.1, Precision, on page 108 of the freshwater chronic
method manual.
11.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below
(Subsections 11.14.1.1 and 11.14.1.2). Single-laboratory precision is a measure of the reproducibility of
test results when tests are conducted using a specific method under reasonably constant conditions in the
same laboratory. Single-laboratory precision is synonymous with the terms within-laboratory precision
and intralaboratory precision. Multilaboratory precision is a measure of the reproducibility of test results
from different laboratories using the same test method and analyzing the same test material.
Multilaboratory precision is synonymous with the term interlaboratory precision. Interlaboratory
precision, as used in this document, includes both within-laboratory and between-laboratory components
of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined
(termed total interlaboratory variability). The total interlaboratory variability that is reported from these
studies is synonymous with interlaboratory variability reported from other studies where individual
variability components are not separated.
4. Insert the following into Subsection 11.14.1.1, Single-Laboratory Precision, on page 108 of the
freshwater chronic method manual.
11.14.1.1.2 EPA evaluated within-laboratory precision of the Fathead Minnow, Pimephales promelas,
Larval Survival and Growth Test using a database of routine reference toxicant test results from 19
laboratories (USEPA, 2000b). The database consisted of 205 reference toxicant tests conducted in 19
laboratories using a variety of reference toxicants including: cadmium, chromium, copper, potassium
chloride, sodium chloride, sodium pentachlorophenate, and sodium dodecyl sulfate. Among the 19
laboratories, the median within-laboratory CV calculated for routine reference toxicant tests was 26% for
the IC25 growth endpoint. In 25% of laboratories, the within-laboratory CV was less than 21%; and in
75% of laboratories, the within-laboratory CV was less than 38%.
5. Insert the following into Subsection 11.14.1.2, Multilaboratory Precision, on page 111 of the
freshwater chronic method manual.
11.14.1.2.2 In 2000, EPA conducted an interlaboratory variability study of the Fathead Minnow,
Pimephales promelas, Larval Survival and Growth Test (USEPA, 200 la; USEPA, 200 Ib). In this study,
each of 27 participant laboratories tested 3 or 4 blind test samples that included some combination of
blank, effluent, reference toxicant, and receiving water sample types. The blank sample consisted of
moderately-hard synthetic freshwater, the effluent sample was a municipal wastewater spiked with KC1,
the receiving water sample was a river water spiked with KC1, and the reference toxicant sample
consisted of moderately-hard synthetic freshwater spiked with KC1. Of the 101 Fathead Minnow Larval
Survival and Growth tests conducted in this study, 98.0% were successfully completed and met the
required test acceptability criteria. Of 24 tests that were conducted on blank samples, none showed false
26
-------
positive results for survival endpoints, and only one resulted in false positive results for the growth
endpoint, yielding a false positive rate of 4.35%. Results from the reference toxicant, effluent, and
receiving water sample types were used to calculate the precision of the method. Table 22 shows the
precision of the IC25 for each of these sample types. Averaged across sample types, the total
interlaboratory variability (expressed as a CV%) was 20.9% for IC25 results. Table 23 shows the
frequency distribution of survival and growth NOEC endpoints for each sample type. For the survival
endpoint, NOEC values spanned four concentrations for the reference toxicant sample type and two
concentrations for the effluent and receiving water sample types. The percentage of values within one
concentration of the median was 97.2%, 100%, and 100% for the reference toxicant, effluent, and
receiving water sample types, respectively. For the growth endpoint, NOEC values spanned five
concentrations for the reference toxicant sample type and four concentrations for the effluent and
receiving water sample types. The percentage of values within one concentration of the median was
86.1%, 91.7%, and 76.9% for the reference toxicant, effluent, and receiving water sample types,
respectively.
6. Replace the footnotes to Table 20 on page 112 of the freshwater chronic method manual with
the following.
1 From DeGraeve et al., 1988.
*—Percent of values within one concentration intervals of the median.
*—Percent of values within two or more concentrations intervals of the median.
2 Percent of values at one concentration interval above or below the median. Adding this percentage to
the percent of values at the median yields the percent of values within one concentration interval of
the median.
3 Percent of values two or more concentration intervals above or below the median.
7. Replace the footnotes to Table 21 on page 113 of the freshwater chronic method manual with
the following.
1 From DeGraeve et al., 1988.
*—Percent of values within one concentration intervals of the median.
*—Percent of values within two or more concentrations intervals of the median.
2 Percent of values at one concentration interval above or below the median. Adding this percentage to
the percent of values at the median yields the percent of values within one concentration interval of
the median.
3 Percent of values two or more concentration intervals above or below the median.
8. Insert the following tables after Table 21 on page 113 of the freshwater chronic method manual.
TABLE 22. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES'.
Test Endpoint Sample Type
Within-lab3 Between-lab4 Total5
IC25 Reference toxicant 10.0 17.2 19.9
27
-------
Effluent
Receiving water
19.1
12.9
23.1
19.8
Average
14.6
15.0
20.9
1 From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
2 CVs were calculated based on the within-laboratory component of variability, the between-laboratory
component of variability, and total interlaboratory variability (including both within-laboratory and
between-laboratory components). For the receiving water sample type, within-laboratory and
between-laboratory components of variability could not be calculated since the study design did not
provide within-laboratory replication for this sample type.
3 The within-laboratory component of variability for duplicate samples tested at the same time in the
same laboratory.
4 The between-laboratory component of variability for duplicate samples tested at different
laboratories.
5 The total interlaboratory variability, including within-laboratory and between-laboratory components
of variability. The total interlaboratory variability is synonymous with interlaboratory variability
reported from other studies where individual variability components are not separated.
TABLE 23. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR
VARIOUS SAMPLE TYPES1.
Test Endpoint
Survival
NOEC
Growth
NOEC
Sample Type
Reference toxicant
Effluent
Receiving water
Reference toxicant
Effluent
Receiving water
Median
NOEC
Value
50%
12.5%
25%
50%
12.5%
12.5%
% of Results
at the Median
75.0
76.9
69.2
58.3
66.7
30.8
% of Results
±12
22.2
23.1
30.8
27.8
25.0
46.1
% of Results
>23
2.78
0.00
0.00
13.9
8.33
23.1
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
28
-------
2 Percent of values at one concentration interval above or below the median. Adding this percentage to
the percent of values at the median yields the percent of values within one concentration interval of
the median.
3 Percent of values two or more concentration intervals above or below the median.
9. Insert the following into Subsection 13.14.1, Precision, on page 189 of the freshwater chronic
method manual.
13.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below
(Subsections 13.14.1.1 and 13.14.1.2). Single-laboratory precision is a measure of the reproducibility of
test results when tests are conducted using a specific method under reasonably constant conditions in the
same laboratory. Single-laboratory precision is synonymous with the terms within-laboratory precision
and intralaboratory precision. Multilaboratory precision is a measure of the reproducibility of test results
from different laboratories using the same test method and analyzing the same test material.
Multilaboratory precision is synonymous with the term interlaboratory precision. Interlaboratory
precision, as used in this document, includes both within-laboratory and between-laboratory components
of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined
(termed total interlaboratory variability). The total interlaboratory variability that is reported from these
studies is synonymous with interlaboratory variability reported from other studies where individual
variability components are not separated.
10. Insert the following into Subsection 13.14.1.1, Single-Laboratory Precision, on page 189 of the
freshwater chronic method manual.
13.14.1.1.3 EPA evaluated within-laboratory precision of the Daphnid, Ceriodaphnia dubia, Survival
and Reproduction Test using a database of routine reference toxicant test results from 33 laboratories
(USEPA, 2000b). The database consisted of 393 reference toxicant tests conducted in 33 laboratories
using a variety of reference toxicants including: cadmium, copper, potassium chloride, sodium chloride,
and sodium pentachlorophenate. Among the 33 laboratories, the median within-laboratory CV calculated
for routine reference toxicant tests was 27% for the IC25 reproduction endpoint. In 25% of laboratories,
the within-laboratory CV was less than 17%; and in 75% of laboratories, the within-laboratory CV was
less than 45%.
11. Insert the following into Subsection 13.14.1.2, Multilaboratory Precision, on page 192 of the
freshwater chronic method manual.
13.14.1.2.3 In 2000, EPA conducted an interlaboratory variability study of the Daphnid, Ceriodaphnia
dubia, Survival and Reproduction Test (USEPA, 2001a; USEPA, 2001b). In this study, each of 34
participant laboratories tested 3 or 4 blind test samples that included some combination of blank,
effluent, reference toxicant, and receiving water sample types. The blank sample consisted of
moderately-hard synthetic freshwater, the effluent sample was a municipal wastewater spiked with KC1,
the receiving water sample was a river water spiked with KC1, and the reference toxicant sample
consisted of moderately-hard synthetic freshwater spiked with KC1. Of the 122 Ceriodaphnia dubia
Survival and Reproduction tests conducted in this study, 82.0% were successfully completed and met the
required test acceptability criteria. Of 27 tests that were conducted on blank samples, none showed false
positive results for survival endpoints, and only one resulted in false positive results for the growth
endpoint, yielding a false positive rate of 3.70%. Results from the reference toxicant, effluent, and
29
-------
receiving water sample types were used to calculate the precision of the method. Table 20 shows the
precision of the IC25 for each of these sample types. Averaged across sample types, the total
interlaboratory variability (expressed as a CV%) was 35.0% for IC25 results. Table 21 shows the
frequency distribution of survival and growth NOEC endpoints for each sample type. For the survival
endpoint, NOEC values spanned three concentrations for the reference toxicant and effluent sample types
and two concentrations for the receiving water sample type. The percentage of values within one
concentration of the median was 97.2%, 91.3%, and 100% for the reference toxicant, effluent, and
receiving water sample types, respectively. For the growth endpoint, NOEC values spanned five
concentrations for the reference toxicant sample type, three concentrations for the effluent sample type,
and two concentrations for the receiving water sample type. The percentage of values within one
concentration of the median was 83.3%, 100%, and 100% for the reference toxicant, effluent, and
receiving water sample types, respectively.
12. Insert the following tables after Table 19 on page 195 of the freshwater chronic method
manual.
TABLE 20. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES'.
Test Endpoint Sample Type
Within-lab3 Between-lab4 Total5
IC25 Reference toxicant ...
Effluent 17.4 27.6 32.6
Receiving water - - 37.4
Average 17.4 27.6 35.0
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
; CVs were calculated based on the within-laboratory component of variability, the between-
laboratory component of variability, and the total interlaboratory variability (including both
within-laboratory and between-laboratory components). For the reference toxicant sample type a
majority of the results were outside of the test concentration range, so precision estimates were
not calculated. For the receiving water sample type, within-laboratory and between-laboratory
components of variability could not be calculated since the study design did not provide within-
laboratory replication for this sample type.
The within-laboratory (intralaboratory) component of variability for duplicate samples tested at
the same time in the same laboratory.
1 The between-laboratory component of variability for duplicate samples tested at different
laboratories.
The total interlaboratory variability, including within-laboratory and between-laboratory
components of variability. The total interlaboratory variability is synonymous with
interlaboratory variability reported from other studies where individual variability components
are not separated.
30
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TABLE 21. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR
VARIOUS SAMPLE TYPES1.
Test Endpoint Sample Type
Median
NOEC
Value
% of Results % of Results
at the Median ±12
% of Results
>23
Survival
NOEC
Reference toxicant
100%
97.2
0.00
2.78
Effluent
Receiving water
25%
25%
65.2
90.0
26.1
10.0
8.70
0.00
Growth
NOEC
Reference toxicant
100%
72.2
11.1
16.7
Effluent
Receiving water
12.5%
25%
70.8
70.0
29.2
30.0
0.00
0.00
1 From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
2 Percent of values at one concentration interval above or below the median. Adding this
percentage to the percent of values at the median yields the percent of values within one
concentration interval of the median.
3 Percent of values two or more concentration intervals above or below the median.
13. Insert the following into Subsection 14.14.1, Precision, on page 225 of the freshwater chronic
method manual.
14.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below
(Subsections 14.14.1.1 and 14.14.1.2). Single-laboratory precision is a measure of the reproducibility of
test results when tests are conducted using a specific method under reasonably constant conditions in the
same laboratory. Single-laboratory precision is synonymous with the terms within-laboratory precision
and intralaboratory precision. Multilaboratory precision is a measure of the reproducibility of test results
from different laboratories using the same test method and analyzing the same test material.
Multilaboratory precision is synonymous with the term interlaboratory precision. Interlaboratory
precision, as used in this document, includes both within-laboratory and between-laboratory components
of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined
(termed total interlaboratory variability). The total interlaboratory variability that is reported from these
studies is synonymous with interlaboratory variability reported from other studies where individual
variability components are not separated.
31
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14. Insert the following into Subsection 14.14.1.1, Single-Laboratory Precision, on page 225 of the
freshwater chronic method manual.
14.14.1.1.2 EPA evaluated within-laboratory precision of the green alga, Selenastrum capricornutum,
Growth Test using a database of routine reference toxicant test results from nine laboratories (USEPA,
2000b). The database consisted of 85 reference toxicant tests conducted in 9 laboratories using a variety
of reference toxicants including: copper, sodium chloride, and zinc. Among the 9 laboratories, the
median within-laboratory CV calculated for routine reference toxicant tests was 26% for the IC25 growth
endpoint. In 25% of laboratories, the within-laboratory CV was less than 25%; and in 75% of
laboratories, the within-laboratory CV was less than 39%.
15. Replace Subsection 14.14.1.2.1 on page 225 of the freshwater chronic method manual with the
following.
1^4. 1^4. i .2*. i Ucitci on tiic inuitiiciDorciiory precision or tins test circ HOT yet 3-V3.ii3.Dic. in .zuuu.,
conducted an interlaboratory variability study of the green alga, Selenastrum capricornutum, Growth
Test (USEPA, 2001a; USEPA, 2001b). In this study, each of 1 1 participant laboratories tested 4 blind
test samples that included some combination of blank, effluent, reference toxicant, and receiving water
sample types. The blank sample consisted of moderately-hard synthetic freshwater, the effluent sample
was a municipal wastewater spiked with KC1, the receiving water sample was a river water spiked with
KC1, and the reference toxicant sample consisted of moderately-hard synthetic freshwater spiked with
KC1. Each sample was tested with and without the addition of EDTA. Of the 44 Selenastrum
capricornutum Growth tests conducted with EDTA, 63.6% were successfully completed and met the
required test acceptability criteria. Of the 44 tests conducted without EDTA, 65.9% were successfully
completed and met the required test acceptability criteria. Of five tests that were conducted on blank
samples with the addition of EDTA, none showed false positive results for the growth endpoint. Of 6
tests that were conducted on blank samples without the addition of EDTA, 2 showed false positive
results for the growth endpoint, yielding a false positive rate of 33.3%. Results from the reference
toxicant, effluent, and receiving water sample types were used to calculate the precision of the method.
Table 13 shows the precision of the IC25 for each of these sample types. Averaged across sample types,
the total interlaboratory variability (expressed as a CV%) was 34.3% and 58.5% for IC25 results in tests
with EDTA and without EDTA, respectively. Table 14 shows the precision of growth NOEC endpoints
for each sample type. NOEC values for tests with EDTA spanned three concentrations for the effluent
sample type and four concentrations for the reference toxicant and receiving water sample types. NOEC
values for tests without EDTA, spanned six concentrations for the reference toxicant sample type, four
concentrations for the effluent sample type, and two concentrations for the receiving water sample type.
The percentage of values within one concentration of the median for tests conducted with EDTA was
85.7%, 100%, and 85.7% for the reference toxicant, effluent, and receiving water sample types,
respectively. The percentage of values within one concentration of the median for tests conducted
without EDTA was 40.0%, 50.0%, and 100% for the reference toxicant, effluent, and receiving water
sample types, respectively.
32
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16. Insert the following tables after Table 12 on page 228 of the freshwater chronic method
manual.
TABLE 13. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPESl.
Test Endpoint
IC25
(with EDTA)
IC25
(without EDTA)
Sample Type
Reference toxicant
Effluent
Receiving water
Average
Reference toxicant
Effluent
Receiving water
Average
Within-lab3
10.9
39.5
25.2
25.6
21.0
23.3
CV (%)2
Between-lab4
20.8
8.48
14.6
83.6
60.3
72.0
Total5
23.5
40.4
38.9
34.3
87.5
63.9
24.1
58.5
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
CVs were calculated based on the within-laboratory component of variability, the between-
laboratory component of variability, and the total interlaboratory variability (including both
within-laboratory and between-laboratory components). For the receiving water sample type,
within-laboratory and between-laboratory components of variability could not be calculated since
the study design did not provide within-laboratory replication for this sample type.
The within-laboratory (intralaboratory) component of variability for duplicate samples tested at
the same time in the same laboratory.
The between-laboratory component of variability for duplicate samples tested at different
laboratories.
The total interlaboratory variability, including within-laboratory and between-laboratory
components of variability. The total interlaboratory variability is synonymous with
interlaboratory variability reported from other studies where individual variability components
are not separated.
33
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TABLE 14. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR
VARIOUS SAMPLE TYPES1.
Test Endpoint Sample Type
Median
NOEC
Value
% of Results % of Results
at the Median ±12
% of Results
>23
Growth NOEC
(with EDTA)
Reference toxicant
Effluent
Receiving water
25%
6.25%
12.5%
57.1
42.9
28.6
28.6
57.1
57.1
14.3
0.00
14.3
Growth NOEC Reference toxicant 18.8%
(without EDTA) Effluent 18.8%
Receiving water 6.25%
75.0
40.0
50.0
25.0
60.0
50.0
0.00
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
Percent of values at one concentration interval above or below the median. Adding this
percentage to the percent of values at the median yields the percent of values within one
concentration interval of the median.
Percent of values two or more concentration intervals above or below the median.
The median NOEC fell between test concentrations, so no test results fell precisely on the
median.
C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Replace Subsection 4.14.3 on page 17 of the marine chronic method manual with the following.
4.14.3 Interlaboratory precision data from a 1991 study of chronic toxicity tests using two reference
toxicants with the mysid, Mysidopsis bahia, and the inland silverside, Menidia beryllina, is listed in
Table 1. Table 2 shows interlaboratory precision data from a study of three chronic toxicity test methods
using effluent, receiving water, and reference toxicant sample types (USEPA, 200la; USEPA, 200Ib).
For the Mysidopsis bahia and the Cyprinodon variegatus test methods, the effluent sample was a
municipal wastewater spiked with KC1, the receiving water sample was a river water spiked with KC1,
and the reference toxicant sample was bioassay-grade Forty Fathoms® synthetic seawater spiked with
KC1. For the Menidia beryllina test method, the effluent sample was an industrial wastewater spiked
with CuSO4, the receiving water sample was a natural seawater spiked with CuSO4, and the reference
toxicant sample was bioassay-grade Forty Fathoms® synthetic seawater spiked with CuSO4. Additional
precision data for each of the tests described in this manual are presented in the sections describing the
individual test methods.
34
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2. Insert the following table after Table 1 on page 18 of the marine chronic method manual.
TABLE 2. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 2000: PRECISION OF RESPONSES USING EFFLUENT, RECEIVING
WATER, AND REFERENCE TOXICANT SAMPLE TYPES1.
Organism
Cyprinodon variegatus
Menidia beryllina
Mysidopsis bahia
Endpoint
Growth, IC25
Growth, IC25
Growth, IC25
Number of Tests2
21
30
36
CV (%)3
10.5
43.8
41.3
1 From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
2 Represents the number of valid tests (i.e., those that met test acceptability criteria) that were used
in the analysis of precision. Invalid tests were not used.
3 CVs based on total interlaboratory variability (including both within-laboratory and between-
laboratory components of variability) and averaged across sample types. IC25s or ICSOs were
pooled for all laboratories to calculate the CV for each sample type. The resulting CVs were
then averaged across sample types.
3. Insert the following into Subsection 11.14.1, Precision, on page 116 of the marine chronic
method manual.
11.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below
(Subsections 11.14.1.1 and 11.14.1.2). Single-laboratory precision is a measure of the reproducibility of
test results when tests are conducted using a specific method under reasonably constant conditions in the
same laboratory. Single-laboratory precision is synonymous with the terms within-laboratory precision
and intralaboratory precision. Multilaboratory precision is a measure of the reproducibility of test results
from different laboratories using the same test method and analyzing the same test material.
Multilaboratory precision is synonymous with the term interlaboratory precision. Interlaboratory
precision, as used in this document, includes both within-laboratory and between-laboratory components
of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined
(termed total interlaboratory variability). The total interlaboratory variability that is reported from these
studies is synonymous with interlaboratory variability reported from other studies where individual
variability components are not separated.
4. Insert the following into Subsection 11.14.1.1, Single-Laboratory Precision, on page 116 of the
marine chronic method manual.
11.14.1.1.2 EPA evaluated within-laboratory precision of the Sheepshead Minnow, Cyprinodon
variegatus, Larval Survival and Growth Test using a database of routine reference toxicant test results
35
-------
from five laboratories (USEPA, 2000b). The database consisted of 57 reference toxicant tests conducted
in 5 laboratories using reference toxicants including: cadmium and potassium chloride. Among the 5
laboratories, the median within-laboratory CV calculated for routine reference toxicant tests was 13% for
the IC25 growth endpoint. In 25% of laboratories, the within-laboratory CV was less than 9%; and in
75% of laboratories, the within-laboratory CV was less than 14%.
5. Insert the following into Subsection 11.14.1.2, Multilaboratory Precision, on page 116 of the
marine chronic method manual.
11.14.1.2.2 In 2000, EPA conducted an interlaboratory variability study of the Sheepshead Minnow,
Cyprinodon variegatus, Larval Survival and Growth Test (USEPA, 200la; USEPA, 200Ib). In this
study, each of 7 participant laboratories tested 4 blind test samples that included blank, effluent,
reference toxicant, and receiving water sample types. The blank sample consisted of bioassay-grade
Forty Fathoms® synthetic seawater, the effluent sample was a municipal wastewater spiked with KC1, the
receiving water sample was a natural seawater spiked with KC1, and the reference toxicant sample
consisted of bioassay-grade Forty Fathoms® synthetic seawater spiked with KC1. Of the 28 Sheepshead
Minnow Larval Survival and Growth tests conducted in this study, 100% were successfully completed
and met the required test acceptability criteria. Of 7 tests that were conducted on blank samples, none
showed false positive results for the survival endpoint or the growth endpoint. Results from the
reference toxicant, effluent, and receiving water sample types were used to calculate the precision of the
method. Table 28 shows the precision of the IC25 for each of these sample types. Averaged across
sample types, the total interlaboratory variability (expressed as a CV%) was 10.5% for IC25 results.
Table 29 shows the frequency distribution of survival and growth NOEC endpoints for each sample type.
For the survival endpoint, NOEC values spanned two concentrations for the reference toxicant sample
type and one concentration for the effluent and receiving water sample types. The percentage of values
within one concentration of the median was 100% for each of the sample types. For the growth endpoint,
NOEC values spanned one concentration for the reference toxicant sample type and two concentrations
for the effluent and receiving water sample types. The percentage of values within one concentration of
the median was 100% for each of the sample types.
6. Insert the following tables after Table 27 on page 123 of the marine chronic method manual.
TABLE 28. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES'.
Test Endpoint Sample Type CV
0/\2
IC25 Reference toxicant 18.4
Effluent 6.12
Receiving water 7.15
Average 10.5
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
36
-------
CVs were calculated based on the total interlaboratory variability (including both within-
laboratory and between-laboratory components of variability). Individual within-laboratory and
between-laboratory components of variability could not be calculated since the study design did
not provide within-laboratory replication for this sample type.
TABLE 29. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR
VARIOUS SAMPLE TYPES1.
„ +TJ , . _ , „ % of Results % of Results % of Results
TestEndpomt Sample Type NOEC ... A/f ,. , 12 ,3
,, , at the Median ±1 >2J
Value
Reference toxicant 25% 57.1 42.9 0.00
Effluent
Receiving water
25%
25%
100
100
0.00
0.00
0.00
0.00
Reference toxicant 25% 100 0.00 0.00
Effluent
Receiving water
12.5%
12.5%
57.1
71.4
42.9
28.6
0.00
0.00
1 From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
2 Percent of values at one concentration interval above or below the median. Adding this
percentage to the percent of values at the median yields the percent of values within one
concentration interval of the median.
3 Percent of values two or more concentration intervals above or below the median.
7. Insert the following into Subsection 13.14.1, Precision, on page 216 of the marine chronic
method manual.
13.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below
(Subsections 13.14.1.1 and 13.14.1.2). Single-laboratory precision is a measure of the reproducibility of
test results when tests are conducted using a specific method under reasonably constant conditions in the
same laboratory. Single-laboratory precision is synonymous with the terms within-laboratory precision
and intralaboratory precision. Multilaboratory precision is a measure of the reproducibility of test results
from different laboratories using the same test method and analyzing the same test material.
Multilaboratory precision is synonymous with the term interlaboratory precision. Interlaboratory
precision, as used in this document, includes both within-laboratory and between-laboratory components
of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined
(termed total interlaboratory variability). The total interlaboratory variability that is reported from these
37
-------
studies is synonymous with interlaboratory variability reported from other studies where individual
variability components are not separated.
8. Insert the following into Subsection 13.14.1.1, Single-Laboratory Precision, on page 216 of the
marine chronic method manual.
13.14.1.1.2 EPA evaluated within-laboratory precision of the Inland Silverside, Menidia beryllina,
Larval Survival and Growth Test using a database of routine reference toxicant test results from 16
laboratories (USEPA, 2000b). The database consisted of 193 reference toxicant tests conducted in 16
laboratories using a variety of reference toxicants including: chromium, copper, potassium chloride, and
sodium dodecyl sulfate. Among the 16 laboratories, the median within-laboratory CV calculated for
routine reference toxicant tests was 27% for the IC25 growth endpoint. In 25% of laboratories, the
within-laboratory CV was less than 18%; and in 75% of laboratories, the within-laboratory CV was less
than 43%.
9. Replace Subsection 13.14.1.2.1 on page 216 of the marine chronic method manual with the
following.
13. 1^4. i .2*. i Ucitci on tiic inuitiiciDorciiory precision or TUG 11113.110. siivcrsiQC itirvcii survival emu. ^rovvrii test
arc not yet available. In 2000, EPA conducted an interlaboratory variability study of the Inland
Silverside, Menidia beryllina, Larval Survival and Growth Test (USEPA, 200la; USEPA, 200Ib). In
this study, each of 10 participant laboratories tested 4 blind test samples that included some combination
of blank, effluent, reference toxicant, and receiving water sample types. The blank sample consisted of
bioassay-grade Forty Fathoms® synthetic seawater, the effluent sample was an industrial wastewater
spiked with CuSO4, the receiving water sample was a natural seawater spiked with CuSO4, and the
reference toxicant sample consisted of bioassay-grade Forty Fathoms® synthetic seawater spiked with
CuSO4. Of the 40 Menidia beryllina Larval Survival and Growth tests conducted in this study, 100%
were successfully completed and met the required test acceptability criteria. Of seven tests that were
conducted on blank samples, none showed false positive results for survival endpoints or for the growth
endpoint. Results from the reference toxicant, effluent, and receiving water sample types were used to
calculate the precision of the method. Table 23 shows the precision of the IC25 for each of these sample
types. Averaged across sample types, the total interlaboratory variability (expressed as a CV%) was
43.8% for IC25 results. Table 24 shows the frequency distribution of survival and growth NOEC
endpoints for each sample type. For the survival endpoint, NOEC values spanned five concentrations for
the effluent, four concentrations for the reference toxicant sample type, and three concentrations for the
receiving water sample type. The percentage of values within one concentration of the median was
90.9%, 84.6%, and 85.7% for the reference toxicant, effluent, and receiving water sample types,
respectively. For the growth endpoint, NOEC values spanned four concentrations for the reference
toxicant and effluent sample types and three concentrations for the receiving water sample type. The
percentage of values within one concentration of the median was 90.9%, 91.7%, and 85.7% for the
reference toxicant, effluent, and receiving water sample types, respectively.
38
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10. Insert the following tables after Table 22 on page 221 of the marine chronic method manual.
TABLE 23. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPESl.
Test Endpoint
IC25
Sample Type
Reference toxicant
Effluent
Receiving water
Within-lab3
22.0
7.24
Average 14.6
CV (%)2
Between-lab4
29.1
55.5
42.3
Total5
36.4
56.0
39.1
43.8
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
CVs were calculated based on the within-laboratory component of variability, the between-
laboratory component of variability, and the total interlaboratory variability (including both
within-laboratory and between-laboratory components). For the receiving water sample type,
within-laboratory and between-laboratory components of variability could not be calculated since
the study design did not provide within-laboratory replication for this sample type.
The within-laboratory (intralaboratory) component of variability for duplicate samples tested at
the same time in the same laboratory.
The between-laboratory component of variability for duplicate samples tested at different
laboratories.
The total interlaboratory variability, including within-laboratory and between-laboratory
components of variability. The total interlaboratory variability is synonymous with
interlaboratory variability reported from other studies where individual variability components
are not separated.
TABLE 24. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR
VARIOUS SAMPLE TYPES1.
Test Endpoint
Survival
NOEC
Sample Type
Reference toxicant
Effluent
Receiving water
Median
NOEC
Value
12.5%
25%
25%
% of Results
at the Median
72.7
38.5
57.1
% of Results
±12
18.2
46.1
28.6
% of Results
>23
9.09
15.4
14.3
39
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NOE? Reference toxicant 12.5% 72.7 18.2 9.09
Effluent
Receiving water
25%
25%
41.7
57.1
50.0
28.6
8.33
14.3
1 From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
2 Percent of values at one concentration interval above or below the median. Adding this
percentage to the percent of values at the median yields the percent of values within one
concentration interval of the median.
3 Percent of values two or more concentration intervals above or below the median.
11. Insert the following into Subsection 14.14.1, Precision, on page 294 of the marine chronic
method manual.
14.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below
(Subsections 14.14.1.1 and 14. 14. 1 .2). Single-laboratory precision is a measure of the reproducibility of
test results when tests are conducted using a specific method under reasonably constant conditions in the
same laboratory. Single-laboratory precision is synonymous with the terms within-laboratory precision
and intralaboratory precision. Multilaboratory precision is a measure of the reproducibility of test results
from different laboratories using the same test method and analyzing the same test material.
Multilaboratory precision is synonymous with the term interlaboratory precision. Interlaboratory
precision, as used in this document, includes both within-laboratory and between-laboratory components
of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined
(termed total interlaboratory variability). The total interlaboratory variability that is reported from these
studies is synonymous with interlaboratory variability reported from other studies where individual
variability components are not separated.
12. Insert the following into Subsection 14.14.1.1, Single-Laboratory Precision, on page 294 of the
marine chronic method manual.
14.14.1.1.2 EPA evaluated within-laboratory precision of the Mysid, Mysidopsis bahia, Survival,
Growth, and Fecundity Test using a database of routine reference toxicant test results from 10
laboratories (USEPA, 2000b). The database consisted of 130 reference toxicant tests conducted in 10
laboratories using a variety of reference toxicants including: chromium, copper, and potassium chloride.
Among the 10 laboratories, the median within-laboratory CV calculated for routine reference toxicant
tests was 28% for the IC25 growth endpoint. In 25% of laboratories, the within-laboratory CV was less
than 24%; and in 75% of laboratories, the within-laboratory CV was less than 32%.
13. Replace Subsection 14.14.1.2.1 on page 294 of the marine chronic method manual with the
following.
1^4. 1^4. i .2*. i i lie inuitiiciDorciiory precision or TUG test iicis HOT yet DCCH CLCTcrtrnticci. in .zuuu,
conducted an interlaboratory variability study of the Mysid, Mysidopsis bahia, Survival, Growth, and
40
-------
Fecundity Test (USEPA, 200la; USEPA, 200Ib). In this study, each of 11 participant laboratories
tested 4 blind test samples that included some combination of blank, effluent, reference toxicant, and
receiving water sample types. The blank sample consisted of bioassay-grade Forty Fathoms® synthetic
seawater, the effluent sample was a municipal wastewater spiked with KC1, the receiving water sample
was a natural seawater spiked with KC1, and the reference toxicant sample consisted of bioassay-grade
Forty Fathoms® synthetic seawater spiked with KC1. Of the 44 Mysidopsis bahia Survival, Growth, and
Fecundity tests conducted in this study, 97.7% were successfully completed and met the required test
acceptability criteria. Of seven tests that were conducted on blank samples, none showed false positive
results for survival, growth, or fecundity endpoints. Results from the reference toxicant, effluent, and
receiving water sample types were used to calculate the precision of the method. Table 34 shows the
precision of the IC25 for each of these sample types. Averaged across sample types, the total
interlaboratory variability (expressed as a CV%) was 41.3% for growth IC25 results. Table 35 shows the
frequency distribution of survival and growth NOEC endpoints for each sample type. For the survival
endpoint, NOEC values spanned three concentrations for the reference toxicant, effluent, and receiving
water sample types. The percentage of values within one concentration of the median was 100% for each
of the sample types. For the growth endpoint, NOEC values spanned four concentrations for the
reference toxicant sample type and three concentrations for the effluent and receiving water sample
types. The percentage of values within one concentration of the median was 92.3%, 100%, and 100% for
the reference toxicant, effluent, and receiving water sample types, respectively. For the fecundity
endpoint, NOEC values spanned three concentrations for the reference toxicant, the effluent, and the
receiving water sample types. The percentage of values within one concentration of the median was
75.0%, 87.5%, and 66.7% for the reference toxicant, effluent, and receiving water sample types,
respectively.
14. Insert the following tables after Table 33 on page 299 of the marine chronic method manual.
TABLE 34. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES'.
Test Endpoint Sample Type
Within-lab3 Between-lab4 Total5
JP5f°r Reference toxicant 8.69 40.0 40.9
Growth
Effluent 5.26 36.6 37.0
Receiving water - - 45.9
Average 6.98 38.3 41.3
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
CVs were calculated based on the within-laboratory component of variability, the between-
laboratory component of variability, and the total interlaboratory variability (including both
within-laboratory and between-laboratory components). For the receiving water sample type,
within-laboratory and between-laboratory components of variability could not be calculated since
the study design did not provide within-laboratory replication for this sample type.
41
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The within-laboratory (intralaboratory) component of variability for duplicate samples tested at
the same time in the same laboratory.
The between-laboratory component of variability for duplicate samples tested at different
laboratories.
The total interlaboratory variability, including within-laboratory and between-laboratory
components of variability. The total interlaboratory variability is synonymous with
interlaboratory variability reported from other studies where individual variability components
are not separated.
TABLE 35. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR
VARIOUS SAMPLE TYPES1.
Test Endpoint Sample Type
Median
NOEC
Value
% of Results % of Results
at the Median ±12
% of Results
>23
Survival
NOEC
Growth
NOEC
Reference toxicant
25%
Reference toxicant
25%
46.2
38.5
0.00
Effluent
Receiving water
12.5%
12.5%
46.7
37.5
53.3
62.5
0.00
0.00
7.69
Effluent
Receiving water
12.5%
12.5%
46.7
50.0
53.3
50.0
0.00
0.00
Fecundity
NOEC
Reference toxicant
18.8%
75.0
25.0
Effluent
Receiving water
25%
9.38%
62.5
_4
25.0
66.7
12.5
33.3
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).
Percent of values at one concentration interval above or below the median. Adding this
percentage to the percent of values at the median yields the percent of values within one
concentration interval of the median.
Percent of values two or more concentration intervals above or below the median.
The median NOEC fell between test concentrations, so no test results fell precisely on the
median.
42
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15. Insert the following into Subsection 16.14.1.1, Single-Laboratory Precision, on page 373 of the
marine chronic method manual.
16.14.1.1.2 EPA evaluated single-laboratory (within-laboratory) precision of the Red Macroalga,
Champia parvula, Reproduction Test using a database of routine reference toxicant test results from two
laboratories (USEPA, 2000b). The database consisted of 23 reference toxicant tests conducted in 2
laboratories using reference toxicants including: copper and sodium dodecyl sulfate. The within-
laboratory CVs calculated for routine reference toxicant tests at these 2 laboratories were 58% and 59%
for the IC25 reproduction endpoint.
43
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IV. Blocking by Known Parentage
A. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Replace Figure 1 on page 156 of the freshwater chronic method manual with the following.
In each of
the 10 cups, »
is one »
culture »
parent with »
at least 8 :
young per I
female/cup I
EH*©©©©©©
DO©©©©
EH*©©©©©©
©©©©©©
©©©©©©
©©©©©©
©©©©©
©©©©©
o©©@©
1
6
2
4
4
2
4
3
3
5
6
4
5
2
6
5
2
6
5
1
4
5
6
1
3
3
6
4
6
2
2
2
3
6
5
1
1
2
4
4
5
1
1
3
1
4
3
5
1
6
3
3
4
5
2
6
5
1
2
3
'0
b
'0
b
0
b
b
o
b
b
b
0
18
O
17
O
p
o
14
O
O
'b
b
'b
o
28
O
27
O
p
o
24
O
O
'b
'b
'b
o
38
O
37
O
p
o
34
O
O
'b
'b
'b'b
00
48 58
oo
47 57
OO
oo
45 55
OO
44 54
OO
OO
'b'b
'b'b
Figure 1. Examples of a test board and randomizing template: {¥} 1) test board with positions for
six columns often replicate test chambers with each position numbered for recording
results on data sheets, f2) 2) cardboard randomizing template prepared by tin-owing a
single die randomly drawing numbers (1-6) for each position in each- a row across the
board, and f3} 3) test board (1) placed on top of the randomizing template (2) for the
purpose of assigning the position of treatment solutions (cups) test treatments (1-6)
within each row on the board block (row on the test board). Following placement of test
chambers, test organisms are allocated using blocking by known parentage. Test
organisms from a single brood cup are distributed to each treatment within a given block
(row on the test board).
2. Replace Subsection 13.10.2, Start of the Test, on page 160 of the freshwater chronic method
manual with the following.
13.10.2 START OF THE TEST
13.10.2.1 Label the test chambers with a marking pen. Use of color-coded tape to identify each treatment
and replicate is helpful. A minimum of five effluent concentrations and a control are used for each
effluent test. Each treatment (including the control) must have ten replicates.
44
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13. 10.2.2 The test solutions chambers -cart must be randomly assigned to a board using a template
(Figure 1) or by using a table of random numbers (see Appendix A). When using the randomized block
design, test ciicirriDGrs circ rcUiuorriizGCi only once, cit TUG Dc^innin^ or tuc test. rvtinciornizin^ trie position
of test chambers as described in Figure 1 (or equivalent) will assist in assigning test organisms using
blocking by known parentage (Subsection 13.10.2.4). A number of different templates should be
prepared, so that the same template is not used for every test, and the template used for each test should
be identified on the data sheet. The same template must not be used for every test.
13. 10.2.3 Neonates less than 24 h old, and all within 8 h of the same age, are required to begin the test.
The neonates are must be obtained from individual cultures using brood boards, as described above in
Subsection 13.6.16.6, Individual Culture (also see Section 6, Test Organisms). Neonates are must be
taken only from adults in individual cultures that have eight or more young in their third or subsequent
broods. These adults can be used as brood stock until they are 14 days old. If the neonates are held more
than one or two hours before using in the test, they should be fed (0.1 mL YCT and 0.1 mL algal
concentrate/ 15 mL of media). Record the age of test organisms, source, and feeding of neonates on test
data sheets.
13. 10.2.4 Ten brood cups, each with 8 or more young, are randomly selected from a brood board for use
in setting up a test. To start the test, neonates from these ten brood cups are distributed to each test
chamber on the test board (one per test chamber). Test organisms must be assigned to test chambers
using a block randomization procedure, such that offspring from a single female are distributed evenly
among the treatments, appearing once in every test concentration. This arrangement is referred to as
"blocking by known parentage". The technique used to achieve blocking by known parentage should be
recorded in the test data report. One effective technique is to block randomize the test board as described
in Figure 1 and transfer one neonate from the first brood cup is transferred to each of the six test
chambers in the first row on the test board (Figure 1). One neonate from the second brood cup is then
transferred to each of the six test chambers in the second row on the test board. This process is continued
until each of the 60 test chambers contains one neonate. The set of six test chambers (one for each test
treatment) containing organisms derived from a single female parent is referred to as a block. When
using the technique described in Figure 1, each row of the test board will represent a block.
13.10.2.4.1 The brood cups and test chambers may be placed on a light table to facilitate counting the
neonates. However, care must be taken to avoid temperature increase due to heat from the light table.
13. 10.2.4.2 Following the allocation of test organisms to the test board, additional neonates might
remain in the ten brood cups that were selected for test setup. These additional neonates may be
discarded or used as future culture organisms if needed.
13. 10.2.5 This blocking procedure Blocking by known parentage allows the performance of each female
test organism to be tracked to its parent culture organism. This technique ensures that any brood effects
(i.e., differences in test organism fecundity or sensitivity attributable to the source of parentage) are
evenly distributed among the test treatments. If a female produces one weak offspring or male, the
i or producing till "WGciK orrsprin^ or till niellos is ^reciter. JJy usin^ tins
technique Also, by knowing the parentage of each test organism, poor performance of young from a
given female blocks consisting largely of males can be omitted from all concentrations, test treatments at
the end of the test (see Subsection 13.13.1.4), decreasing variability among replicates.
45
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3. Insert the following into Subsection 13.10.9, Termination of the Test, on page 165 of the
freshwater chronic method manual.
13.10.9.3.1 In general, the occurrence of males in healthy, well-maintained individual cultures is rare. In
interlaboratory testing of the Ceriodaphnia dubia Survival and Reproduction Test, males were identified
in only 7% (9 of 126 tests) of tests conducted (USEPA, 2001a). The number of males identified in these
tests ranged from 1 to 12. In five tests containing a large number of males (4-12), laboratories
conducting those tests also noted that organism cultures were experiencing or recovering from some
stress. Since male production in cladoceran populations is generally associated with conditions of
environmental stress (Pennak, 1989), culture conditions should be examined whenever males are
identified in a test.
4. Replace item 10 in Table 3 on page 168 of the freshwater chronic method manual with the
following.
10. No. neonates per test chamber 1. Assigned using blocking by known
parentage (Subsection 13.10.2.4).
5. Insert the following into Subsection 13.13.1, General, on page 170 of the freshwater chronic
method manual.
13.13.1.4 At the end of the test, if 50% or more of the surviving organisms in a block are identified as
males, the entire block must be excluded from data analysis for the reproduction endpoint (i.e.,
calculation of the reproduction NOEC and IC25 as described in Subsection 13.13.3), but may be used in
the analysis of the survival endpoint (i.e., calculation of the survival NOEC and LC50 as described in
Subsection 13.13.2). For blocks having fewer than 50% of surviving organisms identified as males, the
males (not the entire block) must be excluded from the analysis of reproduction (i.e., calculation of the
reproduction NOEC and IC25 as described in Subsection 13.13.3), but may be used in the analysis of
survival (i.e., calculation of the survival NOEC and LC50 as described in Subsection 13.13.2). Note that
the exclusion of males from the analysis of reproduction may create unequal sample sizes among the
concentrations, influencing the statistical methods chosen for analysis of reproduction (Figure 6).
Determinations regarding test acceptability criteria for survival and reproduction (Subsection 13.12) must
be made prior to exclusion of any blocks. In addition to these test acceptability criteria, if fewer than
eight replicates in the control remain after excluding males and blocks with 50% or more of surviving
organisms identified as males, the test is invalid and must be repeated with a newly collected sample.
46
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V. pH Drift
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Replace Subsection 9.5.9 on page 49 of the acute method manual with the following.
9.5.9 pH drift Increases in pi I may occur in test solutions during acute, static-renewal, or non-renewal
toxicity tests may contribute to artifactual toxicity when ammonia or other pH-dependent toxicants (such
as metals) are present of pollutants such as ammonia. This problem can be minimized by conducting a
test in a static-renewal mode rather than a non-renewal mode, or the problem can be avoided by
conducting a test in a static renewal or flow through mode rather than a static-renewal or non-renewal
mode.
B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 11.3, Interferences, on page 58 of the freshwater chronic
method manual.
11.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. 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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 11.3.6.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 11.3.6.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
11.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 11.3.6.2. The pHto be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after warming to test temperature.
11.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
47
-------
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.2 pH units in pH-controlled tests (USEPA,
1992).
11.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
11.3.6.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 11.3.6.2) is applied routinely
to subsequent testing of the effluent.
11.3.6.2 The pHcan be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.8). pH can be controlled using the CO2-controlled atmosphere
technique by placing test solutions and test organisms in closed headspace test chambers, and then
injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b;
USEPA, 1992). Prior experimentation will be needed to determine the appropriate volume of CO2 to
inject. This volume will depend upon the sample pH, sample volume, container volume, and sample
constituents. If more than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the
headspace with no more than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine
the toxicity of the effluent in the receiving water, CO2 is injected to maintain the test pH at the pH of the
receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the
effluent, CO2 is injected to maintain the test pH at the initial pH of the sample after the sample has
warmed to test temperature. USEPA (1991b; 1992) and Mount and Mount (1992) provide techniques
and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-controlled testing,
control treatments must be subjected to all manipulations that sample treatments are subjected to. These
manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-
controlled testing, the pH also must be measured in each treatment at the beginning and end of each 24-h
exposure period to confirm that pH was effectively controlled at the target pH level.
11.3.6.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 11.3.6.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 11.3.6.2.
2. Insert the following into Subsection 12.3, Interferences, on page 114 of the freshwater chronic
method manual.
12.3.5 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
48
-------
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. 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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 12.3.5.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 12.3.5.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
12.3.5.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 12.3.5.2. The pH to be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after warming to test temperature.
12.3.5.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.2 pH units in pH-controlled tests (USEPA,
1992).
12.3.5.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
12.3.5.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 12.3.5.2) is applied routinely
to subsequent testing of the effluent.
12.3.5.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.8). pH can be controlled using the CO2-controlled atmosphere
technique by placing test solutions and test organisms in closed headspace test chambers, and then
injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b;
USEPA, 1992). Prior experimentation will be needed to determine the appropriate volume of CO2 to
inject. This volume will depend upon the sample pH, sample volume, container volume, and sample
constituents. If more than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the
headspace with no more than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine
49
-------
the toxicity of the effluent in the receiving water, CO2 is injected to maintain the test pH at the pH of the
receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the
effluent, CO2 is injected to maintain the test pH at the initial pH of the sample after the sample has
warmed to test temperature. USEPA (1991b; 1992) and Mount and Mount (1992) provide techniques
and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-controlled testing,
control treatments must be subjected to all manipulations that sample treatments are subjected to. These
manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-
controlled testing, the pH also must be measured in each treatment at the beginning and end of each 24-h
exposure period to confirm that pH was effectively controlled at the target pH level.
12.3.5.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 12.3.5.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 12.3.5.2.
3. Insert the following into Subsection 13.3, Interferences, on page 144 of the freshwater chronic
method manual.
13.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. 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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 13.3.6.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 13.3.6.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
13.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 13.3.6.2. The pH to be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after warming to test temperature.
13.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.2 pH units in pH-controlled tests (USEPA,
1992).
50
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13.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
13.3.6.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 13.3.6.2) is applied routinely
to subsequent testing of the effluent.
13.3.6.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.8). pH can be controlled using the CO2-controlled atmosphere
technique by placing test solutions and test organisms in closed headspace test chambers, and then
injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b;
USEPA, 1992). Prior experimentation will be needed to determine the appropriate volume of CO2 to
inject. This volume will depend upon the sample pH, sample volume, container volume, and sample
constituents. If more than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the
headspace with no more than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine
the toxicity of the effluent in the receiving water, CO2 is injected to maintain the test pH at the pH of the
receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the
effluent, CO2 is injected to maintain the test pH at the initial pH of the sample after the sample has
warmed to test temperature. USEPA (1991b; 1992) and Mount and Mount (1992) provide techniques
and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-controlled testing,
control treatments must be subjected to all manipulations that sample treatments are subjected to. These
manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-
controlled testing, the pH also must be measured in each treatment at the beginning and end of each 24-h
exposure period to confirm that pH was effectively controlled at the target pH level.
13.3.6.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 13.3.6.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 13.3.6.2.
C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Insert the following into Subsection 11.3, Interferences, on page 61 of the marine chronic
method manual.
11.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity
may increase or decrease with increasing pH. Lead and copper were found to be more acutely toxic at
51
-------
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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 11.3.6.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 11.3.6.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
11.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 11.3.6.2. The pHto be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after adjusting the sample salinity for use in marine
testing.
11.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.3 pH units in pH-controlled tests (USEPA,
1996).
11.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
11.3.6.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 11.3.6.2) is applied routinely
to subsequent testing of the effluent.
11.3.6.2 The pHcan be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.9). pH can be controlled using the CO2-controlled atmosphere
technique by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and
air (USEPA, 1996). Prior experimentation will be needed to determine the appropriate CO2/air ratio to
control pH at the target level. If the objective of the WET test is to determine the toxicity of the effluent
in the receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity
of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the initial pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and
52
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Mount (1992) provide techniques and guidance for controlling test pH using a CO2-controlled
atmosphere. In pH-controlled testing, control treatments must be subjected to all manipulations that
sample treatments are subjected to. These manipulations must be shown to cause no lethal or sublethal
effects on control organisms. In pH-controlled testing, the pH also must be measured in each treatment at
the beginning and end of each 24-h exposure period to confirm that pH was effectively controlled at the
target pH level.
11.3.6.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 11.3.6.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 11.3.6.2.
2. Insert the following into Subsection 12.3, Interferences, on page 124 of the marine chronic
method manual.
12.3.5 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. 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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 12.3.5.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 12.3.5.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
12.3.5.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 12.3.5.2. The pH to be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after adjusting the sample salinity for use in marine
testing.
12.3.5.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.3 pH units in pH-controlled tests (USEPA,
1996).
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12.3.5.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
12.3.5.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 12.3.5.2) is applied routinely
to subsequent testing of the effluent.
12.3.5.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.9). pH can be controlled using the CO2-controlled atmosphere
technique by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and
air (USEPA, 1996). Prior experimentation will be needed to determine the appropriate CO2/air ratio to
control pH at the target level. If the objective of the WET test is to determine the toxicity of the effluent
in the receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity
of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the initial pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and
Mount (1992) provide techniques and guidance for controlling test pH using a CO2-controlled
atmosphere. In pH-controlled testing, control treatments must be subjected to all manipulations that
sample treatments are subjected to. These manipulations must be shown to cause no lethal or sublethal
effects on control organisms. In pH-controlled testing, the pH also must be measured in each treatment at
the beginning and end of each 24-h exposure period to confirm that pH was effectively controlled at the
target pH level.
12.3.5.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 12.3.5.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 12.3.5.2.
3. Insert the following into Subsection 13.3, Interferences, on page 163 of the marine chronic
method manual.
13.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. 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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 13.3.6.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 13.3.6.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
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13.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 13.3.6.2. The pH to be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after adjusting the sample salinity for use in marine
testing.
13.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.3 pH units in pH-controlled tests (USEPA,
1996).
13.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
13.3.6.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 13.3.6.2) is applied routinely
to subsequent testing of the effluent.
13.3.6.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.9). pH can be controlled using the CO2-controlled atmosphere
technique by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and
air (USEPA, 1996). Prior experimentation will be needed to determine the appropriate CO2/air ratio to
control pH at the target level. If the objective of the WET test is to determine the toxicity of the effluent
in the receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity
of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the initial pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and
Mount (1992) provide techniques and guidance for controlling test pH using a CO2-controlled
atmosphere. In pH-controlled testing, control treatments must be subjected to all manipulations that
sample treatments are subjected to. These manipulations must be shown to cause no lethal or sublethal
effects on control organisms. In pH-controlled testing, the pH also must be measured in each treatment at
the beginning and end of each 24-h exposure period to confirm that pH was effectively controlled at the
target pH level.
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13.3.6.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 13.3.6.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 13.3.6.2.
4. Insert the following into Subsection 14.3, Interferences, on page 222 of the marine chronic
method manual.
14.3.4 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-
dependent toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also
increases (see Subsection 8.8.6), so upward pH drift may increase sample toxicity. 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). In situations where sample toxicity is confirmed to be artifactual and due to pH drift (as
determined by parallel testing as described in Subsection 14.3.4.1), the regulatory authority may allow
for control of sample pH during testing using procedures outlined in Subsection 14.3.4.2. It should be
noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1
pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for
toxicity.
14.3.4.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one
with controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to
drift during the test. In the controlled-pH treatment, the pH is maintained using the procedures described
in Subsection 14.3.4.2. The pH to be maintained in the controlled-pH treatment (or target pH) will
depend on the objective of the test. If the objective of the WET test is to determine the toxicity of the
effluent in the receiving water, the pH should be maintained at the pH of the receiving water. If the
objective of the WET test is to determine the absolute (end-of-pipe) toxicity of the effluent, the pH
should be maintained at the initial pH of the sample after adjusting the sample salinity for use in marine
testing.
14.3.4.1.1 During parallel testing, the pH must be measured in each treatment at the beginning and end
of each 24-h exposure period. pH measurements taken during the test must confirm that (1) pH was
effectively maintained at the target pH in the controlled-pH treatment, and (2) pH drift in the
uncontrolled-pH treatment was substantially greater than in the controlled-pH treatment. In general, the
range in pH (i.e., drift) occurring in the uncontrolled-pH treatment must be at least twice that of the range
observed in the controlled-pH treatment. Test procedures for conducting toxicity identification
evaluations (TIEs) recommend maintaining pH within ±0.3 pH units in pH-controlled tests (USEPA,
1996).
14.3.4.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing.
Total ammonia concentrations greater than 5 mg/L in the 100% effluent may indicate ammonia toxicity
(USEPA, 1992).
14.3.4.1.3 Following parallel testing, the toxicity observed in the controlled and uncontrolled-pH
treatments is compared. If toxicity is removed or reduced in the pH-controlled treatment, artifactual
toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of artifactual
toxicity is representative of a given effluent, the regulatory authority may require additional information
56
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or additional parallel testing before pH control (as described in Subsection 14.3.4.2) is applied routinely
to subsequent testing of the effluent.
14.3.4.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-
controlled atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding
IN NaOH or IN HC1 (see Subsection 8.8.9). pH can be controlled using the CO2-controlled atmosphere
technique by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and
air (USEPA, 1996). Prior experimentation will be needed to determine the appropriate CO2/air ratio to
control pH at the target level. If the objective of the WET test is to determine the toxicity of the effluent
in the receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the receiving water. If the objective of the WET test is to determine the absolute (end-of-pipe) toxicity
of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the initial pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and
Mount (1992) provide techniques and guidance for controlling test pH using a CO2-controlled
atmosphere. In pH-controlled testing, control treatments must be subjected to all manipulations that
sample treatments are subjected to. These manipulations must be shown to cause no lethal or sublethal
effects on control organisms. In pH-controlled testing, the pH also must be measured in each treatment at
the beginning and end of each 24-h exposure period to confirm that pH was effectively controlled at the
target pH level.
14.3.4.3 In rare circumstances, the daily cycle of upward pH drift and renewal (which returns the test pH
to the initial sample pH) may cause artifactual toxicity even in the absence of pH-dependent toxicants. If
toxicity is confirmed to be artifactual and due to pH drift (as determined by parallel testing as described
in Subsection 14.3.4.1), the regulatory authority may allow for control of sample pH during testing using
procedures outlined in Subsection 14.3.4.2.
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VI. Concentration-response Relationships
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Replace Section 12, Report Preparation, on page 119 of the acute method manual with the
following.
SECTION 12
REPORT PREPARATION AND TEST REVIEW
12.1 REPORT PREPARATION
The following general format and content are recommended for the report:
12.1.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body
5. Contract Laboratory (if the tests are performed under contract)
a Name of firm
b. Phone number
c. Address
12.1.2 PLANT OPERATIONS
1. Product(s)
2. Raw materials
3. Operating schedule
4. Description of waste treatment
5. Schematic of waste treatment
6. Retention time (if applicable)
7. Volume of waste flow (MOD, CFS, GPM)
8. Design flow of treatment facility at time of sampling
12.1.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Effluent Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Mean daily discharge on sample collection date
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f. Lapsed time from sample collection to delivery
g. Sample temperature when received at the laboratory
2. Receiving Water Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Streamflow (at 7Q10 and at time of sampling)
f. Sample temperature when received at the laboratory
g. Lapsed time from sample collection to delivery
3. Dilution Water Samples
a. Source
b. Collection date(s) and time(s)
c. Pretreatment
d. Physical and chemical characteristics
12.1.4 TEST METHODS
1. Toxicity test method used (title, number, source)
2. Endpoint(s) of test
3. Deviation(s) from reference method, if any, and the reason(s)
4. Date and time test started
5. Date and time test terminated
6. Type and volume of test chambers
7. Volume of solution used per chamber
8. Number of organisms per test chamber
9. Number of replicate test chambers per treatment
10. Acclimation of test organisms (temperature mean and range)
11. Test temperature (mean and range)
12. Specify if aeration was needed
13. Feeding frequency, and amount and type of food
12.1.5 TEST ORGANISMS
1. Scientific name and how determined
2. Age
3. Life stage
4. Mean length and weight (where applicable)
5. Source
6. Diseases and treatment (where applicable)
7. Taxonomic key used for species identification
12.1.6 QUALITY ASSURANCE
1. Reference toxicant used routinely: source
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2. Date and time of most recent reference toxicant test, test results, and current control
(cusum) chart
3. Dilution water used in reference toxicant test
4. Results (LC50 or where applicable, NOAEC)
5. Physical and chemical methods used
12.1.7 RESULTS
1. Provide raw toxicity data in tabular form, including daily records of affected organisms
in each concentration (including controls) and replicate, and in graphical form (plots of
toxicity data)
2. Provide table of LC50, NOAEC, Pass/Fail
3. Indicate statistical methods used to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
12.1.8 CONCLUSIONS AND RECOMMENDATIONS
1. Relationship between test endpoints and permit limits
2. Actions to be taken
12.2 TEST REVIEW
12.2.1 Test review is an important part of an overall quality assurance program (Section 4) and is
necessary for ensuring that all test results are reported accurately. Test review should be conducted on
each test by both the testing laboratory and the regulatory authority.
12.2.2 SAMPLING AND HANDLING
12.2.2.1 The collection and handling of samples are reviewed to verify that the sampling and handling
procedures given in Section 8 were followed. Chain-of-custody forms are reviewed to verify that
samples were tested within allowable sample holding times (Subsection 8.5.4). Any deviations from the
procedures given in Section 8 should be documented and described in the data report (Subsection 12.1).
12.2.3 TEST ACCEPTABILITY CRITERIA
12.2.3.1 Test data are reviewed to verify that test acceptability criteria (TAC) requirements for a valid
test have been met. Any test not meeting the minimum test acceptability criteria is considered invalid.
All invalid tests must be repeated with a newly collected sample.
12.2.4 TEST CONDITIONS
12.2.4.1 Test conditions are reviewed and compared to the specifications listed in the summary of test
condition tables provided for each method. Physical and chemical measurements taken during the test
(e.g., temperature, pH, and DO) also are reviewed and compared to specified ranges. Any deviations
from specifications should be documented and described in the data report (Subsection 12.1).
12.2.4.2 Deviations in test conditions (from the specifications listed in the summary of test condition
tables) must be evaluated to determine the validity of test results. Test condition deviations may or may
not invalidate a test result depending on the degree of the departure and the objective of the test. The
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reviewer should consider the degree of the deviation and the potential or observed impact of the
deviation on the test result before rejecting or accepting a test result as valid.
12.2.43 Whereas slight deviations in test conditions may not invalidate an individual test result, test
condition deviations that continue to occur frequently in a given laboratory may indicate the need for
improved quality control in that laboratory.
12.2.5 STATISTICAL METHODS
12.2.5.1 The statistical methods used for analyzing test data are reviewed to verify that the
recommended flowcharts for statistical analysis were followed. Any deviation from the recommended
flowcharts for selection of statistical methods should be noted in the data report. Statistical methods other
than those recommended in the statistical flowcharts may be appropriate (see Subsection 11.1.4),
however, the laboratory must document the use of and provide the rationale for the use of any alternate
statistical method. In all cases (flowchart recommended methods or alternate methods), reviewers should
verify that the necessary assumptions are met for the statistical method used.
12.2.6 CONCENTRATION-RESPONSE RELATIONSHIPS
12.2.6.1 The concept of a concentration-response, or more classically, a dose-response relationship is
"the most fundamental and pervasive one in toxicology" (Casarett and Doull, 1975). This concept
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, however, the particular shape of the concentration-
response curve may differ for each coupled toxicant and response pair. In general, more severe responses
(such as acute effects) occur at higher concentrations of the toxicant, and less severe responses (such as
chronic effects) occur at lower concentrations. A single toxicant also may produce multiple responses,
each characterized by a concentration-response relationship. 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 possesses toxicity and in identifying
anomalous test results.
12.2.6.2 The concentration-response relationship generated for each multi-concentration test must be
reviewed to ensure that calculated test results are interpreted appropriately. USEPA (2000a) provides
guidance on evaluating concentration-response relationships to assist in determining the validity of WET
test results. All WET test results (from multi-concentration tests) reported under the NPDES program
should be reviewed and reported according to USEPA guidance on the evaluation of concentration-
response relationships (USEPA, 2000a). This guidance provides review steps for 10 different
concentration-response patterns that may be encountered in WET test data. Based on the review, the
guidance provides one of three determinations: that calculated effect concentrations are reliable and
should be reported, that calculated effect concentrations are anomalous and should be explained, or that
the test was inconclusive and the test should be repeated with a newly collected sample. It should be
noted that the determination of a valid concentration-response relationship is not always clear cut. Data
from some tests may suggest consultation with professional toxicologists and/or regulatory officials.
Tests that exhibit unexpected concentration-response relationships also may indicate a need for further
investigation and possible retesting.
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12.2.7 REFERENCE TOXICANT TESTING
12.2.7.1 Test review of a given effluent or receiving water test should include review of the associated
reference toxicant test and current control chart. Reference toxicant testing and control charting is
required for documenting the quality of test organisms (Subsection 4.7) and ongoing laboratory
performance (Subsection 4.15). The reviewer should verify that a quality control reference toxicant test
was conducted according to the specified frequency required by the permitting authority or recommended
by the method (e.g., monthly). The test acceptability criteria, test conditions, concentration-response
relationship, and test sensitivity of the reference toxicant test are reviewed to verify that the reference
toxicant test conducted was a valid test. The results of the reference toxicant test are then plotted on a
control chart (see Subsection 4.15) and compared to the current control chart limits (± 2 standard
deviations).
12.2.7.2 Reference toxicant tests that fall outside of recommended control chart limits are evaluated to
determine the validity of associated effluent and receiving water tests (see Subsection 4.15). An out of
control reference toxicant test result does not necessarily invalidate associated test results. The reviewer
should consider the degree to which the reference toxicant test result fell outside of control chart limits,
the width of the limits, the direction of the deviation (toward increasing test organism sensitivity or
toward decreasing test organism sensitivity), the test conditions of both the effluent test and the reference
toxicant test, and the objective of the test. More frequent and/or concurrent reference toxicant testing
may be advantageous if recent problems (e.g., invalid tests, reference toxicant test results outside of
control chart limits, reduced health of organism cultures, or increased within-test variability) have been
identified in testing.
12.2.8 TEST VARIABILITY
12.2.8.1 The within-test variability of individual tests should be reviewed. Excessive within-test
variability may invalidate a test result and warrant retesting. For evaluating within-test variability,
reviewers should consult EPA guidance on upper and lower percent minimum significant difference
(PMSD) bounds (USEPA, 2000b).
12.2.8.2 In an effort to reduce the variability of WET test methods, USEPA guidance on WET
variability recommends implementing upper and lower bounds on the PMSD calculated in a test for the
sublethal endpoint (USEPA, 2000b). The minimum significant difference (MSB) is the smallest
difference between the control and another test treatment that can be determined as statistically
significant in a given test, and the PMSD is the MSB represented as a percentage of the control response.
The equation and examples of MSB calculations are shown in Subsection 11.3.7.4.4.
12.2.8.3 To assist in reviewing within-test variability, EPA recommends maintaining control charts of
PMSBs calculated for successive effluent tests (USEPA, 2000b). A control chart of PMSB values
characterizes the range of variability observed within a given laboratory, and allows comparison of
individual test PMSBs with the laboratory's typical range of variability. Control charts of other
variability and test performance measures, such as the MSB, standard deviation or CV of control
responses, or average control response, also may be useful for reviewing tests and minimizing variability.
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B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Replace Section 10, Report Preparation, on page 55 of the freshwater chronic method manual
with the following.
SECTION 10
REPORT PREPARATION AND TEST REVIEW
10.1 REPORT PREPARATION
The following general format and content are recommended for the report:
10.1.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body
5. Contract Laboratory (if the tests are performed under contract)
a Name of firm
b. Phone number
c. Address
10.1.2 PLANT OPERATIONS
1. Product(s)
2. Raw materials
3. Operating schedule
4. Description of waste treatment
5. Schematic of waste treatment
6. Retention time (if applicable)
7. Volume of waste flow (MOD, CFS, GPM)
8. Design flow of treatment facility at time of sampling
10.1.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Effluent Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Mean daily discharge on sample collection date
f Lapsed time from sample collection to delivery
g. Sample temperature when received at the laboratory
2. Receiving Water Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
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c. Sample collection method
d. Physical and chemical data
e. Streamflow (at 7Q10 and at time of sampling)
f. Sample temperature when received at the laboratory
g. Lapsed time from sample collection to delivery
3. Dilution Water Samples
a. Source
b. Collection date(s) and time(s)
c. Pretreatment
d. Physical and chemical characteristics
10.1.4 TEST METHODS
1. Toxicity test method used (title, number, source)
2. Endpoint(s) of test
3. Deviation(s) from reference method, if any, and the reason(s)
4. Date and time test started
5. Date and time test terminated
6. Type and volume of test chambers
7. Volume of solution used per chamber
8. Number of organisms per test chamber
9. Number of replicate test chambers per treatment
10. Acclimation of test organisms (temperature mean and range)
11. Test temperature (mean and range)
12. Specify if aeration was needed
13. Feeding frequency, and amount and type of food
14. Specify if (and how) pH control measures were implemented
10.1.5 TEST ORGANISMS
1. Scientific name and how determined
2. Age
3. Life stage
4. Mean length and weight (where applicable)
5. Source
6. Diseases and treatment (where applicable)
7. Taxonomic key used for species identification
10.1.6 QUALITY ASSURANCE
1. Reference toxicant used routinely; source
2. Date and time of most recent reference toxicant test, test results, and current control
(cusum) chart
3. Dilution water used in reference toxicant test
4. Results (NOEC or, where applicable, LOEC, LC50, EC50, IC25 and/or IC50); report
percent minimum significant difference (PMSD) calculated in reference toxicant test
5. Physical and chemical methods used
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10.1.7 RESULTS
1. Provide raw toxicity data in tabular form, including daily records of affected organisms
in each concentration (including controls) and replicate, and in graphical form (plots of
toxicity data)
2. Provide table of LCSOs, NOECs, IC25, IC50, etc.
3. Indicate statistical methods used to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
6. Provide percent minimum significant difference (PMSD) calculated for sublethal
endpoints
10.1.8 CONCLUSIONS AND RECOMMENDATIONS
1. Relationship between test endpoints and permit limits
2. Actions to be taken
10.2 TEST REVIEW
10.2.1 Test review is an important part of an overall quality assurance program (Section 4) and is
necessary for ensuring that all test results are reported accurately. Test review should be conducted on
each test by both the testing laboratory and the regulatory authority.
10.2.2 SAMPLING AND HANDLING
10.2.2.1 The collection and handling of samples are reviewed to verify that the sampling and handling
procedures given in Section 8 were followed. Chain-of-custody forms are reviewed to verify that
samples were tested within allowable sample holding times (Subsection 8.5.4). Any deviations from the
procedures given in Section 8 should be documented and described in the data report (Subsection 10.1).
10.2.3 TEST ACCEPTABILITY CRITERIA
10.2.3.1 Test data are reviewed to verify that test acceptability criteria (TAC) requirements for a valid
test have been met. Any test not meeting the minimum test acceptability criteria is considered invalid.
All invalid tests must be repeated with a newly collected sample.
10.2.4 TEST CONDITIONS
10.2.4.1 Test conditions are reviewed and compared to the specifications listed in the summary of test
condition tables provided for each method. Physical and chemical measurements taken during the test
(e.g., temperature, pH, and DO) also are reviewed and compared to specified ranges. Any deviations
from specifications should be documented and described in the data report (Subsection 10.1).
10.2.4.2 Deviations in test conditions (from the specifications listed in the summary of test condition
tables) must be evaluated to determine the validity of test results. Test condition deviations may or may
not invalidate a test result depending on the degree of the departure and the objective of the test. The
reviewer should consider the degree of the deviation and the potential or observed impact of the
deviation on the test result before rejecting or accepting a test result as valid.
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10.2.4.3 Whereas slight deviations in test conditions may not invalidate an individual test result, test
condition deviations that continue to occur frequently in a given laboratory may indicate the need for
improved quality control in that laboratory.
10.2.5 STATISTICAL METHODS
10.2.5.1 The statistical methods used for analyzing test data are reviewed to verify that the
recommended flowcharts for statistical analysis were followed. Any deviation from the recommended
flowcharts for selection of statistical methods should be noted in the data report. Statistical methods other
than those recommended in the statistical flowcharts may be appropriate (see Subsection 9.4.1.2),
however, the laboratory must document the use of and provide the rationale for the use of any alternate
statistical method. In all cases (flowchart recommended methods or alternate methods), reviewers should
verify that the necessary assumptions are met for the statistical method used.
10.2.6 CONCENTRATION-RESPONSE RELATIONSHIPS
10.2.6.1 The concept of a concentration-response, or more classically, a dose-response relationship is
"the most fundamental and pervasive one in toxicology" (Casarett and Doull, 1975). This concept
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, however, the particular shape of the concentration-
response curve may differ for each coupled toxicant and response pair. In general, more severe responses
(such as acute effects) occur at higher concentrations of the toxicant, and less severe responses (such as
chronic effects) occur at lower concentrations. A single toxicant also may produce multiple responses,
each characterized by a concentration-response relationship. 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 possesses toxicity and in identifying
anomalous test results.
10.2.6.2 The concentration-response relationship generated for each multi-concentration test must be
reviewed to ensure that calculated test results are interpreted appropriately. USEPA (2000a) provides
guidance on evaluating concentration-response relationships to assist in determining the validity of WET
test results. All WET test results (from multi-concentration tests) reported under the NPDES program
should be reviewed and reported according to USEPA guidance on the evaluation of concentration-
response relationships (USEPA, 2000a). This guidance provides review steps for 10 different
concentration-response patterns that may be encountered in WET test data. Based on the review, the
guidance provides one of three determinations: that calculated effect concentrations are reliable and
should be reported, that calculated effect concentrations are anomalous and should be explained, or that
the test was inconclusive and the test should be repeated with a newly collected sample. It should be
noted that the determination of a valid concentration-response relationship is not always clear cut. Data
from some tests may suggest consultation with professional toxicologists and/or regulatory officials.
Tests that exhibit unexpected concentration-response relationships also may indicate a need for further
investigation and possible retesting.
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10.2.7 REFERENCE TOXICANT TESTING
10.2.7.1 Test review of a given effluent or receiving water test should include review of the associated
reference toxicant test and current control chart. Reference toxicant testing and control charting is
required for documenting the quality of test organisms (Subsection 4.7) and ongoing laboratory
performance (Subsection 4.16). The reviewer should verify that a quality control reference toxicant test
was conducted according to the specified frequency required by the permitting authority or recommended
by the method (e.g., monthly). The test acceptability criteria, test conditions, concentration-response
relationship, and test sensitivity of the reference toxicant test are reviewed to verify that the reference
toxicant test conducted was a valid test. The results of the reference toxicant test are then plotted on a
control chart (see Subsection 4.16) and compared to the current control chart limits (± 2 standard
deviations).
10.2.7.2 Reference toxicant tests that fall outside of recommended control chart limits are evaluated to
determine the validity of associated effluent and receiving water tests (see Subsection 4.16). An out of
control reference toxicant test result does not necessarily invalidate associated test results. The reviewer
should consider the degree to which the reference toxicant test result fell outside of control chart limits,
the width of the limits, the direction of the deviation (toward increasing test organism sensitivity or
toward decreasing test organism sensitivity), the test conditions of both the effluent test and the reference
toxicant test, and the objective of the test. More frequent and/or concurrent reference toxicant testing
may be advantageous if recent problems (e.g., invalid tests, reference toxicant test results outside of
control chart limits, reduced health of organism cultures, or increased within-test variability) have been
identified in testing.
10.2.8 TEST VARIABILITY
10.2.8.1 The within-test variability of individual tests should be reviewed. Excessive within-test
variability may invalidate a test result and warrant retesting. For evaluating within-test variability,
reviewers should consult EPA guidance on upper and lower percent minimum significant difference
(PMSD) bounds (USEPA, 2000b).
10.2.8.2 In an effort to reduce the variability of WET test methods, USEPA guidance on WET
variability recommends implementing upper and lower bounds on the PMSD calculated in a test for the
sublethal endpoint (USEPA, 2000b). The minimum significant difference (MSB) is the smallest
difference between the control and another test treatment that can be determined as statistically
significant in a given test, and the PMSD is the MSB represented as a percentage of the control response.
The equation and examples of MSB calculations are shown in Appendix C.
10.2.8.3 To assist in reviewing within-test variability, EPA recommends maintaining control charts of
PMSBs calculated for successive effluent tests (USEPA, 2000b). A control chart of PMSB values
characterizes the range of variability observed within a given laboratory, and allows comparison of
individual test PMSBs with the laboratory's typical range of variability. Control charts of other
variability and test performance measures, such as the MSB, standard deviation or CV of control
responses, or average control response, also may be useful for reviewing tests and minimizing variability.
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C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Replace Section 10, Report Preparation, on page 58 of the marine chronic method manual with
the following.
SECTION 10
REPORT PREPARATION AND TEST REVIEW
10.1 REPORT PREPARATION
The following general format and content are recommended for the report:
10.1.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body
5. Contract Laboratory (if the tests are performed under contract)
a Name of firm
b. Phone number
c. Address
10.1.2 PLANT OPERATIONS
1. Product(s)
2. Raw materials
3. Operating schedule
4. Description of waste treatment
5. Schematic of waste treatment
6. Retention time (if applicable)
7. Volume of waste flow (MOD, CFS, GPM)
8. Design flow of treatment facility at time of sampling
10.1.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Effluent Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Mean daily discharge on sample collection date
f Lapsed time from sample collection to delivery
g. Sample temperature when received at the laboratory
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2. Receiving Water Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Tide stages
f. Sample temperature when received at the laboratory
g. Lapsed time from sample collection to delivery
3. Dilution Water Samples
a. Source
b. Collection date(s) and time(s)
c. Pretreatment
d. Physical and chemical characteristics
10.1.4 TEST METHODS
1. Toxicity test method used (title, number, source)
2. Endpoint(s) of test
3. Deviation(s) from reference method, if any, and the reason(s)
4. Date and time test started
5. Date and time test terminated
6. Type and volume of test chambers
7. Volume of solution used per chamber
8. Number of organisms per test chamber
9. Number of replicate test chambers per treatment
10. Acclimation of test organisms (temperature and salinity mean and range)
11. Test temperature (mean and range)
12. Specify if aeration was needed
13. Feeding frequency, and amount and type of food
14. Test salinity (mean and range)
15. Specify if (and how) pH control measures were implemented
10.1.5 TEST ORGANISMS
1. Scientific name and how determined
2. Age
3. Life stage
4. Mean length and weight (where applicable)
5. Source
6. Diseases and treatment (where applicable)
7. Taxonomic key used for species identification
10.1.6 QUALITY ASSURANCE
1. Reference toxicant used routinely; source
2. Date and time of most recent reference toxicant test, test results, and current control
(cusum) chart
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3. Dilution water used in reference toxicant test
4. Results (NOEC or, where applicable, LOEC, LC50, EC50, IC25 and/or IC50); report
percent minimum significant difference (PMSD) calculated in reference toxicant test
5. Physical and chemical methods used
10.1.7 RESULTS
1. Provide raw toxicity data in tabular form, including daily records of affected organisms
in each concentration (including controls) and replicate, and in graphical form (plots of
toxicity data)
2. Provide table of LCSOs, NOECs, IC25, IC50, etc.
3. Indicate statistical methods used to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
6. Provide percent minimum significant difference (PMSD) calculated for sublethal
endpoints
10.1.8 CONCLUSIONS AND RECOMMENDATIONS
1. Relationship between test endpoints and permit limits
2. Actions to be taken
10.2 TEST REVIEW
10.2.1 Test review is an important part of an overall quality assurance program (Section 4) and is
necessary for ensuring that all test results are reported accurately. Test review should be conducted on
each test by both the testing laboratory and the regulatory authority.
10.2.2 SAMPLING AND HANDLING
10.2.2.1 The collection and handling of samples are reviewed to verify that the sampling and handling
procedures given in Section 8 were followed. Chain-of-custody forms are reviewed to verify that
samples were tested within allowable sample holding times (Subsection 8.5.4). Any deviations from the
procedures given in Section 8 should be documented and described in the data report (Subsection 10.1).
10.2.3 TEST ACCEPTABILITY CRITERIA
10.2.3.1 Test data are reviewed to verify that test acceptability criteria (TAC) requirements for a valid
test have been met. Any test not meeting the minimum test acceptability criteria is considered invalid.
All invalid tests must be repeated with a newly collected sample.
10.2.4 TEST CONDITIONS
10.2.4.1 Test conditions are reviewed and compared to the specifications listed in the summary of test
condition tables provided for each method. Physical and chemical measurements taken during the test
(e.g., temperature, pH, and DO) also are reviewed and compared to specified ranges. Any deviations
from specifications should be documented and described in the data report (Subsection 10.1).
10.2.4.2 Deviations in test conditions (from the specifications listed in the summary of test condition
tables) must be evaluated to determine the validity of test results. Test condition deviations may or may
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not invalidate a test result depending on the degree of the departure and the objective of the test. The
reviewer should consider the degree of the deviation and the potential or observed impact of the
deviation on the test result before rejecting or accepting a test result as valid.
10.2.4.3 Whereas slight deviations in test conditions may not invalidate an individual test result, test
condition deviations that continue to occur frequently in a given laboratory may indicate the need for
improved quality control in that laboratory.
10.2.5 STATISTICAL METHODS
10.2.5.1 The statistical methods used for analyzing test data are reviewed to verify that the
recommended flowcharts for statistical analysis were followed. Any deviation from the recommended
flowcharts for selection of statistical methods should be noted in the data report. Statistical methods other
than those recommended in the statistical flowcharts may be appropriate (see Subsection 9.4.1.2),
however, the laboratory must document the use of and provide the rationale for the use of any alternate
statistical method. In all cases (flowchart recommended methods or alternate methods), reviewers should
verify that the necessary assumptions are met for the statistical method used.
10.2.6 CONCENTRATION-RESPONSE RELATIONSHIPS
10.2.6.1 The concept of a concentration-response, or more classically, a dose-response relationship is
"the most fundamental and pervasive one in toxicology" (Casarett and Doull, 1975). This concept
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, however, the particular shape of the concentration-
response curve may differ for each coupled toxicant and response pair. In general, more severe responses
(such as acute effects) occur at higher concentrations of the toxicant, and less severe responses (such as
chronic effects) occur at lower concentrations. A single toxicant also may produce multiple responses,
each characterized by a concentration-response relationship. 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 possesses toxicity and in identifying
anomalous test results.
10.2.6.2 The concentration-response relationship generated for each multi-concentration test must be
reviewed to ensure that calculated test results are interpreted appropriately. USEPA (2000a) provides
guidance on evaluating concentration-response relationships to assist in determining the validity of WET
test results. All WET test results (from multi-concentration tests) reported under the NPDES program
should be reviewed and reported according to USEPA guidance on the evaluation of concentration-
response relationships (USEPA, 2000a). This guidance provides review steps for 10 different
concentration-response patterns that may be encountered in WET test data. Based on the review, the
guidance provides one of three determinations: that calculated effect concentrations are reliable and
should be reported, that calculated effect concentrations are anomalous and should be explained, or that
the test was inconclusive and the test should be repeated with a newly collected sample. It should be
noted that the determination of a valid concentration-response relationship is not always clear cut. Data
from some tests may suggest consultation with professional toxicologists and/or regulatory officials.
Tests that exhibit unexpected concentration-response relationships also may indicate a need for further
investigation and possible retesting.
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10.2.7 REFERENCE TOXICANT TESTING
10.2.7.1 Test review of a given effluent or receiving water test should include review of the associated
reference toxicant test and current control chart. Reference toxicant testing and control charting is
required for documenting the quality of test organisms (Subsection 4.7) and ongoing laboratory
performance (Subsection 4.16). The reviewer should verify that a quality control reference toxicant test
was conducted according to the specified frequency required by the permitting authority or recommended
by the method (e.g., monthly). The test acceptability criteria, test conditions, concentration-response
relationship, and test sensitivity of the reference toxicant test are reviewed to verify that the reference
toxicant test conducted was a valid test. The results of the reference toxicant test are then plotted on a
control chart (see Subsection 4.16) and compared to the current control chart limits (± 2 standard
deviations).
10.2.7.2 Reference toxicant tests that fall outside of recommended control chart limits are evaluated to
determine the validity of associated effluent and receiving water tests (see Subsection 4.16). An out of
control reference toxicant test result does not necessarily invalidate associated test results. The reviewer
should consider the degree to which the reference toxicant test result fell outside of control chart limits,
the width of the limits, the direction of the deviation (toward increasing test organism sensitivity or
toward decreasing test organism sensitivity), the test conditions of both the effluent test and the reference
toxicant test, and the objective of the test. More frequent and/or concurrent reference toxicant testing
may be advantageous if recent problems (e.g., invalid tests, reference toxicant test results outside of
control chart limits, reduced health of organism cultures, or increased within-test variability) have been
identified in testing.
10.2.8 TEST VARIABILITY
10.2.8.1 The within-test variability of individual tests should be reviewed. Excessive within-test
variability may invalidate a test result and warrant retesting. For evaluating within-test variability,
reviewers should consult EPA guidance on upper and lower percent minimum significant difference
(PMSD) bounds (USEPA, 2000b).
10.2.8.2 In an effort to reduce the variability of WET test methods, USEPA guidance on WET
variability recommends implementing upper and lower bounds on the PMSD calculated in a test for the
sublethal endpoint (USEPA, 2000b). The minimum significant difference (MSB) is the smallest
difference between the control and another test treatment that can be determined as statistically
significant in a given test, and the PMSD is the MSB represented as a percentage of the control response.
The equation and examples of MSB calculations are shown in Appendix C.
10.2.8.3 To assist in reviewing within-test variability, EPA recommends maintaining control charts of
PMSBs calculated for successive effluent tests (USEPA, 2000b). A control chart of PMSB values
characterizes the range of variability observed within a given laboratory, and allows comparison of
individual test PMSBs with the laboratory's typical range of variability. Control charts of other
variability and test performance measures, such as the MSB, standard deviation or CV of control
responses, or average control response, also may be useful for reviewing tests and minimizing variability.
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VII. Nominal Error Rates
A. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 9.4.6, Recommended Alpha Levels, on page 49 of the
freshwater chronic method manual.
9.4.6.2 Under specific circumstances, the alpha level used for hypothesis testing may be reduced from
0.05 to 0.01. These circumstances apply when sublethal endpoints from Ceriodaphnia dubia or fathead
minnow tests are reported under NPDES permit requirements, or when WET permit limits are derived
without allowing for receiving water dilution. Even under these specific circumstances, the alpha level in
a test may only be reduced if adequate test sensitivity can be maintained. USEPA guidance on nominal
error rate adjustments (USEPA, 2000a) provides procedures for determining adequate test sensitivity and
for determining the appropriateness of reductions in the alpha level.
B. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Insert the following into Subsection 9.4.6, Recommended Alpha Levels, on page 52 of the
marine chronic method manual.
9.4.6.2 Under specific circumstances, the alpha level used for hypothesis testing may be reduced from
0.05 to 0.01. These circumstances apply when WET permit limits are derived without allowing for
receiving water dilution. Even under these specific circumstances, the alpha level in a test may only be
reduced if adequate test sensitivity can be maintained. USEPA guidance on nominal error rate
adjustments (USEPA, 2000a) provide procedures for determining adequate test sensitivity and for
determining the appropriateness of reductions in the alpha level.
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VIII. Confidence Intervals
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Insert the following into Subsection 11.2, Determination of the LC50 from Definitive, Multi-
effluent-concentration Acute Toxicity Tests, on page 76 of the acute method manual.
11.2.1.2 The Probit Method, the Spearman-Karber Method, and the Trimmed Spearman-Karber Method
are designed to produce LC50 values and associated 95% confidence intervals. It should be noted that
software used to calculate point estimates occasionally may not provide associated 95% confidence
intervals. This situation may arise when test data do not meet 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. USEPA (2000a) provides guidance on confidence
intervals under these circumstances.
B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 9.3.2, Point Estimation Techniques, on page 47 of the
freshwater chronic method manual.
9.3.2.2 It should be noted that software used to calculate point estimates occasionally may not provide
associated 95% confidence intervals. This situation may arise when test data do not meet 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. USEPA
(2000a) provides guidance on confidence intervals under these circumstances.
C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Insert the following into Subsection 9.3.2, Point Estimation Techniques, on page 50 of the
marine chronic method manual.
9.3.2.2 It should be noted that software used to calculate point estimates occasionally may not provide
associated 95% confidence intervals. This situation may arise when test data do not meet 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. USEPA
(2000a) provides guidance on confidence intervals under these circumstances.
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IX. Dilution Series
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Insert the following into Subsection 9.3, Multi-concentration (Definitive) Effluent Toxicity
Tests, on page 47 of the acute method manual.
9.3.2.1 USEPA recommends the use of a >0.5 dilution factor for selecting effluent test concentrations.
Effluent test concentrations of 6.25%, 12.5%, 25%, 50%, and 100% are commonly used, however, test
concentrations should be selected independently for each test based on the objective of the study, the
expected range of toxicity, the receiving water concentration, and any available historical testing
information on the effluent. USEPA (2000a) provides additional guidance on choosing appropriate test
concentrations.
B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 8.10, Multi-concentration (Definitive) Effluent Toxicity
Tests, on page 42 of the freshwater chronic method manual.
8.10.2.1 USEPA recommends the use of a >0.5 dilution factor for selecting effluent test concentrations.
Effluent test concentrations of 6.25%, 12.5%, 25%, 50%, and 100% are commonly used, however, test
concentrations should be selected independently for each test based on the objective of the study, the
expected range of toxicity, the receiving water concentration, and any available historical testing
information on the effluent. USEPA (2000a) provides additional guidance on choosing appropriate test
concentrations.
C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Insert the following into Subsection 8.10, Multi-concentration (Definitive) Effluent Toxicity
Tests, on page 45 of the marine chronic method manual.
8.10.2.1 USEPA recommends the use of a >0.5 dilution factor for selecting effluent test concentrations.
Effluent test concentrations of 6.25%, 12.5%, 25%, 50%, and 100% are commonly used, however, test
concentrations should be selected independently for each test based on the objective of the study, the
expected range of toxicity, the receiving water concentration, and any available historical testing
information on the effluent. USEPA (2000a) provides additional guidance on choosing appropriate test
concentrations.
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X. Dilution Waters
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Insert the following into Subsection 7.1, Types of Dilution Water, on page 33 of the acute
method manual.
7.1.2 An acceptable dilution water is one which is appropriate for the objectives of the test; supports
adequate performance of the test organisms with respect to survival, growth, reproduction, or other
responses that may be measured in the test (i.e., consistently meets test acceptability criteria for control
responses); is consistent in quality; and does not contain contaminants that could produce toxicity.
USEPA (2000a) provides additional guidance on selecting appropriate dilution waters.
B. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 7.1, Types of Dilution Water, on page 31 of the freshwater
chronic method manual.
7.1.2 An acceptable dilution water is one which is appropriate for the objectives of the test; supports
adequate performance of the test organisms with respect to survival, growth, reproduction, or other
responses that may be measured in the test (i.e., consistently meets test acceptability criteria for control
responses); is consistent in quality; and does not contain contaminants that could produce toxicity.
USEPA (2000a) provides additional guidance on selecting appropriate dilution waters.
C. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Insert the following into Subsection 7.1, Types of Dilution Water, on page 32 of the marine
chronic method manual.
7.1.2 An acceptable dilution water is one which is appropriate for the objectives of the test; supports
adequate performance of the test organisms with respect to survival, growth, reproduction, or other
responses that may be measured in the test (i.e., consistently meets test acceptability criteria for control
responses); is consistent in quality; and does not contain contaminants that could produce toxicity.
USEPA (2000a) provides additional guidance on selecting appropriate dilution waters.
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XI. Pathogen Interference
A. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Replace Subsection 11.3.4 on page 59 of the freshwater chronic method manual with the
following.
11.3.4 Pathogenic and/or predatory organisms in the dilution water and effluent effluent samples or
receiving water that is used for dilution may affect test organism survival and confound test results. A
typical indication that pathogen interference has occurred in a WET test is when test organisms exhibit
"sporadic mortality". This sporadic morality phenomenon is characterized by 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
(USEPA, 2000a). The observed sporadic mortality among replicates may occur in receiving water
controls and lower effluent concentrations on day 3 or day 4 of the chronic test and occasionally in the
full-strength effluent.
11.3.4.1 Observations of sporadic mortality must be documented. When sporadic mortality occurs, a
fungal growth may appear directly on the fish, especially in the gill area. The fungal growth has been
attributed to Saprolegnia sp. (Downey etal., 2000). Microbiological evaluations on receiving waters, the
fish, and the food indicated the ubiquitous nature of pathogenic organisms (e.g., Flexibacter spp.,
Aeromonas hydrophild), and eradicating them from the test through the decontamination of the fish and
their food has not been practical (Geis et al., 2000).
11.3.4.2 When pathogen interference is suspected, a series of data evaluations are required. The test
data must be reviewed 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 dose response curves are not considered indicators of test interference by pathogenic bacteria
(USEPA, 2000a). Each treatment (including the control) should be evaluated for an unusually high
mortality response and unevenness of mortalities among replicates. If the within-treatment coefficient of
variation (CVs) for survival in an effluent treatment is >40% but relatively small for control replicates in
a standard reconstituted water, pathogen interference should be considered. Receiving water controls
from improper preparation or collection also should be evaluated.
11.3.4.3 Because of the ubiquitous nature of the pathogens or predatory organisms, all test equipment,
glassware, and pipettes must be kept clean and dry when not in use. Use of separate glassware, pipettes,
and siphons for each concentration is recommended to minimize cross contaminating replicates of all
treatments.
11.3.4.4 When filtration through a 2-4 mm mesh opening (Subsection 8.8.2) and proper laboratory
hygiene (Subsection 11.3.4.3) do not reduce the interference, the analyst will need to determine if the
toxicity test results could be due to pathogens. Parallel tests should be conducted using reconstituted
water and receiving water as diluents with the effluent to confirm that the test results are due to pathogen
interference. When the effluent exhibits the interference, both the intake water and effluent samples
must be tested to determine the source of pathogens and to determine if the intake water is contributing
the interference observed in the toxicity test of the final effluent. When the dilution water exhibits the
interference, reconstituted laboratory water instead of receiving waters should be used to minimize the
77
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interference. However, if receiving water is required, modify the test design to minimize the effects of
the interference (Subsection 11.3.4.5) prior to treating the receiving water diluent or effluent sample
(Subsection 11.3.4.6).
11.3.4.5 When data evaluation indicates that sporadic mortality has occurred as described in Subsection
11.3.4, the test design can be modified as described below to minimize pathogen interference. The use of
2 fish per 20 ml in each 1 ounce plastic cup test solution or 2 fish per 50 ml in each 4 ounce plastic cup
can be used rather than 10 fish per test chamber. The total number offish tested remains unchanged (i.e.,
40 per treatment). At test initiation, for each test concentration and replicate, the test cups must be
labeled to easily recombine the fish to the original replicate at the end of the test. For example, for
replicate A, each of the five plastic test cups would be identified as subreplicate Al, A2, A3, A4, and A5
repeating the pattern for subsequent replicates (e.g., for replicate B, each cup would be identified as
subreplicate Bl, B2, B3, B4, and B5). At test termination, all test organisms from the five A
subreplicates are combined for a survival and weight determination. Document the recombination of
replicates in records.
11.3.4.5.1 All test chambers must be randomized using a template for randomization or by using a table
of random numbers. For either a randomized or randomized block design (see Subsection 13.10.2), test
chambers are randomized once at the beginning of the test. When using templates, a number of different
templates should be prepared, so that the same template is not used for every test. Randomization
procedures must be documented with daily records.
11.3.4.5.2 When adding or transferring the larvae to test chambers, the amount of excess water added to
the chambers should be kept to a minimum to avoid unnecessary dilution of the test concentrations. The
fish in each test chamber should be fed 0.1 ml of a concentrated suspension of newly hatched (less than
24-h old) brine shrimp nauplii three times daily at 4 h intervals, or 0.15 ml should be fed twice daily at an
interval of 6 h. (NOTE: to prevent low dissolved oxygen levels, the amount of food added to cups should
be adjusted to account for the modified test design that uses smaller test chambers). Dead test organisms
should be removed as soon as they are observed.
11.3.4.5.3 Fish are transferred to new or clean test chambers daily. At the time of the daily renewal of
the test solutions, the fish are transferred to a new test chamber containing fresh test solution using a
pipette which has at least a 5mm bore diameter. Separate pipettes should be used for each treatment.
Water transfer is kept to a minimum by allowing the fish to swim out of the pipette into the new test
chamber. Any potential injury to individual fish should be recorded on the test sheets.
11.3.4.5.4 At test termination, the surviving larvae in each chamber must be counted and all
subreplicates within a replicate (e.g., Al, A2, A3, A4, and A5) combined. For example, all test cups
(within a treatment) labeled A would be combined for a survival and dry weight determination.
11.3.4.6 When parallel testing has confirmed pathogen interference and the above test design
modification (Section 11.3.4.5) does not reduce the pathogen interference, the regulatory authority may
allow modifications of the effluent samples or receiving water diluent to remove or inactivate the
pathogens. TIE approaches (USEPA, 199 Ib; USEPA, 1992) and various sterilization techniques can be
applied to assess the source of the sporadic mortality. The following procedures can be used alone or in
combination to ascertain the adverse influence on tests caused by pathogens in the intake or receiving
waters used for dilution. The effects of pathogenic bacteria must be confirmed by parallel and
simultaneous testing of the following procedures with altered and unaltered samples.
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11.3.4.6.1 Use of ultra-violet light to irradiate the sample. The rate of pumping specified by the
manufacturer of the apparatus should be used, and the life of the UV light source must follow
manufacturers' recommendations and be documented. For example, one liter of water can be irradiated
for 20 min using an 8 watt UV light (Aquatic Ecosystems, Apopka, FL) prior to use each day of the test.
Light sources have limited lifetimes and their effectiveness will decrease with age. The delivery pump
and the light source should be on the same electrical circuit to ensure that when power is interrupted both
terminate operation. QA/QC procedures should be put into place to assure that the light source is on at
the beginning and at the end of the procedure. Treatment of the large volumes of water necessary for test
dilution also may be impractical. Caution: Since the effluent or receiving water samples must be passed
through the UV sterilizer and then test treatments prepared, there may be potential effects of UV light on
the sample. UV exposure may increase or decrease toxicity from other pollutants in the sample. UV
treatment is known to cause photoactivation of some organic compounds, which may increase toxicity.
UV treatment also is known to cause the photochemical breakdown of certain organic compounds, which
could decrease toxicity (if the parent compound is toxic) or increase toxicity (if reaction products are
toxic). The effectiveness of UV for sterilization may decrease with turbid or stained samples. Bacteria
can escape exposure by being lodged in crevices of particulate matter in the sample. All toxicity tests
using a sterilized sample must include a blank preparation consisting of similarly sterilized laboratory
water.
11.3.4.6.2 Ultra-filtration through a 0.22 |im pore diameter filter (such as Gelman Suprocap®) may be
conducted on sample aliquots before daily use. Samples may need to be filtered through a glass fiber
filter prior to the 0.22 |im filter. This is time consuming and volume restricted. Treatment of the large
volumes of water necessary for test dilution may be impractical. Caution: Since the effluent or receiving
water samples must be passed through the filter, the effect of filtering must be evaluated. Filtration can
remove toxicity if toxic components of the sample are bound to particles (USEPA, 1991b; 1992). The
removal of suspended solids also may influence the bioavailability of chemical pollutants. The removal
of toxicity by filtration must be evaluated for each sample by testing samples before and after filtration.
All toxicity tests using a sterilized sample also must include a blank preparation consisting of similarly
sterilized reconstituted laboratory water.
11.3.4.6.3 Use of chlorination and dechlorination. In some cases, pathogens can survive the
chlorination/dechlorination process and the pathogenic effects may increase due to lack of competition
from other organisms. Sufficient data must be collected and documented to determine the effective
dosage required. Toxicity tests conducted with the addition of chlorine and subsequent dechlorination
(USEPA, 1991b; 1992) to either effluent or receiving water samples also must include a blank
preparation consisting of similarly treated laboratory water.
11.3.4.6.4 Use of antibiotics. The addition of wide spectrum antibiotics has been effective in removing
the pathogen effect (Downey et al., 2000). Antibacterial treatment such as those commonly used in
aquaculture or home aquarium maintenance (e.g., oxytetracycline, chloramphenicol, and actinomycin)
may be effective. Sufficient data must be collected to determine the effective dosage required. Caution:
While antibiotics are effective, easy to use, inexpensive, and readily available, the antibiotic treatment
may alter the sample in unknown or undesirable ways and may make the sample too cloudy. Large
volumes of a sample may need to be treated. All toxicity tests using antibiotic treatments also must
include treatment blanks of similarly prepared laboratory water.
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XII. Selenastrum capricornutum Growth Test Method
A. Freshwater Chronic Method
The following are proposed changes to the freshwater chronic method manual.
1. Replace Subsection 14.6.16.2 on page 199 of the freshwater chronic method manual with the
following.
14.6.16.2 Algal Culture Medium is prepared as follows:
14.6.16.2.1? Prepare (five) stock nutrient solutions using reagent grade chemicals as described in Table
i. v^ctutioiiciry notG \ i_i/u IA. iriciy tirrGct rriGtcii toxicity. it is rGCOiYiiYiGuciGCi tiicit tests DG coiiciuctGCi \vitn
cUlCl \VltllOUt I_!/U 1 A. Ill tllG CUltUl'G HlGCLlcl It HlGtcllS tU~G SUSpGCtGQ 111 tllG GltlUGllt Ol" 1'GCGIVIU^ VvcttGl".
14.6.16.2.2? Add ImL of each stock solution, in the order listed in Table 1, ....
14.6.16.2.3? Immediately filter the pH-adjusted medium through a 0.45 [impore....
14.6.16.2.4? If the filtration is carried out with sterile apparatus, ....
14.6.16.2.5? Unused sterile medium should not be stored more than one week....
14.6.16.2.6 When prepared according to Table 1, the micronutrient stock solution contains
ethylenediaminetetraacetic acid (EDTA). This nutrient stock formulation containing EDTA is
recommended for culturing and testing with Selenastrum capricornutum. EDTA should be included in
the nutrients added to algal culture media, test dilution water (see Subsection 14.10.1.3.1), and samples
prior to testing (see Subsection 14.10.1.2.7). The use of EDTA improves test method performance by
reducing the incidence of false positives and increasing test method precision. In interlaboratory testing
of split samples analyzed with and without the addition of EDTA, false positive rates were 0.00% with
EDTA and 33.3% without EDTA (USEPA, 200la). Interlaboratory variability, expressed as the CV for
IC25 values, was 34.3% with EDTA and 58.5% without EDTA (USEPA, 200la). While the use of
EDTA is recommended, testing without the addition of EDTA may be appropriate in some cases. EDTA
effectively binds some metals, so the use of EDTA in the test procedure may cause tests to underestimate
toxicity if metals are the primary contributor to sample toxicity. In cases where metals are known to
contribute to sample toxicity, testing without the addition of EDTA may be conducted if the testing
laboratory has demonstrated success in the use of the without EDTA procedure. Demonstrated success
should include documentation of meeting appropriate test acceptability criteria and control charts of
reference toxicant tests conducted without the addition of EDTA.
2. Replace Subsection 14.12, Acceptability of Test Results, on page 209 of the freshwater chronic
method manual with the following.
14.12 ACCEPTABILITY OF TEST RESULTS
14.12.1 For the test results to be acceptable, the mean algal cell density in the control flasks must exceed
1 X 106 cells/mL with EDTA or 2X10^ cclls/mL without EDTA at the end of the test, and not vai-> more
than 20% among replicates the coefficient of variation (CV, calculated as standard deviation X 100 /
mean) for algal cell density among the control replicates must not exceed 20%.
80
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14.12.2 If the test is conducted without including EDTA in the nutrient stock solution that is added to
the dilution water and sample (see Subsection 14.6.16.2.6), the test acceptability criteria for mean algal
cell density may be reduced. For the results of tests conducted without EDTA to be acceptable, the mean
algal cell density in the control flasks must exceed 2 X 105 cells/mL at the end of the test, and the CV
(calculated as standard deviation X 100 / mean) for algal cell density among the control replicates must
not exceed 20%.
3. Replace Table 3 (continued) on page 211 of the freshwater chronic method manual with the
following.
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
GREEN ALGA, SELENASTRUM CAPRICORNUTUM, GROWTH TOXICITY TESTS
WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
14. Test concentrations:
Effluents: Minimum of 5 and a control
Receiving Water: 100% receiving water or minimum of 5
and a control
15. Test dilution factor:
16. Test duration:
17. Endpoint:
18. Te st acceptability
criteria:2
19. Sampling requirements:
20. Sample volume required:
Effluents: >0.5
Receiving Waters: None or >0.5
96 h
Growth (cell counts, chlorophyll fluorescence,
absorbance, biomass)
Mean cell density of at least 1 X 106 cells/mL with EDTA
or 2X10^ colla/mLwithout EDTA in the controls'; and
¥variability (CV%) among of controls replicates less than
or equal to should not exceed 20%
For on-site tests, one sample collected at test initiation,
and used within 24 h of the time it is removed from the
sampling device. For off-site tests, holding time must not
exceed 36 h (see Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample Preparation for
Toxicity Tests, Subsection 8.5.4)
1 or 2 L depending on test volume
2 If the test is conducted without including EDTA in the nutrient stock solution that is added to the
dilution water and the sample, the test acceptability criteria are a mean cell density of at least 2 X 105
cells/mL in the controls, and variability (CV%) among control replicates less than or equal to 20%.
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XIII. Mysidopsis bahia Survival, Growth, and Fecundity Test Method
A. Marine Chronic Method
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 14.6.13.2, Test Organisms, on page 233 of the marine
chronic manual.
14.6.13.2.11 The pre-test holding conditions of test organisms (as well as the test conditions) have been
shown to significantly influence the success of achieving the test acceptability criteria for the fecundity
endpoint (egg production by 50% or more of control females). Temperature, feeding, and organism
density are important factors in the rate of mysid development. Laboratories should optimize these
factors (within the limits of the test procedure) during both the pre-test holding period and the testing
period to encourage achieving the test acceptability criteria for the fecundity endpoint. If test organisms
are purchased, the testing laboratory should also confer with the supplier to ensure that pre-test holding
conditions are optimized to successfully achieve the fecundity endpoint. Lussier et al. (1999) found that
by increasing holding temperature and test temperature from 26°C ± 1°C to 26°C - 27°C and maintaining
holding densities to < 10 organisms / L, the percentage of tests meeting the test acceptability criteria for
fecundity increased from 60% to 97%. While the fecundity endpoint is an optional endpoint, it is often
the most sensitive measure of toxicity, and the 7-d mysid test estimates the chronic toxicity of effluents
most effectively when all three endpoints (survival, growth, and fecundity) are measured (Lussier et al.
1999).
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XIV. Holmesimysis costata Acute Test Method
A. Acute Method Manual
The following are proposed changes to the acute method manual.
1. Replace Subsections 6.1.2 and 6.1.3 on page 27 of the acute method manual with the following.
6.1.2 Toxicity test conditions and culture methods are provided in this manual for the following
principal test organisms:
Freshwater Organisms:
1. Ceriodaphnia dubia (daphnid) (Table 11).
2. Daphnia pulex and D. magna (daphnids) (Table 12).
3. Pimephalespromelas (fathead minnow) (Table 13).
4. Oncorhynchus mykiss (rainbow trout) and Salvelinus fontinalis (brook trout) (Table 14).
Estuarine and Marine Organisms:
1. Mysidopsis bahia (mysid) (Table 15).1
2. Cyprinodon variegatus (sheepshead minnow) (Table 16).
3. Menidia beryllina (inland silverside), M. menidia (Atlantic silverside), andM peninsulae (tidewater
silverside) (Table 17).
4. Holmesimysis costata (mysid) (Table 18).
6.1.3 The test species listed in Subsection 6.1.2 are the recommended acute toxicity test organisms.
They are easily cultured in the laboratory, are sensitive to a variety of pollutants, and are generally
available throughout the year from commercial sources. Summaries of test conditions for these species
are provided in Tables 11-1?8. Guidelines for culturing and/or holding the organisms are provided in
Appendix A.
2. Insert the following footnote on page 27 of the acute method manual.
1 The genus name of this organism was formally changed to Americamysis (Price et al., 1994), however,
the method manual will continue to refer to Mysidopsis bahia to maintain consistency with previous
versions of the method.
3. Replace Subsection 9.17, Summary of Test Conditions for the Principal Test Organisms, on
page 56 of the acute method manual with the following.
9.17 SUMMARY OF TEST CONDITIONS FOR THE PRINCIPAL TEST ORGANISMS
9.17.1 Summaries of the test conditions for the daphnids, Ceriodaphnia dubia, Daphnia pulex, and D.
magna, fathead minnows, Pimephales promelas, rainbow trout, Oncorhynchus mykiss, brook trout,
Salvelinus fontinalis, the mysids, Mysidopsis bahia and Holmesimysis costata, sheepshead minnows,
Cyprinodon variegatus, and silversides, Menidia beryllina, M. menidia, andM peninsulae, are provided
in Tables 11-1?8.
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4. Replace Table 15 on page 65 of the acute method manual with the following.
TABLE 15. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
MYSID, MYSIDOPSIS BAHIA, ACUTE TOXICITY TESTS WITH EFFLUENTS AND
RECEIVING WATERS*
1. Test type:
2. Test duration:
3. Temperature:31
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test
solutions:
10. Age of test organisms:
11. No. organisms per
test chamber:
12. No. replicate chambers
per concentration:
13. No. organisms per
concentration:
14. Feeding regime:
15. Test chamber cleaning:
Static non-renewal, static-renewal, or flow-through
24, 48, or 96 h
20°C±rC;or25°C±rC
Ambient laboratory illumination
10-20 |iE/m2/s (50-100 ft-c)
(ambient laboratory levels)
16 h light, 8 h darkness
250 mL (minimum)
200 mL (minimum)
Minimum, after 48 h
1-5 days; 24-h range in age
Minimum, 10 for effluent and receiving water tests
Minimum, 2 for effluent tests
Minimum, 4 for receiving water tests
Minimum, 20 for effluent tests
Minimum, 40 for receiving water tests
Artemia nauplii are made available while holding prior to the
test; feed 0.2 mL of concentrated suspension of Artemia
nauplii <24-h old, daily (approximately 100 nauplii per
mysid)
Cleaning not required
*Honwsiinysis costata (mysid) can be used with the test conditions in this table, except at a
temperature of 12°C, instead of 20°C or 25°C, and a salinity of 32-34%u, instead of 5-30%u, where it is
tiic rccjuircci test or^ctmsrri in Qisciictr^c permits.
S1 Acute and chronic toxicity tests performed simultaneously to obtain acute/chronic ratios must use the
same temperature and salinity.
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5. Insert the following table after Table 17 on page 70 of the acute method manual.
TABLE 18. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
WEST COAST MYSID, HOLMESIMYSIS COSTATA, ACUTE TOXICITY TESTS WITH EFFLUENTS
AND RECEIVING WATERS1
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. organisms per
test chamber:
12. No. replicate chambers
per concentration:
13. No. organisms per
concentration:
14. Feeding regime:
Static non-renewal or static-renewal
24, 48, or 96 h
15°C ± 1°C for organisms collected South of Pt
Conception, CA
13°C ± 1°C for organisms collected North of Pt
Conception, CA
Ambient laboratory illumination
10-20 |iE/m2/s (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
1000 mL (minimum)
200 mL (minimum)
Minimum, at 48 h
3 to 4 days post-hatch juveniles
Minimum, 5 for effluent and receiving water tests
Minimum, 5 for effluent tests and receiving water tests
Minimum, 25 for effluent tests and receiving water tests
Artemia nauplii are made available while holding prior to
the test; feed 0.2 mL of concentrated suspension of
Artemia nauplii <24-h old, daily approximately 40
nauplii per mysid)
1 Acute and chronic toxicity tests performed simultaneously to obtain acute/chronic ratios must use
the same temperature and salinity.
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TABLE 18. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
WEST COAST MYSID, HOLMESIMYSIS COSTATA, ACUTE TOXICITY TESTS WITH EFFLUENTS
AND RECEIVING WATERS (CONTINUED)
15. Test chamber cleaning:
16. Test solution aeration:
17. Dilution water:
18. Te st concentrations:
19. Dilution series:
20. Endpoint:
21. Sampling and sample
holding requirements:
22. Sample volume required:
23. Test acceptability
criterion:
Cleaning not required
None, unless DO concentration falls below 4.0 mg/L; rate
should not exceed 100 bubbles/min
34 + 2%o salinity; Uncontaminated seawater (1 jam
filtered) or hypersaline brine or equivalent (see Section
7, Dilution Water)
Effluents: Minimum of five effluent concentrations and a
control
Receiving Waters: 100% receiving water and a control
Effluents: > 0.5 dilution series
Receiving Waters: None, or > 0.5 dilution series
Effluents: Mortality (LC50 orNOAEC)
Receiving Waters: Mortality (Significant difference from
control)
Effluents and Receiving Waters: Grab or composite
samples are used within 36 h of completion of the
sampling period.
1 L for effluents
2 L for receiving waters
90% or greater survival in controls
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6. Replace the text of Appendix A.3 on page 172 of the acute method manual with the following.
APPENDIX A
DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS
A.3. MYSIDS (MYSIDOPSIS BAHIA AND HOLMESIMYSIS COST AT A}
1 DISTRIBUTION
1.1 Mysids (Figure 1) are small shrimp-like crustaceans found in both the marine and freshwater
environments. The mysid(s) that currently are is of primary interest in the NPDES program are is the
estuarine species, Mysldopsls bahia (now identified as Americamysis bahia; Price et al, 1994) and
Holmesimysis costata.
1.2 M. bahia occurs primarily at salinities above 15%o; Stuck et al. (1979a) and Price (1982) found
greatest abundances at salinities near 30%o. Three sympatric species ofMysidopsis, M. almyra,
M. bahia, andM bigelowi, have been cultured and used in toxicity testing. The distribution of
Mysldopsls species has been reported by Stuck etal. (1979b), Price (1982), and Heard etal. (1987).
1.3 H. costata (Holmes 1900; previously referred to as Acanthomysis sculpta) is a west coast species that
lives in the surface canopy of the giant kelp Macrocystis pyrifera where it feeds on zooplankters, kelp,
epiphytes, and detritus. There are few references to the ecology of this mysid species (Holmquist, 1979;
Clutter, 1967, 1969; Green, 1970; Turpen etal., 1994). H. costata is numerically abundant in kelp forest
habitats and is considered to be an important food source for kelp forest fish (Clark 1971, Mauchline
1980). H. costata eggs develop for about 20 days in their marsupium (abdominal pouch) before the young
are released as juveniles; broods are released at night during molting. Females release their first brood at
55 to 70 days post-release (at 12°C), and may have multiple broods throughout their approximately 120-
day life.
1.42- Other marine mysids that have been used in toxicity testing and held or cultured in the lab include
Metamysidopsis elongata, Neomysis americana, Neomysis awatschensis, Neomysis intermedia, and
recently for the Pacific coast, Holmesimysis sculpta and Neomysis merceidis. A freshwater species,
My sis relicta, presently not used in toxicity testing, but found in the same habitat as Daphnia pulex,
might be considered in the future for toxicity testing.
2 MYSIDOPSIS BAHIA
2.1 LIFE CYCLE
2.1.1 In laboratory culture, Mysldopsls bahia reach sexual maturity in 12 to 20 days, depending on water
temperature and diet (Nimmo etal., 1977). Normally, the female will have eggs in the ovary at
approximately 12 days of age. The lamellae of the marsupium pouch have formed or are in the process
of forming when the female is approximately 4 mm in length (Ward, 1993). Unlike Daphnia, the eggs
will not develop unless fertilized. Mating takes place at night and lasts only a few minutes (Mauchline,
1980).
2.1.2 Brood pouches are normally fully formed at approximately 15 days (approximately 5 mm in body
length), and young are released in 17 to 20 days (Ward, 1993). The number of eggs deposited in the
87
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brood and the number of young produced per brood are a direct function of body length as well as
environmental conditions. Mature females have produced as many as 25 Stage I larvae (egg-shaped
embryo) per brood (8-9 mm in body length) in natural and artificial seawater (FORTY FATHOMS®) but
average 1 1 ± 6 Stage III larvae (final stage before larvae are released), with increasing numbers
correlated with increasing body length (Ward, 1993). A new brood is produced every 4 to 7 days.
2.1.3 At time of emergence, juveniles are immobile, making them susceptible to predation by adult
mysids. The juveniles are planktonic for the first 24-48 h and then settle to the bottom, orient to the
current, and actively pursue food organisms such as Artemia. Carr et al. (1980) reported that the stage in
the life cycle of M. almyra most sensitive to drilling mud was the juvenile molt, which occurs between
24 and 48 h after release from the brood pouch. Ward (1989) found a relationship between CaCO3 level
and growth and reproduction and that M. bahia were more sensitive to cadmium during molting (24-72 h
post release) in high or low levels of CaCO3. Work done by Lee and Buikema (1979) for Daphnia pulex
also showed increased sensitivity during molting.
2.23. MORPHOLOGY AND TAXONOMY
2.2.1 3d- Since Mysidopsis bahia occur with two other species ofMysidopsis, an understanding of the
taxonomy ofM almyra, M. bahia, andM bigelowi is important for culturing and testing practices. The
taxonomic key of Heard et al. (1987) is suggested (see Table 1 for morphological guide to Mysidopsis).
2.2.2 35 Adults of M bahia range in length from 4.4 mm to 9.4 mm (Molenock, 1969), measured from
the anterior margin of the carapace to the end of uropods. The mature females are normally larger than
the males and the pleopods of the female are smaller than those of the male (Ward, 1993)
(Figure 2). Mysidopsis bahia can be positively identified as male or female when they are 4 mm in body
length (Ward, 1993). Living organisms are usually transparent, but may be tinted yellow, brown or
black. Mysidopsis bigelowi can be readily distinguished fromM almyra andM bahia by the
morphology of the second thoracic leg. Mysidopsis bigelowi has a greatly enlarged endopod of the
thoracic limb 2 ("first leg") and the limb has a distinctive row of 6 to 12 spiniform setae on the inner
margin of the sixth segment (Heard etal., 1987). Mysidopsis bahia can also be distinguished from other
species ofMysidopsis by the number of apical spines on the telson (4-5 pairs) and the number of spines
on the inner uropods distal to the statocyst (normally 2-3) (Figure 2).
2.2.3 33 Heard etal. (1987) state that the most reliable character for separating adultM almyra andM
bahia is the number of spines on the inner uropods (M. almyra will always have a single spine). Further,
Price (1982) found that for all stages of development for both species, the shape of the anterior margin of
the carapace (rostral plate) could be used to distinguish M. almyra (broadly rounded) from M. bahia
(more produced). Figure 2 illustrates the morphological features most useful in identifying M. bahia
(redrawn Molenock, 1969; Heard etal., 1987).
2.3 4Hr CULTURE METHODS
2.3. 14-+ SOURCE OF ORGANISMS
2.3.1.1 4.1.1 Starter cultures of mysids can be obtained from commercial sources, particularly in the
Gulf of Mexico region forM almyra andM bahia.
2.3.1.2 4.1.2 Mysids of different species can also be collected by plankton tows or dip nets
(approximately 1.0 mm mesh size) in estuarine systems. Heard etal. (1987) have identified specimens of
M. bahia along the eastern coast, however, it has been principally identified as a subtropical species
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found in the Gulf of Mexico and along the east coast of Florida. Since many species of mysids may be
present at a given collection site, the identification of the organisms selected for culture should be
verified by an experienced taxonomist. The permittee should consult the permitting authority for
guidance on the source of test organisms (indigenous or laboratory reared) before use.
2.4 4-2 CULTURING SYSTEM
2.4.1 4.2.1 Stock cultures can be maintained in continuous-flow or closed recirculating systems. In
laboratory culture of M. bahia, recirculating systems are probably the most common practice. During the
past ten years, a number of closed recirculating systems have been described (Nimmo etal, 1978; Leger
and Sorgeloos, 1982; Ward 1984; 1991). Since no single recirculating technique is the best in all
respects, the system adopted will depend on the facilities and equipment available and the objectives of
the culturing activities. Two other species of mysid, M. almyra andM bigelowi, have also been
successfully reared in the system described in this section (Ward, 1991). Further, there now exist a
number of review papers (Venables, 1987 and Lussier etal, 1988) that describe in detail techniques
developed by others that will be very helpful in culturing Mysidopsis.
2.4.2 4.2.2 Closed recirculating systems are unique because the re-used seawater they contain develops
an unusual set of characteristics caused primarily by metabolic waste produced by the mysids. The
accumulation of waste products and suspended particles in the water column is prevented by passing the
seawater through a biological filtration system, in which ammonia and nitrite are oxidized by nitrifying
bacteria.
2.54-3 CULTURE TANKS
2.5.1 4.3.1 Stock cultures of mysids are maintained in a closed recirculating system. The system should
consist of four 200-L glass aquaria. However, smaller tanks, such as 80-L glass aquaria, can be used.
When setting up a system, it is important to consider surface to volume ratio since this will determine
how many mysids can be held in each aquarium. If smaller tanks must be used, the 20-gallon "high"
form is recommended. Figure 3 (Ward, 1984; 1991) illustrates the main components of the biological
filtration system. The flow rate through the filter is controlled by the water valve and is maintained
between 4-5 L/min. This flow will be sufficient to establish a moderate current (from the filter return
line) in the aquarium to allow the mysids (which are positively rheotactic) to align themselves with the
current formed.
2.5.2 4.3.2 The filtration system consists of commercially-available under-gravel filter plates and
external power filter. Each aquarium has two filter plates, forming a false bottom on each side of the
tank, on which 2 cm of crushed coral are placed. The external power filter (Eheim, model 2017) canister
is layered as shown in Figure 3 with a thin layer of filter fiber between each layer of carbon and crushed
oyster shells. There has been some modification of the original filtration system (Ward, 1984), with
crushed coral instead of oyster shells used on the filter bed, because crushed coral does not dissolve in
seawater as readily as crushed oyster shells. If the system described above cannot be used, an acceptable
alternative is an airlift pumping arrangement (Spotte, 1979). Crushed coral and oyster shells are
commercially available and should be washed with deionized water and autoclaved before use.
2.64-4 CULTURE MEDIA
2.6.1 4.4.1 A clean source of filtered natural seawater (0.45 /mi pore diameter) should be used to culture
Mysidopsis bahia, however, artificial seasalts (FORTY FATHOMS®) have also been successfully used
(Ward, 1993). A salinity range between 20 and 30%o can be used (25%o is suggested) to culture M.
bahia. Leger and Sorgeloos (1982) reported success in culturing M bahia in a formula following
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Dietrich and Kalle (Kalle, 1971), and still report continued use of this formula (Leger etal., 1987b).
Other commercial brands have also been used (Reitsema and Neff, 1980; Nimmo and Iley, 1982; Nimmo
et al., 1988) with varying degrees of success. The culture methods presented in Ward (1984; 1991) have
been tried with a number of commercial brands of artificial seawater listed in Bidwell and Spotte (1985).
Commercial brands of seasalts can be extremely variable in the amount of NaHCO3 they provide, which,
if not controlled, can affect growth and reproduction (Ward; 1989, 1991). In a comparative study, Ward
(1993) found normal larval development within the marsupium using both natural seawater and FORTY
FATHOMS® (i.e., Stage I - embryo; Stage II - eyeless larva; Stage III - eyed larva which is the final stage
before release) and stressed the importance of proper preparation of the seasalts and monitoring of
conditions in the tank.
2.6.2 4.4.2 The culture media should be aged to allow the build-up of nitrifying bacteria in the filter
substrate. To expedite the aging process, 15 mL of a concentrated suspension ofArtemia should be
added daily. If using natural or artificial seawater, the carbonate alkalinity level should be maintained
between 90 and 120 mg/L. It is also important to establish an algal community, Spirulina subsalsa, in the
filter bed (Ward, 1984) and a healthy surface dwelling diatom community, Nitzchia sp., on the walls
(Ward, 1991) in conjunction with the transfer of part of the biological filter from a healthy tank, when
possible. After seven days, the suitability of the medium is checked by adding 20 adult mysids. If the
organisms survive for 96 h, the culture should be suitable for stocking.
2.6.3 4.4.3 If brine solutions are used, 100%o salinity must not be exceeded. This corresponds to a
carbonate alkalinity value of approximately 50 mg/L, which will allow relatively normal physiological
mechanisms associated with CaCO3 to occur during certain phases of the life cycle forM bahia (Ward,
1989).
2.74-5 ENVIRONMENTAL FACTORS
2.7.1 4r6- Temperature must be maintained within a range of 24°C to 26°C. Twelve to sixteen h
illumination should be provided daily at 50 to 100 ft-c. The daily light cycle can be provided by
combining overhead room lights, cool-white fluorescent bulbs (approx. 50 ft-c, 12L: 12D), with
individual Grow-lux fluorescent bulbs placed horizontally over each tank (approx. 65 ft-c, 10L: 14D).
This procedure will avoid acute illumination changes by allowing the room lights to turn on 1 h before
and 1 h after the aquaria lights. A timing device, such as an electronic microprocessor-based timer
(ChronTrol®, model CD, or equivalent) can be used to control the light cycle. These procedures are fully
outlined in Ward (1984; 1991).
2.7.2 4.5.2 Good aeration (>60% saturation by vigorous aeration with an air stone), a 10-20 percent
exchange of seawater per week, and carbonate in the filtration system are essential in helping to control
pH drops caused by oxidation of NH4-N and NO2-N by bacteria.
2.7.3 4.5.3 The single most important environmental factor when culturingMysidopsis bahia or other
organisms in recirculators is the conversion of ammonia to nitrite, and nitrite to nitrate by nitrifying
bacteria. Spotte (1979) has suggested upper limits of 0.1 mg total NH4-N/L, 0.1 mg NO2-N/L and 20 mg
NO3-N/L for good laboratory operation of recirculating systems. For the recirculating system and
techniques described here for mysids, the levels of ammonia, nitrite and nitrate never exceeded 0.05 mg
of total ammonia-N/L (NH3(aq)and NH4+), 0.08 mg NO2-N/L and 18 mg NO3-N/L (Ward, 1991). The
toxicity of ammonia is based primarily on unionized ammonia (NH3) and the proportion of NH3 species
to NH4+ species is dependent on pH, ionic strength and temperature. It is strongly recommended that the
concentrations of total ammonia, nitrite and nitrate do not exceed those reported here. The ammonia,
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nitrite, and nitrate levels can be checked by using color comparison test kits such as those made by
LaMotte Chemical or equivalent methods.
2.7.4 4.5.4 Bacterial oxidation of excreted ammonia by two groups of autotrophic nitrifying bacteria
(Nitrosomonas and Nitrobacter), results in an increase of hydrogen ions, which causes a drop in pH and
subsequent loss of buffering capacity. Typically, the culturist responds to the change in pH by adding
Na2CO3 or NaHCO3. However, such efforts to buffer against a drop in pH will result in an increase in
alkalinity and the uncontrolled use of carbonates can affect reproduction, especially at higher alkalinity
values (Ward; 1989, 1991). Therefore, when using carbonates to buffer against pH changes, alkalinity
values should not exceed 120 mg/L, which is easily measured by using a titrator kit such as that available
from LaMotte Chemical or equivalent methods.
2.7.5 4.5.5 Figure 4 (from Ward, 1991) depicts juvenile production per aquarium, no buffer added, over
a period of 24 weeks. A regression line was calculated for these data and the slope and correlation
coefficient were analyzed by Student's t test. The data showed that even when the pH dropped as low of
7.5, there was a significant increase (P < 0.001) in juvenile production. However, the pH should be
maintained above 7.8 by the controlled use of NaHCO3 and frequent water exchanges.
2.8 4-6 FEEDING
2.8.1 4.G.I Frequent feeding with live food is necessary to prevent cannibalism of the young by the
adults. McKenny (1987) suggests feeding densities of 2-3 Artemia per mL of seawater and Lussier et al.
(1988) suggest a feeding rate of 150 Artemia nauplii per mysid daily.
2.8.2 4.G.2 In the M. bahia-Artemia predator-prey relationship, it is also important to provide sufficient
quantities of nutritionally viable free-swimming stage-I nauplii (Ward, 1987); final hatching from the
membranous-sac (pre-nauplii) into stage-I nauplii does not always occur. Artemia cysts that have been
incubated for 24 h should be periodically examined with a stereozoom microscope to enumerate free-
swimming stage-I nauplii and prenauplii (membranous-sac stage).
2.8.3 4.G.3 It has also been found that heavy metals can affect the hatchability of Artemia (Rafiee et al.,
1986; Liu and Chen, 1987), therefore, when using natural seawater the level of metals should always be
checked.
2.8.4 4.G.4 Ward (1987; 1991) has tried different brands of Artemia from different geographic origins
and lot numbers; many achieved stage I nauplii and still caused variability in production of mysids which
suggests that they were nutritionally lacking. Leger et al. (1985; 1987) have drawn attention to poor
larval survival of M bahia and low levels of certain polyunsaturated fatty acids found in the Artemia fed.
The enhancement of Artemia has also been studied and there are numerous techniques that have been
successful (Leger et al, 1986).
2.8.5 4-^5 Ward (1987; 1991) has found that it is important to control the flow of seawater in
recirculating systems (keep below 5 L/min) so that Artemia does not become limiting to the mysid.
Newly hatched Artemia should be fed to mysids at least twice a day. To supply Artemia to the mysid
population on the weekend and prevent cannibalism of newly released mysids, an automatic feeder such
as described by Schimmel and Hansen (1975) or Ward (1984; 1991) could be used. Ward (1991)
designed a system to hatch Artemia when personnel were not available to set up Artemia for the
following morning and afternoon feeding, such as Monday. Cysts were placed in two 4-L Erlenmeyer
flasks (dry), an airstone was placed in each flask, and two vessels overhead were filled with 3.5 L of
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30%o seawater each. The previously described timer (ChronTrol®, Model CD) was used to open the
normally closed solenoids, allowing the seawater to gravity feed and hydrate the cysts.
2.8.6 4.G.G It is possible that a surface dwelling diatom community acts as a secondary food that
supplements deficient brands ofArtemia, especially for newly released juveniles. Ward (1991) has
observed that a strong fertilizing action is caused by the excretory products of the mysid population. As
the concentration of nitrate increases (nitrification) to about 5 mg/L (in approximately 7-10 weeks in an
aquarium), a bloom of surface dwelling diatoms, principally Nitzschia, but including Amphora and
Cocconeis, occurs in natural or artificial seawater (Ward, 1993). It is interesting to note that, at the same
time, there is a dramatic increase in the number of juveniles observed in the aquaria (Figure 4). The
diatoms form layers on the walls of the aquarium and swarms of newly released juveniles have been
found among them, possibly feeding upon them.
2.8.7 4.G.7 Nitzschia has been identified as a food source for the marine mud snail, Ilyanassa obsoleta
(Collier, 1981), and the sea urchin, Lytechinus pictus (Hinegardner and Tuzzi, 1981). The diatom,
Skeletonema, has also been used as a supplemental food forM bahia (Venables, 1987). De Lisle and
Roberts (1986) reported on the use of rotifers, Branchionus plicatilis, as a superior food for juvenile
mysids. Rotifers are active swimmers, ranging in size from 100-175 jwm as compared to 420-520 jwm for
Anemia, and would provide a good alternative food source if their fatty acid profile is adequate.
2.94-? CULTURE MAINTENANCE
2.9.1 4.7.1 To avoid an excessive accumulation of algal growth on the internal surfaces of the aquaria,
the walls and internal components should be scraped periodically and the shell substrate (coral or oyster)
turned over weekly. Also, the filter plates must be completely covered so that the biological filter
functions properly. After a culture tank has been in operation for approximately 2-3 months, detritus
builds up on the bottom, which is removed with a fish net after first removing the mysids. The rate of
water flow through the tanks should be maintained between 4-5 L/min, and 10-20% of the seawater in
each aquarium should be exchanged weekly.
2.9.2 4.7.2 Some culturists have noted problems with hydrozoan pests in their cultures and there are
procedures for their eradication, if necessary (Lawlerand Shepard, 1978; Hutton etal, 1986).
2.104* PRODUCTION LEVEL
2.10.1 4.8.1 At least four aquaria should be maintained to insure a sufficient number of organisms on a
continuing basis. If each 200-L aquarium is initially stocked with between 200 and 500 adults (do not
exceed 500 adults), they will provide sufficient numbers of test organisms (Figure 4) each month. If the
cultures are correctly maintained, at least 20 percent of the adult population should consist of gravid
females (have a visible oostegite brood pouch with young). It is also advantageous to cull older mysids
in the population every 4-6 weeks and to move mysids among the four aquaria to diversify the gene pool.
2.115- VIDEO TRAINING TAPE AVAILABLE FOR CULTURING METHODS
2.11.1 5d- A video training tape and supplemental report (USEPA, 1990) on culturing Mysidopsis bahia
are available from the National Audiovisual Center, Customer Services Section, 8700 Edgeworth Drive,
Capitol Heights, MD 20743-3701, (Phone 301-763-1891), as part of a video package on culturing and
short-term chronic toxicity test methods (Order No. A18657; cost $75.00).
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2.126 TEST ORGANISMS
2.12.1 6rt Juvenile Mysidopsis bahia, one to five days old, are used in the acute toxicity test and the
survival, growth and fecundity test (USEPA, 1994). To obtain the necessary number for a test, there are
a number of techniques available. A mysid generator such as the one described by Reistsema and Neff
(1980) has been successfully used. Another method to obtain juveniles is to take approximately 200
adult females (bearing embryos in their brood pouches) from the stock culture and place them in a large
(10 cm X 15 cm) standard fish transfer net (2.0 to 3.0 mm openings) that is partially submerged in an 8-L
aquarium containing 4 L of clean culture medium. As the juveniles are released from the brood pouches,
they drop through the fish net into the aquarium. The adults and juveniles in the aquarium are fed twice
daily 24-h post hydrated Artemia. The adults are allowed to remain in the net for 48 h, and are then
returned to the stock tanks. The juveniles that are produced in the small tank may be used in the toxicity
tests over a five-day period. Another method for obtaining juveniles (Ward 1987; 1989) is simply to
remove juveniles from the stock culture with a fine mesh net, place them in 2-L Pyrex® crystalline dishes
with media, positioned on a light table that has an attached viewing plate (2 mm squares), and remove
juveniles less than 2 mm in length (approximately 24 h old).
3. HOLMESIMYSIS COSTATA
3.1 MORPHOLOGY AND TAXONOMY
3.1.1 Laboratories unfamiliar with the test organism should collect preliminary samples to verify species
identification. Refer to Holmquist (1979) or send samples of mysids and any similar co-occurring
organisms to a qualified taxonomist. Request certification of species identification from any organism
supplier. Records of verification should be maintained along with a few preserved specimens. A review
by Holmquist (1979) considered previous references to Acanthomysis sculpta in California to be
synonymous with Holmesimysis costata and this is considered definitive at this time.
3.2 SOURCE OF BROODSTOCK AND TRANSPORT
3.2.1 Broodstock of H. costata are collected by sweeping a small-mesh (0.5-1 mm) hand net through the
water just under the surface canopy blades of giant kelp Macrocystis pyrifera. Although this method
collects mysids of all sizes, attention should be paid to the number of gravid females collected because
these are used to produce the juvenile mysids used in toxicity testing. Gravid females are identified by
their large, extended marsupia filled with young. Mysids should be collected from waters remote from
sources of pollution to minimize the possibility of physiological or genetic adaptation to toxicants.
3.2.2 Mysids can be transported for a short time (< 3 h) in tightly covered 20 L plastic buckets. The
buckets should be filled to the top with seawater from the collection site, and should be gently aerated or
oxygenated to maintain dissolved oxygen above 60% saturation. Transport temperatures should remain
within 3°C of the temperature at the collection site.
3.2.3 For longer transport times of up to 36 h, mysids can be shipped in sealed plastic bags filled with
seawater. The following transport procedure has been used successfully:
1) fill the plastic bag with one L of dilution water seawater,
2) saturate the seawater with oxygen by bubbling pure oxygen for at least 10 minutes,
3) place 25-30 adult mysids, or up to 100 juvenile mysids in each bag,
4) for adults add about 20 Artemia nauplii per mysid, for 100 juveniles add a pinch (10 to 20 mg)
of ground Tetramin® flake food and 200 newly-hatched Artemia nauplii,
5) seal the bag securely, eliminating any airspace, and
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6) place it within a second sealed bag in an ice chest.
Do not overfeed mysids in transport, as this may deplete dissolved oxygen, causing stress or mortality in
transported mysids. A well-insulated ice chest should be cooled to approximately 15°C by adding one 1-
L blue ice block for every five 1-L bags of mysids (organisms will tolerate the temperature range of 12 to
16°C). Wrap the ice in newspaper and a plastic bag to insulate it from the mysid bags. Pack the bags
tightly to avoid shifting within the cooler.
3.3 HOLDING AND CULTURING
3.3.1 After collection, the mysids should be transported directly to the laboratory and placed in seawater
tanks or aquaria equipped with flowing seawater or adequate aeration and filtration. Initial flow rates
should be adjusted so that any temperature change occurs gradually (0.5°C per h). Broodstock will be
collected and maintained at two temperatures as follows: 1) maintain the water temperature of 15 ± 1 C
for mysids collected south of Pt. Conception, CA and 2) maintain the water temperatures of 13 ± 1 C for
mysids collected north of Pt. Conception, CA. Mysids can be cultured in tanks ranging from 4 to 1000
L. Tanks should be equipped with gentle aeration and blades ofMacrocystis to provide habitat. Static
culture tanks can be used if there is constant aeration, temperature control, and frequent water changes
(one half the water volume changed at least twice a week). Maintain culture density below 20 animals
per L by culling out adult males or juveniles.
3.4 FEEDING
3.4.1 Adult mysids should be fed 100 Artemia nauplii per mysid per day. Juveniles should be fed 5 to 10
newly released Artemia nauplii per juvenile per day and a pinch (10 to 20 mg) of ground TetraminT flake
food per 100 juveniles per day. Static chambers should be carefully monitored and rations adjusted to
prevent overfeeding and fouling of culture water.
3.5 TEST ORGANISMS
3.5.1 Juvenile Holmesimysis costata three to four days old, are used in the acute toxicity test and the
survival and growth test (Hunt et a/., 1997; USEPA, 1995). To obtain the necessary number for a test,
there are a number of techniques available for the acute toxicity test and the survival and growth test
(USEPA, 1995). Approximately 150 gravid female mysids will typically produce approximately 400
juveniles. Gravid females can be identified by their large, extended marsupia filled with (visible) eyed
juveniles. Marsupia appear distended and gray when females are ready to release young, due to presence
of the juveniles. Gravid females are easily isolated from other mysids using the following technique: 1)
use a small dip net to capture about 100 mysids from the culture tank, 2) transfer the mysids to a screen-
bottomed plastic tube (150 |im-mesh, 25-cm diameter) partly immersed in a water bath or bucket, 3) lift
the screen-tube out of the water to immobilize mysids on the damp screen, 4) gently draw the gravid
females off the screen with a suction bulb and fire-polished glass tube (5-mm I.D.), and 5) collect gravid
females in a separate screen tube. Re-immerse the screen continuously during the isolation process;
mysids should not be exposed to air for more than a few seconds at a time.
3.5.2 Four to five days before a toxicity test begins, transfer gravid females into a removable, 2-mm-
mesh screened cradle suspended within an aerated 80-L aquarium. Before transfer, make sure there are
no juveniles in with the adult females. Extraneous juveniles are excluded to avoid inadvertently mixing
them with the soon-to-be released juveniles used in testing. Provide the gravid females with newly
hatched Artemia nauplii (approximately 200 per mysid) to help stimulate juvenile release. Artemia can
be provided continuously throughout the night from an aerated reservoir holding approximately 75,000
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Anemia. Direct the flow from the feeder into the screened compartment with the females, and add a few
blades ofMacrocystis for habitat. The females are placed within the screened compartment so that as the
juveniles are released, they can swim through the mesh into the bottom of the aquarium. Outflows on
flow-through aquaria should be screened (150-|im-mesh) to retain juveniles and allow some Anemia to
escape.
3.5.3 Juveniles are generally released at night, so it is important to turn off all lights at night to promote
release. In the morning, the screened compartment containing the females should be removed and
placed in a separate aquarium. Juveniles should be slowly siphoned through a wide-diameter hose into
al50-|im-mesh screen-bottom tube (25 cm diam.) immersed in a bucket filled with clean seawater. Once
the release aquarium is emptied, it should be washed with hot fresh water to eliminate stray juveniles that
might mix with the next cohort.
3.5.4 After collection, the number of juveniles should be estimated visually or by counting subsamples
with a small beaker. If there are not enough juveniles, the juveniles from previous or subsequent releases
can be combined so that the test is initiated with three and/or four-day old juveniles. Mysids 2-days old
and younger have higher mortality rates, while mysids older than four days may vary in their toxicant
sensitivity or survival rate (Hunt etal, 1989; Martin etal., 1989).
3.5.5 Test juveniles should be transferred to additional screen-tubes (or to 4-L static beakers if flowing
seawater is unavailable). The screen-tubes are suspended in a 15-L bucket so that dilution water
seawater (0.5 L/min) can flow into the tube, through the screen, and overflow from the bucket. Check
water flow rates (< 1 L/min) to make sure that juveniles orArtemia nauplii are not forced down onto the
screen. The height of the bucket determines the level of water in the screen tube. About 200 to 300
juveniles can be held in each screen-tube (200 juveniles per static 4-L beaker). Juveniles should be fed
40 newly hatched Anemia nauplii per mysid per day and a pinch (10 to 20 mg) of ground TetraminT
flake food per 100 juveniles per day. A blade ofMacrocystis (well rinsed in seawater) should be added to
each chamber. Chambers should be gently aerated and temperature controlled at 15 ± 1 C (or 13 ± 1 C if
collected north of Pt. Conception). Half of the seawater in static chambers should be changed at least
once between isolation and test initiation.
3.5.6 The day juveniles are isolated is designated day 0 (the morning after their nighttime release). The
toxicity test should begin on day three or four. For example, if juveniles are isolated on Friday, the
toxicity test would begin on the following Monday or Tuesday. Pool all of the test juveniles into a 1-L
beaker. Using a 10-mL wide-bore pipet or fire-polished glass tube (approximately 2-3 mm I.D), place
one or two juveniles into as many plastic cups (one for each test chamber). These cups should contain
enough clean dilution seawater to maintain water quality and temperature during the transfer process
(approximately 50 mL per cup). When each of the cups contains one or two juveniles, repeat the process,
adding mysids until each cup contains 10 organisms. Carefully pour or pipet off excess water in the
cups, leaving less than 5 mL with the test mysids. This 5 mL volume can be estimated visually after
initial measurements. Carefully pour or pipet the juveniles into the test chambers immediately after
reducing the water volume. Gently rocking the water back and forth before pouring may help prevent
juveniles from clinging to the walls of the randomization cups. Juveniles can become trapped in drops;
have a squirt bottle ready to gently rinse down any trapped mysids. If more than 5 mL of water is added
to the test solution with the juveniles, report the amount on the data sheet. Be sure that all water used in
culture, transfer, and test solutions is within 1°C of the test temperature. Because of the small volumes
involved in the transfer process, temperature control is best accomplished in a constant-temperature
room.
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3.5.7 Immobile mysids that do not respond to a stimulus are considered dead. The stimulus should be
two or three gentle prods with a disposable pipet. Mysids that exhibit any response clearly visible to the
naked eye are considered living. The most commonly observed movement in moribund mysids is a quick
contraction of the abdomen. This or any other obvious movement qualifies a mysid as alive.
7. Insert the following references into the Selected References section of Appendix A.3 on page 184
of the acute method manual.
SELECTED REFERENCES
Clark, W. 1971. Mysids of the southern kelp region. In: W. North, ed., Biology of Giant Kelp Beds
(Macrocystis) in California. J. Cramer Publisher, Lehre, Germany: p 369-380.
Clutter, R.I. 1967. Zonation of nearshore mysids. Ecology. 48(2): 200-208.
Clutter, R.I. 1969. The microdistribution and social behavior of some pelagic mysids. J. Exp. Mar. Biol.
Ecol. 3: 125-155.
Green, J.M. 1970. Observations on the behavior and larval development ofAcanthomysis sculpta
(Tattersall), (Mysidacea). Can. J. Zool. 48: 289-292.
Holmquist, C. 1979. Mysis costata and its relations (Crustacea, Mysidacea). Zool. Jb. Syst. 106: 471-499.
Hunt, J.W., B.S. Anderson, S.L. Turpen, A.R. Coulen, M. Martin, F.H. Palmer, and J.J. Janik. 1989.
Marine bioassay project fourth report: Experimental evaluation of effluent toxicity testing
protocols with giant kelp, mysids, red abalone, and topsmelt. Report 89-5WQ. California State
Water Resources Control Board, Sacramento, CA.
Hunt, J.W., B.S. Anderson, S.L.Turpen, M.A. Englund, and W.J. Piekarski. 1997. Precision and
sensitivity of a seven-day growth and survival toxicity test using the west coast marine mysid
crustacean Holmesimysis costata. Environ. Toxicol. Chem. 16: 824-834.
Martin, M., J.W. Hunt, B.S. Anderson, S.L. Turpen, and F.H. Palmer. 1989. Experimental evaluation of
the mysid Holmesimysis costata as a test organism for effluent toxicity testing. Environ. Toxicol.
Chem. 8: 1003-1012.
Turpen, S.L, J.W. Hunt, B.S. Anderson, and J.S. Pearse. 1994. Population structure, growth and
fecundity of the kelp forest mysid, Holmesmysis costata in Monterey Bay, California. J. Crust.
Biol. 14(4): 657-664.
USEPA. 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving waters
to marine and estuarine organisms, 2nd ed. Environmental Monitoring Systems Laboratory, U.S.
Environmental Protection Agency, Cincinnati, OH. EPA/600/4-91/003.
USEPA, 1995. Short-term methods for estimating the chronic toxicity of effluents and receiving waters
to west coast marine and estuarine organisms. National Exposure Research Laboratory, U.S.
Environmental Protection Agency, Cincinnati, OH. EPA/600/R-95-136.
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8. Replace footnote 2 in Appendix B on page 266 of the acute method manual with the following.
2 Test conditions for Holmesimvsis costata are found in Table 158
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XV. Percent Minimum Significant Difference (PMSD)
A. Freshwater Chronic Method Manual
The following are proposed changes to the freshwater chronic method manual.
1. Insert the following into Subsection 11.13.1, General, on page 81 of the freshwater chronic
method manual.
11.13.1.4 For hypothesis tests, the percent reduction from the control corresponding to the minimum
significant difference (MSB) (see Subsection 9.6.1.4) is abbreviated herein as PMSD. Upper and lower
bounds for the PMSD identified in USEPA (2000b) must be applied to the determination of NOEC and
LOEC for the growth endpoint, as described in Subsections 11.13.3.7.13 and 11.13.3.7.14. The upper
PMSD bound for the fathead minnow growth endpoint is 35% and the lower bound is 9.4%. The PMSD
bounds are intended for use in determining the NOEC and LOEC in multi-concentration tests.
2. Insert the following into Subsection 11.13.3.7, Dunnett's Procedure, on page 102 of the
freshwater chronic method manual.
11.13.3.7.11 Upper and lower bounds for the PMSD (USEPA, 2000b) must be applied to the
determination of NOEC and LOEC for the growth endpoint, as described below in Subsections
11.13.3.7.13 and 11.13.3.7.14. The upper PMSD bound is 35% and the lower PMSD bound is 9.4%.
11.13.3.7.12 To make comparisons with upper and lower bounds for PMSD (USEPA, 2000b), calculate
MSD as described in Appendix C, using Dunnett's critical value (d), and calculate the PMSD as
described in Appendix C, Subsection 1.11.2.2. Calculate these whether or not the Dunnett's test will be
used for hypothesis testing. The upper and lower bounds for PMSD (USEPA, 2000b) apply to
untransformed growth data for a large sample of toxicity tests, including those in which Dunnett's test
would not have been used for hypothesis testing. For the purposes of calculating PMSDs, use of
Dunnett's test also is acceptable when there is variation among treatments in the number of replicates.
11.13.3.7.13 When the MSD represents a percent reduction from the control that is smaller than the
lower PMSD bound for the growth endpoint of this test method, 9.4% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which differ significantly from the control (using whichever statistical test was selected as
appropriate), but which represent a percent reduction from the control smaller than 9.4%. These test
concentrations will be considered as not differing significantly from the control for the purpose of
determining the NOEC and LOEC.
11.13.3.7.14 When the MSD represents a percent reduction from the control that is larger than the upper
PMSD bound for the growth endpoint of this test method, 35% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which do not differ significantly from the control (using whichever statistical test was
selected as appropriate), but which represent a percent reduction from the control larger than 35%.
These test concentrations will be considered as differing significantly from the control for the purpose of
determining the NOEC and LOEC.
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3. Insert the following into Subsection 13.13.1, General, on page 170 of the freshwater chronic
method manual.
13.13.1.5 For hypothesis tests, the percent reduction from the control corresponding to the minimum
significant difference (MSB) (see Subsection 9.6.1.4) is abbreviated herein as PMSD. Upper and lower
bounds for the PMSD identified in USEPA (2000b) must be applied to the determination of NOEC and
LOEC for the reproduction endpoint, as described in Subsections 13.13.3.7.13 and 13.13.3.7.14. The
upper PMSD bound for the Ceriodaphnia dubia reproduction endpoint is 37%, and the lower bound is
11%. The PMSD bounds are intended for use in determining the NOEC and LOEC in multi-
concentration tests.
4. Insert the following into Subsection 13.13.3.7, Dunnett's Procedure, on page 182 of the
freshwater chronic method manual.
13.13.3.7.11 Upper and lower bounds for the PMSD (USEPA, 2000b) must be applied to the
determination of NOEC and LOEC for the reproduction endpoint, as described below in Subsections
13.13.3.7.13 and 13.13.3.7.14. The upper PMSD bound is 37% and the lower PMSD bound is 11%.
13.13.3.7.12 To make comparisons with upper and lower bounds for PMSD (USEPA, 2000b), calculate
MSD as described in Appendix C, using Dunnett's critical value (d), and calculate the PMSD as
described in Appendix C, Subsection 1.11.2.2. Calculate these whether or not the Dunnett's test will be
used for hypothesis testing. The upper and lower bounds for PMSD (USEPA, 2000b) apply to
untransformed reproduction data for a large sample of toxicity tests, including those in which Dunnett's
test would not have been used for hypothesis testing. For the purposes of calculating PMSDs, use of
Dunnett's test also is acceptable when there is variation among treatments in the number of replicates.
13.13.3.7.13 When the MSD represents a percent reduction from the control that is smaller than the
lower PMSD bound for the reproduction endpoint of this test method, 11% (USEPA, 2000b), the
following modification must be used when determining the NOEC and LOEC. Identify and document
any concentrations which differ significantly from the control (using whichever statistical test was
selected as appropriate), but which represent a percent reduction from the control smaller than 11%.
These test concentrations will be considered as not differing significantly from the control for the
purpose of determining the NOEC and LOEC.
13.13.3.7.14 When the MSD represents a percent reduction from the control that is larger than the upper
PMSD bound for the reproduction endpoint of this test method, 37% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which do not differ significantly from the control (using whichever statistical test was
selected as appropriate), but which represent a percent reduction from the control larger than 37%.
These test concentrations will be considered as differing significantly from the control for the purpose of
determining the NOEC and LOEC.
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B. Marine Chronic Method Manual
The following are proposed changes to the marine chronic method manual.
1. Insert the following into Subsection 13.13.1, General, on page 186 of the marine chronic method
manual.
13.13.1.4 For hypothesis tests, the percent reduction from the control corresponding to the minimum
significant difference (MSB) (see Subsection 9.6.1.4) will be abbreviated herein as PMSD. Upper and
lower bounds for the PMSD identified in USEPA (2000b) must be applied to the determination of NOEC
and LOEC for the growth endpoint, as described in Subsections 13.13.3.7.13 and 13.13.3.7.14. The
upper PMSD bound for the inland silverside growth endpoint is 35% and the lower bound is 12%. The
PMSD bounds are intended for use in determining the NOEC and LOEC in multi-concentration tests.
2. Insert the following into Subsection 13.13.3.7, Dunnett's Procedure, on page 210 of the marine
chronic method manual.
13.13.3.7.11 Upper and lower bounds for the PMSD (USEPA, 2000b) must be applied to the
determination of NOEC and LOEC for the growth endpoint, as described below in Subsections
13.13.3.7.13 and 13.13.3.7.14. The upper PMSD bound is 35% and the lower PMSD bound is 12%.
13.13.3.7.12 To make comparisons with upper and lower bounds for PMSD (USEPA, 2000b), calculate
MSD as described in Appendix C, using Dunnett's critical value (d), and calculate PMSD as described in
Appendix C, Subsection 1.11.2.2. Calculate these whether or not the Dunnett's test will be used for
hypothesis testing. The upper and lower bounds for PMSD (USEPA, 2000b) apply to untransformed
growth data for a large sample of toxicity tests, including those in which Dunnett's test would not have
been used for hypothesis testing. For the purposes of calculating PMSDs, use of Dunnett's test also is
acceptable when there is variation among treatments in the number of replicates.
13.13.3.7.13 When the MSD represents a percent reduction from the control that is smaller than the
lower PMSD bound for the growth endpoint of this test method, 12% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which differ significantly from the control (using whichever statistical test was selected as
appropriate), but which represent a percent reduction from the control smaller than 12%. These test
concentrations will be considered as not differing significantly from the control for the purpose of
determining the NOEC and LOEC.
13.13.3.7.14 When the MSD represents a percent reduction from the control that is larger than the upper
PMSD bound for the growth endpoint of this test method, 35% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which do not differ significantly from the control (using whichever statistical test was
selected as appropriate), but which represent a percent reduction from the control larger than 35%.
These test concentrations will be considered as differing significantly from the control for the purpose of
determining the NOEC and LOEC.
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3. Insert the following into Subsection 14.13.1, General, on page 243 of the marine chronic method
manual.
14.13.1.4 For hypothesis tests, the percent reduction from the control corresponding to the minimum
significant difference (MSB) (see Sebsection 9.6.1.4) will be abbreviated herein as PMSD. Upper and
lower bounds for the PMSD identified in USEPA (2000b) must be applied to the determination of NOEC
and LOEC for the growth endpoint, as described in Subsections 14.13.3.7.13 and 14.13.3.7.14. The
upper PMSD bound for the mysid growth endpoint is 32% and the lower bound is 12%. The PMSD
bounds are intended for use in determining the NOEC and LOEC in multi-concentration tests.
4. Insert the following into Subsection 14.13.3.7, Dunnett's Procedure, on page 269 of the marine
chronic method manual.
14.13.3.7.11 Upper and lower bounds for the PMSD (USEPA, 2000b) must be applied to the
determination of NOEC and LOEC for the growth endpoint, as described below in Subsections
14.13.3.7.13 and 14.13.3.7.14. The upper PMSD bound is 32% and the lower PMSD bound is 12%.
14.13.3.7.12 To make comparisons with upper and lower bounds for PMSD (USEPA, 2000b), calculate
MSD as described in Appendix C, using Dunnett's critical value (d), and calculate PMSD as described in
Appendix C, Subsection 1.11.2.2. Calculate these whether or not the Dunnett's test will be used for
hypothesis testing. The upper and lower bounds for PMSD (USEPA, 2000b) apply to untransformed
growth data for a large sample of toxicity tests, including those in which Dunnett's test would not have
been used for hypothesis testing. For the purposes of calculating PMSDs, use of Dunnett's test also is
acceptable when there is variation among treatments in the number of replicates.
14.13.3.7.13 When the MSD represents a percent reduction from the control that is smaller than the
lower PMSD bound for the growth endpoint of this test method, 12% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which differ significantly from the control (using whichever statistical test was selected as
appropriate), but which represent a percent reduction from the control smaller than 12%. These test
concentrations will be considered as not differing significantly from the control for the purpose of
determining the NOEC and LOEC.
14.13.3.7.14 When the MSD represents a percent reduction from the control that is larger than the upper
PMSD bound for the growth endpoint of this test method, 32% (USEPA, 2000b), the following
modification must be used when determining the NOEC and LOEC. Identify and document any
concentrations which do not differ significantly from the control (using whichever statistical test was
selected as appropriate), but which represent a percent reduction from the control larger than 32%.
These test concentrations will be considered as differing significantly from the control for the purpose of
determining the NOEC and LOEC.
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