EPA
United States
Environmental Protection
Agency
Research And Development
(MD-591)
EPA-600-4-91-003
July 1994
Shprt-Term Methods For
Estimating The Chronic Toxicity
Of Effluents And Receiving Water
To Marine And Estuaririe
Organisms
Second Edition
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EPA/600/4-91 /003
July 1994
9CRT-TBMIVE1KC6 FCR ESTIMtTIISG Tl-E OflCNIC TOXICITY OF EFFLJUBMT5
RECEIVIN3 WATERS TO IvPRIIVE tbD ESTU4RINE CR3NI5M6
(Second Edition)
Edited by
Donald J. Klenm1 and George E. IVbrrison2
Teresa J. Nor berg-King3, Wi I I ian H. Peltier4
and IVbrgarete A. Heber5
2Envirormsntal MbhStoring Systems Laboratory, Cincinnati, Ohio
EnvirorrnentaI Research Laboratory, IMarragansett, Rhode Island
Environmental Research Laboratory, Duluth, Minnesota
Environmental Services Division, Athens, Georgia
^Office of \A6ter, Washington, D.C.
EH/IR3WBMTAL M3MITORIN3 SvSTHvB LftBCRATCRY-CIISCINWI
CFFICE OF RESEARCH A\D DEVELORVENT
U.S. BWIR3WENTAL PRDTECTICM
ClrCihhATI, OHIO 45268
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DISCLAIMER
This document has been reviewed by the Environmental Monitoring Systems
Laboratory-Cincinnati (EMSL-Cincinnati), U. S. Environmental Protection Agency
(USEPA), and approved for publication. The mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
The results of data analyses by computer programs described in the section on
data analysis were verified using data commonly obtained from effluent
toxicity tests. However, these computer programs may not be applicable to all
data, and the USEPA assumes no responsibility for their use.
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FOREWORD
Environmental measurements are required to determine the quality of ambient
waters and the character of waste effluent. The Environmental Monitoring
Systems Laboratory-Cincinnati (EMSL-Cincinnati) conducts research to:
i
• Develop and evaluate analytical methods to identify and measure the
.concentration of chemical pollutants in drinking waters, surface waters,
groundwaters, wastewaters, sediments, sludges, and solid wastes.
• Investigate methods for the identification and measurement of viruses,
bacteria and other microbiological organisms in aqueous samples and to
determine the responses of aquatic organisms to water quality.
• Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of pollutants in
drinking water, surface water, groundwater, wastewater, sediment and
solid waste.
• Develop methods and models to detect and quantify responses in aquatic
and terrestrial organisms exposed to environmental stressors and to
correlate the exposure with effects on chemical and biological
indicators.
i
The Federal Water Pollution Control Act Amendments of 1972 (PL 92-500), the
Clean Water Act (CWA) of 1977 (PL 95-217) and the Water Quality Act of 1987
(PL 100-4) explicitly state that it is the national policy that the discharge
of toxic substances in toxic amounts be prohibited. Thus., the detection of
chronically toxic effluents plays an important role in identifying and
controlling toxic discharges to surface waters. This manual is the second
edition of the marine and estuarine chronic toxicity test manual for
effluents, first published (EPA/600/4-87/028) by EMSL-Cincinnati in May 1988.
It provides updated and standardized methods for estimating the chronic
toxicity of effluents and receiving waters to estuarine arid marine organisms
for use by the U. S. Environmental Protection Agency (USEPA) regional
programs, the state programs, and the National Pollutant Discharge Elimination
System (NPDES) permittees.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory-Cincinnati
m
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1
PREFACE
This manual represents the second edition of the Agency's methods manual
for estimating the chronic toxicity of effluents and receiving waters to
marine and estuarine organisms initially published by USEPA's Office of
Research and Development, Environmental Monitoring and Support Laboratory
(EMSL-Cincinnati) in May 1988. This edition reflects changes recommended by ,
the Toxicity Assessment Subcommittee of the Biological Advisory Committee,
USEPA headquarters, program offices, and regional staff, other Federal
agencies, state and interstate water pollution control programs, environmental
protection groups, trade associations, major industries, consulting firms,
academic institutions engaged in aquatic toxicology research, and other
interested parties in the private sector.
The membership of the Toxicity Assessment Subcommittee, USEPA's Biological
Advisory Committee is as follows:
William Peltier, Subcommittee Chairman,
Environmental Services Division, Region 4
Peter Nolan, Environmental Services Division, Region 1
Jim Ferretti, Environmental Services Division, Region 2
Ronald Preston, Environmental Services Division, Region 3
Charles Steiner, Environmental Services Division, Region 5
Evan Hornig, Environmental Services Division, Region 6
Terry Hollister, Environmental Services Division, Region 6
Michael Tucker, Environmental Services Division, Region 7
Loys Parrish, Environmental Services Division, Region 8
Peter Husby, Environmental Services Division, Region 9
Joseph Cummins, Environmental Services Division, Region 10
Gretchen Hayslip, Water Monitoring and Analysis Section, Region 10
Bruce Binkley, National Enforcement Investigations Center, Denver
Wesley Kinney, Environmental Monitoring Systems Laboratory-Las Vegas
James Lazorchak, Environmental Monitoring Systems Laboratory-Cincinnati
Douglas Middaugh, Environmental Research Laboratory-Gulf Breeze
George Morrison, Environmental Research Laboratory-Narragansett
Teresa Norberg-King, Environmental Research Laboratory-Duluth
Donald Klemm, Environmental Monitoring Systems Laboratory-Cincinnati
Philip Lewis, Environmental Monitoring Systems Laboratory-Cincinnati
Cornelius I. Weber, Environmental Monitoring Systems Laboratory-
Cincinnati
Richard Swartz, Environmental Research Laboratory-Newport
Margarete Heber, Human Health and Ecological Criteria Division, Office
of Science & Technology (OST), Office of Water (OW)
Bruce Newton, Assessment and Watershed Protection Division, Office
of Wetlands, Oceans, and Watersheds, OW
Christopher Zarba, Health and Ecological Criteria Division, Office
of Science & Technology, OW
Daniel Rieder, Hazard Evaluation Division, Office of Pesticide
Programs
Jerry Smrchek, Health and Environmental Review Division, Office
of Toxic Substances
iv
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Gail Hansen, Office of Solid Waste
Royal Nadeau, Emergency Response Team, Edison
Teresa J. Norberg-King
Chairman, Biological Advisory Committee
Regulatory Ecotoxicology Branch, ERL-Duluth
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ABSTRACT
This manual describes six short-term (one hour to nine days) estuarine and
marine methods for measuring the chronic toxicity of effluents and receiving
waters to five species: the sheepshead minnow, Cypn'nodon van'egatus; the
inland silverside, Menidia beryllina-, the mysid, Mysidopsis bahia; the sea
urchin, Arbacia punctulata; and the red macroalga, Champia parvula. The
methods include single and multiple concentration static renewal and static
nonrenewal toxicity tests for effluents and receiving waters. Also included
are guidelines on laboratory safety, quality assurance, facilities, and
equipment and supplies; dilution water; effluent and receiving water sample
collection, preservation, shipping, and holding; test conditions; toxicity
test data analysis; report preparation; and organism culturing, holding, and
handling. Examples of computer input and output for Dunnett's Procedure,
Probit Analysis, Trimmed Spearman-Karber Method, and the Linear Interpolation
Method are provided in the Appendices.
VI
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Foreword
Preface
Abstract
CONTENTS i
Page
Tables . . .
Acknowledgments
vi
'xii
xviii
xxix
Section Number Page
1. Introduction 1
2. Short-Term Methods for Estimating Chronic Toxicity ! ....... 4
Introduction 4
Types of Tests . . . . i 7
Static Tests 8
Advantages and Disadvantages of Toxicity Test Types ..... 8
3. Health and Safety . . . . 10
General Precautions 10
Safety Equipment 10
General Laboratory and Field Operations . . . . 10
Disease Prevention 11
Safety Manuals 11
Waste Disposal '• 11
4. Quality Assurance 12
Introduction
12
Facilities, Equipment, and Test Chambers . . . 12
Test Organisms 13
Laboratory Water Used for Culturing and
and Test Dilution Water . 13
Effluent and Receiving Water Sampling and
Handling 13
Test Conditions 13
Quality of Test Organisms 14
Food Quality 14
Acceptability of Chronic Toxicity Tests 15
Analytical Methods ....... 16
Calibration and Standardization 16
Replication and Test Sensitivity 16
Variability in Toxicity Test Results i. 16
Test Precision 17
Demonstrating Acceptable Laboratory Performance !. 18
Documenting Ongoing Laboratory Performance ... 18
Reference Toxicants 21
Record Keeping „ 21
Video Tapes of USEPA Culture and Toxicity
Test Methods
21
Supplemental Reports for Training Video Tapes ......... 22
5. Facilities, Equipment, and Supplies ..... 23
General Requirements . 23
Test Chambers .... i....... 24
vn
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CONTENTS (CONTINUED)
Section Number Page
Cleaning Test Chambers and Laboratory Apparatus ....... 24
Apparatus and Equipment for Culturing and
Toxicity Tests 25
Reagents and Consumable Materials 25
Test Organisms 25
Supplies 25
6. Test Organisms 27
Test Species 27
Sources of Test Organisms 28
Life Stage 29
Laboratory Culturing 29
Holding and Handling of Test Organisms 29
Transportation to the Test Site .30
Test Organism Disposal 31
7. Dilution Water 32
Types of Dilution Water 32
, Standard, Synthetic Dilution Water 32
Use of Receiving Water as Dilution Water . 34
Use of Tap Water as Dilution Water 36
Dilution Water Holding 36
8. Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests 37
Effluent Sampling 37
Effluent Sample Types 37
Effluent Sampling Recommendations 38
Receiving Water Sampling 39
Effluent and Receiving Water Sample Handling,
Preservation, and Shipping 40
Sample Receiving 41
Persistence of Effluent Toxicity During Sample
Shipment and Holding 41
Preparation of Effluent and Receiving Water Samples
for Toxicity Tests 41
Preliminary Toxicity Range-finding Tests . 45
Multiconcentration (Definitive) Effluent
Toxicity Tests 45
Receiving Water Tests 46
9. Chronic Toxicity Test Endpoints and Data Analysis . . . . . . .47
Endpoints 47
Relationship between Endpoints Determined
by Hypothesis Testing and Point Estimation Techniques ... 48
Precision 50
Data Analysis 50
Choice of Analysis 52
Hypothesis Tests 54
Point Estimation Techniques . . 56
viii
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CONTENTS (CONTINUED)
•
Section Number ' Page
10. Report Preparation j 58
Introduction ....... 58
Plant Operations 58
Source of Effluent, .Receiving Water, and Dilution Water ... 58
Test Methods 59
Test Organisms 59
Quality Assurance 59
Results 59
Conclusions and Recommendations 60
11. Test Method: Sheepshead Minnow, Cyprinodon van'eg&tus, Larval
Survival and Growth Test Method 1004.0 ..*... 61
Scope and Application ............ 61
Summary of Method . 61
Interferences 61
Safety 62
Apparatus and Equipment 62
Reagents and Consumable Materials [ 64
Effluent and Receiving Water Collection, Preservation, and
Storage \ . 74
Calibration and Standardization ! 74
Quality Control , 74
Test Procedures ; .' 74
Summary of Test Conditions and Test Acceptability Criteria . 84
Acceptability of Test Results j ....... 84
Data Analysis J . 84
Precision and Accuracy « ' 116
12. Test Method: Sheepshead Minnow, Cyprinodon variegntus, Embryo-
larval Survival and Teratogenicity Test Method 1005.0 .... 124
Scope and Application 1 124
Summary of Method 1 124
Interferences '. » 124
Safety * 125
Apparatus and Equipment i 125
Reagents and Consumable Materials . 127
Effluent and Receiving Water Collection, Preservation, and
Storage I 137
Calibration and Standardization I 137
Quality Control ....... L 138
Test Procedures L 138
Acceptability of Test Results L 144
Summary of Test Conditions and Test Acceptability Criteria 144
Data Analysis L ...... 144
Precision and Accuracy . L 160
13. Test Method: Inland Silverside, Menidia beryTlina, Larval
Survival and Growth Method 1006.0 I 163
Scope and Application L 163
Summary of Method L 163
ix
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CONTENTS (CONTINUED)
Section Number Page
Interferences 163
Safety 164
Apparatus and Equipment 164
Reagents and Consumable Materials 167
Effluent and Receiving Water Collection, Preservation, and
Storage 177
Calibration and Standardization 177
Quality Control 177
Test Procedures 177
Summary of Test Conditions and Test Acceptability Criteria 186
Acceptability of Test Results 186
Data Analysis 186
Precision and Accuracy 216
14. Test Method: Mysid, Mysidopsis bahia, Survival, Growth, and
Fecundity Test Method 1007.0 222
Scope and Application 222
Summary of Method 222
Interferences 222
Safety [ [ 223
Apparatus and Equipment 223
Reagents and Consumable Materials 225
Effluent and Receiving Water Collection, Preservation, and
Storage . . 235
Calibration and Standardization ..... ... 235
Quality Control ...... 235
Test Procedures 235
Summary of Test Conditions and Test Acceptability Criteria 243
Acceptability of Test Results 243
Data Analysis 243
Precision and Accuracy ' 294
15. Test Method: Sea Urchin, Arbacia punctulata, Fertilization Test
Method 1008.0 300
Scope and Application . 300
Summary of Method 300
Interferences 300
Safety ' [ 301
Apparatus and Equipment 301
Reagents and Consumable Materials 302
Effluent and Receiving Water Collection, Preservation, and
Storage 311
Calibration and Standardization 311
Quality Control 311
Test Procedures ; 311
Summary of Test Conditions and Test Acceptability Criteria 316
Acceptability of Test Results 316
Data Analysis 319
Precision and Accuracy '.'.'. 333
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CONTENTS (CONTINUED)
I
Section Number Page
16. Test Method: Red Macroalga, Champia parvuTa, Reproduction Test
Method 1009.0 . 341
Scope and Application 341
Summary of Method 341
Interferences 341
Safety i 342
Apparatus and Equipment 342
Reagents and Consumable Materials 343
Effluent and Receiving Water Collection, Preservation, and
Storage 350
Calibration and Standardization 351
Quality Control 351
Test Procedures 351
Summary of Test Conditions and Test Acceptability Criteria 356
Acceptability of Test Results 360
Data Analysis 360
Precision and Accuracy 373
Cited References 380
Bibliography 391
Appendices 401
A. Independence, Randomization, and Outliers ..... 403
B. Validating Normality and Homogeneity of Variance
Assumptions 409
C. Dunnett's Procedure 419
D. T test with Bonferroni's Adjustment 432
E. Steel's Many-one Rank Test ' 438
F. Wilcoxon Rank Sum Test 443
G. Single Concentration Toxicity Test - Comparison
of Control with 100% Effluent or Receiving Water 450
H. Probit Analysis 454
I. Spearman-Karber Method 457
J. Trimmed Spearman-Karber Method 462
K. Graphical Method 467
L. Linear Interpolation Method 471
Cited References 482
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FIGURES
SECTION 1-10
Number Page
1. Control (cusum) charts 20
2. Flowchart for statistical analysis of test data 53
SECTION 11
Number Page
1. Embryonic development of sheepshead minnow, Cypn'nodon
variegatus 72
2. Data form for the sheepshead minnow, Cypn'nodon variegatus,
larval survival and growth test. Daily record of
larval survival and test conditions 80
3. Data form for the sheepshead minnow, Cypn'nodon variegatus,
larval survival and growth test. Dry weights of
larvae 83
4. Data form for the sheepshead minnow, Cypn'nodon variegatus,
larval survival and growth test. Summary of test
results 85
5. Flowchart for statistical analysis of the sheepshead minnow,
Cypn'nodon variegatus, larval survival data by hypothesis
testing 89
6. Flowchart for statistical analysis of the sheepshead minnow,
Cypn'nodon variegatus, larval survival data by
point estimation 90
7. Plot of mean survival proportion data in Table 5 93
8. Flowchart for statistical analysis of the sheepshead
minnow, Cypn'nodon variegatus, larval growth data 101
9. Plot of weight data from sheepshead minnow, Cypn'nodon
variegatus, larval survival and growth test 103
10. Plot of raw data, observed means and smoothed means
for the sheepshead minnow, Cypn'nodon variegatus,
growth data from Tables 4 and 21 112
xn
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FIGURES (CONTINUED)
SECTION 11 (Continued)
Number j Page
11. ICPIN program output for the IC25 114
12. ICPIN program output for the IC50 i 115
SECTION 12
Number Page
!
1. Data form for sheepshead-minnow, Cyprinodon variegatus,
embryo-larval survival and teratogenicity test. Daily record
of embryo-larval survival/terata and test conditions 128
i
2. Embryonic development of sheepshead minnow, Cyprinodon
variegatus 142
3. Flowchart for statistical analysis of sheepshead minnow,
Cyprinodon variegatus, embryo-larval survival and
teratogenicity test. Survival and terata data 148
4. Plot of sheepshead minnow, Cyprinodon variegatus, total
mortality data from the embryo-larval test 150
5. Output for USEPA Probit Analysis program, Version 1.5 159
SECTION 13
i
Number • ' Page
1. Glass chamber with sump area
166
2. Inland silverside, Menidia beryllina 176
i
3. Data form for the inland silverside, Menidia beryllina,
larval survival and growth test. Daily record of larval
survival and test conditions j 182
4. Data form for the inland silverside, Menidia beryllina,
larval survival and growth test. Dry weights of
larvae i 185
i
5. Data form for the inland silverside, Menidia beryllina, larval
survival and growth test. Summary of test results 187
xm
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FIGURES (CONTINUED)
SECTION 13 (Continued)
Number Page
6. Flowchart for statistical analysis of the inland silverside,
Henida beryl Una, survival data by hypothesis testing 191
7. Flowchart for statistical analysis of the inland silverside,
Nenida beryllina, survival data by point estimation 192
8. Plot of mean survival proportion of the inland silverside,
Menidia beryllina, larvae 193
9. Output for USEPA Probit Analysis program, Version 1.5 203
10. Flowchart for statistical analysis of the inland silverside,
Menidia beryllina, growth data 204
11. Plot of mean weights of inland silverside, Menidia beryllina,
larval survival and growth test 206
12. Plot of the raw data, observed means, and smoothed
means from Tables 13 and 19 215
13. ICPIN program output for the IC25 217
14. ICPIN program output for the IC50 218
SECTION 14
Number Page
1. Apparatus (brood chamber) for collection of juvenile mysids,
Mysidopsis bahia 234
2. Data form for the mysid, Mysidopsis bahia, water quality
measurements 240
3. Mature female mysid, Mysidopsis bahia, with eggs in oviducts . 241
4. Mature female mysid, Mysidopsis bahia, with eggs in oviducts
and developing embryos in the brood sac 242
5. Mature male mysid, Mysidopsis bahia . . 243
6. Immature mysid, Mysidopsis bahia, (A) lateral view,
(B) dorsal view 244
xiv
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FIGURES (CONTINUED)
SECTION 14 (Continued)
Number Page
7. Data form for the mysid, Mysidopsis bahia, survival and
fecundity data 245
8. Data form for the mysid, Mysidopsis bahia, dry weight
measurements 247
9. Flowchart for statistical analysis of mysid, Mysidopsis bahia,
survival data by hypothesis testing j ....... 253
10. Flowchart for statistical analysis of mysid, Mysidopsis bahia,
survival data by point estimation i 254
11. Plot of survival proportions of mysids, Mysidopsis hahia,
at each treatment level . 255
12. Output for USEPA Probit Analyis program, Version 1.5' 262
13. Flowchart for statistical analysis of mysid, Mysidopsis bahia,
growth data 264
14. Plot of mean growth data for mysid, Mysidopsis bahia, test . . 265
i
15. Plot of raw data, observed means, and smoothed means
for the mysid, Mysidopsis bahia, growth data from
Tables 13 and 20 274
16. ICPIN program output for the IC25 j 276
17. ICPIN program output for the IC50 277
i
18. Flowchart for statistical analysis of mysid, Mysidopsis
bahia, fecundity data j ...... 278
19. Proportion of female mysids, Mysidopsis bahia, with eggs ... 280
i
20. Plot of the mean proportion of females mysids,
Mysidopsis bahia, with eggs . 290
i
21. ICPIN program output for the IC25 292
22. ICPIN program output for the IC50 293
xv
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FIGURES (CONTINUED)
SECTION 15
Number Page
1. Data form for (1) fertilization test using sea urchin,
Arbacia punctulata 304
2. Data form (2) for fertilization test using sea urchin,
Arbacia punctulata 305
3. Data form (3) for fertilization test using sea urchin,
Arbacia punctulata 306
4. Flowchart for statistical analysis of sea urchin,
Arbacia punctulata, by point estimation .... 321
5. Plot of mean percent of fertilized sea urchin,
Arbacia punctulata, eggs . . 322
6. ICPIN program output for the IC25 '. 332
7. ICPIN program output for the IC50 334
SECTION 16
Number Page
1. Life history of the red macroalga, Champia parvula 347
2. Apex of branch of female plant, showing sterile
hairs and reproductive hairs (trichogynes) 349
3. A portion of the male thai 1 us showing spermatial sori .... 349
4. A magnified portion of a spermatial sorus 350
5. Apex of a branch on a mature female plant that was
exposed to spermatia from a male plant 350
6. Data form for the red macroalga, Champia parvula, sexual
reproduction test. Receiving water summary sheet 352
7. A mature cystocarp 355
8. Comparison of a very young branch and an immature
cystocarp 355
xvi
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FIGURES (CONTINUED)
SECTION 16 (Continued)
Number Page
9. An aborted cystocarp 356
i
10. Data form for the red macroalga, Champia parvula, sexual
reproduction test.. Cystocarp data sheet 357
11. Flowchart for statistical analysis of the red macroalga,
Champia parvula, data 362
12. Plot of the number of cystocarps per plant . . . 364
13. ICPIN program output for the IC25 374
" !
14. ICPIN program output for the IC50 375
xvii
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TABLES
SECTION 1-10
Number Page
1. Rational Inter!aboratory study of chronic toxicity test
precision, 1991: Summary of responses using two reference
toxicants 18
2. Commercial suppliers of brine shrimp (Artemia) cysts 26
3. Preparation of 6P2 artificial seawater using reagent
grade chemicals 34
4. Oxygen solubility (mg/L) in water at equilibrium with
air at 760 mm Hg 43
5. Percent unionized NH3 in aqueous ammonia solutions:
temperature 15-26°C and pH 6.0-8.9 44
SECTION 11
Number Page
1. Reagent grade chemicals used in the preparation of GP2
artificial seawater for the sheepshead minnow, Cypn'nodon
van'egatus, toxicity test 67
2. Preparation of test solutions at a salinity of 20%o, using
20%o salinity dilution water prepared from natural seawater,
hypersaline brine, or artificial sea salts 76
3. Summary of test conditions and test acceptability criteria
for sheepshead minnow, Cypn'nodon van'egatus, larval survival
and growth test with effluents and receiving waters 86
4. Summary of survival and growth data for sheepshead minnow,
Cypn'nodon van'egatus, larvae exposed to an effluent for
seven days 88
5. Sheepshead minnow, Cypn'nodon van'egatus, survival data .... 92
6. Centered observations for Shapiro-Wilk's example 92
7. Ordered centered observations for the Shapiro-Wilk's
example 94
8. Coefficients and differences for Shapiro-Wilk's example .... 95
xvm
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TABLES (CONTINUED)
SECTION 11 (Continued)
Number Page
9. Assigning ranks to the control and 6.25% effluent
concentration for Steel's Many-one Rank Test 96
10. Table of ranks .....' 96
11. Rank sums ; 96
12. Data for example of Spearman-Karber Analysis . . 98
i
13. Sheepshead minnow, Cyprinodon variegatus, growth data 102
14. Centered observations for Shapiro-Milk's example . i 102
15. Ordered centered observations for Shapiro-Wilk's example '. . . 104
16. Coefficients and differences for Shapiro-Wilk's example . . . 105
17. ANOVA table . . 107
18. ANOVA table for Dunnett's Procedure example . . 109
19. Calculated t values 110
20. Sheepshead minnow, Cyprinodon variegatus, mean growth
response after smoothing Ill
21. Single-laboratory precision of the sheepshead minnow,
Cyprinodon variegatus, larval survival and growth test
performed in FORTY FATHOMS® artificial seawater, using
larvae from fish maintained and spawned in FORTY FATHOMS®
artificial seawater, using copper (Cu) sulfate as a reference
toxicant ...... 117
i
22. Single-laboratory precision of the sheepshead minnow,
Cyprinodon variegatus, larval survival and growth test
performed in FORTY FATHOMS® artificial seawater, using
larvae from fish maintained and spawned in FORTY FATHOMS®
artificial seawater, using sodium dodecyl sulfate (SDS) as
a reference toxicant 118
xix
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TABLES (CONTINUED)
SECTION 11 (Continued)
Number Page
23. Single-laboratory precision of the sheepshead minnow,
Cypn'nodon variegatus, larval survival and growth test
performed in natural seawater, using larvae from fish
maintained and spawned in natural seawater, using copper
(Cu) sulfate as a reference toxicant 119
24. Single-laboratory precision of the sheepshead minnow,
Cypn'nodon variegatus, larval survival and growth test
performed in natural seawater, using larvae from fish
maintained and spawned in natural seawater, using sodium
dodecyl sulfate (SDS) as a reference toxicant 120
25. Single-laboratory precision of the sheepshead minnow,
Cypn'nodon variegatus, larval survival and growth test
performed in FORTY FATHOMS® artificial seawater, using
larvae from fish maintained and spawned in FORTY FATHOMS®
artificial seawater, and hexavalent chromium as a reference
toxicant 121
26. Comparison of larval survival (LC50) and growth (IC50)
values for the sheepshead minnow, Cypn'nodon variegatus,
exposed to sodium dodecyl sulfate (SDS) and copper (Cu)
sulfate in 6P2 artificial seawater medium or natural
seawater ". . . . 122
27. Data from interlaboratory study of the sheepshead minnow,
Cypn'nodon variegatus, larval survival and growth test,
using an industrial effluent as a reference toxicant 123
SECTION 12
Number Page
1. Preparation of test solutions at a salinity of 20%o,
using 20%o natural or artificial seawater, hypersaline
brine, or artificial sea salts 132
2. Summary of test conditions and test acceptability criteria
for sheepshead minnow, Cypn'nodon variegatus, embryo-larval
survival and teratogenicity test with effluents and receiving
waters 145
3. Sheepshead minnow, Cypn'nodon variegatus, embryo-larval
total mortality data 149
xx
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TABLES (CONTINUED)
SECTION 12 (Continued)
Number Page
4. Centered observation for Shapiro-Wilk's example . . 149
5. Ordered centered observations for Shapiro-Wilk's example . . . t 151
6. Coefficients and differences for Shapiro-Wilk's example .... 152
i
7. ANOVA table !...... 154
8. ANOVA table for Dunnett's Procedure example . . 156
9. Calculated t values 157
.
10. Data for Probit Analysis 158
11. Single-laboratory precision of the sheepshead minnow,
Cyprinodon variegatus, embryo-larval survival and
teratogenicity test performed in HW MARINEMIX® artificial
seawater, using embryos from fish maintained and spawned
in HW MARINEMIX® artificial seawater using copper (Cu)
sulfate as a reference toxicant 161
12. Single-laboratory precision of the sheepshead minnow',
Cyprinodon variegatus, embryo-larval survival and
teratogenicity test performed in HW MARINEMIX® artificial
seawater, using embryos from fish maintained and spawned
in HW MARINEMIX® artificial seawater using sodium dodecyl
sulfate (SDS) as a reference toxicant 162
i
SECTION 13
Number i Page
1. Preparation of 3 L saline water from deionized water and a
hypersaline brine of 100%o needed for test solutions at
20%o salinity | 170
2. Reagent grade chemicals used in the preparation of GP2
artificial seawater for the inland silverside, Menidia
beryTlina, toxicity test ...... 171
3. Summary of test conditions and test acceptability criteria
for inland silverside, Menidia beryTTina, larval survival
and growth test with effluents and receiving waters
xxi
188
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TABLES (CONTINUED)
SECTION 13 (Continued)
Number Pa9e
4. Inland silverside, Mem'dia beryllina, larval survival data . . 194
5. Centered observations for Shapiro-WiIk's example 194
6. Ordered centered observations for Shapiro-WiIk's example ... 195
7. Coefficients and differences for Shapiro-WiIk's example ... 196
8. ANOVA table 198
9. ANOVA table for Dunnett's Procedure example 199
10. Calculated t values 200
11. Data for Probit Analysis 202
12. Inland silverside, Mem'dia beryllina, growth data 205
13. Centered observations for Shapiro-WiIk's example 205
14. Ordered centered observations for Shapiro-WiIk's example . . . 207
15. Coefficients and differences for Shapiro-WiIk's example ... 208
16. ANOVA table 210
17. ANOVA table for Dunnett's Procedure example 211
18. Calculated t values 212
19. Inland silverside mean growth response after smoothing .... 214
20. Single-laboratory precision of the inland silverside,
Nenidia beryllina, survival and growth test performed
in natural seawater, using larvae from fish maintained
and spawned in natural seawater, and copper (Cu) as a
reference toxicant 219
21. Single-laboratory precision of the inland silverside,
Mem'dia beryllina, survival and growth test performed
in natural seawater, using larvae from fish maintained
and spawned in natural seawater, and sodium dodecyl
sulfate (SDS) as a reference toxicant 220
xxn
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TABLES (CONTINUED) i
i
SECTION 13 (Continued) !
Number Page
22. Comparison of the single-laboratory precision of the inland
silverside, Mem'dia beryllina, larval survival (LC50) and
growth (IC50) values exposed to sodium dodecyl sulfate
(SDS) or copper (Cu) sulfate, in GP2 artificial seawater
medium or natural seawater 221
SECTION 14 - i
Number Page
1. Reagent grade chemicals used in the preparation of GP2
artificial seawater for the mysid, Mysidopsis bahia, toxicity
test 228
2. Quantities of effluent, deionized water, and hypersaline
brine (100%o) needed to prepare 1800 ml volumes of test
solution with a salinity of 20%o 229
3. Summary of test conditions and test acceptability criteria
for the mysid, Mysidopsis bahia, seven day survival, growth,
and fecundity test with effluents and receiving waters .... 249
4. Data for Mysidopsis bahia 7-day survival, growth, and
fecundity test 251
i
5. Mysid, Mysidopsis bahia, survival data 256
6. Centered observations for Shapiro-Wilk's example 257
7. Ordered centered observations for Shapiro-Wilk's example . . . 258
8. Coefficients and differences for Shapiro-Wilk's example . . . 259
9. Assigning ranks to the control and 50 ppb concentration level
for Steel's Many-one Rank Test 260
10. Table of ranks i. ...... 261
I
11. Rank sums , 261
.
12. Data for Probit Analysis 263
xxm
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TABLES (CONTINUED)
SECTION 14 (Continued)
Number Page
13. Hysid, Mysidopsis bahia, growth data 263
14. Centered observations for Shapiro-Wilk's example 266
15. Ordered centered observations for Shapiro-Wilk's example . . . 267
16. Coefficients and differences for Shapiro-Wilk's example . . . 268
17. ANOVA table . 270
18. ANOVA table for Dunnett's Procedure example 271
19. Calculated t values 272
20. Mysid, Mysidopsis bahia, mean growth response after
smoothing 273
21. Hysid, Mysidopsis bahia, fecundity data: Percent females
with eggs 281
22. Centered observations for Shapiro-Wilk's example 282
23. Ordered centered observations for Shapiro-Wilk's example . . . 283
24. Coefficients and differences for Shapiro-Wilk's example . . . 283
25. ANOVA table 285
26. ANOVA table for the t test with Bonferroni's
Adjustment example 287
27. Calculated t values . . . 288
28. Mysid, Mysidopsis bahia, mean proportion of females
with eggs 289
29. Single-laboratory precision of the mysid, Mysidopsis bahia,
survival, growth, and fecundity test performed in natural
seawater, using juveniles from mysids cultured and spawned
in natural seawater and copper (Cu) sulfate as a reference
toxicant 295
xxiv
-------
TABLES (CONTINUED)
.
SECTION 14 (Continued) j
Number Page
,
30. Single-laboratory precision of the mysid, Mysidopsis bahia,
survival, growth, and fecundity test performed in natural
seawater, using juveniles from mysids cultured and spawned
in natural seawater and sodium dodecyl sulfate (SDS) as a
reference toxicant i, 296
31. Comparison of survival (LC50), growth and fecundity (IC50)
results from 7-day tests with the mysid, Mysidopsis bah fa,
using natural seawater (NSW) and artificial seawater (GP2) as
dilution water and sodium dodecyl sulfate (SDS) as a
reference toxicant i 297
32. Comparison of survival (LC50), growth and fecundity (IC50)
results from 7-day tests with the mysid, Mysidopsis bahia,
using natural seawater (NSW) and artificial seawater (GP2)
as dilution water and copper (Cu) sulfate as a reference
toxicant ; 298
I
33. Control results from 7-day survival, growth, and fecundity
tests with the mysid, Mysidopsis bahia, using natural
seawater and artificial seawater (GP2) as a dilution
water ,, 299
SECTION 15
Number Page
1. Preparation of test solutions at a salinity of 30%o using
natural seawater, hypersaline brine, or artificial sea salts . 309
2. Reagent grade chemicals used in the preparation of GP2
artificial seawater for the sea urchin, Arbacia punctulata
toxicity test ,; 310
•
3. Summary of test conditions and test acceptability criteria
for sea urchin, Arbacia punctulata, fertilization test with
effluent and receiving waters ,; 317
4. Data from sea urchin, Arbacia punctulata, fertilization test . 319
i
5. Sea urchin, Arbacia punctulata, fertilization data i 323
6. Centered observations for Shapiro-Wilk's example .1 323
7. Ordered centered observations for Shapiro-Wilk's example . . . 324
xxv
-------
TABLES (CONTINUED)
SECTION 15 (Continued)
Number Page
8. Coefficients and differences for Shapiro-Wilk's example . . . 325
9. ANOVA table 326
10. ANOVA table for Dunnett's Procedure example 328
11.. Calculated t values 329
12. Sea urchin, Arbacia punctulata, mean proportion of
fertilized eggs 331
13. Single-laboratory precision of the sea urchin, Arbacia
punctulata, fertilization test performed in FORTY FATHOMS®
artificial seawater, using gametes from adults maintained
in FORTY FATHOMS® artificial seawater, or obtained directly
from natural sources, and copper (Cu) sulfate as reference
toxicant 335
14. Single-laboratory precision of the sea urchin,
Arbacia punctulata, fertilization test performed
in FORTY FATHOMS® artificial seawater, using gametes
from adults maintained in FORTY FATHOMS® artificial
seawater, or obtained directly from natural sources,
and sodium dodecyl sulfate (SDS) as a reference toxicant ... 336
15. Single-laboratory precision of the sea urchin,
Arbacia punctulata, fertilization test performed
in natural seawater, using gametes from adults
maintained in natural seawater and copper (Cu)
sulfate as a reference toxicant 337
16. Single-laboratory precision of the sea urchin,
Arbacia punctulata, fertilization test performed
in natural seawater, using gametes from adults
maintained in natural seawater and sodium dodecyl
sulfate (SDS) as a reference toxicant 338
17. Single-laboratory precision of the sea urchin,
Arbacia punctulata, fertilization test performed
in 6P2, using gametes from adults maintained in
6P2 artificial seawater and copper (Cu) sulfate and
sodium dodecyl sulfate (SDS) as reference toxicants ...... 339
xxvi
-------
TABLES (CONTINUED)
SECTION 15 (Continued)
Number Page
.
18. Single-laboratory precision of the sea urchin, Arbacia
punctulata, fertilization test performed in natural sea.water,
using gametes from adults maintained in natural seawater
and copper (Cu) sulfate and sodium dodecyl sulfate (SDS)
as reference toxicants 340
SECTION 16
Number
Page
1. Nutrients to be added to natural seawater and to
artificial seawater (GP2) described in Table 2 345
2. Reagent grade chemicals used in the preparation of GP2
artificial seawater for use in conjunction with natural
seawater for the red macroalga, Champia parvula, culturing
and toxicity testing 346
3. Summary of test conditions and test acceptability criteria
for red macroalga, Champia parvula, sexual reproduction
test with effluents and receiving waters ' 358
i
4. Data from the red macroalga, Champia parvula, effluent
toxicity test. Cystocarp counts for individual plants and
mean count per test chamber for each effluent concentration . 361
5. The red macroalga, Champia parvula, sexual reproduction data . 363
6. Centered observations for Shapiro-Wilk's example 365
7. Ordered centered observations for Shapiro-Wilk's example . . . 366
I
8. Coefficients and differences for Shapiro-Wilk's example . . . 366
9. ANOVA table ...... 368
10. ANOVA table for Dunnett's Procedure example 370
11. Calculated t values ...... 371
12. Red macroalga, Champia parvula, mean number of cystocarps . . 372
xxv
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TABLES (CONTINUED)
SECTION 16 (Continued)
Number Page
13. Single-laboratory precision of the red macroalga, Champia
parvula, reproduction test performed in a 50/50 mixture
of natural seawater and GP2 artificial seawater, using
gametes from adults cultured in natural seawater. The
reference toxicant used was copper (Cu) sulfate 376
14. Single-laboratory precision of the red macroalga, Champia
parvula, reproduction test performed in a 50/50 mixture of
natural seawater and GP2 artificial seawater, using gametes
from adults cultured in natural seawater. The reference
toxicant used was sodium dodecyl sulfate (SDS) 377
15. Single-laboratory precision of the red macroalga,
Champia parvula, reproduction test in natural seawater
(30%o salinity). The reference toxicant used was copper
(Cu) sulfate 378
16. Single-laboratory precision of the red macroalga,
Champia parvula, reproduction test in natural seawater
(30%o salinity). The reference toxicant used was sodium
dodecyl sulfate (SDS) 379
xxv m
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ACKNOWLEDGMENTS
The principal authors of this document are Donald Klemm, Cornelius Weber,
Philip Lewis, James Lazorchak, Florence Fulk, and Timothy Neiheisel,
Environmental Monitoring Systems Laboratory-Cincinnati, Ohio; George Morrison,
Environmental Research Laboratory, Narragansett, Rhode Island; Dennis M.
McMullen, Technology Applications Incorporated, Cincinnati, Ohio and Cathy
Poore, Computer Sciences Corporation, Cincinnati, Ohio. Contributors to
specific sections of this manual are listed below.
1. Sections 1-10; General Guidelines i
Margarete Heber, OST, Office of Water
Donald Klemm, EMSL-Cincinnati
James Lazorchak, EMSL-Cincinnati
Philip Lewis, EMSL-Cincinnati
George Morrison, ERL-Narragansett
Teresa Norberg-King, ERL - Duluth
William Peltier, ESD, Region 4
- Cornelius Weber, EMSL - Cincinnati
2. Sections 11-16; Toxicity Test Methods
Margarete Heber, OST, Office of Water
Donald Klemm, EMSL-Cincinnati
George Morrison, ERL-Narragansett
William Peltier, ESD, Region 4
Quentin Pickering, EMSL-Cincinnati
3. Data Analysis (Section 9, 11-16, and Appendices)
Florence Fulk, EMSL-Cincinnati
Laura Gast, Technology Applications, Inc. (TAI)
Cathy Poore, Computer Sciences Corporation (CSC)
Review comments from the following persons are gratefully acknowledged:
i
Pam Comeleo, Science Application International Corporation, Environmental
Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Randy Comeleo, Science Application International Corporation, Environmental
Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Philip Crocker, Water Quality Management Branch, U.S. Environmental
Protection Agency, Region 6, Dallas, TX
Dan Fisher, John Hopkins University, Queenstown, MD
Terry Hollister, Environmental Services Division, Houston Branch, U.S.
Environmental Protection Agency, Region 6, Houston, TX
Glen Modica, Science Application International Corporation, Environmental
Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Michael Morton, Permits Branch, U.S. Environmental Protection Agency,
Region 6, Dallas, TX I
Peter Nolan, Environmental Services Division, U.S. Environmental Protection
xxix
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ACKNOWLEDGMENTS (CONTINUED)
Agency, Region 1, Lexington, MA
Mark Tagliabue, Science Application International Corporation, Environmental
Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Glenn Thursby, Environmental Research Laboratory, U.S. Environmental
Protection Agency, Narragansett, Rhode Island
Jerry Smrchek, Office of Pesticides and Toxic Substances, Environmental
Effects Branch, Health and. Environmental Review Division, U.S. Environmental\
Protection Agency, Washington, DC
Amy Wagner, Laboratory Support Section, U.S. Environmental Protection
Agency, San Francisco, CA
Audrey Weber, Virginia Water Control Board, Glen Allen, VA
Many useful public comments on the first edition of the marine and
estuarine toxicity test methods "Short-term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Freshwater Organisms, EPA/600/4-
87/028 (USEPA, 1988a) were received in response to the proposed rule,
published in the Federal Register, December 4, 1989 [FR 54(231):50216-§0224],
regarding the Agency's intent to include short-term chronic toxicity tests in
Table IA, 40 CFR Part 136. These comments were carefully considered in
preparation of the second edition of the manual, and are included in the
Public Docket for the rulemaking, located at room 2904, USEPA Headquarters,
Washington, D.C.
Materials in this manual were taken in part from the following sources:
Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and
Amphibians, Environmental Research Laboratory, U.S. Environmental Protection
Agency, Duluth, Minnesota, EPA-660/3-75/009 (USEPA, 1975); Handbook for
Analytical Quality Control in Water and Wastewater Laboratories, Environmental
Monitoring and Support Laboratory - Cincinnati, U.S. Environmental Protection
Agency, Cincinnati, Ohio, EPA-600/4-79/019 (USEPA, 1979a); Methods for
chemical analysis of water and wastes, Environmental Monitoring and Support
Laboratory-Cincinnati, U. S. Environmental Protection Agency, Cincinnati, OH,
EPA-600/4-79-020 (USEPA, 1979b); Interim NPDES Compliance Biomonitoring
Inspection Manual, Enforcement Division, Office of Water Enforcement, U.S.
Environmental Protection Agency, Washington, D.C. (USEPA, 1979c); Methods for
Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms,
Environmental Monitoring and Support Laboratory - Cincinnati, U.S.
Environmental Protection Agency, Cincinnati, Ohio, EPA-600/4-85/013 (USEPA,
1985a); Short-term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Freshwater Organisms, Environmental Monitoring and
Support Laboratory - Cincinnati, U.S. Environmental Protection Agency,
Cincinnati, Ohio, EPA-600/4-85/014 (USEPA, 1985b); Users Guide to the Conduct
and Interpretation of Complex Effluent Toxicity Tests at Estuarine/Marine
Sites, Environmental Research Laboratory-Narragansett (ERL-N), U.S.
Environmental Protection Agency, Narragansett, Rhode Island (USEPA, 1987a);
NPDES Compliance Inspection Manual, Office of Water Enforcement and Permits
(EN-338), U.S. Environmental Protection Agency, Washington, D.C. (USEPA,
1988b); Short-term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Marine and Estuarine Organisms, Environmental
Monitoring and Support Laboratory-Cincinnati, U.S. Environmental Protection
xxx
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ACKNOWLEDGMENTS (CONTINUED)
Agency, Cincinnati, Ohio, EPA-600/4-87/028 (USEPA, 1988a); Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms, Environmental Monitoring Systems'Laboratory-Cincinnati,
U.S. Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-89/001
(USEPA, 1989a); Methods for Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms, Environmental Monitoring Systems Laboratory-
Cincinnati, U.S. Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-
90/027F (USEPA, 1993a); Short-term Methods for Estimating the Chronic Toxicity
of Effluents and Receiving Waters to Freshwater Organisms (Third Edition),
Environmental Monitoring Systems Laboratory-Cincinnati, U.S. Environmental
Protection Agency, Cincinnati, Ohio, EPA/600/4-91/002 (USEPA, 1993b); and the
Technical Support Document for Water Quality-based Toxic Control, Office of
Water Enforcement and Permits and Office of Water Regulations and Standards,
U.S. Environmental Protection Agency, Washington, D.C., EPA/505/2-90-001
(USEPA, 1991a).
Five of the six methods in the manual were adapted from methods developed
at the Environmental Research Laboratory-Narragansett. Individuals
responsible for specific methods are as follows: Melissa Hughes, Margarete
Heber, and Walter Berry, Science Applications International Corporation
(SAIC), and Steven Schimmel, USEPA, developed the sheepshead minnow larval
survival and growth test; Margarete Heber and Melissa Hughes, SAIC, Steven
Schimmel, USEPA, and David Bengtson, University of Rhode Island, developed the
inland silverside, Mem'dia beryllina, larval survival and growth test; Suzanne
Lussier, USEPA, Anne Kuhn and John Sewall, SAIC, developed the mysid,
Mysidopsia bahia, survival, growth, and fecundity test; Diane Nacci and
Raymond Walsh, SAIC, and Eugene Jackim, USEPA, developed the sea urchin,
Arbacia punctulata, fertilization test; and Glen Thursby, University of Rhode
Island, and Richard Steele, USEPA, developed the red macroalga, Champia
parvula, reproduction test.
Terry Hollister, U.S. Environmental Protection Agency, Region 6, Houston,
Texas, adapted the sheepshead minnow, Cyprinodon van'egatus, embryo-larval
survival and teratogenicity test from the fathead minnow, ipimephales promelas,
embryo-larval test in EPA/660/4-85/014 (USEPA, 1985b).
Debbie Hall, Bioassessment and Ecotoxicology Branch, and Mary Sullivan,
Quality Assurance Research Division, provided valuable secretarial assistance,
and Betty Thomas, Technical Information Manager, EMSL-Cincinnati, provided an
editorial review.
xxxi
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SECTION 1
INTRODUCTION
j
1.1 This manual describes chronic toxicity tests for use in the National
Pollutant Discharge Elimination System (NPDES) Permits Program to identify
effluents and receiving waters containing toxic materials in chronically toxic
concentrations. The methods included in this manual are referenced in Table
IA, 40 CFR Part 136 regulations and, therefore, constitute approved methods
for chronic toxicity tests. They are also suitable for determining the
toxicity of specific compounds contained in discharges. The tests may be
conducted in a central laboratory or on-site, by the regulatory agency or the
permittee.
i
1.2 The data are used for NPDES permits development and to determine
compliance with permit toxicity limits. Data can also be used to predict
potential acute and chronic toxicity in the receiving water, based on the
LC50, NOEC, IC25, or IC50 (see Section 9, Chronic Toxicity Test Endpoints and
Data Analysis) and appropriate dilution, application, and persistence factors.
The tests are performed as a part of self-monitoring permit requirements,
compliance biomonitoring inspections, toxics sampling inspections, and special
investigations. Data from chronic toxicity tests performied as part of permit
requirements are evaluated during compliance evaluation inspections and
performance audit inspections.
1.3 Modifications of these tests are also used in toxicity reduction
evaluations and toxicity identification evaluations to identify the toxic
components of an effluent, to aid in the development and implementation of
toxicity reduction plans, and to compare and control the effectiveness of
various treatment technologies for a given type of industry, irrespective of
the receiving water (USEPA, 1988c; USEPA, 1989b; USEPA, 1989c; USEPA 1989d*
USEPA, 1989e; USEPA, 1991a; USEPA, 1991b; and USEPA, 1992).
1.4 This methods manual serves as a companion to the acute toxicity test
methods for freshwater and marine organisms (USEPA, 1993a), the short-term
chronic toxicity test methods for freshwater organisms (USEPA, 1993b), and the
manual for evaluation of laboratories performing aquatic toxicity tests
(USEPA, 1991c).
1.5 Guidance for the implementation of toxicity tests in the NPDES program is
provided in the Technical Support Document for Water Quality-Based Toxics
Control (USEPA, 1991a).
1.6 These marine and estuarine short-term toxicity tests are similar to those
developed for the freshwater organisms to evaluate the toxicity of effluents
discharged to estuarine and coastal marine waters under the NPDES permit
program. Methods are presented in this manual for five species from four
phylogenetic groups. Five of the six methods were developed and extensively
field tested by Environmental Research Laboratory-Narragansett (ERL-N). The
methods vary in duration from one hour and 20 minutes to nine days.
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1.7 The five species for which toxicity test methods are provided are: the
sheepshead minnow, Cyprinodon variegatus; the inland silverside, Mem'dia
beryllina-, the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata;
and the red macroalga, Champia parvula.
1.7.1 The tests included in this document are based on the following methods:
1. "Guidance manual for conducting complex effluent and receiving water
larval fish growth/survival studies with the sheepshead minnow,
Cyprinodon variegatus," by Melissa M. Hughes, Margarete A. Heber, Steven
C. Schimmel and Walter J. Berry, 1987, Contribution No. X104,
Environmental Research Laboratory, U.S. Environmental Protection Agency,
Narragansett, RI (USEPA, 1987b).
2. "Guidance manual for rapid chronic toxicity test on effluents and
receiving waters with larval inland silversides, Mem'dia beryllina,1 by
Margarete A. Heber, Melissa M. Hughes, Steven C. Schimmel, and David
Bengtson, 1987, Contribution No. 792, Environmental Research Laboratory,
U.S. Environmental Protection Agency, Narragansett, RI (USEPA, 1987c).
3. "Guidance manual for conducting seven-day, mysid survival/growth/
reproduction study using the estuarine mysid, Mysidopsis bahia," by
Suzanne M. Lussier, Anne Kuhn, and John Sewall, 1987, Contribution No.
X106, Environmental Research Laboratory, U.S. Environmental Protection
Agency, Narragansett, RI (USEPA, 1987d).
4. "Guidance manual for conducting sperm cell tests with the sea urchin,
Arbacia punctulata, for use in testing complex effluents," by Diane E.
Nacci, Raymond Walsh, and Eugene Jackim, 1987, Contribution No. X105,
Environmental Research Laboratory, U.S. Environmental Protection Agency,
Narragansett, RI (USEPA, 1987e).
5. "Guidance manual for conducting sexual reproduction tests with the
marine macroalga, Champia parvula, for use in testing complex
effluents," by Glenn B. Thursby and Richard L. Steele, 1987,
Contribution No. X103, Environmental Research Laboratory, U.S.
Environmental Protection Agency, Narragansett, RI (USEPA, 1987f).
6. A nine-day, sheepshead minnow, Cyprinodon variegatus, static-renewal,
embryo-larval survival and teratogenicity test, developed by Terry
Hollister, USEPA, Region 6, Houston, TX.
1.7.2 Four of the methods incorporate the chronic endpoints of growth or
reproduction (or both) in addition to lethality. The sheepshead minnow 9-day
embryo-larval survival and teratogenicity test incorporates teratogenic
effects in addition to lethality. The sea urchin sperm cell test uses
fertilization as an endpoint and has the advantage of an extremely short
exposure period (1 h and 20 min).
1.8 The validity of the marine/estuarine methods in predicting adverse
ecological impacts of toxic discharges was demonstrated in field studies
(USEPA, 1986d).
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1.9 The use of any test species or test conditions other than those described
in the methods summary tables in this manual shall be subject to application
and approval of alternate test procedures under 40 CFR 136.4 and 40 CFR 136.5.
1.10 These methods are restricted to use by or under the supervision of
analysts experienced in the use or conduct of aquatic toxicity testing and the
interpretation of data from aquatic toxicity testing. Each analyst must
demonstrate the ability to generate acceptable test results with these methods
using the procedures described in this methods manual.
/MCCD The manual was PrePared in the established EMSL-Cincinnati format
(UotrA, 1983). i
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SECTION 2
SHORT-TERM METHODS FOR ESTIMATING CHRONIC TOXICITY
2.1 INTRODUCTION
2.1.1 The objective of aquatic toxicity tests with effluents or pure
compounds is to estimate the "safe" or "no-effect" concentration of these
substances, which is defined as the concentration which will permit normal
propagation of fish and other aquatic life in the receiving waters. The
endpoints that have been considered in tests to determine the adverse effects
of toxicants include death and survival, decreased reproduction and growth,
locomotor activity, gill ventilation rate, heart rate, blood chemistry,
histopathology, enzyme activity, olfactory function, and terata. Since it is
not feasible to detect and/or measure all of these (and other possible)
effects of toxic substances on a routine basis, observations in toxicity tests
generally have been limited to only a few effects, such as mortality, growth,
and reproduction.
2.1.2 Acute lethality is an obvious and easily observed effect which accounts
for its wide use in the early period of evaluation of the toxicity of pure
compounds and complex effluents. The results of these tests were usually
expressed as the concentration lethal to 50% of the test organisms (LC50) over
relatively short exposure periods (one-to-four days).
2.1.3 As exposure periods of acute tests were lengthened, the LC50 and lethal
threshold concentration were observed to decline for many compounds. By
lengthening the tests to include one or more complete life cycles and
observing the more subtle effects of the toxicants, such as a reduction in
growth and reproduction, more accurate, direct, estimates of the threshold or
safe concentration of the toxicant could be obtained. However, laboratory
life cycle tests may not accurately estimate the "safe" concentration of
toxicants because they are conducted with a limited number of species under
highly controlled, steady state conditions, and the results do not include the
effects of the stresses to which the organisms would ordinarily be exposed in
the natural environment.
2.1.4 An early published account of a full life cycle, fish toxicity test was
that of Mount and Stephan (1967). In this study, fathead minnows, Pimephales
promelas, were exposed to a graded series of pesticide concentrations
throughout their life cycle, and the effects of the toxicant on survival,
growth, and reproduction were measured and evaluated. This work was soon
followed by full life cycle tests using other toxicants and fish species.
2.1.5 McKim (1977) evaluated the data from 56 full life cycle tests, 32 of
which used the fathead minnow, Pimephales promelas, and concluded that the
embryo-larval and early juvenile life stages were the most sensitive stages.
He proposed the use of partial life cycle toxicity tests with the early life
stages (ELS) of fish to establish water quality criteria.
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2.1.6 Macek and Sleight (1977) found that exposure of critical life stages of
fish to toxicants provides estimates of chronically safe concentrations
remarkably similar to those derived from full life cycle toxicity tests. They
reported that "for a great majority of toxicants, the concentration which will
not be acutely toxic to the most sensitive life stages is the chronically safe
concentration for fish, and that the most sensitive life stages are the
embryos and fry." Critical life stage exposure was considered to be exposure
of the embryos during most, preferably all, of the embryogenic (incubation)
period, and exposure of the fry for 30 days post-hatch for warm water fish
with embryogenic periods ranging from one-to-fourteen days, and for 60 days
post-hatch for fish with longer embryogenic periods. They: concluded that in
the majority of cases, the maximum acceptable toxicant concentration (MATC)
could be estimated from the results of exposure of the embryos during
incubation, and the larvae for 30 days post-hatch.
2.1.7 Because of the high cost of full life-cycle fish toxicity tests and the
emerging consensus that the ELS test data usually would be adequate for
estimating chronically safe concentrations, there was a rapid shift by aquatic
toxicologists to 30- to 90-day ELS toxicity tests for estimating chronically
safe concentrations in the late 1970s. In 1980, USEPA adopted the policy that
ELS test data could be used in establishing water quality criteria if data
from full life-cycle tests were not available (USEPA, 1980a).
i
2.1.8 Published reports of the results of ELS tests indicate that the
relative sensitivity of growth and survival as endpoints may be species
dependent, toxicant dependent, or both. Ward and Parrish (1980) examined the
literature on ELS tests that used embryos and juveniles of the sheepshead
minnow, Cyprinodon variegatus, and found that growth was not a statistically
sensitive indicator of toxicity in 16 of 18 tests. They suggested that the
ELS tests be shortened to. 14 days posthatch and that growth be eliminated as
an indicator of toxic effects.
.
2.1.9 In a review of the literature on 173 fish full life-cycle and ELS tests
performed to determine the chronically safe concentrations of a wide variety
of toxicants, such as metals, pesticides, organics, inorganics, detergents,
and complex effluents, Weltering (1984) found that at the lowest effect
concentration, significant reductions were observed in fry survival in 57%,
fry growth in 36%, and egg hatchability in 19% of the tests. He also found
that fry survival and growth were very often equally sensitive, and concluded
that the growth response could be deleted from routine application of the ELS
tests. The net result would be a significant reduction in the duration and
cost of screening tests with no appreciable impact on estimating MATCs for
chemical hazard assessments. Benoit et al. (1982), however, found larval
growth to be the most significant measure of effect and survival to be equally
or less sensitive than growth in early life-stage tests with four organic
chemicals.
j
2.1.10 Efforts to further reduce the length of partial life-cycle toxicity
tests for fish without compromising their predictive value have resulted in
the development of an eight-day, embryo-larval survival and teratogenicity
test for fish and other aquatic vertebrates (USEPA, 1981; Birge et al.,
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1985), and a seven-day larval survival and growth test (Norberg and Mount,
1985).
2.1.11 The similarity of estimates of chronically safe concentrations of
toxicants derived from short-term, embryo-larval survival and teratogenicity
tests to those derived from full life-cycle tests has been demonstrated by
Birge et al. (1981), Birge and Cassidy (1983), and Birge et al. (1985).
2.1.12 Use of a seven-day, fathead minnow, Pimephales promelas, larval
survival and growth test was first proposed by Norberg and Mount at the 1983
annual meeting of the Society for Environmental Toxicology and Chemistry
(Norberg and Mount, 1983). This test was subsequently used by Mount and
associates in field demonstrations at Lima, Ohio (USEPA, 1984), and at many
other locations (USEPA, 1985c, USEPA, 1985d; USEPA, 1985e; USEPA, 1986a;
USEPA, 1986b; USEPA, 1986c; USEPA, 1986d). Growth was frequently found to be
more sensitive than survival in determining the effects of complex effluents.
2.1.13 Norberg and Mount (1985) performed three single toxicant fathead
minnow larval growth tests with zinc, copper, and DURSBAN®, using dilution
water from Lake Superior. The results were comparable to, and had confidence
intervals that overlapped with, chronic values reported in the literature for
both ELS and full life-cycle tests.
2.1.14 USEPA (1987b) and USEPA (1987c) adapted the fathead minnow larval
growth and survival test for use with the sheepshead minnow and the inland
silverside, respectively. When daily renewal 7-day sheepshead minnow larval
growth and survival tests and 28-day ELS tests were performed with industrial
and municipal effluents, growth was more sensitive than survival in seven out
of 12 larval growth and survival tests, equally sensitive in four tests, and
less sensitive in only one test. In four cases, the ELS test may have been
three to 10 times more sensitive to effluents than the larval growth and
survival test. In tests using copper, the No Observable Effect Concentrations
(NOECs) were the same for both types of test, and growth was the most
sensitive endpoint for both. In a four laboratory comparison, six of seven
tests produced identical NOECs for survival and growth (USEPA, 1987a). Data
indicate that the inland silverside is at least equally sensitive or more
sensitive to effluents and single compounds than the sheepshead minnow, and
can be tested over a wider salinity range, 5-30%> (USEPA, 1987a).
2.1.15 Lussier et al. (1985) and USEPA (1987e) determined that survival and
growth are often as sensitive as reproduction in 28-day life-cycle tests with
the mysid, Mysidopsis bahia.
2.1.16 Nacci and Jackim (1985) and USEPA (1987g) compared the results from
the sea urchin fertilization test, using organic compounds, with results from
acute toxicity tests using the freshwater organisms, fathead minnows,
Pimphales promelas, and Daphnia magna. The test was also compared to acute
toxicity tests using Atlantic silverside, Menidia menidia^ and the mysid,
Mysidopsis bahia, and five metals. For six of the eight organic compounds,
the results of the fertilization test and the acute toxicity test correlated
well (r = 0.85). However, the results of the fertilization test with the
five metals did not correlate well with the results from the acute tests.
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2.1.17 USEPA (1987f) evaluated two industrial effluents containing heavy
metals, five industrial effluents containing organic chemicals (including dyes
and pesticides), and 15 domestic wastewaters using the two-day red macroalga,
Champia parvuTa, sexual reproduction test. Nine single compounds were used to
compare the effects on sexual reproduction using a two-week exposure and a
two-day exposure. For six of the nine compounds tested, the chronic values
were the same for both tests.
2.1.18 The use of short-term toxicity tests in the NPDES Program is
especially attractive because they provide a more direct estimate of the safe
concentrations of effluents in receiving waters than was provided by acute
toxicity tests, at an only slightly increased level of effort, compared to the
fish full life-cycle chronic and 28-day ELS tests and the 28-day mysid life-
cycle test. ;
2.2 TYPES OF TESTS
2.2.1 The selection of the test type will depend on the NPDES permit
requirements, the objectives of the test, the available resources, the
requirements of the test organisms, and effluent characteristics such as
fluctuations in effluent toxicity.
2.2.2 Effluent chronic toxicity is generally measured using a multi-
concentration, or definitive test, consisting of a control and a minimum of
five effluent concentrations. The tests are designed to provide dose-response
information, expressed as the percent effluent concentration that affects the
hatchability, gross morphological abnormalities, survival, growth, and/or
reproduction within the prescribed period of time (one hour and 20 minutes to
nine days). The results of the tests are expressed in terms of either the
highest concentration that has no statistically significant observed effect on
those responses when compared to the controls or the estimated concentration
that causes a specified percent reduction in responses versus the controls.
2.2.3 Use of pass/fail tests consisting of a single effluent concentration
(e.g., the receiving water concentration or RWC) and a control is not
recommended. If the NPDES permit has a whole effluent toxicity limit for
acute toxicity at the RWC, it is prudent to use that permit limit as the
midpoint of a series of five effluent concentrations. This will ensure that
there is sufficient information on the dose-response relationship. For
example, the effluent concentrations utilized in a test may be:
(1) 100% effluent, (2) (RWC + 100)/2, (3) RWC, (4) RWC/2, and (5) RWC/4. More
specifically, if the RWC = 50%, the effluent concentrations used in the
toxicity test would be 100%, 75%, 50%, 25%, and 12.5%.
2.2.4 Receiving (ambient) water toxicity tests commonly employ two
treatments, a control and the undiluted receiving water, but may also consist
of a series of receiving water dilutions.
2.2.5 A negative result from a chronic toxicity test does not preclude the
presence of toxicity. Also, because of the potential temporal variability in
the toxicity of effluents, a negative test result with a particular sample
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does not preclude the possibility that samples collected at some other time
might exhibit chronic toxicity.
2.2.6 The frequency with which chronic toxicity tests are conducted under a
given NPDES permit is determined by the regulatory agency on the basis of
factors such as the variability and degree of toxicity of the'waste,
production schedules, and process changes.
2.2.7 Tests recommended for use in this methods manual may be static non-
renewal or static renewal. Individual methods specify which static type of
test is to be conducted.
2.3 STATIC TESTS
2.3.1 Static non-renewal tests - The test organisms are exposed to the same
test solution for the duration of the test.
2.3.2 Static-renewal tests - The test organisms are exposed to a fresh
solution of the same concentration of sample every 24 h or other prescribed
interval, either by transferring the test organisms from one test chamber to
another, or by replacing all or a portion of solution in the test chambers.
2.4 ADVANTAGES AND DISADVANTAGES OF TOXICITY TEST TYPES
2.4.1 STATIC NON-RENEWAL, SHORT-TERM TOXICITY TESTS:
Advantages:
1. Simple and inexpensive
2. Very cost effective in determining compliance with permit conditions.
3. Limited resources (space, manpower, equipment) required; would permit
staff to perform many more tests in the same amount of time.
4. Smaller volume of effluent required than for static renewal or flow-
through tests.
Disadvantages:
1. Dissolved oxygen (DO) depletion may result from high chemical oxygen
demand (COD), biological oxygen demand (BOD), or metabolic wastes.
2. Possible loss of toxicants through volatilization and/or adsorption to
the exposure vessels.
3. Generally less sensitive than static renewal because the toxic
substances may degrade or be adsorbed, thereby reducing the apparent
toxicity. Also, there is less chance of detecting slugs of toxic
wastes, or other temporal variations in waste properties.
2.4.2 STATIC RENEWAL, SHORT-TERM TOXICITY TESTS:
Advantages:
1. Reduced possibility of DO depletion from high COD and/or BOD, or ill
effects from metabolic wastes from organisms in the test solutions.
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2. Reduced possibility of loss of toxicants through volatilization and/or
adsorption to the exposure vessels.
3. Test organisms that rapidly deplete energy reserve:; are fed when the
test solutions are renewed, and are maintained in a healthier state.
Disadvantages:
1. Require greater volume of effluent than non-renewal tests.
2. Generally less chance of temporal variations in waste properties.
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SECTION 3
HEALTH AND SAFETY
3.1 GENERAL PRECAUTIONS
3.1.1 Each laboratory should develop and maintain an effective health and
safety program, requiring an ongoing commitment by the laboratory management
and includes: (1) a safety officer with the responsibility and authority to
develop and maintain a safety program; (2) the preparation of a formal,
written, health and safety plan, which is provided to the laboratory staff;
(3) an ongoing training program on laboratory safety; and (4) regularly
scheduled, documented, safety inspections.
3.1.2 Collection and use of effluents in toxicity tests may involve
significant risks to personal safety and health. Personnel collecting
effluent samples and conducting toxicity tests should take all safety
precautions necessary for the prevention of bodily injury and illness which
might result from ingestion or invasion of infectious agents, inhalation or
absorption of corrosive or toxic substances through skin contact, and
asphyxiation due to a lack of oxygen or the presence of noxious gases.
3.1.3 Prior to sample collection and laboratory work, personnel should
determine that all necessary safety equipment and materials have been obtained
and are in good condition.
3.1.4 Guidelines for the handling and disposal of hazardous materials must be
strictly followed.
3.2 SAFETY EQUIPMENT
3.2.1 PERSONAL SAFETY GEAR
3.2.1.1 Personnel must use safety equipment, as required, such as rubber
aprons, laboratory coats, respirators, gloves, safety glasses, hard hats, and
safety shoes. Plastic netting on glass beakers, flasks and other glassware
minimizes breakage and subsequent shattering of the glass.
3.2,2 LABORATORY SAFETY EQUIPMENT
3.2.2.1 Each laboratory (including mobile laboratories) should be provided
with safety equipment such as first aid kits, fire extinguishers, fire
blankets, emergency showers, chemical spill clean-up kits, and eye fountains.
3.2.2.2 Mobile laboratories should be equipped with a telephone to enable
personnel to summon help in case of emergency.
3.3 GENERAL LABORATORY AND FIELD OPERATIONS
3.3.1 Work with effluents should be performed in compliance with accepted
rules pertaining to the handling of hazardous materials (see safety manuals
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listed in Section 3, Health and Safety, Subsection 3.5). It is recommended
that personnel collecting samples and performing toxicity tests should not
work alone.
3.3.2 Because the chemical composition of effluents is usually only poorly
known, they should be considered as potential health hazards, and exposure to
them should be minimized. Fume and canopy hoods over the toxicity test areas
must be used whenever possible.
3.3.3 It is advisable to cleanse exposed parts of the body immediately after
collecting effluent samples. [
3.3.4 All containers should be adequately labeled to indicate their contents.
3.3.5 Staff should be familiar with safety guidelines on Material Safety Data
Sheets for reagents and other chemicals purchased from suppliers.
Incompatible materials should not be stored together. Good housekeeping
contributes to safety and reliable results.
3.3.6 Strong acids and volatile.organic solvents employed in glassware
cleaning must be used in a fume hood or under an exhaust canopy over the work
area.
3.3.7 Electrical equipment or extension cords not bearing the approval of
Underwriter Laboratories must not be used. Ground-fault interrupters must be
installed in all "wet" laboratories where electrical equipment is used.
'
3.3.8 Mobile laboratories should be properly grounded to protect against
electrical shock.
3.4 DISEASE PREVENTION
3.4.1 Personnel handling samples which are known or suspected to contain
human wastes should be immunized against tetanus, typhoid fever, polio, and
hepatitis B.
3.5 SAFETY MANUALS
3.5.1 For further guidance on safe practices when collecting effluent samples
and conducting toxicity tests, check with the permittee and consult general
safety manuals, including USEPA (1986e), and Walters and Jameson (1984).
3.6 WASTE DISPOSAL j
3.6.1 Wastes generated during toxicity testing must be properly handled and
disposed of in an appropriate manner. Each testing facility will have its own
waste disposal requirements based on local, state and Federal rules and
regulations. It is extremely important that these rules and regulations be
known, understood, and complied with by all persons responsible for, or
otherwise involved in, performing toxicity testing activities. Local fire
officials should be notified of any potentially hazardous conditions.
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SECTION 4
QUALITY ASSURANCE
4.1 INTRODUCTION
4.1.1 Development and maintenance of a toxicity test laboratory quality
assurance (QA) program (USEPA, 1991b) requires an ongoing commitment by
laboratory management. Each toxicity test laboratory should (1) appoint a
quality assurance officer with the responsibility and authority to develop and
maintain a QA program, (2) prepare a quality assurance plan with stated data
quality objectives (DQOs), (3) prepare written descriptions of laboratory
standard operating procedures (SOPs) for culturing, toxicity testing,
instrument calibration, sample chain-of-custody procedures, laboratory sample
tracking system, glassware cleaning, etc., and (4) provide an adequate,
qualified technical staff for culturing and toxicity testing the organisms,
and suitable space and equipment to assure reliable data.
4.1.2 QA practices for toxicity .testing laboratories must address all
activities that affect the quality of the final effluent toxicity data, such
as: (1) effluent sampling and handling; (2) the source and condition of the
test organisms; (3) condition of equipment; (4) test conditions; (5)
instrument calibration; (6) replication; (7) use of reference toxicants; (8)
record keeping; and (9) data evaluation.
4.1.3 Quality control practices, on the other hand, consist of the more
focused, routine, day-to-day activities carried out within the scope of the
overall QA program. For more detailed discussion of quality assurance and
general guidance on good laboratory practices and laboratory evaluation
related to toxicity testing, see FDA (1978); USEPA (1979d); USEPA (1980b);
USEPA (1980c); USEPA (1991c); DeWoskin (1984); and Taylor (1987).
4.1.4 Guidelines for the evaluation of laboratory performing toxicity tests
and laboratory evaluation criteria are found in USEPA (1991c).
4.2 FACILITIES, EQUIPMENT, AND TEST CHAMBERS
4.2.1 Separate test organism culturing and toxicity testing areas should be
provided to avoid possible loss of cultures due to cross-contamination.
Ventilation systems should be designed and operated to prevent recirculation
or leakage of air from chemical analysis laboratories or sample storage and
preparation areas into organism culturing or testing areas, and from testing
and sample preparation areas into culture rooms.
4.2.2 Laboratory and toxicity test temperature control equipment must be
adequate to maintain recommended test water temperatures. Recommended
materials must be used in the fabrication of the test equipment which comes in
contact'with the effluent (see Section 5, Facilities, Equipment, and Supplies;
and specific toxicity test method).
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4.3 TEST ORGANISMS
4.3.1 The test organisms used in the procedures described in this manual are
the sheepshead minnow, Cypn'nodon van'egatus; the inland silverside, Menidia
beryTlina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata-,
•and the red macroalga, Champia parvula. The organisms used should be disease-
free and appear healthy, behave normally, feed well, and Have low mortality in
cultures, during holding, and in test control. Test organisms should be
positively identified to species (see Section 6, Test Organisms).
4.4 LABORATORY WATER USED FOR CULTURING AND TEST DILUTION WATER
4.4.1 The quality of water used for test organism culturing and for dilution
water used in toxicity tests is extremely important. Water for these two uses
should come from the same source. The dilution water used in effluent
toxicity tests will depend on the objectives of the study and logistical
constraints, as discussed in Section 7, Dilution Water. The dilution water
used in the toxicity tests may be natural seawater, hypersaline brine (100%o)
prepared from natural seawater, or artificial seawater prepared from
commercial sea salts, such as FORTY FATHOMS® or HW MARINEMIX®, if recommended
in the method. GP2 synthetic seawater, made from reagent grade chemical salts
(30%o) in conjunction with natural seawater, may also be used if recommended.
Hypersaline brine and artificial seawater can be used with Champia parvula
only if they are accompanied by at least 50% natural seawater. Types of water
are discussed in Section 5, Facilities, Equipment, and Supplies. Water used
for culturing and test dilution water should be analyzed for toxic metals and
organics at least annually or whenever difficulty is encountered in meeting
minimum acceptability criteria for control survival and reproduction or
growth. The concentration of the metals, Al, As, Cr, Co, Cu, Fe, Pb, Ni, Zn,
expressed as total metal, should not exceed 1 /jg/L each, and Cd, Hg, and Ag,
expressed as total metal, should not exceed 100 ng/L each. Total
organochlorine pesticides plus PCBs should be less than 50 ng/L (APHA, 1992).
Pesticide concentrations should not exceed USEPA's National Ambient Water
Quality chronic criteria values where available. ,
4.5 EFFLUENT AND RECEIVING WATER SAMPLING AND HANDLING
4.5.1 Sample holding times and temperatures of effluent samples collected for
on-site and off-site testing must conform to conditions described in
Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests.
4.6 TEST CONDITIONS
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I
4.6.1 Water temperature and salinity must be maintained within the limits
specified for each test. The temperature ofttest solutions must be measured
by placing the thermometer or probe directly into the test solutions, or by
placing the thermometer in equivalent volumes of water in surrogate vessels
positioned at appropriate locations among the test vessels. Temperature
should be recorded continuously in at least one vessel during the duration of
each test. Test solution temperatures must be maintained .within the limits
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specified for each test. DO concentrations and pH should be checked at the
beginning of the test and daily throughout the test period.
4.7 QUALITY OF TEST ORGANISMS
4.7.1 If the laboratory performs short-term chronic toxicity tests routinely
but does not have an ongoing test organism culturing program and must obtain
the test organisms from an outside source, the sensitivity of a batch of test
organisms must be determined with a reference toxicant in a short-term chronic
toxicity test performed monthly (see Section 4, Quality Assurance, Subsections
4.14, 4.15, 4.16, and 4.17). Where acute or short-term chronic toxicity tests
are performed with effluents or receiving waters using test organisms obtained
from outside the test laboratory, concurrent toxicity tests of the same type
must be performed with a reference toxicant, unless the test organism supplier
provides control chart data from at least the last five monthly short-term
chronic toxicity tests using the same reference toxicants and test conditions
(see Section 6, Test Organisms).
4.7.2 The supplier should certify the species identification of the test
organisms, and provide the taxonomic reference (citation and page) or name(s)
of the taxonomic expert(s) consulted.
4.7.3 If the laboratory maintains breeding cultures, the sensitivity of the
offspring should be determined in a short-term chronic toxicity test performed
with a reference toxicant at least once each month (see Section 4, Quality
Assurance, Subsection 4.14, 4.15, 4.16, and 4.17). If preferred, this
reference toxicant test may be performed concurrently with an effluent
toxicity test. However, if a given species of test organism produced by
inhouse cultures is used only monthly, or less frequently in toxicity tests, a
reference toxicant test must be performed concurrently with each short-term
chronic effluent and/or receiving water toxicity test.
4.7.4 If a routine reference toxicant test fails to meet acceptability
criteria, the test must be immediately repeated. If the failed reference
toxicant test was being performed concurrently with an effluent or receiving
water toxicity test, both tests must be repeated (For exception, see
Section 4, Quality Assurance, Subsection 4.16.5).
4.8 FOOD QUALITY
4.8.1 The nutritional quality of the food used in culturing and testing fish
and invertebrates is an important factor in the quality of the toxicity test
data. This is especially true for the unsaturated fatty acid content of brine
shrimp nauplii, Artemia. Problems with the nutritional suitability of the
food will be reflected in the survival, growth, and reproduction of the test
organisms in cultures and toxicity tests. Artemia cysts and other foods must
be obtained as described in Section 5, Facilities, Equipment, and Supplies.
4.8.2 Problems with the nutritional suitability of food will be reflected in
the survival, growth, and reproduction of the test organisms in cultures and
toxicity tests. If a batch of food is suspected to be defective, the
performance of organisms fed with the new food can be compared with the
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performance of organisms fed with a food of known quality in side-by-side
tests. If the food is used for culturing, its suitability should be
determined using a short-term chronic test which will determine the affect of
food quality on growth or reproduction of each of the relevant test species in
culture, using four replicates with each food source. Where applicable, foods
used only in chronic toxicity tests can be compared with a food of known
quality in side-by-side, multi-concentration chronic tests, using the
reference toxicant regularly employed in the laboratory QA program. For list
of commercial, sources of Artemia cysts, see Table 2 of Section 5, Facilities,
Equipment, and Supplies.
i
4.8.3 New batches of food used in culturing and testing should be analyzed
for toxic organics and metals or whenever difficulty is encountered in meeting
minimum acceptability criteria for control survival and reproduction or
growth. If the concentration of total organochlorine pesticides exceeds
0.15 fj,g/g wet weight, or the concentration of total organochlorine pesticides
plus PCBs exceeds 0.30 ^g/g wet weight, or toxic metals (Al, As, Cr, Cd, Cu,
Pb, Ni, Zn, expressed as total metal) exceed 20 /j.g/g wet weight, the food
should not be used (for analytical methods, see AOAC, 1990; and USDA, 1989).
For foods (e.g., YCT) which are used to culture and test organisms, the
quality of the food should meet the requirements for the laboratory water used
for culturing and test dilution water as described in Section 4.4 above.
4.9 ACCEPTABILITY OF CHRONIC TOXICITY TESTS
i
4.9.1 The results of the sheepshead minnow, Cyprinodon variegatus, inland
silverside, Mem'dia beryl Una, or mysid, Mysidopsis bahia,, tests are
acceptable if survival in the controls is 80% or greater. The sea urchin,
Arbacia punctulata, test requires control egg fertilization equal to or
exceeding 50%. However, greater than 90% fertilization may result in masking
toxic responses. The red macroalga, Champia parvula, test is acceptable if
survival is 100%, and the mean number of cystocarps per plant should equal or
exceed 10. If the sheepshead minnow, Cypn'ndon van'egatus, larval survival
and growth test is begun with less-than-24-h old larvae, the mean dry weight
of the surviving larvae in the control chambers at the end of the test should
equal or exceed 0.60 mg, if the weights are determined immediately, or 0.50 mg
if the larvae are preserved in a 4% formalin or 70% ethanol solution. If the
inland silverside, Mem'dia beryllina, larval survival and growth test is begun
with larvae seven days old, the mean dry weight of the surviving larvae in the
control chambers at the end of the test should equal or exceed 0.50 mg, if the
weights are determined immediately, or 0.43 mg if the larvae are preserved in
a 4% formalin or 70% ethanol solution. The mean mysid dry weight of survivors
should be at least 0.20 mg. Automatic or hourly feeding will generally
provide control mysids with a dry weight.of 0.30 mg. At least 50% of the
females should bear eggs at the end of the test, but mysid fecundity is not a
factor in test acceptability. However, fecundity must equal or exceed 50% to
be used as an endpoint in the test. If these criteria are not met, the test
must be repeated.
.
4.9.2 An individual test may be conditionally acceptable if temperature, DO,
and other specified conditions fall outside specification.';, depending on the
I
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degree of the departure and the objectives of the tests (see test conditions
and test acceptability criteria summaries). The acceptability of the test
will depend on the experience and professional judgment of the laboratory
investigator and the reviewing staff of the regulatory authority. Any
deviation from test specifications must be noted when reporting data from a
test.
4.10 ANALYTICAL METHODS
4.10.1 Routine chemical and physical analyses for culture and dilution water,
food, and test solutions must include established quality assurance practices
outlined in USEPA methods manuals (USEPA, 1979a and USEPA, 1979b).
4.10.2 Reagent containers should be dated and catalogued when received from
the supplier, and the shelf life should not be exceeded. Also, working
solutions should be dated when prepared, and the recommended shelf life should
be observed.
4.11 CALIBRATION AND STANDARDIZATION
4.11.1 Instruments used for routine measurements of chemical and physical
parameters, such as pH, DO, temperature, conductivity, and salinity, must be
calibrated and standardized according to instrument manufacturers procedures
as indicated in the general section on quality assurance (see USEPA Methods
150.1, 360.1, 170.1, and 120.1 in USEPA, 1979b). Calibration data are
recorded in a permanent log book.
4.11.2 Wet chemical methods used to measure hardness, alkalinity, and total
residual chlorine, must be standardized prior to use each day according to the
procedures for those specific USEPA methods (see USEPA Methods 130.2 and 310.1
in USEPA, 1979b).
4.12 REPLICATION AND TEST SENSITIVITY
4.12.1 The sensitivity of the tests will depend in part on the number of
replicates per concentration, the significance level selected, and the type of
statistical analysis. If the variability remains constant, the sensitivity of
the test will increase as the number of replicates is increased. The minimum
recommended number of replicates varies with the objectives of the test and
the statistical method used for analysis of the data.
4.13 VARIABILITY IN TOXICITY TEST RESULTS
4.13.1 Factors which can affect test success and precision include: (1) the
experience and skill of the laboratory, analyst; (2) test organism age,
condition, and sensitivity; (3) dilution water quality; (4) temperature
control; (5) and the quality and quantity of food provided. The results will
depend upon the species used and the strain or source of the test organisms,
and test conditions, such as temperature, DO, food, and water quality. The
repeatability or precision of toxicity tests is also a function of the number
of test organisms used at each toxicant concentration. Jensen (1972)
discussed the relationship between sample size (number of fish) and the
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standard error of the test, and considered 20 fish per concentration as
optimum for Probit Analysis.' :
4.14 TEST PRECISION
4.14.1 The ability of the laboratory .personnel to obtain consistent, precise
results must be demonstrated with reference toxicants before they attempt to
measure effluent toxicity. The single-laboratory precision of each type of
test to be used in a laboratory should be determined by performing at least
five or more tests with a reference toxicant.
.
4.14.2 Test precision can be estimated by using the same strain of organisms
under the same test conditions, and employing a known toxicant, such as a
reference toxicant.
4.14.3 Interlaboratory precision of chronic toxicity tests using two
reference toxicants with the mysid, Mysidopsis bahia, and the inland
silverside, Mem'dia beryllina, is listed in Table 1. Additional precision
data for each of the tests described in this manual are presented in the
sections describing the individual test methods.
4.14.4 Additional information on toxicity test precision is provided in the
Technical Support Document for Water Quality-based Toxic Control (see pp. 2-4,
and 11-15 in USEPA, 1991a).
4.14.5 In cases where the test data are used in Probit Analysis or other
point estimation techniques (see Section 9, Chronic Toxicity Test Endpoints
and Data Analysis), precision can be described by the mean, standard
deviation, and relative standard deviation (percent coefficient of variation,
or CV) of the calculated .endpoints from the replicated tests. In cases where
the test data are used in the Linear Interpolation Method, precision can be
estimated by empirical confidence intervals derived by using the ICPIN Method
(see Section 9, Chronic Toxicity Test Endpoints and Data Analysis). However,
in cases where the results are reported in terms of the Mo-Observed-Effect-
Concentration (NOEC) and Lowest-Observed-Effect-Concentration (LOEC) (see
Section 9, Chronic Toxicity Test Endpoints and Data Analysis), precision can
only be described by listing the NOEC-LOEC interval for each test. It is not
possible to express precision in terms of a commonly used statistic. However,
when all tests of the same toxicant yield the same NOEC-LOEC interval, maximum
precision has been attained. The "true" no effect concentration could fall
anywhere within the interval, NOEC ± (LOEC minus NOEC).
I
4.14.6 It should be noted here that the dilution factor selected for a test
determines the width of the NOEC-LOEC interval and the inherent maximum
precision of the test. As the absolute value of the dilution factor
decreases, the width of the NOEC-LOEC interval increases, and the inherent
maximum precision of the test decreases. When a dilution factor of 0.3 is
used, the NOEC could be considered to have a relative uncertainty as high as ±
300%. With a dilution factor of 0.5, the NOEC could be considered to have a
relative variability of ± 100%. As a result of the variability of different
dilution factors, USEPA recommends the use of a > 0.5 dilution factor. Other
factors which can affect test precision include: test organism age,
condition, and sensitivity; temperature control; and feeding.
i
17
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TABLE 1. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 1991: SUMMARY OF RESPONSES USING TWO REFERENCE
TOXICANTS1'2
Organism
Organism
Endpoint
No. Labs KCl(mg/L)
SD
Endpoint
No. Labs Cu(mg/L)
SD
CV(%)3
Hysidopsis
bahia
Survival, NOEC
Growth, IC25
Growth, IC50
Growth, NOEC
Fecundity, NOEC
34
26
22
32
25
NA
480
656
NA
NA
NA
3.47
3.17
NA
NA
NA
28.9
19.3
NA
NA
CV(%)
Nenidia
beryl 1 ina
Survival, NOEC
Growth, IC25
Growth, IC50
Growth, NOEC
19
13
12
17
NA
0.144
0.180
NA
NA
1.56
1.87
NA
NA
43.5
41.6
NA
From a national study of inter!aboratory precision of toxicity test
data performed in 1991 by the Environmental Monitoring Systems
Laboratory-Cincinnati, U.S. Environmental Protection Agency,
Cincinnati, OH 45268. Participants included federal, state, and
private laboratories engaged in NPDES permit compliance monitoring.
* Static renewal test, using 25%o modified GP2 artificial seawater.
Percent coefficient of variation = (standard deviation X 100)/mean.
Expressed as mean.
4.15 DEMONSTRATING ACCEPTABLE LABORATORY PERFORMANCE
4.15.1 It is a laboratory's responsibility to demonstrate its ability to
obtain consistent, precise results with reference toxicants before it performs
toxicity tests with effluents for permit compliance purposes. To meet this
requirement, the intralaboratory precision, expressed as percent coefficient
of variation (CV%), of each type of test to be used in a laboratory should be
determined by performing five or more tests with different batches of test
organisms, using the same reference toxicant, at the same concentrations, with
the same test conditions (i.e., the same test duration, type of dilution
water, age of test organisms, feeding, etc.), and same data analysis methods.
A reference toxicant concentration series (0.5 or higher) should be selected
that will consistently provide partial mortalities at two or more
concentrations.
4.16 DOCUMENTING ONGOING LABORATORY PERFORMANCE
4.16.1 Satisfactory laboratory performance is demonstrated by performing at
least one acceptable test per month with a reference toxicant for each
18
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toxicity test method commonly used in the laboratory. For a given test
method, successive tests must be performed with the same reference toxicant,
at the same concentrations, in the same dilution water, using the same data
analysis methods,. Precision may vary with the test species, reference
toxicant, and type of test.
*
4.16.2 A control chart should be prepared for each combination of reference
toxicant, test species, test conditions, and endpoints. Toxicity endpoints
from five or six tests are adequate for establishing the control charts.
Successive toxicity endpoints (NOECs, IC25s, LCSOs, etc.) should be plotted
and examined to determine if the results (X,) are within prescribed limits
(Figure 1). The types of control charts illustrated (see USEPA, 1979a) are
used to evaluate the cumulative trend of results from a series of samples.
For endpoints that are point estimates (LCSOs and IC25s), the cumulative
mean (X) and upper and lower control limits (± 2S) are recalculated with each
successive test result. Endpoints from hypothesis tests (NOEC, NOAEC) from
each test are plotted directly on the control chart. The control limits would
consist of one concentration interval above and below the concentration
representing the central tendency. After two years of data collection, or a
minimum of 20 data points, the control (cusum) chart should be maintained
using only the 20 most recent data points.
I
4.16.3 The outliers, which are values falling outside the upper and lower
control limits, and trends of increasing or decreasing sensitivity, are
readily identified. In the case of endpoints that are point estimates (LCBOs
and IC25s), at the Pp 05 probability level, one in 20 tests would be expected
to fall outside of the control limits by chance alone. If more than one out
of 20 reference toxicant tests fall outside the control limits, the effluent
toxicity tests conducted during the month in which the second reference
toxicant test failed are suspect, and should be considered as provisional and
subject to careful review. Control limits for the NOECs will also be exceeded
occasionally, regardless of how well a laboratory performs.
4.16.4 If the toxicity value from a given test with a reference toxicant fall
well outside the expected range for the test organisms when using the standard
dilution water and other test conditions, the sensitivity of the organisms and
the overall credibility of the test system are suspect. In this case, the
test procedure should be examined for defects and should be repeated with a
different batch of test organisms.
4.16.5 Performance should improve with experience, and the control limits for
endpoints that are point estimates should gradually narrow. However, control
limits of ± 2S will be exceeded 5% of the time by chance alone, regardless of
how well a laboratory performs. Highly proficient laboratories which develop
very narrow control limits may be unfairly penalized if a test result which
falls just outside the control limits is rejected de facto. For this reason,
the width of the control limits should be considered by the permitting
authority in determining whether the outliers should be rejected.
19
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o
UJ
O
UPPER CONTROL LIMIT
CENTRAL TENDENCY
LOWER CONTROL LIMIT
I , ,
I I I I I I 1 I 1 III
10
15 20
O
O
HI
O
UPPER CONTROL LIMIT (X •*• 2 S)
CENTRAL TENDENCY
LOWER CONTROL LIMIT (X - 2 S)
0 5 10 15 20
TOXICITY TESTS WITH REFERENCE TOXICANTS
_
y = j=i
n
S =
\
n
n-1
Where: X± = Successive toxicity values from toxicity tests.
n = Number of tests.
X = Mean toxicity value.
S = Standard deviation.
Figure 1. Control (cusum) charts. (A) hypothesis testing results; (B) point
estimates (LC, EC, or 1C).
20
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4.17 REFERENCE TOXICANTS
i
I
4.17.1 Reference toxicants such as sodium chloride (NaClj, potassium chloride
(KC1), cadmium chloride (CdClJ, copper sulfate (CuS04), sodium dodecyl
sulfate (SDS), and potassium dichromate (K2Cr207), are suitable for use in the
NPDES Program and other Agency programs requiring aquatic toxicity tests.
EMSL-Cincinnati plans to release USEPA-certified solutions of cadmium and
copper for use as reference toxicants, through cooperative; research and
development agreements with commercial suppliers, and will continue to develop
additional reference toxicants for future release. Interested parties can
determine the availability of "EPA Certified" reference toxicants by checking
the EMSL-Cincinnati electronic bulletin board, using a modem to access the
following telephone number: 513-569-7610. Standard reference materials also
can be obtained from commercial supply houses, or can be prepared inhouse
using reagent grade chemicals. The regulatory agency should be consulted
before reference toxicant(s) are selected and used.
4.18 RECORD KEEPING
4.18.1 Proper record keeping is important. A complete file must be
maintained for each individual toxicity test or group of tests on closely
related samples. This file must contain a record of the sample chain-of-
custody; a copy of the sample log sheet; the original bench sheets for the
test organism responses during the toxicity test(s); chemical analysis data on
the sample(s); detailed records of the test organisms used in the test(s),
such as species, source, age, date of receipt, and other pertinent information
relating to their history and health; information on the calibration of
equipment and instruments; test conditions employed; and results of reference
toxicant tests. Laboratory data should be recorded on a real-time basis to
prevent the loss of information or inadvertent introduction of errors into the
record. Original data sheets should be signed and dated by the laboratory
personnel performing the tests.
I
4.18.2 The regulatory authority should retain records pertaining to discharge
permits. Permittees are required to retain records pertaining to permit
applications and compliance for a minimum of 3 years [40 CFR 122.41(j)(2)].
4.19 VIDEO TAPES OF USEPA CULTURE AND TOXICITY TEST METHODS
Three video-based training packages are available from National Technical
Information Service (NTIS), Department of Commerce, 5285 Port. Royal Road,
Springfield, VA 22161. Credit card orders can be placed by calling toll-free
(800) 788-6282, or by FAX at 703-321-8547, or by mail at the above address.
For other information call 703-487-4650.
1. Order # A18545: Toxicity Test Methods for the Red nriacroalga,
Champia parvula; the Sheepshead Minnow, Cypn'nodon van'egatus; the
inland silverside, Mem'dia beryllina; and the Sea Urchin, Arbacia
punctuTata. Price $85.00.
21
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2. Order # A18657: Mysids, Mysidopsis bahia, Culture and Toxicity Test.
Trice $75.00.
4.20 SUPPLEMENTAL REPORTS FOR TRAINING VIDEO TAPES
4.20.1 Ordering information: USEPA, Center for Environmental Research
Information, Cincinnati, OH 45268.
4.20.1.1 Sheepshead minnow, Cyprinodon van'egatus, and inland silverside,
Henidia beryllina, larval survival and growth toxicity tests (EPA/600/3-
90/075), 1990.
4.20.1.2 Red algae, Champia parvula, sexual reproduction (EPA/600/3-90/076),
1990.
4.20.1.3 Sperm cell test using the sea urchin, Arbacia punctulata,
(EPA/600-3-90/077), 1990.
4.20.2 Ordering information: USEPA, Office of Water (EN-336), Washington,
D.C. 20460.
4.20.2.1 Mysids, Mysidopsis bahia, survival, growth, and fecundity test
(EPA/505/8-90-006a), 1990.
22
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SECTION 5
FACILITIES, EQUIPMENT, AND SUPPLIES
5.1 GENERAL REQUIREMENTS
5.1.1 Effluent toxicity tests may be performed in a fixed or mobile
laboratory. Facilities must include equipment for rearing and/or holding
organisms. Culturing facilities for test organisms may be desirable in fixed
laboratories which perform large numbers of tests. Temperature control can be
achieved using circulating water baths, heat exchangers, or environmental
chambers. Water used for rearing, holding, acclimating, and testing organisms
may be natural seawater or water made up from hypersaline brine derived from
natural seawater, or water made up from reagent grade chemicals (GP2) or
commercial (FORTY FATHOMS® or HW MARINEMIX®) artificial sea salts when
specifically recommended in the method. Air used for aeration must be free of
oil and toxic vapors. Oil-free air pumps should be used where possible.
Particulates can be removed from the air using BALSTON® Grade BX or equivalent
filters (Balston, Inc., Lexington, Massachusetts), and oil and other organic
vapors can be removed using activated carbon filters (BALSTOM®, C-l filter, or
equivalent).
•
5.1.2 The facilities must be well ventilated and free of fumes. Laboratory
ventilation systems should be checked to ensure that return air from chemistry
laboratories and/or sample handling areas is not circulated to test organism
culture rooms or toxicity test rooms, or that air from toxicity test rooms
does not contaminate culture areas. Sample preparation, culturing, and
toxicity testing areas should be separated to avoid cross-contamination of
cultures or toxicity test solutions with toxic fumes. Air pressure
differentials between such rooms should not result in a net flow of
potentially contaminated air to sensitive areas through open or loosely-
fitting doors. Organisms should be shielded from external disturbances.
i
5.1.3 Materials used for exposure chambers, tubing, etc., which come in
contact with the effluent and dilution water, should be carefully chosen.
Tempered glass and perfluorocarbon plastics (TEFLON®) should be used whenever
possible to minimize sorption and leaching of toxic substances. These
materials may be reused following decontamination. Containers made of
plastics, such as polyethylene, polypropylene, polyvinyl chloride, TYGON®,
etc., may be used as test chambers or to ship, store, and transfer effluents
and receiving waters, but they should not be reused unless absolutely
necessary, because they might carry over adsorbed toxicants from one test to
another, if reused. However, these containers may be repeatedly reused for
storing uncontaminated waters such as deionized or laboratory-prepared
dilution waters and receiving waters. Glass or disposable polystyrene
containers can be used as test chambers. The use of large (> 20 L) glass
carboys is discouraged for safety reasons. j
5.1.4 New plastic products of a type not previously used should be tested for
toxicity before initial use by exposing the test organisms in the test system
where the material is used. Equipment (pumps, valves, etc.) which cannot be
23
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discarded after each use because of cost, must be decontaminated according to
the cleaning procedures listed below (see Section 5, Facilities, Equipment,
and Supplies, Subsection 5.3.2). Fiberglass, in addition to the previously
mentioned materials, can be used for holding, acclimating, and dilution water
storage tanks, and in the water delivery system, but once contaminated with
pollutants the fiberglass should not be reused. All material should be
flushed or rinsed thoroughly with the test media before using in the test.
5.1.5 Copper, galvanized material, rubber, brass, and lead must not come in
contact with culturing, holding, acclimation, or dilution water, or with
effluent samples and test solutions. Some materials, such as several types of
neoprene rubber (commonly used for stoppers) may be toxic and should be tested
before use.
5.1.6 Silicone adhesive used to construct glass test chambers absorbs some
organochlorine and organophosphorus pesticides, which are difficult to remove.
Therefore, as little of the adhesive as possible should be in contact with
water. Extra beads of adhesive inside the containers should be removed.
5.2 TEST CHAMBERS
5.2.1 Test chamber size and shape are varied according to size of the test
organism. Requirements are specified in each toxicity test method.
5.3 CLEANING TEST CHAMBERS AND LABORATORY APPARATUS
5.3.1 New plasticware used for sample collection or organism exposure vessels
generally does not require thorough cleaning before use. It is sufficient to
rinse new sample containers once with dilution water before use. New,
disposable, plastic test chambers may have to be rinsed with dilution water
before use. New glassware must be soaked overnight in 10% acid (see below)
and also should be rinsed well in deionized water and seawater.
5.3.2 All non-disposable sample containers, test vessels, pumps, tanks, and
other equipment that has come in contact with effluent must be washed after
use to remove surface contaminants, as described below.
1. Soak 15 minutes in tap water and scrub with detergent, or clean in an
automatic dishwasher.
2. Rinse twice with tap water.
3. Carefully rinse once with fresh dilute (10% V:V) hydrochloric acid or
nitric acid to remove scale, metals and bases. To prepare a 10%
solution of acid, add 10 ml of concentrated acid to 90 ml of
deionized water.
4. Rinse twice with deionized water.
5. Rinse once with full-strength, pesticide-grade acetone to remove
organic compounds (use a fume hood or canopy).
6. Rinse three times with deionized water.
5.3.3 All test chambers and equipment must be thoroughly rinsed with the
dilution water immediately prior to use in each test.
24
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5.4 APPARATUS AND EQUIPMENT FOR CULTURIN6 AND TOXICITY TESTS
5.4.1 Apparatus and equipment requirements for culturing and toxicity tests
are specified in each toxicity test method. Also, see USIEPA, 1993a.
5.4.2 WATER PURIFICATION SYSTEM
5.4.2.1 A good quality deionized water, providing 18 mega-ohm, laboratory
grade water, should be available in the laboratory and with sufficient
capacity 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
Culligen®,. Continental®, or equivalent.
• ;
5.5 REAGENTS AND CONSUMABLE MATERIALS
i
5.5.1 SOURCE'S OF FOOD FOR CULTURE AND TOXICITY TESTS
1. Brine Shrimp, Artemia sp. cysts -- A list of commercial sources is
provided in Table 2.
2. Frozen Adult Brine Shrimp, Artemia -- Available from most pet supply
shops-or from San Francisco Bay Brand, 8239 Enterprise Dr., Newark, CA
94560 (415-792-7200).
3. Flake Food -- TETRAMIN® and BIORIL® or equivalent are available at most
pet supply shops.
4. Feeding requirements and other specific foods are indicated; in the
specific toxicity test method. I
5.5.1.1 All food should be tested for nutritional suitability ahd chemically
analyzed for organochlorine pesticides, PCBs, and toxic metals (see Section 4,
Quality Assurance). <
i
5.5.2 Reagents and consumable materials are specified in each toxicity test
method. Also, see Section 4, Quality Assurance.
5.6 TEST ORGANISMS
5.6.1 Test organisms are obtained from inhouse cultures or commercial
suppliers (see specific toxicity test method; Sections 4, Quality Assurance
and 6, Test Organisms).
5.7 SUPPLIES
i
5.7.1 See toxicity test methods (see Sections 11-16) for specific supplies.
25
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TABLE 2. COMMERCIAL SUPPLIERS OF BRINE SHRIMP (ARTEMIA) CYSTS1'2
Aquafauna Biomarine
P.O. Box 5
Hawthorne CA 90250
Tel.
Fax.
(310
310
(Great Sa
San Francisco Bay)
Argent Chemical
8702 152nd Ave. NE
Redmond, WA 98052
973-5275
676-9387
t Lake North Arm,
Tel.
Tel.
Fax.
800)
206,
206
426-6258
855-3777
885-2112
(Plat num Label - San Francisco Bay;
Gold Label - San Francisco Bay,
Brazil; Silver Label - Great
Salt Lake, Australia; Bronze
Label - China, Canada, other)
Bonneville Artemia International, Inc.
P.O. Box 511113
Salt Lake City, UT 84151-1113
Tel. (801) 972-4704
Fax. (801) 972-4795
Ocean Star International
P.O. Box 643
Snowville, UT 84336
Tel. (801) 872-8217
Fax (801) 872-8272
(Great Salt Lake)
Sanders Brine Shrimp Co.
3850 South 540 West
Ogden, UT 84405
Tel. (801) 393-5027
(Great Salt Lake)
Sea Critters Inc.
P.O. Box 1508
Tavernier, FL 33070
Tel. (305) 367-2672
Aquarium Products
180L Penrod Court
Glen Burnie, MD 21061
Tel.
Fax.
Tel.
800
410
301
(Columbia
368-2507
761-6458
761-2100
I.NVE Artemia Systems
Oeverstraat 7
B-9200 Baasrode, Belgium
Tel. 011-32-52-331320
Fax. 011-32-52-341205
(For marine species - AF grade)
[smal 1 naupl i i ], UL grade [1 arge
nauplii], for freshwater species
-HI grade [small nauplii], EG
[large nauplii]
Golden West Artemia .
411 East 100 South
Salt Lake City, UT 84111
Tel. (801) 975-1222
Fax. (801) 975-1444
San Francisco Bay Brand
8239 Enterprise Drive
Newark, CA 94560
Tel. (510) 792-7200
Fax. (510) 792-5360
(Great Salt Lake,
San Francisco Bay)
Western Brine Shrimp
957 West South Temple
Salt Lake City, UT 84104
Tel. (801) 364-3642
Fax. (801) 534-0211
(Great Salt Lake)
List from David A. Bengtson, University of Rhode Island, Narragansett,
RI.
The geographic sources from which the vendors obtain the brine shrimp
cysts are shown in parentheses.
26
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SECTION 6
TEST ORGANISMS
6.1 TEST SPECIES
I
6.1.1 .The species used in characterizing the chronic toxicity of effluents
and/or receiving waters will depend on the requirements of the regulatory
authority and the objectives of the test. It is essential that good quality
test organisms be readily available throughout the year from inhouse or
commercial sources to meet NPDES monitoring requirements. The organisms used
in toxicity tests must be identified to species. If there is any doubt as to
the identity of the test organisms, representative specimens should be sent to
a taxonomic expert to confirm the identification.
6.1.2 Toxicity test conditions and culture methods for the species listed in
Subsection 6.1.3 are provided in this manual (also, see USEPA, 1993a).
6.1.3 The organisms used in the short-term tests described in this manual are
the sheepshead minnow, Cyprinodon variegatus; the inland silverside, Menidia
berynina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata;
and the red macroalga, Champia parvuTa.
i
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.
6.1.5 Where states have developed culturing and testing methods for
indigenous species other than those recommended in this manual, data comparing
the sensitivity of the substitute species and one or more of the recommended
species must be obtained in side-by-side toxicity tests with reference
toxicants and/or effluents, to ensure that the species selected are at least
as sensitive as the recommended species. These data must be submitted to the
permitting authority (State or Region) if required. USEPA acknowledges that
reference toxicants prepared from pure chemicals may not always be
representative of effluents. However, because of the observed and/or
potential variability in the quality and toxicity of effluents, it is not
possible to specify a representative effluent.
6.1.6 Guidance for the selection of test organisms where the salinity of the
effluent and/or receiving water requires special consideration is provided in
the Technical Support Document for Water Quality-based Toxics Control (USEPA,
1991a).
,
1. Where the salinity of the receiving water is < l%o, freshwater organisms
are used regardless of the salinity of the effluent.
i
27
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2. Where the salinity of the receiving water is > l%o, the choice of
organisms depends on state water quality standards and/or permit
requirements.
6.2 SOURCES OF TEST ORGANISMS
6.2.1 The test organisms recommended in this manual can be cultured in the
laboratory using culturing and handling methods for each organism described in
the respective test method sections. Also, see USEPA (1993a).
6.2.2 Inhouse cultures should be established wherever it is cost effective.
If inhouse cultures cannot be maintained or it is not cost effective, test
organisms should be purchased from experienced commercial suppliers (see
USEPA, 1993c).
6.2.3 Sheepshead minnows, inland silversides, mysids, and sea urchins may be
purchased from commercial suppliers. However, some of these organisms (e.g.,
adult sheepshead minnows or adult inland silversides) may not always be
available from commercial suppliers and may have to be collected in the field
and brought back to the laboratory for spawning to obtain eggs and larvae.
6.2.4 If, because of their source, there is any uncertainty concerning the
identity of the organisms, it is advisable to have them examined by a
taxonomic specialist to confirm their identification. For detailed guidance
on identification, see the individual toxicity test methods.
6.2.5 FERAL (NATURAL OCCURRING, WILD CAUGHT) ORGANISMS
6.2.5.1 The use of test organisms taken from the receiving water has strong
appeal, and would seem to be the logical approach. However, it is generally
impractical and not recommended for the following reasons:
1. Sensitive organisms may not be present in the receiving water because of
previous exposure to the effluent or other pollutants.
2. It is often difficult to collect organisms of the required age and
quality from the receiving water.
3. Most states require collection permits, which may be difficult to
obtain. Therefore, it is usually more cost effective to culture the
organisms in the laboratory or obtain them from private, state, or
Federal sources. Fish such as sheepshead minnows and silversides, and
invertebrates such as mysids, are easily reared in the laboratory or
purchased.
4. The required QA/QC records, such as the single-laboratory precision
data, would not be available.
5. Since it is mandatory that the identity of test organisms is known to
the species level, it would be necessary to examine each organism caught
in the wild to confirm its identity, which would usually be impractical
or, at the least, very stressful to the organisms.
6. Test organisms obtained from the wild must be observed in the
laboratory for a minimum of one week prior to use, to ensure that they
are free of signs of parasitic or bacterial infections and other adverse
28
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effects. Fish captured by electroshocking must not be used in toxicity
testing.
6.2.5.2 Guidelines for collection of natural occurring organisms are provided
in USEPA (1973); USEPA (1990a); and USEPA (1993c).
6.2.6 Regardless of their source, test organisms should be carefully observed
to ensure that they are free of, signs of stress and disease, and in good
physical condition. Some species of test organisms, such as trout, can be
obtained from stocks certified as "disease-free." |
6.3 LIFE STAGE |
6.3.1 Young organisms are often more sensitive to toxicants than are adults.
For this reason, the use of early life stages, such as juvenile mysids and
larval fish, is required for all tests. There may be special cases, however,
where the limited availability of organisms will require some deviation from
the recommended life stage. In a given test, all organisms should be
approximately the same age and should be taken from the same source. Since
age may affect the results of the tests, it would enhance the value and
comparability of the data if the same species in the same^life stages were
used throughout a monitoring program at a given facility.!
6.4 LABORATORY CULTURING
6.4.1 Instructions for culturing and/or holding the recommended test
organisms are included in specified test methods (also, see USEPA, 1993a).
6.5 HOLDING AND HANDLING TEST ORGANISMS
6.5.1 Test organisms should not be subjected to changes of more than 3°C in
water temperature or 3%o in salinity in any 12 h period.
6.5.2 Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as .possible to
minimize stress. Organisms that are dropped or touch dry surfaces or are
injured during handling must be discarded. Dipnets are best for handling
larger organisms. These nets are commercially available or can be made from
small-mesh nylon netting, silk bolting cloth, plankton netting, or similar
material. Wide-bore, smooth glass tubes (4 to 8 mm ID) with rubber bulbs or
pipettors (such as a PROPIPETTE® or other pipettor) should be used for
transferring smaller organisms such as mysids, and larval fish.
6.5.3 Holding tanks for fish are supplied with a good quality water (see
Section 5, Facilities, Equipment, and Supplies) with a flow-through rate of at
least two tank-volumes per day. Otherwise, use a recirculation system where
the water flows through an activated carbon or undergravel filter to remove
dissolved metabolites. Culture water can also be piped through high intensity
ultraviolet light sources for disinfection, and to photo-degrade dissolved
organics.
29
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6.5.4 Crowding should be avoided because it will stress the organisms and
lower the DO concentrations to unacceptable levels. The DO must be maintained
at a minimum of 4.0 mg/L. The solubility of oxygen depends on temperature,
salinity, and altitude. Aerate gently if necessary.
6.5.5 The organisms should be observed carefully each day for signs of
disease, stress, physical damage, or mortality. Dead and abnormal organisms
should be removed as soon as observed. It is not uncommon for some fish
mortality (5-10%) to occur during the first 48 h in a holding tank because of
individuals that refuse to feed on artificial food and die of starvation.
Organisms in the holding tanks should generally be fed as in the cultures (see
'culturing methods in the respective methods).
6.5.6 Fish should be fed as much as they will eat at least once a day with
live brine shrimp nauplii, Artemia, or frozen adult brine shrimp or dry food
(frozen food should be completely thawed before use). Adult brine shrimp can
be supplemented with commercially prepared food such as TETRAMIN® or BIORIL®
flake food, or equivalent. Excess food and fecal material should be removed
from the bottom of the tanks at least twice a week by siphoning.
6.5.7 Fish should be observed carefully each day for signs of disease,
stress, physical damage, and mortality. Dead and abnormal specimens should be
removed as soon as observed. It is not uncommon to have some fish (5-10%)
mortality during the first 48 h in a holding tank because of individuals that
refuse to feed on artificial food and die of starvation. Fish in the holding
tanks should generally be fed as in the cultures (see culturing methods in the
respective methods).
6.5.8 A daily record of feeding, behavioral observations, and mortality
should be maintained.
6.6 TRANSPORTATION TO THE TEST SITE
6.6.1 Organisms are transported from the base or supply laboratory to a
remote test site in culture water or standard dilution water in plastic bags
or large-mouth screw-cap (500 ml) plastic bottles in styrofoam coolers.
Adequate DO is maintained by replacing the air above the water in the bags
with oxygen from a compressed gas cylinder, and sealing the bags. Another
method commonly used to maintain sufficient DO during shipment is to aerate
with an airstone which is supplied from a portable pump. The DO concentration
must not fall below 4.0 mg/L.
6.6.2 Upon arrival at the test site, organisms are transferred to receiving
water if receiving water is to be used as the test dilution water. All but a
small volume of the holding water (approximately 5%) is removed by siphoning,
and replaced slowly over a 10 to 15 minute period with dilution water. If
receiving water is used as dilution water, caution must be exercised in
exposing the test organisms to it, because of the possibility that it might be
toxic. For this reason, it is recommended that only approximately 10% of the
test organisms be exposed initially to the dilution water. If this group does
not show excessive mortality or obvious signs of stress in a few hours, the
remainder of the test organisms are transferred to the dilution water.
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A group of organisms must not be used for a test if they appear to be
thy, discolored, or otherwise stressed, or if mortality appears to
6.6.3
unhealthy,
exceed 10% preceding the test. If the organisms fail to meet these criteria,
the entire group must be discarded and a new group obtained. The mortality
may be due to the presence of toxicity, if receiving water is used as dilution
water, rather than a diseased condition of the test organisms. If the
acclimation process is repeated with a new group of test organisms and
excessive mortality occurs, it is recommended that an alternative source of
dilution water be used.
6.6.4 The marine organisms can be used at all concentrations of effluent by
adjusting the salinity of the effluent to salinities specified for the
appropriate species test condition or to the salinity approximating that of
the receiving water, by adding sufficient dry ocean salts, such as FORTY
FATHOMS®, or equivalent, GP2, or hypersaline brine.
6.6.5 Saline dilution water can be prepared with deionized water or a
freshwater such as well water or a suitable surface water. If dry ocean salts
are used, care must be taken to ensure that the added salts are completely
dissolved and the solution is aerated 24 h before the test organisms are
placed in the solutions. The test organisms should be acclimated in synthetic
saline water prepared with the dry salts. Caution: addition of dry ocean
salts to dilution water may result in an increase in pH. (The pH of estuarine
and coastal saline waters is normally 7.5-8.3).
6.6.6 All effluent concentrations and the control(s) used in a test should
have the same salinity. The change in salinity upon acclimation at the
desired test dilution should not exceed 6%o. The required salinities for
culturing and toxicity tests with estuarine and marine species are listed in
the test method sections.
6.7 TEST ORGANISM DISPOSAL
6.7.1 When the toxicity test(s) is concluded, all test organisms (including
controls) should be humanely destroyed and disposed of in an appropriate
manner.
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SECTION 7
DILUTION WATER
7.1 TYPES OF DILUTION WATER
7.1.1 The type of dilution water used in effluent toxicity tests will depend
largely on the objectives of the study.
7.1.1.1 If the objective of the test is to estimate the chronic toxicity of
the effluent, which is a primary objective of NPDES permit-related toxicity
testing, a synthetic (standard) dilution water is used. If the test organisms
have been cultured in water which is different from the test dilution water, a
second set of controls, using culture water, should be included in the test.
7.1.1.2 If the objective of the test is to estimate the chronic toxicity of
the effluent in uncontaminated receiving water, the test may be conducted
using dilution water consisting of a single grab sample of receiving water (if
non-toxic), collected outside the influence of the outfall, or with other
uncontaminated natural water (surface water) or standard dilution water having
approximately the same salinity as the receiving water. Seasonal variations
in the quality of receiving waters may affect effluent toxicity. Therefore,
the salinity of saline receiving water samples should be determined before
each use. If the test organisms have been cultured in water which is
different from the test dilution water, a second set of controls, using
culture water, should be included in the test.
7.1.1.3 If the objective of the test is to determine the additive or
mitigating effects of the discharge on already contaminated receiving water,
the test is performed using dilution water consisting of receiving water
collected outside the influence of the outfall. A second set of controls,
using culture water, should be included in the test.
7.2 STANDARD, SYNTHETIC DILUTION WATER
7.2.1 Standard, synthetic, dilution water is prepared with deionized water
and reagent grade chemicals (GP2) or commercial sea salts (FORTY FATHOMS®, HW
HARINEMIX®) (Table 3). The source water for the deionizer can be ground water
or tap water.
7.2.2 DEIONIZED WATER USED TO PREPARE STANDARD, SYNTHETIC, DILUTION WATER
7.2.2.1 Deionized water is obtained from a MILLIPORE MILLI-Q®, MILLIPORE®
QPAK™2 or equivalent system. It is advisable to provide a preconditioned
(deionized) feed water by using a Culligan®, Continental®, or equivalent
system in front of the MILLI-Q® System to extend the life of the MILLI-Q®
cartridges (see Section 5, Facilities, Equipment, and Supplies).
7.2.2.2 The recommended order of the cartridges in a four-cartridge deionizer
(i.e., MILLI-Q® System or equivalent) is: (1) ion exchange, (2) ion exchange,
(3) carbon, and (4) organic cleanup (such as ORGANEX-Q®, or equivalent),
32
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followed by a final bacteria filter. The QPAK™2 water system is a sealed
system which does not allow for the rearranging of the cartridges. However,
the final cartridge is an ORGANEX-Q® filter, followed by a final bacteria
filter. Commercial laboratories using this system have not experienced any
difficulty in using the water for culturing or testing. Reference to the
MILLI-Q® systems throughout the remainder of the manual includes all
MILLIPORE® or equivalent systems.
7.2.3 STANDARD, SYNTHETIC SEAWATER .
7.2.3.1 To prepare 20 L of a standard, synthetic, reconstituted seawater
(modified GP2), using reagent grade chemicals (Table 3), with a salinity of
31%o, follow the instructions below. Other salinities can be prepared by
making the appropriate dilutions. Larger or smaller volumes of modified GP2
can be prepared by using proportionately larger or smaller amounts of salts
and dilution water.
1. Place 20 L of MILLI-Q® or equivalent deion.ized water in a properly
cleaned plastic carboy.
2. Weigh reagent grade salts listed in Table 3 and add, one at a time, to
the deionized water. Stir well after adding each salt..
3. Aerate the final solution at a rate of 1 L/h for 24 h.
4. Check the pH and salinity.
7.2.3.2 Synthetic seawater can also be prepared by adding commercial sea
salts, such as FORTY FATHOMS®, HW MARINEMIX®, or equivalent, to deionized
water. For example, thirty-one parts per thousand (31%o) FORTY FATHOMS® can
be prepared by dissolving 31 g of sea salts per liter of deionized water. The
salinity of the resulting solutions should be checked with a refractometer.
7.2.4 Artificial seawater is to be used only if specified in the method.
EMSL-Cincinnati has found FORTY FATHOMS® artificial sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204, 301-321-1189)
suitable for maintaining and spawning the sheepshead minnow, Cyprinodon
variegatus, and for its use in the sheepshead minnow larval survival and
growth test, suitable for maintaining and spawning the inland silverside,
Menidia beryllina, and for its use in the inland silverside larval survival
and growth test, suitable for culturing and maintaining mysid shrimp,
Mysidopsis bahia, and its use in the mysid shrimp survival, growth, and
fecundity test, and suitable for maintaining sea urchins, Arbacia punctulata,
and for its use in the sea urchin fertilization test. The USEPA Region 6
Houston Laboratory has successfully used HW MARINEMIX® (Hawaiian Marine
Imports Inc., P.O. Box 218687, Houston, TX 77218, 713-492^7864) sea salts to
maintain and spawn sheepshead minnows, and perform the larval survival and
growth test and the embryo-larval survival and teratogenicity test. Also, HW
MARINEMIX® sea salts has been used successfully to culture and maintain the
mysid brood stock and perform the mysid survival, growth, fecundity test. An
artificial seawater formulation, GP2 (Spotte et al., 1984), Table 3, has been
used by the Environmental Research Laboratory-Narragansett, RI for all but the
embryo-larval survival and teratogenicity test. The suitability of GP2 as a
medium for culturing organisms has not been determined.
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TABLE 3. PREPARATION OF GP2 ARTIFICIAL SEAWATER USING REAGENT GRADE
CHEMICALS1'2'3
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 • 6 H20
CaCl2 • 2 H20
SrCl2 • 6 H20
NaHC03
Concentration
(g/L)
21.03
3.52
0.61
0.088
0.034
9.50
1 .32
0.02
0.17
Amount (g)
Required for
20 L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
J Modified GP2 from Spotte et al. (1984).
The constituent salts and concentrations were taken from USEPA (1993a).
The salinity is 30.89 g/L.
6P2 can be diluted with deionized (DI) water to the desired test
salinity.
7.3 USE OF RECEIVING WATER AS DILUTHON WATER
7.3.1 If the objectives of the test require the use of uncontaminated
receiving water as dilution water, and the receiving water is uncontaminated,
it may be possible to collect a sample of the receiving water close to the
outfall, but should be away from or beyond the influence of the effluent.
However, if the receiving water is contaminated, it may be necessary to
collect the sample in an area "remote" from the discharge site, matching as
closely as possible the physical and chemical characteristics of the receiving
water near the outfall.
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7.3.2 The sample should be collected immediately prior to-the test, but never
•more than 96 h before the test begins. Except where it is used within 24 h,
or in the case where large volumes are required for flow through tests, the
sample should be chilled to 4°C during or immediately following collection,
and maintained at that temperature prior to use in the test.
1
7.3.3 The investigator should collect uncontaminated water having a salinity
as near as possible to the salinity of the receiving water at the discharge
site. Water should be collected at slack high tide, or within one hour after
•high tide. If there is reason to suspect contamination of the water in the
estuary, it is advisable to collect uncontaminated water from an adjacent
estuary. At times it may be necessary to collect water at a location closer
to the open sea, where the salinity is relatively high. In such cases,
deionized water or uncontaminated freshwater is added to the saline water to
dilute it to the required test salinity. Where necessary, the salinity of a
surface water can be increased by the addition of artificial sea salts, such
as FORTY FATHOMS®, HW MARINEMIX®, or equivalent, GP2, a natural seawater of
higher salinity, or hypersaline brine. Instructions for the preparation of
hypersaline brine by concentrating natural seawater are provided below.
7.3.4 Receiving water containing debris or indigenous organisms, that may be
confused with or attack the test organisms, should be filtered through a sieve
having 60 ^m mesh openings prior to use.
7.3.5 HYPERSALINE BRINE
I
7.3.5.1 Hypersaline brine (HSB) has several advantages that make it desirable
for use in toxicity testing. It can be made from any high quality, filtered
seawater by evaporation, and can be added to deionized water to prepare
dilution water, or to effluents or surface waters to increase their salinity.
7.3.5.2 The ideal container for making HSB from natural sieawater is one that
(1) has a high surface to volume ratio, (2) is made of a rioncorrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine. If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that would contaminate the brine. One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass. If aeration is used, use only oil-free air compressors to
prevent contamination.
7.3.5.3 Before adding seawater to the brine generator, thoroughly clean the
generator, aeration supply tube, heater, and any other materials that will be
in direct.contact with the brine. A good quality biodegradable detergent
should be used, followed by several thorough deionized water rinses. High
quality (and preferably high salinity) seawater should be filtered to at least
10 /um before placing into the brine generator. Water should be collected on
an incoming tide to minimize the possibility of contamination.
7.3.5.4 The temperature of the seawater is increased slowly to 40°C. The
water should be aerated to prevent temperature stratification and to increase
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water evaporation. The brine should be checked daily (depending on the volume
being generated) to ensure that the salinity does not exceed 100%o and that
the temperature does not exceed 40°C. Additional seawater may be added to the
brine to obtain the volume of brine required.
7.3.5.5 After the required salinity is attained, the MSB should be filtered a
second time through a l-/zm filter and poured directly into portable containers
(20-L CUBITAINERS® or polycarbonate water cooler jugs are suitable). The
containers should be -capped and labelled with the date the brine was generated
and its salinity. Containers of HSB should be stored in the dark and
maintained under room temperature until used.
7.3.5.6 If a source of HSB is available, test solutions can be made by
following the directions below. Thoroughly mix together the deionized water
and brine before mixing in the effluent.
7.3.5.7 Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine. For example, if the
salinity of the brine is 100%o and the test is to be conducted at 25%o, 100%o
divided, by 25%o = 4.0. The proportion of brine is 1 part in 4 (one part brine
to three parts deionized water).
7.3.5.8 To make 1 L of seawater at 25%o salinity from a hypersaline brine of
100%o, 250 ml of brine and 750 ml of deionized water are required.
7.4 USE OF TAP WATER AS DILUTION WATER
7.4.1 The use of tap water in the reconstituting of synthetic (artificial)
seawater as dilution water is discouraged unless it is dechlorinated and fully
treated. Tap water can be dechlorinated by deionization, carbon filtration,
or the use of sodium thiosulfate. Use of 3.6 mg/L (anhydrous) sodium
thiosulfate will reduce 1.0 mg chlorine/L (APHA, 1992). Following
dechlorination, total residual chlorine should not exceed 0.01 mg/L. Because
of the possible toxicity of thiosulfate to test organisms, a control lacking
thiosulfate should be included in toxicity tests utilizing thiosulfate-
dechlorinated water.
7.4.2 To be adequate for general laboratory use following dechlorination, the
tap water is passed through a deionizer and carbon filter to remove toxic
metals and organics, and to control hardness and alkalinity.
7.5 DILUTION WATER HOLDING
7.5.1 A given batch of dilution water should not be used for more than 14
days following preparation because of the possible build up of bacterial,
fungal, or algal slime growth and the problems associated with it. The
container should be kept covered and the contents should be protected from
light.
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SECTION 8
EFFLUENT AND RECEIVING MATER SAMPLING, SAMPLE HANDLING,
AND SAMPLE PREPARATION FOR TOXICITY TESTS
8.1 EFFLUENT SAMPLING j
i
8.1.1 The effluent sampling point should be the same as that specified in the
NPDES discharge permit (USEPA, 1988b). Conditions for exception would be:
(1) better access to a sampling point between the final treatment and the
discharge outfall; (2) if the processed waste is chlorinated prior to
discharge, it may also be desirable to take samples prior to contact with the
chlorine to determine toxicity of the unchlorinated effluent; or (3) in the
event there is a desire to evaluate the toxicity of the influent to municipal
waste treatment plants or separate wastewater streams in industrial facilities
prior to their being combined with other wastewater streams or non-contact
cooling water, additional sampling points may be chosen.
8.1.2 The decision on whether to collect grab or composite samples is based
on the objectives of the test and an understanding of the short and long-term
operations and schedules of the discharger. If the effluent quality varies
considerably with time, which can occur where holding times are short, grab
samples may seem preferable because of the ease of collection and the
potential of observing peaks (spikes) in toxicity. However, the sampling
duration of a grab sample is so short that full characterization of an
effluent over a 24-h period would require a prohibitively large number of
separate samples and tests. Collection of a 24-h composite sample, however,
may dilute toxicity spikes, and average the quality of the effluent over the
sampling period. Sampling recommendations are provided below (also see USEPA,
1993a).
j
8.1.3 Aeration during collection and transfer of effluents should be
minimized to reduce the loss of volatile chemicals. i
8.1.4 Details of date, time, location, duration, and procedures used for
effluent sample and dilution water collection should be recorded.
8.2 EFFLUENT SAMPLE TYPES
8.2.1 The advantages and disadvantages of effluent grab and composite samples
are listed below:
8.2.1.1 GRAB SAMPLES
Advantages:
1. Easy to collect; require a minimum of equipment and !on-site time.
2. Provide a measure of instantaneous toxicity. Toxicity spikes are not
masked by dilution.
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Disadvantages:
1. Samples are collected over a very short period of time and on a
relatively infrequent basis. The chances of detecting a spike in
toxicity would depend on the frequency of sampling, and the probability
of missing spikes is high.
8.2.1.2 COMPOSITE SAMPLES:
Advantages:
1. A single effluent sample is collected over a 24-h period.
2. The sample is collected over a much longer period of time than grab
samples and contains all toxicity spikes.
Disadvantages:
1. Sampling equipment is more sophisticated and expensive, and must be
placed on-site for at least 24 h.
2. Toxicity spikes may not be detected because they are masked by dilution
with less toxic wastes.
8.3 EFFLUENT SAMPLING RECOMMENDATIONS
8.3.1 When tests are conducted on-site, test solutions can be renewed daily
with freshly collected samples.
8.3.2 When tests are conducted off-site, a minimum of three samples are
collected. If these samples are collected on Test Days 1,3, and 5, the first
sample would be used for test initiation, and for test solution renewal on Day
2. The second sample would be used for test solution renewal on Days 3 and 4.
The third sample would be used for test solution renewal on Days 5, 6, and 7.
8.3.3 Sufficient sample must be collected to perform the required toxicity
and chemical tests. A 4-L (1-gal) CUBITAINER® will provide sufficient sample
volume for most tests.
8.3.4 THE FOLLOWING EFFLUENT SAMPLING METHODS ARE RECOMMENDED:
8.3.4.1 Continuous Discharges
1. If the facility discharge is continuous, but the calculated retention
time of the continuously discharged effluent is less than 14 days and
the variability of the effluent toxicity is unknown, at a minimum, four
grab samples or four composite samples are collected over a 24-h period.
For example, a grab sample is taken every 6 h (total of four samples)
and each sample is used for a separate toxicity test, or four
successive 6-h composite samples are taken and each is used in a
separate test.
2. If the calculated retention time of a continuously discharged effluent
is greater than 14 days, or if it can be demonstrated that the
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wastewater does not vary more than 10% in toxicity over a 24-h period,
regardless of retention time, a single grab sample is collected for a
single toxicity test.
3. The retention time of the effluent in the wastewater treatment facility
may be estimated from calculations based on the volume of the retention
basin and rate of wastewater inflow. However, the calculated retention
time may be much greater than the actual time because of
short-circuiting in the holding basin. Where short-circuiting is
suspected, or sedimentation may have reduced holding basin capacity, a
more accurate estimate of the retention time can be obtained by carrying
out a dye study.
8.3.4.2 Intermittent Discharges I
8.3.4.2.1 If the facility discharge is intermittent, a grab sample is
collected midway during each discharge period. Examples of intermittent
discharges are:
1. When the effluent is continuously discharged during a single 8-h work
shift (one sample is collected), or two successive 8-h work shifts (two
samples are collected).
2. When the facility retains the wastewater during an 8-h work shift, and
then treats and releases the wastewater as a batch discharge (one sample
is collected).
3. When the facility discharges wastewater to an estuary only during an
outgoing tide, usually during the 4 h following slack high tide (one
sample is collected).
4. At the end of a shift, clean up activities may result in the
discharge of a slug of toxic waste (one sample is collected).
8.4 RECEIVING WATER SAMPLING
8.4.1 Logistical problems and difficulty in securing sampling equipment
generally preclude the collection of composite receiving water samples for
toxicity tests. Therefore, based on the requirements of the test, a single
grab sample or daily grab samples of receiving water is collected for use in
the test.
I
I
8.4.2 The sampling point is determined by the objectives of the test. At
estuarine and marine sites, samples should be collected at mid-depth.
8.4.3 To determine the extent of the zone of toxicity in the receiving water
at estuarine and marine effluent sites, receiving water samples are collected
at several distances away from the discharge. The time required for the
effluent-receiving-water mixture to travel to sampling points away from the
effluent, and the rate and degree .of mixing, may be difficult to ascertain.
Therefore, it may not be possible to correlate receiving water toxicity with
effluent toxicity at the discharge point unless a dye study is performed. The
toxicity of receiving water samples from five stations in the discharge plume
can be evaluated using the same number of test vessels and test organisms as
used in one effluent toxicity test with five effluent dilutions.
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8.5 EFFLUENT AND RECEIVING WATER SAMPLE HANDLING, PRESERVATION, AND SHIPPING
8.5.1 Unless the samples are used in an on-site toxicity test the day of
collection, it is recommended that they be held at 4°C until used to inhibit
microbial degradation, chemical transformations, and loss of highly volatile
toxic substances.
8.5.2 Composite samples should be chilled as they are collected. Grab
samples should be chilled immediately following collection.
8.5.3 If the effluent has been chlorinated, total residual chlorine must be
measured immediately following sample collection.
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 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 sample may also be used to prepare test
solutions for renewal at 24 h and 48 h after test initiation, if stored at
4°C, with minimum head space, as described in Paragraph 8.5. Guidance for
determining the persistence of the sample is provided in Subsection 8.7.
8.5.5 To minimize the loss of toxicity due to volatilization of toxic
constituents, all sample containers should be "completely" filled, leaving no
air space between the contents and the lid.
8.5.6 SAMPLES USED IN.ON-SITE TESTS
8.5.6.1 Samples collected for on-site tests should be used within 24 h.
8.5.7 SAMPLES SHIPPED TO OFF SITE FACILITIES
8.5.7.1 Samples collected for off site toxicity testing are to be chilled to
4°C during or immediately after collection, and shipped iced to the performing
laboratory. Sufficient ice should be placed with the sample in the shipping
container to ensure that ice will still be present when the sample arrives at
the laboratory and is unpacked. Insulating material must not be placed
between the ice and the sample in the shipping container.
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8.5.7.2 Samples may be shipped in one or more 4-L (1-gal) CUBITAINERS® or new
plastic "milk" jugs. All sample containers should be rinsed with source water
before being filled with sample. After use with receiving water or effluents,
CUBITAINERS® and plastic jugs are punctured to prevent reuse.
8.5.7.3 Several sample shipping options are available, including Express
Mail, air express, bus, and courier service. Express Mai] is delivered seven
days a week. Saturday and Sunday shipping and receiving schedules of private
carriers vary with the carrier.
8.6 SAMPLE RECEIVING
8.6.1 Upon arrival at the laboratory, samples are logged in and the
temperature is measured and recorded. If the samples are not
immediately prepared for testing, they are stored at 4°C until used.
8.6.2 Every effort must be made to initiate the test with an effluent sample
on the day of arrival in the laboratory, and the sample holding time should
not exceed 36 h unless a variance has been granted by the NPDES permitting
authority.
8.7 PERSISTENCE OF EFFLUENT TOXICITY DURING SAMPLE SHIPMENT AND HOLDING
8.7.1 The persistence of the .toxicity of an effluent prior to its use in a
toxicity test is of interest in assessing the validity of toxicity test data,
and in determining the possible effects of allowing an extension of the
holding time. Where a variance in holding time (> 36 h, but < 72 h) is
requested by a permittee (See subsection 8.5.4), information on the effects of
the extension in holding time on the toxicity of the samples must be obtained
by comparing the results of multi-concentration chronic toxicity tests
performed on effluent samples held 36 h with toxicity test results using the
same samples after they were held for the requested, longer period. The
portion of the sample set aside for the second test must be held under the
same conditions as during shipment and holding.
8.8 PREPARATION OF EFFLUENT AND RECEIVING WATER SAMPLES FOR TOXICITY TESTS
8.8.1 Adjust the samp-le salinity to the level appropriate for objectives of
the study using hypersaline brine or artificial sea salts,
8.8.2 When aliquots are removed from the sample container, the head space
above the remaining sample should be held to a minimum. Air which enters a
container upon removal of sample should be expelled by compressing the
container before reelosing, if possible (i.e., where a CUBITAINER® used), or
by using an appropriate discharge valve (spigot).
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 be filtered through a sieve
with 60 urn mesh openings. Since filtering may increase the dissolved oxygen (DO)
in an effluent, the DO should be determined prior to filtering. Low dissolved
41
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oxygen concentrations will indicate a potential problem in performing the test.
Caution: filtration may remove some toxicity.
8.8.4 If the samples must be warmed to bring them to the prescribed test
temperature, supersaturation of the dissolved oxygen and nitrogen may become a
problem. To avoid this problem, the effluent and dilution water are checked with
a DO probe after reaching test temperature and, if the DO is greater than 100%
saturation or lower than 4.0 mg/L, based on temperature and salinity, the
solutions are aerated moderately (approximately 500 mL/min) for a few minutes,
using an airstone, until the DO is lowered to 100% saturation (Table 4) or until
the DO is within the prescribed range (> 4.0 mg/L). Caution: avoid excessive
aeration.
8.8.4.1 Aeration during the test may alter the results and should be used only
as a last resort to maintain the required DO. Aeration can reduce the apparent
toxicity of the test solutions by stripping them of highly volatile toxic
substances, or increase their toxicity by altering the pH. However, the DO in
the test solution must not be permitted to fall below 4.0 mg/L.
8.8.4.2 In static tests (non-renewal or renewal) low DOs may commonly occur in
the higher concentrations of wastewater. Aeration is accomplished by bubbling
air through a pipet at the rate of 100 bubbles/min. If aeration is necessary,
all test solutions must be aerated. It is advisable to monitor the DO closely
during the first few hours of the test. Samples with a potential DO problem
generally show a downward trend in DO within 4 to 8 h after the test is started.
Unless aeration is initiated during the first 8 h of the test, the DO may be
exhausted during an unattended period, thereby invalidating the test.
8.8.5 At a minimum, pH, conductivity or salinity, and total residual chlorine
are measured in the undiluted effluent or receiving water, and pH and
conductivity are measured in the dilution water.
8.8.5.1 It is recommended that total alkalinity and total hardness also be
measured in the undiluted effluent test water and the dilution water.
8.8.6 Total ammonia is measured in effluent and receiving water samples where
toxicity may be contributed by unionized ammonia (i.e., where total ammonia
•&. 5 mg/L). The concentration (mg/L) of unionized (free) ammonia in a sample is a
function of temperature and pH, and is calculated using the percentage value
obtained from Table 5, under the appropriate pH and temperature, and multiplying
it by the concentration (mg/L) of total ammonia in the sample.
8.8.7 Effluents and receiving waters can be dechlorinated using 6.7 mg/L
anhydrous sodium thiosulfate to reduce 1 mg/L chlorine (APHA, 1992). Note that
the amount of thiosulfate required to dechlorinate effluents is greater than the
amount needed to dechlorinate tap water, (see Section 7, Dilution Water). Since
thiosulfate may contribute to sample toxicity, a thiosulfate control should be
used in the test in addition to the normal dilution water control.
8.8.8 The DO concentration in the samples should be near saturation prior to
use. Aeration will bring the DO and other gases into equilibrium with air,
minimize oxygen demand, and stabilize the pH. However, aeration during
42
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TABLE 4. OXYGEN SOLUBILITY (MG/L) IN WATER AT EQUILIBRIUM WITH AIR AT 760 MM
HG (AFTER Richards and Corwin, 1956)
TEMP
(C°)
0
1
2
3
4
5
6
8
10
12
14
16
18
20
22
24
26
28
30
32
SALINITY (%o)
0
14.2
13.8
13.4
13.1
12.7
12.4
12.1
11.5
10.9
10.5
10.0
9.6
9.2
8.9
8.6
8.3
8.1
7.8
7.6
7.3.
5
13.8
13.4
13.0
12.7
12.3
12.0
11.7
11.2
10.7
10.2
9.7
9.3
9.0
8.6
8.4
8.1
7.8
7.6
7.4
7.1
10
13.4
13.0
12.6
12.3
12.0
11.7
11.4
10.8
10.3
9.9
9.5
9.1
8.7
8.4
8.1
7.8
7.6
7.4
7.1
6.9
15
12.9
12.6
12.2
11.9
11.6
11.3
11.0
10.5
10.0
9.6
9.2
8.8
8.5
8.1
7.9
7.6
7.4
7.2
6.9
6.7
20
12.5
12.2
11.9
11.6
11.3
11.0
10.7
10.2
9.7
9.3
8.9
8.5
8.2
7.9
7.6
7.4
7.2
7.0
6.7
6.5
25
12.1
11.8
11.5
11.2
10.9
10.6
10.3
9.8
9.4
9.0
8.6
8.3
8.0
7.7
7.4
7.2
7.0
6.8
6.5
6.3
30
11.7
11.4
11.1
10.8
10.5
10.2
10.0
9.5
9.1
8.7
8.3
8.0
7.7
7.4
7.2
6.9
6.7
6.5
6.3
6.1
35
11.2
11.0
10.7
10.4
10.1
9.8
9.6
9.2
8.8
8.4
8.1
7.7
7.5
7.2
6.9
6.7
6.5
6.3
6.1
5.9
40
10.8
10.6
10.3
10.0
9.8
9.5
9.3
8.9
8.5
8.1
7.8
7.5
7.2
6.9
6.7
6.5
6.3
6.1
5.9
5.7
43
10.6
10.3
10.0
9.8
9.5
9.3
9.1
8.7
8.3
7.9
7.6
7.3
7.1
6.8
6.6
6.4
6.1
6.0
5.8
5.6
43
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TABLE 5. PERCENT UNIONIZED NH, IN AQUEOUS AMMONIA SOLUTIONS:
15-26°C AND pH 6.0-8.91
TEMPERATURE
pH
TEMPERATURE <°C)
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
15
0.0274
0.0345
0.0434
0.0546
0.0687
0.0865
0.109
0.137
0.172
0.217
0.273
0.343
0.432
0.543
0.683
0.858
1.08
1.35
1.70
2.13
2.66
3.33
4.16
5.18
6.43
7.97
9.83
12.07
14.7
17.9
16
0.0295
0.0372
0.0468
0.0589
0.0741
0.0933
0.117
0.148
0.186
0.234
0.294
0.370
0.466
0.586
0.736
0.925
1.16
1.46
1.83
2.29
2.87
3.58
4.47
5.56
6.90
8.54
10.5
12.9
15.7
19.0
17
0.0318
0.0400
0.0504
0.0634
0.0799
0.1005
0.127
0.159
0.200
0.252
0.317
0.399
0.502
0.631
0.793
0.996
1.25
1.57
1.97
2.46
3.08
3.85
4.80
5.97
7.40
9.14
11.2
13.8
16.7
20.2
18
0.0343
0.0431
0.0543
0.0683
0.0860
0.1083
0.136
0.171
0.216
0.271
0.342
0.430
0.540
0.679
0.854
1.07
1.35
1.69
2.12
2.65
3.31
4.14
5.15
6.40
7.93
9.78
12.0
14.7
17.8
21.4
•19
0.0369
0.0464
0.0584
0.0736
0.0926
0.1166
0.147
0.185
0.232
0.292
0.368
0.462
0.581
0.731
0.918
1.15
1.45
1.82
2.28
2.85
3.56
4.44
5.52
6.86
8.48
10.45
12.8
15.6
18.9
22.7
20
0.0397
0.0500
0.0629
0.0792
0.0996
0.1254
0.158
0.199
0.250
0.314
0.396
0.497
0.625
0.786
0.988
1.24
1.56
1.95
2.44
3.06
3.82
4.76
5.92
7.34
9.07
11.16
13.6
16.6
20.0
24.0
21
0.0427
0.0537
0.0676
0.0851
0.107
0.135
0.170
0.214
0.269
0.338
0.425
0.535
0.672
0.845
1.061
1.33
1.67
2.10
2.62
3.28
4.10
5.10
6.34
7.85
9.69
11.90
14.5
17.6
21.2
25.3
22
0.0459
0.0578
0.0727
0.0915
0.115
0.145
0.182
0.230
0.289
0.363
0.457
0.575
0.722
0.908
1.140
1.43
1.80
2.25
2.82
3.52
4.39
5.46
6.78
8.39
10.3
12.7
15.5
18.7
22.5
26.7
23
0.0493
0.0621
0.0781
0.0983
0.124
0.156
0.196
0.247
0.310
0.390
0.491
0.617
0.776
0.975
1.224
1.54
1.93
2.41
3.02
3.77
4.70
5.85
7.25
8.96
11.0
13.5
16.4
19.8
23.7
28.2
24 25
0.0530 0.0568
0.0667 0.0716
0.0901 0.0901
0.1134 0.1134
0.133 0.143
0.167 0.180
0.210 0.226
0.265 0.284
0.333 0.358
0.419 0.450
0.527 0.566
0.663 0.711
0.833 0.893
1.05 1.12
1.31 1.41
1.65 1.77
2.07 2.21
2.59 2.77
3.24 3.46
4.04 4.32
5.03 5.38
6.25 6.68
7.75 8.27
9.56 10.2
11.7 12.5
14.4 15.2
17.4 18.5
21.0 22.2
25.1 26,4
29.6 31.1
26
0.0610
0.0768
0.0966
0.1216
0.153
0.193
0.242
0.305
0.384
0.482
0.607
0.762
0.958
1.20
1.51
1.89
2.37
2.97
3.71
4.62
5.75
7.14
8.82
10.9
13.3
16.2
19.5
23.4
27.8
32.6
Table provided by Teresa Norberg-King, Duluth, Minnesota. Also
Emerson et al. (1975), Thurston et al. (1974), and USEPA (1985a).
see
44
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collection, transfer, and preparation of samples should be minimized to reduce
the loss of volatile chemicals.
8.8.9 Mortality or impairment of growth or reproduction due to pH alone may
occur if the pH of the sample falls outside the range of 6.0 - 9.0. Thus, the
presence of other forms of toxicity (metals and organics) in the sample may be
masked by the toxic effects of low or high pH. The question about the presence
of other toxicants can be answered only by performing two parallel tests, one
with an adjusted pH, and one without an adjusted pH. Freshwater samples are
adjusted to pH 7.0, and marine samples are adjusted to pH 8.0, by adding IN NaOH
or IN HC1 dropwise, as required, being careful to avoid overadjustment.
8.9 PRELIMINARY TOXICITY RANGE-FINDING TESTS
8.9.1 USEPA Regional and State personnel generally have observed that it is not
necessary to conduct a toxicity range-finding test prior to initiating a static,
chronic, definitive toxicity test. However, when preparing to perform a static
test with a sample of completely unknown quality, or before initiating a flow-
through test, it is advisable to conduct a preliminary toxicity range-finding
test.
8.9.2 A toxicity range-finding test ordinarily consists of a down-scaled,
abbreviated static acute test in which groups of five organisms are exposed to
several widely-spaced sample dilutions in a logarithmic series, such as 100%,
10.0%, 1.00%, and 0.100%, and a control, for 8-24 h. Cautiom: if the sample
must also be used for the full-scale definitive test, the 36-h limit on holding
time (see Subsection 8.5.4) must not be exceeded before the definitive test is
initiated. •
8.9.3 It should be noted that the toxicity (LC50) of a sample observed in a
range-finding test may be significantly different from the toxicity observed in
the follow-up, chronic, definitive test because: (1) the definitive test is
longer; and (2) the test may be performed with a sample collected at a different
time, and possibly differing significantly in the level of toxicity.
8.10 MULTICONCENTRATION (DEFINITIVE) EFFLUENT TOXICITY TESTS
8.10.1 The tests recommended for use in determining discharge permit compliance
in the NPDES program are multiconcentration, or definitive, tests which provide
(1) a point estimate of effluent toxicity in terms of an IC25, IC50, or LC50, or
(2) a no-observed-effect-concentration (NOEC) defined in terms of mortality,
growth, reproduction, and/or teratogenicity and obtained by hypothesis testing.
The tests may be static renewal or static non-renewal. i
8.10.2 The tests consist of a control and a minimum of five;effluent
concentrations commonly selected to approximate a geometric series, such as 100%,
50%, 25%, 12.5%, and 6.25%, using a > 0.5 dilution series.
8.10.3 These tests are also to be used in determining compliance with permit
limits on the mortality of the receiving water concentration (RWC) of effluents
by bracketing the RWC with effluent concentrations in the following manner: (1)
100% effluent, (2) [RWC + 100J/2, (3) RWC, (4) RWC/2, and (5) RWC/4. For
45
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example, where the RWC = 50%, the effluent concentrations used in the test would
be 100%, 75%, 50%, 25%, and 12.5%.
8.10.4 If acute/chronic ratios are to be determined by simultaneous acute and
short-term chronic tests with a single species, using the same sample, both types
of tests must use the same test conditions, i.e., pH, temperature, water
hardness, salinity, etc.
8.11 RECEIVING WATER TESTS
8.11.1 Receiving water toxicity tests generally consist of 100% receiving water
and a control. The total salinity of the control should be comparable to the
receiving water.
8.11.2 The data from the two treatments are analyzed by hypothesis testing to
determine if test organism survival in the receiving water differs significantly
from the control. Four replicates and 10 organisms per replicate are required
for each treatment (see Summary of Test Conditions and Test Acceptability
Criteria in the specific test method).
8.11.3 In cases where the objective of the test is to estimate the degree of
toxicity of the receiving water, a definitive, multiconcentration test is
performed by preparing dilutions of the receiving water, using a > 0.5 dilution
series, with a suitable control water.
46
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SECTION 9
CHRONIC TOXICITY TEST ENDPOINTS AND DATA ANALYSIS
9.1 ENDPOINTS
9 1.1 The objective of chronic aquatic toxicity tests with effluents and pure
compounds is to estimate the highest "safe" or "no-effect concentration" of these
substances. For practical reasons, the responses observed in these tests are
usually limited to hatchability, gross morphological abnormalities, survival,
growth, and reproduction, and the results of the tests are usually expressed in
terms of the highest toxicant concentration that has no statistically significant
observed effect on these responses, when compared to the controls. The terms
currently used to define the endpoints employed in the rapid, chronic and
sub-chronic toxicity tests have been derived from the terms previously used for
full life-cycle tests. As shorter chronic tests were developed, it became common
practice to apply the same terminology to the endpoints. The terms used in this
manual are as follows:
9.1.1.1 Safe Concentration - The highest concentration of toxicant that will
permit normal propagation of fish and other aquatic life in receiving waters.
The concept of a "safe concentration" is a biological concept, whereas the
"no-observed-effect concentration" (below) is a statistically defined
concentration.
9.1.1.2 No-Observed-Effect-Concentration (NOEC) - The highest concentration of
toxicant to which organisms are exposed in a full life-cycle or partial life-
cycle (short-term) test, that causes no observable adverse effects on the test
organisms (i.e., the highest concentration of toxicant in which the values for
the observed responses are not statistically significantly different from the
controls). This value is used, along with other factors, to determine toxicity
limits in permits.
9.1.1.3 Lowest-Observed-Effect-Concentration (LOEC) - The "lowest concentration
of toxicant to which organisms are exposed in a life-cycle or partial life-cycle
(short-term) test, which causes adverse effects on the test organisms (i.e.,
where the values for the observed responses are statistically significantly
different from the controls).
9.1.1.4 Effective Concentration (EC) - A point estimate of the toxicant
concentration that would cause an observable adverse affect on a quanta!, "all or
nothing," response (such as death, immobilization, or serious incapacitation) in
a given percent of the test organisms, calculated by point estimation techniques.
If the observable effect is death or immobility, the term, Lethal Concentration
(LC), should be used (see Subsection 9.1.1.5). A certain EC or LC value might be
judged from a biological standpoint to represent a threshold concentration, or
lowest concentration that would cause an adverse effect on |:he observed response.
9.1.1.5 Lethal Concentration (LC) - The toxicant concentration that would cause
death in a given percent of the test population. Identical to EC when the
47 !
-------
observable adverse effect is death. For example, the LC50 is the concentration
of toxicant that would cause death in 50% of the test population.
9.1.1.6 Inhibition Concentration (1C) - The toxicant concentration that would
cause a given percent reduction in a nonquantal biological measurement for the
test population. For example, the IC25 is the concentration of toxicant that
would cause a 25% reduction in mean young per female or in growth for the test
population, and the IC50 is the concentration of toxicant that would cause a 50%
reduction in the mean population responses.
9.2 RELATIONSHIP BETWEEN ENDPOINTS DETERMINED BY HYPOTHESIS TESTING AND POINT
ESTIMATION TECHNIQUES
9.2.1 If the objective of chronic aquatic toxicity tests with effluents and pure
compounds is to estimate the highest "safe or no-effect concentration" of these
substances, it is imperative to understand how the statistical endpoints of these
tests are related to the "safe" or "no-effect" concentration. NOECs and LOECs
are determined by hypothesis testing (Dunnett's Test, a t test with the
Bonferroni adjustment, Steel's Many-One Rank Test, or the Wilcoxon Rank Sum Test
with Bonferroni adjustment), whereas LCs, ICs, and ECs are determined by point
estimation techniques (Probit Analysis, the Spearman-Karber Method, the Trimmed
Spearman-Karber Method, the Graphical Method or Linear Interpolation Method).
There are inherent differences between the use of a NOEC or LOEC derived from
hypothesis testing to estimate a "safe" concentration, and the use of a LC, 1C,
EC, or other point estimates derived from curve fitting, interpolation, etc.
9.2.2 Most point estimates, such as the LC, 1C, or EC are derived from a
mathematical model that assumes a continuous dose-response relationship. By
definition, any LC, 1C, or EC value is an estimate of some amount of adverse
effect. Thus the assessment of a "safe" concentration must be made from a
biological standpoint rather than with a statistical test. In this instance, the
biologist must determine some amount of adverse effect that is deemed to be
"safe," in the sense that from a practical biological viewpoint it will not
affect the normal propagation of fish and other aquatic life in receiving waters.
9.2.3 The use of NOECs and LOECs, on the other hand, assumes either (1) a
continuous dose-response relationship, or (2) a non-continuous (threshold) model
of the dose-response relationship.
9.2.3.1 In the case of a continuous dose-response relationship, it is also
assumed that adverse effects that are not "statistically observable" are also not
important from a biological standpoint, since they are not pronounced enough to
test as statistically significant against some measure of the natural variability
of the responses.
9.2.3.2 In the case of non-continuous dose-response relationships, it is assumed
that there exists a true threshold, or concentration below which there is no
adverse effect on aquatic life, and above which there is an adverse effect. The
purpose of the statistical analysis in this case is to estimate as closely as
possible where that threshold lies.
48
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9.2.3.3 In either case, it is important to realize that the amount of adverse
effect that is statistically observable (LOEC) or not observable (NOEC) is highly
dependent on all aspects of the experimental design, such as the number of
concentrations of toxicant, number of replicates per concentration, number of
organisms per replicate, and use of randomization. Other factors that affect the
sensitivity of the test include the choice of statistical analysis, the choice of
an alpha level, and the amount of variability between responses at a given
concentration.
9.2.3.4 Where the assumption of a continuous dose-response relationship is made,
by definition some amount of adverse effect might be present! at the NOEC, but is
not great enough to be detected by hypothesis testing.
9.2.3.5 Where the assumption of a noncontinuous dose-response relationship is
made, the NOEC would indeed be an estimate of a "safe" or "rio-effect"
concentration if the amount of adverse effect that appears at the threshold is
great enough to test as statistically significantly different from the controls
in the face of all aspects of the experimental design mentioned above. If,
however, the amount of adverse effect at the threshold were not great enough to
test as statistically different, some amount of adverse effect might be present
at the NOEC. In any case, the estimate of the NOEC with hypothesis testing is
always dependent on the aspects of the experimental design mentioned above. For
this reason, the reporting and examination of some measure of the sensitivity of
the test (either the minimum significant difference -or the percent change from
the control that this minimum difference represents) is extremely important.
9.2.4 In summary, the assessment of a "safe" or "no-effect" concentration cannot
be made from the results of statistical analysis alone, unless (1) the
assumptions of a strict threshold model are accepted, and (!>) it is assumed that
the amount of adverse effect present at the threshold is statistically detectable
"by hypothesis testing. In this case, estimates obtained from a statistical
analysis are indeed estimates of a "no-effect" concentration. If the assumptions
are not deemed tenable, then estimates from a statistical analysis can only be
used in conjunction with an assessment from a biological standpoint of what
magnitude of adverse effect constitutes a "safe" concentration. In this
instance, a "safe" concentration is not necessarily a truly "no-effect"
concentration, but rather a concentration at which the effects are judged to be
of no biological significance.
9.2.5 A better-understanding of the relationship between endpoints derived by
hypothesis testing (NOECs) and point estimation techniques [LCs, ICs, and ECs)
would be very helpful in choosing methods of data analysis. Norberg-King (1991)
reported that the IC25s were comparable to the NOECs for 23 effluent and
reference toxicant data sets analyzed. The data sets included short-term chronic
toxicity tests for the sea urchin, Arbacia punctulata, the sheepshead minnow,
Cyprinodon van'egatus, and the red macroalga, Champia parvula. Birge et al.
(1985) reported that LCls derived from Probit Analyses of data from short-term
embryo-larval tests with reference toxicants were comparable to NOECs for several
organisms. Similarly, USEPA (1988d) reported that the IC25s were comparable to
the NOECs for a set of daphnia, Cen'odaphm'a dubia chronic tests with a single
reference toxicant. However, the scope of these comparisons was very limited,
and sufficient information is not yet available to establish an overall
49
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relationship between these two types of endpoints, especially when derived from
effluent toxicity test data.
9.3 PRECISION
9.3.1 HYPOTHESIS TESTS
9.3.1.1 When hypothesis tests are used to analyze toxicity test data, it is not
possible to express precision in terms of a commonly used statistic. The results
of the test are given in terms of two endpoints, the No-Observed-Effect
Concentration (NOEC) and the Lowest-Observed-Effect Concentration (LOEC). The
NOEC and LOEC are limited to the concentrations selected for the test. The width
of the NOEC-LOEC interval is a function of the dilution series, and differs
greatly depending on whether a dilution factor of 0.3 or 0.5 is used in the test
design. Therefore, USEPA recommends the use of the > 0.5 dilution factor (see
Section 4, Quality Assurance). It is not possible to place confidence limits on
the NOEC and LOEC derived from a given test, and it is difficult to quantify the
precision of the NOEC-LOEC endpoints between tests. If the data from a series of
tests performed with the same toxicant, toxicant concentrations, and test
species, were analyzed with hypothesis tests, precision could only be assessed by
a qualitative comparison of the NOEC-LOEC intervals, with the understanding that
maximum precision would be attained if all tests yielded the same NOEC-LOEC
interval. In practice, the precision of results of repetitive chronic tests is
considered acceptable if the NOECs vary by no more than one concentration
interval above or below a central tendency. Using these guidelines, the "normal"
range of NOECs from toxicity tests using a 0.5 dilution factor (two-fold
difference between adjacent concentrations), would be four-fold.
9.3.2 POINT ESTIMATION TECHNIQUES
9.3.2.1 Point estimation techniques have the advantage of providing a point
estimate of the toxicant concentration causing a given amount of adverse
(inhibiting) effect, the precision of which can be quantitatively assessed (1)
within tests by calculation of 95% confidence limits, and (2) across tests by
calculating a standard deviation and coefficient of variation.
9.4 DATA ANALYSIS
9.4.1 ROLE OF THE STATISTICIAN
9.4.1.1 The use of the statistical methods described in this manual for routine
data analysis does not require the assistance of a statistician. However, the
interpretation of the results of the analysis of the data from any of the
toxicity tests described in this manual can become problematic because of the
inherent variability and sometimes unavoidable anomalies in biological data. If
the data appear unusual in any way, or fail to meet the necessary assumptions, a
statistician should be consulted. Analysts who are not proficient in statistics
are strongly advised to seek the assistance of a statistician before selecting
the method of analysis and using any of the results.
9.4.1.2 The statistical methods recommended in this manual are not the only
possible methods of statistical analysis. Many other methods have been proposed
50
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and considered. Certainly there are other reasonable and defensible methods of
statistical analysis for this kind of toxicity data. Among alternative
hypothesis tests some, like Williams' Test, require additional assumptions, while
others, like the bootstrap methods, require computer-intensive computations.
Alternative point estimations approaches most probably would require the services
of a statistician to determine the appropriateness of the model (goodness of
fit), higher order linear or nonlinear models, confidence intervals for estimates
generated by inverse regression, etc. In addition, point estimation or
regression approaches would require the specification by biologists or
toxicologists of some low level of adverse effect that would be deemed acceptable
or safe. The statistical methods contained in this manual have been chosen
because they are (1) applicable to most of the different toxicity test data sets
for which they are recommended, (2) powerful statistical tests, (3) hopefully
"easily" understood by nonstatisticians, and (4) amenable to use without a
computer, if necessary. ;
9.4.2 PLOTTING THE DATA
9.4.2.1 The data should be plotted, both as a preliminary step to help detect
problems and unsuspected trends or patterns in the responses, and as an aid in
interpretation of the results. Further discussion and plotted sets of data are
included in the methods and the Appendices.
9.4.3 DATA TRANSFORMATIONS
9.4.3.1 Transformations of the data, (e.g., arc sine square root and logs), are
used where necessary to meet assumptions of the proposed analyses, such as the
requirement for normally distributed data.
9.4.4 INDEPENDENCE, RANDOMIZATION, AND OUTLIERS
i
9.4.4.1 Statistical independence among observations is a critical assumption in
all statistical analysis of toxicity data. One of the best ways to ensure
independence is to properly follow rigorous randomization procedures.
Randomization techniques should be employed at the start of the test, including
the randomization of the placement of test organisms in the test chambers and
randomization of the test chamber location within the array of chambers.
Discussions of statistical independence, outliers and randomization, and a sample
randomization scheme, are included in Appendix A. ;.
9.4.5 REPLICATION AND SENSITIVITY
•
9.4.5.1 The number of replicates employed for each toxicant concentration is an
important factor in determining the sensitivity of chronic toxicity tests. Test
sensitivity generally increases as the number of replicates is increased, but the
point of diminishing returns in sensitivity may be reached rather quickly. The
level of sensitivity required by a hypothesis test or the confidence interval for
a point estimate will determine the number of replicates, arid should be based on
the objectives for obtaining the toxicity data.
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9.4.5.2 In a statistical analysis of toxicity data, the choice of a particular
analysis and the ability to detect departures from the assumptions of the
analysis, such as the normal distribution of the data and homogeneity of
variance, is also dependent on the number of replicates. More than the minimum
number of replicates may be required in situations where it is imperative to
obtain optimal statistical results, such as with tests used in enforcement cases
or when it is not possible to repeat the tests. For example, when the data are
analyzed by hypothesis testing, the nonparametric alternatives cannot be used
unless there are at least four replicates at each toxicant concentration.
9.4.6 RECOMMENDED ALPHA LEVELS
9.4.6.1 The data analysis examples included in the manual specify an alpha level
of 0.01 for testing the assumptions of hypothesis tests and an alpha level of
0.05 for the hypothesis tests themselves. These levels are common and well
accepted levels for this type of analysis and are presented as a recommended
minimum significance level for toxicity data analysis.
9.5 CHOICE OF ANALYSIS
9.5.1 The recommended statistical analysis of most data from chronic toxicity
tests with aquatic organisms follows a decision process illustrated in the
flowchart in Figure 2. An initial decision is made to use point estimation
techniques (the Probit Analysis, the Spearman-Karber Method, the Trimmed
Spearman-Karber Method, the Graphical Method, or Linear Interpolation Method)
and/or to use hypothesis testing (Dunnett's Test, the t test with the Bonferroni
adjustment, Steel's Many-one Rank Test, or Wilcoxon Rank Sum Test with the
Bonferroni adjustment). NOTE: For the NPDES Permit Program, the point estimation
techniques are the preferred statistical methods in calculating end points for
effluent toxicity tests. If hypothesis testing is chosen, subsequent decisions
are made on the appropriate procedure for a given set of data, depending on the
results of tests of assumptions, as illustrated in the flowchart. A specific
flow chart is included in the analysis section for each test.
9.5.2 Since a single chronic toxicity test might yield information on more than
one parameter (such as survival, growth, and reproduction), the lowest estimate
of a "no-observed-effect concentration" for any of the responses.would be used as
the "no-observed-effect concentration" for each test. It follows logically that
in the statistical analysis of the data, concentrations that had a significant
toxic effect on one of the observed responses would not be subsequently tested
for an effect on some other response. This is one reason for excluding
concentrations that have shown a statistically significant reduction in survival
from a subsequent hypothesis test for effects on another parameter such as
reproduction. A second reason is that the exclusion of such concentrations
usually results in a more powerful and appropriate statistical analysis. In
performing the point estimation techniques recommended in this manual, an all-
data approach is used. For example, data from concentrations above the NOEC for
survival are included in determining ICp estimates using the Linear Interpolation
Method.
52
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DATA (SURVIVAL, GROWTH, REPRODUCTION, ETC.)
POINT
ESTIMATION
HYPOTHESIS TESTING
TRANSFORMATION?
ENDPOINT ESTIMATE
LC, EC, 1C
SHAPIRO-WILK-S TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
NO STATISTICAL ANALYSIS
RECOMMENDED
NO
4 OF! MORE
REPLICATES?
YESJ
EQUAL NUMBER OF
REPLICATES?
T-TESTWITH
BONFERRONI
ADJUSTMENT
EQUAL NUMBER OF
REPLICATES?
YES
T
NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONF:ERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 2. Flowchart for statistical analysis of test data.
53
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9.5.3 ANALYSIS OF GROWTH AND REPRODUCTION DATA
9.5.3.1 Growth data from the sheepshead minnow, Cyprinodon van'egatus, and
inland silverside, Menidia beryTlina, larval survival and growth tests, and the
mysid, Mysidopsis bahia, survival, growth, and fecundity test, are analyzed using
hypothesis testing according to the flowchart in Figure 2. The above mentioned
growth data may also be analyzed by generating a point estimate with the Linear
Interpolation Method. Data from effluent concentrations that have tested
significantly different from the control for survival are excluded from further
hypothesis tests concerning growth effects. Growth is defined as the change in
dry weight of the orginal number of test organisms when group weights are
obtained. When analyzing the data using point estimating techniques, data from
all concentrations are included in the analysis.
9.5.3.2 Fecundity data from the mysid, Mysidopsis bahia, test may be analyzed
using hypothesis testing after an arc sine transformation according to the
flowchart in Figure 2. The fecundity data from the mysid test may also be
analyzed by generating a point estimate with the Linear Interpolation Method.
9.5.3.3 Reproduction data from the red macroalga, Champia parvula, test are
analyzed using hypothesis testing as illustrated in Figure 2. The reproduction
data from the red macroalga test may also be analyzed by generating a point
estimate with the Linear Interpolation Method.
9.5.4 ANALYSIS OF THE SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA
9.5.4.1 Data from the sea urchin, Arbacia punctulata, fertilization test may be
analyzed by hypothesis testing after an arc sine transformation according to the
flowchart in Figure 2. The fertilization data from the sea urchin test may also
be analyzed by generating a point estimate with the Linear Interpolation Method.
9.5.5 ANALYSIS OF MORTALITY DATA
9.5.5.1 Mortality data are analyzed by Probit Analysis, if appropriate, or other
point estimation techniques, (i.e., the Spearman-Karber Method, the Trimmed
Spearman-Karber Method, or the Graphical Method) (see Appendices G-I) (see
discussion below). The mortality data can also be analyzed by hypothesis
testing, after an arc sine square root transformation (see Appendices B-F),
according to the flowchart in Figure 2.
9.6 HYPOTHESIS TESTS
9.6.1 DUNNETT'S PROCEDURE
9.6.1.1 Dunnett's Procedure is used to determine the NOEC. The procedure
consists of an analysis of variance (ANOVA) to determine the error term, which is
then used in a multiple comparison procedure for comparing each of the treatment
means with the control mean, in a series of paired tests (see Appendix C) Use
of Dunnett's Procedure requires at least three replicates per treatment to check
the assumptions of the test. In cases where the numbers of data points
(replicates) for each concentration are not equal, a t test may be performed with
54
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Bonferroni's adjustment for multiple comparisons (see Appendix D), instead of
using Dunnett's Procedure.
9.6.1.2 The assumptions upon which the use of Dunnett's Procedure is contingent
are that the observations within.treatments are normally distributed, with
homogeneity of variance. Before analyzing the data, these assumptions must be
tested using the procedures provided in Appendix B.
9.6.1.3 If, after suitable transformations have been carried out, the normality
assumptions have not been met, Steel's Many-one Rank Test should be used if there
are four or more data points (replicates) per toxicant concentration. If the
numbers of data points for each toxicant concentration are not equal, the
Wilcoxon Rank Sum Test with Bonferroni's adjustment should be used (see
Appendix F).
9.6.1.4 Some indication of the sensitivity of the analysis should be provided by
calculating (1) the minimum difference between means that can be detected as
statistically significant, and (2) the percent change from the control mean that
this minimum difference represents for a given test.
9.6.1.5 A step-by-step example of the use of Dunnett's Procedure is provided in
Appendix C.
9.6.2 T TEST WITH THE BONFERRONI ADJUSTMENT
9.6.2.1 The t test with the Bonferroni adjustment is used as an alternative to
Dunnett's Procedure when the number of replicates is not the same for all
concentrations. This test sets an upper bound of alpha on the overall error
rate, in contrast to Dunnett's Procedure, for which the overall error rate is
fixed at alpha. Thus, Dunnett's Procedure is a more powerful test.
9.6.2.2 The assumptions upon which the use of the t test with the Bonferroni
adjustment is contingent are that the observations within treatments are normally
distributed, with homogeneity of variance. These assumptions must be tested
using the procedures provided in Appendix B. j
9.6.2.3 The estimate of the safe concentration derived from this test is
reported in terms of the NOEC. A step-by-step example of the use of a t-test
with the Bonferroni adjustment is provided in Appendix D.
9.6.3 STEEL'S MANY-ONE RANK TEST
9.6.3.1 Steel's Many-one Rank Test is a multiple comparison procedure for
comparing several treatments with a control. This method is similar to Dunnett's
procedure, except that it is not necessary to meet the assumption of normality.
The data are ranked, and the analysis is performed on the ranks rather than on
the data themselves. If the data are normally or nearly normally distributed,
Dunnett's Procedure would be more sensitive (would detect smaller differences
between the treatments and control). For data that are not normally distributed,
Steel's Many-one Rank Test can be much more efficient (Hodges and Lehmann, 1956).
55
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9.6.3.2 It is necessary to have at least four replicates per toxicant
concentration to use Steel's test. Unlike Dunnett's procedure, the sensitivity
of this test cannot be stated in terms of the minimum difference between
treatment means and the control mean that can be detected as statistically
significant.
9.6.3.3 The estimate of the safe concentration is reported as the NOEC A
step-by-step example of the use of Steel's Many-One Rank Test is provided in
Appendix E.
9.6.4 WILCOXON RANK SUM TEST WITH THE BONFERRONI ADJUSTMENT
9.6.4.1 The Wilcoxon Rank Sum Test is a nonparametric test for comparing a
treatment with a control. The data are ranked and the analysis proceeds exactly
as in Steel's Test except that Bonferroni's adjustment for multiple comparisons
is used instead of Steel's tables. When Steel's test can be used (i.e., when
there are equal numbers of data points per toxicant concentration), it will be
more powerful (able to detect smaller differences as statistically significant)
than the Wilcoxon Rank Sum Test with Bonferroni's-adjustment.
9.6.4.2 The estimate of the safe concentration is reported as the NOEC. A
step-by-step example of the use of the Wilcoxon Rank Sum Test with Bonferroni
adjustment is provided in Appendix F.
9.6.5 A CAUTION IN THE USE OF HYPOTHESIS TESTING
9.6.5.1 If in the calculation of an NOEC by hypothesis testing, two tested
concentrations cause statistically significant adverse effects, but an
intermediate concentration did not cause statistically significant effects, the
results should be used with extreme caution.
9.7 POINT ESTIMATION TECHNIQUES
9.7.1 PROBIT ANALYSIS
9.7.1.1 Probit Analysis is used to estimate an LCI, LC50, EC1, or EC50 and the
associated 95% confidence interval. The analysis consists of adjusting the data
for mortality in the control, and then using a maximum likelihood technique to
estimate the parameters of the underlying log tolerance distribution, which is
assumed to have a particular shape.
9.7.1.2 The assumption upon which the use of Probit Analysis is contingent is a
normal distribution of log tolerances. If the normality assumption is not met,
and at least two partial mortalities are not obtained, Probit Analysis should not
be used. It is important to check the results of Probit Analysis to determine if
use of the analysis is appropriate. The chi-square test for heterogeneity
provides a good test of appropriateness of the analysis. The computer program
(see discussion, Appendix H) checks the chi-square statistic calculated for the
data set against the tabular value, and provides an error message if the
calculated value exceeds the tabular value.
56
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9.7.1.3 A discussion of Probit Analysis, and examples of computer program input
and output, are found in Appendix H.
9.7.1.4 In cases where Probit Analysis is not appropriate, the LC50 and
confidence interval may be estimated by the Spearman-Karber Method (Appendix I)
or the Trimmed Spearman-Karber Method (Appendix J). If a test results in 100%
survival and 100% mortality in adjacent treatments (all or nothing effect), the
LC50 may be estimated using the Graphical Method (Appendix K).
9.7.2 LINEAR INTERPOLATION METHOD
9.7.2.1 The Linear Interpolation Method (see Appendix L) is a procedure to
calculate a point estimate of the effluent or other toxicant concentration
[Inhibition Concentration, (1C)] that causes a given percent reduction (e.g.,
25%, 50%, etc.) in the reproduction, growth, fertilization, or fecundity of the
test organisms. The procedure was designed for general applicability in the
analysis of data from short-term chronic toxicity tests.
.
9.7.2.2 Use of the Linear Interpolation Method is based on the assumptions that
the responses (1) are monotonically non-increasing (the mean response for each
higher concentration is less than or equal to the mean response for the previous
concentration), (2) follow a piece-wise linear response function, and (3) are
from a random, independent, and representative sample of test data. The
assumption for piece-wise linear response cannot be tested statistically, and no
defined statistical procedure is provided to test the assumption for
monotonicity. Where the observed means are not strictly monotonic by
examination, they are adjusted by smoothing. In cases where the responses at the
low toxicant concentrations are much higher than in the controls, the smoothing
process may result in a large upward adjustment in the control mean.
j
9.7.2.3 The inability to test the monotonicity and piece wise linear response
assumptions for this method makes it difficult to assess when the method is, or
is not, producing reliable results. Therefore, the method should be used with
caution when the results of a toxicity test approach an "all or nothing" response
from one concentration to the next in the concentration series, and when it
appears that there is a large deviation from monotonicity. See Appendix L for a
more detailed discussion of the use of this method and a computer program
available'for performing calculations. j
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SECTION 10
REPORT PREPARATION
The toxicity data are reported, together with other appropriate data.
following general format and content are recommended for the report:
The
10.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body
5. Contract Laboratory (if the test was performed under contract)
a. Name of firm
b. Phone number
c. Address
10.2 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 (MGD, CFS, GPM)
8. Design flow of treatment facility at time of sampling
10.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Eff1uent Samples
a. Sampling point
Collection dates and times
Sample collection method
Physical and chemical data
Mean daily discharge on sample collection date
Lapsed time from sample collection to delivery
Sample temperature when received at the laboratory
Receiving Water Samples
a. Sampling point
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
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3. Dilution Water Samples
a. Source
b. Collection date and time
c. Pretreatment
d. Physical and chemical characteristics
10.4 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 of volume and test chambers
7. Volume of solution used per chamber
8. Number of organisms used 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)
10.5 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
10.6 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, IC2:5 and/or IC50)
5. Physical and chemical methods used
10.7 RESULTS
1. Provide raw toxicity data in tabular form, including daily records of
affected organisms in each concentration (including controls), and plots of
toxicity data
2. Provide table of LC50s, NOECs, IC25, IC50, etc.
3. Indicate statistical methods to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
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10.8 CONCLUSIONS AND RECOMMENDATIONS
1. Relationship between test endpoints and permit limits.
2. Action to be taken.
60
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SECTION 11
TEST METHOD
SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS
LARVAL SURVIVAL AND GROWTH TEST
METHOD 1004.0
11.1 SCOPE AND APPLICATION
11.1.1 This method, adapted in part from USEPA (1987b), estimates the chronic
toxicity of effluents and receiving waters to the sheepshead minnow,
Cypn'nodon van'egatus, using newly hatched larvae in a seven-day,
static-renewal test. The effects include the synergistic, antagonistic, and
additive effects of all the chemical, physical, and biological components
which adversely affect the physiological and biochemical functions of the test
species. i
11.1.2 Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LC50s).
11.1.3 Detection limits of the toxicity of an effluent or chemical are
organism dependent.
11.1.4 Brief excursions in toxicity may not be detected using 24-h composite
samples. Also, because of the long sample collection period involved in
composite sampling, and because the test chambers are not sealed, highly
volatile and highly degradable toxicants present in the source may not be
detected in the test.
i
11.1.5 This method is commonly used in one of two forms: (1) a definitive
test, consisting of a minimum of five effluent concentration:; and a control,
and (2) a receiving water test(s), consisting of one or more receiving water
concentrations and a control.
11.2 SUMMARY OF METHOD
11.2.1 Sheepshead minnow, Cypn'nodon van'egatus, larvae (preferably less than
24-h old) are exposed in a static renewal system for seven days to different
concentrations of effluent or to receiving water. Test results are based on
the survival and weight of the larvae.
i
11.3 INTERFERENCES
11.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
11.3.2 Adverse effects of low dissolved oxygen concentrations (DO), high
concentrations of suspended and/or dissolved solids, and extremes of pH, may
mask the effects of toxic substances.
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11.3.3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
11.3.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
11.3.5 Food added during the test may sequester metals and other toxic
substances and reduce the apparent toxicity of the test substance. However,
in a growth test the nutritional needs of the organisms must be satisfied,
even if feeding has the potential to confound test results.
11.4 SAFETY
11.4.1 See Section 3, Health and Safety.
11.5 APPARATUS AND EQUIPMENT
11.5.1 Facilities for holding and acclimating test organisms.
11.5.2 Brine shrimp, Artemia, culture unit -- see Subsection 11.6.14 below
and Section 4, Quality Assurance.
11.5.3 Sheepshead minnow culture unit -- see Subsection 11.6.15 below. The
maximum number of larvae required per test will range from a maximum of 360,
if 15 larvae are used in each of four replicates, to a minimum of 180 per
test, if 10 larvae are used in each of three replicates. It is preferable to
obtain the test organisms from an in-house culture unit. If it is not
feasible to culture fish in-house, embryos or newly hatched larvae can be
obtained from other sources if shipped in well oxygenated saline water in
insulated containers.
11.5.4 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.
11.5.5 Environmental chamber or equivalent facility with temperature control
(25 ± 1°C).
11.5.6 Water purification system -- Millipore Milli-Q®, deionized water (DI)
or equivalent.
11.5.7 Balance -- Analytical, capable of accurately weighing to 0.00001 g.
11.5.8 Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of the weighing pans and the
expected weights of the pans plus fish.
11.5.9 Drying oven -- 50-105°C range, for drying larvae.
11.5.10 Air pump -- for oil-free air supply.
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11.5.11 Air lines, and air stones -- for aerating water containing embryos or
larvae, or for supplying air to test solutions with low DO.
11.5.12 Meters, pH and DO -- for routine physical and chemical measurements.
11.5.13 Standard or micro-Winkler apparatus -- for determining DO (optional).
11.5.14 Dissecting microscope -- for checking embryo viability.
11.5.15 Desiccator -- for holding dried larvae.
11.5.16 Light box -- for counting and observing larvae.
11.5.17 Refractometer -- for determining salinity.
11.5.18 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.
11.5.19 Thermometers, bulb-thermograph or electronic-chart type'-- for
continuously recording temperature.
11.5.20 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers..
I
11.5.21 Test chambers -- four (minimum of three) for each concentration and
control. Borosilicate glass 1000 ml beakers or modified Norberg and Mount
(1985) glass chambers used in the short-term inland silverside test may be
used. It is recommended that each chamber contain a minimum of 50 ml/larvae
and allow adequate depth of test solution (5.0 cm). To avoid potential
contamination from the air and excessive evaporation of test solutions during
the test, the chambers should be covered with safety glass plates or sheet
plastic (6 mm thick).
11.5.22 Beakers -- six Class A, borosilicate glass or non-toxic plasticware,
1000 ml for making test solutions.
11.5.23 Wash bottles -- for deionized water, for washing embryos from
substrates and containers, and for rinsing small glassware and instrument
electrodes and probes. i
I
11.5.24 Crystallization dishes, beakers, culture dishes (1 L), or equivalent
-- for incubating embryos.
11.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
11.5.26 Separatory funnels, 2-L -- two to four for cultuiring Artemia nauplii.
11.5.27 Pipets, volumetric — Class A, 1-100 ml. j
11.5.28 Pipets, automatic -- adjustable, 1-100 ml.
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11.5.29 Pipets, serological -- 1-10 ml, graduated.
11.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent.
11.5.31 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for
transferring larvae.
11.5.32 Siphon with bulb and clamp -- for cleaning test chambers.
11.5.33 Forceps -- for transferring dead larvae to weighing boats.
11.5.34 NITEX® or stainless steel mesh sieves (< 150 /zm, 500 A*ITI, 3 to 5 mm)
-- for collecting Artemia nauplii and fish embryos, and for spawning baskets,
respectively. (Nitex® is available from Sterling Marine Products, 18 Label
Street, Montclair, NJ 07042; 201-783-9800).
11.6 REAGENTS AND CONSUMABLE MATERIALS
11.6.1 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
11.6.2 Data sheets (one set per test) -- for data recording.
11.6.3 Vials, marked -- 18-24 per test, containing 4% formalin or 70%
ethanol, to preserve larvae (optional).
11.6.4 Weighing pans, aluminum -- 18-24 per test.
11.6.5 Tape, colored -- for labelling test chambers.
11.6.6 Markers, waterproof -- for marking containers, etc.
11.6.7 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) -- for standards and calibration check (see USEPA Method 150.1,
USEPA, 1979b).
11.6.8 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA, 1979b), or reagents -- for modified Winkler analysis.
11.6.9 Laboratory quality control samples and standards -- for calibration of
the above methods.
11.6.10 Reference toxicant solutions -- see Section 4, Quality Assurance.
11.6.11 Ethanol (70%) or formalin (4%) -- for use as a preservative for the
fish larvae.
11.6.12 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms.
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11.6.13 Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
11.6.13.1 Saline test and dilution water -- The salinity of the test water
must be in the range of 20 to 32%o. The salinity should vary by no more than
± 2%o among the chambers on a given day. If effluent and receiving water
tests are conducted concurrently, the salinities of these tests should be
similar. This test is not recommended for salinities less than 20%o.
I
11.6.13.2 The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts. Exposure of sheepshead minnow larvae to these effluents
will require adjustments in the salinity of the test solutions. It is
important to maintain a constant salinity across all treatments. 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.
11.6.13.3 Hypersaline brine (HSB): (HSB) has several advantages that make it
desirable for use in toxicity testing. It can be made from any high quality,
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity. HSB derived from natural seawater
contains the necessary trace metals, biogenic colloids, and some of the
microbial components necessary for adequate growth, survival, and/or
reproduction of marine and estuarine organisms, and may be stored for
prolonged periods without any apparent degradation. However, if 100%o HSB is
used as a diluent, the maximum concentration of effluent that can be tested
will be 80% at 20%o salinity and 70% at 30%o salinity.
11.6.13.3.1 The ideal container for making brine from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a non-corrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine. If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that would contaminate the brine. One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass. If aeration is used, use only oil-free air compressors to
prevent contamination. |
i
11.6.13.3.2 Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be in direct contact with the brine. A good quality biodegradable detergent
should be used, followed by several (at least three) thorough deionized water
rinses.
11.6.13.3.3 High quality (and preferably high salinity) seawater should be
filtered to at least 10 urn before placing into the brine generator. Water should
be collected on an incoming tide to minimize the possibility of contamination.
65
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11.6.13.3.4 The temperature of the seawater is increased slowly to 40°C. The
water should be aerated to prevent temperature stratification and to increase
water evaporation. The brine should be checked daily (depending on volume being
generated) to ensure that the salinity does not exceed 100%o and that the
temperature does not exceed 40°C. Additional seawater may be added to the brine
to obtain the volume of brine required.
11.6.13.3.5 After the required salinity is attained, the HSB should be filtered
a second time through a 1 //m filter and poured directly into portable containers
(20-L cubitainers or polycarbonate water cooler jugs are suitable). The
containers should be capped and labelled with the date the HSB was generated and
its salinity. Containers of HSB should be stored in the dark and maintained at
room temperature until used.
11.6.13.3.6 If a source of HSB is available, test solutions can be made by
following the directions below. Thoroughly mix together the deionized water and
HSB before adding the effluent.
11.6.13.3.7 Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine. For example, if the
salinity of the brine is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0. The proportion of brine is 1 part in 5 (one part brine to
four parts deionized water). To make 1 L of seawater at 20%o salinity from a HSB
of 100%o, divide 1 L (1000 mL) by 5.0. The result, 200 ml, is the quantity of
brine needed to make 1 L of seawater. The difference, 800 ml, is the quantity of
deionized water required.
11.6.13.4 Artificial sea salts: FORTY FATHOMS® brand sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-321-1189) have
been used successfully at the EMSL-Cincinnati to maintain and spawn sheephead
minnows and perform the larval survival and growth test (see Section 7, Dilution
Water). HW MARINEMIX® (Hawaiian Marine Imports, Inc., P.O. Box 218687, Houston,
TX 77218; 713-492-7864) sea salts have been used successfully at the USEPA Region
6 Houston Laboratory to maintain and spawn sheephead minnows and perform the
larval growth and survival test and the embryo-larval survival and teratogenicity
test. In addition, a slightly modified version of the GP2 medium (Spotte et a!.,
1984) has been successfully used to perform the sheepshead minnow survival and
growth test (Table 1). Artifical sea salts may be used for culturing sheepshead
minnows and for the larval survival and growth test if the criteria for
acceptability of test data are satisfied (see Subsection 11.12).
11.6.13.4.1 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 artificial sea
salts is conditioned (Spotte, 1973; Spotte et a!., 1984; Bower, 1983) before it
is used for culturing or testing. After adding the water, place an air stone in
the container, cover, and aerate the solution mildly for 24 h before use.
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11.6.13.4.2 The GP2 reagent grade chemicals (Table 1) 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 NaHC03 in 500 ml of
deionized water. Add 2.5 ml of this stock solution for each liter of the GP2
artificial seawater.
TABLE 1. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
ARTIFICIAL SEAWATER FOR THE SHEEPSHEAD MINNOW, CYPRINODON
VARIEGATUS, TOXICITY TEST1'2'3
Compound Concentration
NaCl 21.03
Na2S04 3.52
KC1 0.61
KBr 0.088
Na2B407 • 10 H20 0.034
MgCl2 • 6 H20 9.50
CaCl2 • 2 H20 1.32
SrCl2 • 6 H20 0.02
NaHC03 0.17
Amount (g)
Required for
20 L
420.6
70.4 .
12.2
1.76
0.68
190.0
26.4
0.400
3.40
1 Modified GP2 from Spotte et al. (1984). ,
2 The constituent salts and concentrations were taken from USEPA (1990b).
The salinity is 30.89 g/L. !
GP2 can be diluted with deionized (DI) water to the desired test
salinity. ;
11.6.14 BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test
organisms
11.6.14.1 Newly-hatched Artemia nauplii (see USEPA, 1993a) are used as food
for sheepshead minnow larvae in toxicity tests and in the maintenance of
continuous stock cultures. Although there are many commercial sources of
brine shrimp cysts, the Brazilian or Colombian strains are currently preferred
because the supplies examined have had low concentrations of chemical residues
and produce nauplii of suitably small size. For commercial sources of brine
shrimp, Artemia, cysts, see Table 2 of Section 5, Facilities, Equipment, and
Supplies and Section 4, Quality Assurance, Subsection 4.8.
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11.6.14.2 Each new batch of Artemia cysts must be evaluated for size
(Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al., 1980) and nutritional
suitability (Leger et al., 1985, and Leger et al., 1986) against known
suitable reference cysts by performing a side-by-side larval growth test using
the "new" and "reference" cysts. The "reference" cysts used in the
suitability test may be a previously tested and acceptable batch of cysts, or
may be obtained from the Quality Assurance Research Division, Environmental
Monitoring Systems Laboratory, Cincinnati, OH 45268; 513-569-7325. A sample
of newly-hatched Artemia nauplii from each new batch of cysts should be
chemically analyzed. The Artemia cysts should not be used if the
concentration of total organochlorine pesticides exceeds 0.15 /zg/g wet weight
or the total concentration of organochlorine pesticides plus PCBs exceeds
0.30 ng/g wet weight. (For analytical methods see USEPA, 1982.)
11.6.14.3 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or a solution prepared by adding 35.0 g uniodized
salt (NaCl) or artificial sea salts to 1 L of deionized water, to a 2-L
separatory funnel, or equivalent.
2. Add 10 ml Artemia cysts to the separatory funnel and aerate for 24 h at
27°C. (Hatching time varies with incubation temperature and the
geographic strain of Artemia used (USEPA, 1985a; USEPA, 1993a; ASTM,
1993).
3. After 24 h, cut off the air supply in the separatory funnel. Artemia
nauplii are phototactic, and will concentrate at the bottom of the funnel
if it is covered for 5-10 minutes. To prevent mortality, do not leave .
the concentrated nauplii at the bottom of the funnel more than 10 min
without aeration.
4. Drain the nauplii into a beaker or funnel fitted with a < 150 ^m NITEX®
or stainless steel screen, and rinse with seawater or equivalent before
use.
11.6.14.4 Testing Artemia nauplii as food for toxicity test organisms.
11.6.14.4.1 The primary criterion for acceptability of each new supply of
brine shrimp cysts is the ability of the.nauplii to support good survival and
growth of the sheepshead minnow larvae (see Subsection 11.12). The larvae
used to evaluate the suitability of the brine shrimp nauplii must be of the
same geographical origin, species, and stage of development as those used
routinely in the toxicity tests. Sufficient data to detect differences in
survival and growth should be obtained by using three replicate test vessels,
each containing a minimum of 15 larvae, for each type of food.
11.6.14.4.2 The feeding rate and frequency, test vessels, volume of control
water, duration of the test, and age of the nauplii at the start of the test,
should be the same as used for the routine toxicity tests.
"new"
11.6.14.4.3 Results of the brine shrimp nutrition assay, where there are only
two treatments, can be evaluated statistically by use of a t test. The
food is acceptable if there are no statistically significant differences in
the survival and growth of the larvae fed the two sources of nauplii.
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11.6.15 TEST ORGANISMS, SHEEPSHEAD MINNOWS, CYPRINODON VARIEGATUS
11.6.15.1 Brood Stock i
11.6.15.1.1 Adult sheepshead minnows for use as brood stock may be obtained
by seine in Gulf of Mexico and Atlantic coast estuaries, from commercial
sources, or from young fish raised to maturity in the laboratory. Feral brood
stocks and first generation laboratory fish are preferred, to minimize
inbreeding.
11.6.15.1.2 To detect disease and to allow time for acute mortality due to
the stress of capture, field-caught adults are observed in the laboratory a
minimum of two weeks before using as a source of gametes. Injured or diseased
fish are discarded.
i
11.6.15.1.3 Sheepshead minnows can be continuously cultured in the laboratory
from eggs to adults. The larvae, juvenile, and adult fish should be kept in
appropriate size rearing tanks, maintained at ambient laboratory temperature.
The larvae should be fed sufficient newly-hatched Artemia naitplii daily to
assure that live nauplii are always present. Juveniles are fed frozen adult
brine shrimp and a commercial flake food, such as TETRA SM-80®, available from
Tetra Sales (U.S.A.), 201 Tabor Rd, Morris Plains, NJ 07950; 800-526-0650, or
MARDEL AQUARIAN® Tropical Fish Flakes, available from Mardel Laboratories,
Inc., 1958 Brandon Court, Glendale Heights, IL 60139; 312-351-0606, or
equivalent. Adult fish (age one month) are fed flake food three or four times
daily, supplemented with frozen adult brine shrimp.
11.6.15.1.3.1 Sheepshead minnows reach sexual maturity in three-to-five
months after hatch, and have an average standard length of approximately 27 mm
for females and 34 mm for males. At this time, the males begin to exhibit
sexual dimorphism and initiate territorial behavior. When the fish reach
sexual maturity and are to be used for natural spawning, the temperature
should be controlled at 18-20°C.
.
11.6.15.1.4 Adults can be maintained in natural or artificial seawater in a
flow-through or recirculating, aerated system consisting of an all-glass
aquarium, or a "Living Stream" (Figid Unit, Inc., 3214 Sylvania Ave, Toledo,
OH 43613; 419-474-6971), or equivalent.
•
11.6.15.1.5 The system is equipped with an undergravel or outside biological
filter of shells Spotte (1973) or Bower (1983) for conditioning the biological
filter), or a cartridge filter, such as a MAGNUM® Filter, available from
Carolina Biological Supply Co., Burlington, NC 27215; 800-334-5551, or an
EHEIM® Filter, available from Hawaiian Marine Imports Inc., P.O. Box 218687,
Houston, TX 77218; 713-492-7864, or equivalent, at a salinity of 20-30%o and a
photoperiod of 16 h light/8 h dark.
11.6.15.2 Obtaining Embryos for Toxicity Tests (See USEPA, 1978)
i
11.6.15.2.1 Embryos can be shipped to the laboratory from an outside source
or obtained from adults held in the laboratory. Ripe eggs can be obtained
either by natural spawning or by intraperitoneal. injection of the females with
69
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human chorionic gonadotrophin (HCG) hormone, available from United States
Biochemical Corporation, Cleveland, OH 44128; 216-765-5000. If the culturing
system for adults is temperature controlled, natural spawning can be induced.
Natural spawning is preferred because repeated spawnings can be obtained from
the same brood stock, whereas with hormone injection, the brood stock is
sacrificed in obtaining gametes.
11.6.15.2.2 It should be emphasized that the injection and hatching schedules
given below are to be used only as guidelines. Response to the hormone varies
from stock to stock and with temperature. Time to hatch and percent viable
hatch also vary among stocks and among batches of embryos obtained from the
same stock, and are dependent on temperature, DO, and salinity. The
coordination of spawning and hatching is further complicated by the fact that,
even under the most ideal conditions, embryos spawned over a 24-h period may
hatch over a 72-h period. Therefore, it is advisable (especially if natural
spawning is used) to obtain fertilized eggs over several days to ensure that a
sufficient number of newly hatched larvae (less than 24 h old) will be
available to initiate a test.
11.6.15.2.3 Forced Spawning
11.6.15.2.3.1 HCG is reconstituted with sterile saline or Ringer's solution
immediately before use. The standard HCG vial contains 1,000 IU to be
reconstituted in 10 mL of saline. Freeze-dried HCG which comes with
premeasured and sterilized saline is the easiest to use. Use of a 50 IU dose
requires injection of 0.05 ml of reconstituted hormone solution.
Reconstituted HCG may be used for several weeks if kept in the refrigerator.
11.6.15.2.3.2 Each female is injected intraperitoneally with 50 IU HCG on two
consecutive days, starting at least 10 days prior to the beginning of a test.
Two days following the second injection, eggs are stripped from the females
and mixed with sperm derived from excised macerated testes. At least ten
females and five males are used per test to ensure that there is a sufficient
number (400) of viable embryos.
11.6.15.2.3.3 HCG is injected into the peritoneal cavity, just below the
skin, using as small a needle as possible. A 50 IU dose is recommended for
females approximately 27 mm in standard length. A larger or smaller dose may
be used for fish which are significantly larger or smaller than 27 mm. With
injections made on days one and two, females which are held at 25°C should be
ready for stripping on days 4, 5, and 6. Ripe females should show pronounced
abdominal swelling, and release at least a few eggs in response to a gentle
squeeze. Injected females should be isolated from males. It may be helpful
if fish that are to be injected are maintained at 20°C before injection, and
the temperature raised to 25°C on the day of the first injection.
11.6.15.2.3.4 Prepare the testes immediately before stripping the eggs from
the females. Remove the testes from three-to-five males. The testes are
paired, dark grey organs along the dorsal midline of the abdominal cavity. If
the head of the male is cut off and pulled away from the rest of the fish,
most of the internal organs can be pulled out of the body cavity, leaving the
testes behind. The testes are placed in a few mL of seawater until the eggs
are ready.
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11.6.15.2.3.5 Strip the eggs from the females, into a dish containing 50-100
ml of seawater, by firmly squeezing the abdomen. Sacrifice the females and
remove the ovaries if all the ripe eggs do not flow out freely. Break up any
clumps of ripe eggs and remove clumps of ovarian tissue and underripe eggs.
Ripe eggs are spherical, approximately 1 mm in diameter, and almost clear.
11.6.15.2.3.6 While being held over the dish containing the eggs, the testes
are macerated in a fold of NITEX® screen (250-500 n\n mesh) dampened with
seawater. The testes are then rinsed with seawater to remove the sperm from
tissue, and the remaining sperm and testes are washed into the dish. Let the
eggs and milt stand together for 10-15 min, swirling occasionally.
11.6.15.2.3.7 Pour the contents of the dish into a beaker, and insert an
airstone. Aerate gently, such that the water moves slowly over the eggs, and
incubate at 25°C for 60-90 min. After incubation, wash the eggs on a NITEX®
screen and resuspend them in clean seawater. Examine the eggs period.ically
under a dissecting microscope until they are in the 2-8 cell stage. (The
stage at which it is easiest to tell the developing embryos from the abnormal
embryos and unfertilized eggs; see Figure 1). The eggs cam then be gently
rolled on a NITEX® screen and culled (see Section 6, Test Organisms).
I
11.6.15.2.4 Natural Spawning
11.6.15.2.4.1 Cultures of adult fish to be used for spawning are maintained
at 18-20°C until embryos are required. When embryos are required, raise the
temperature to 25°C in the morning, seven or eight days before the beginning
of a test. That afternoon, transfer the adult fish (generally, at least five
females and three males) to a spawning chamber (approximately, 20 x 35 x 22 cm
high; USEPA, 1978), which is a basket constructed of 3-5 mm NITEX® mesh, made
to fit a 57-L (15 gal) aquarium. Spawning generally will begin within 24 h or
less. Embryos will fall through the bottom of the basket and onto a
collecting screen (250-500 urn mesh) or tray below the basket. Allow the
embryos to collect for 24 h. Embryos are washed from the screen, checked for
viability, and placed in incubation dishes. Replace the screens until a
sufficient number of embryos have been collected. One-to-three spawning
aquaria can be used to collect the required number of embryos to run a
toxicity test. To help keep the embryos clean, the adults are fed while the
screens are removed.
11.6.15.2.5 Incubation \
11.6..15.2.5.1 Four hours post-fertilization, the embryos obtained by natural
or forced spawning are rolled gently with a finger on a 250-500 /m Nitex®
screen to remove excess fibers and tissue. The embryos have adhesive threads
and tend to adhere to each other. Gentle rolling on the screen facilitates
the culling process described below. To reduce fungal conrtamination of the
newly spawned embryos after they have been manipulated, they should be placed
in a 250 fan sieve and briskly sprayed with seawater from a squeeze bottle.
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Figure 1. Embryonic development of sheepshead minnow, Cypn'nodon
variegatus: A. Mature unfertilized egg, snowing attachment
filaments and micropyle, X33; B. Blastodisc fully developed;
C,D. Blastodisc, 8 cells; E. Blastoderm, 16 cells;
F. Blastoderm, late cleavage stage; G. Blastoderm with germ ring
formed, embryonic shield developing; H. Blastoderm covers over
3/4 of yolk, yolk noticeably constricted; I. Early embryo. From
Kuntz (1916).
72
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Figure 1. Embryonic development of sheepshead minnow, Cypn'nodon
variegatus: J. Embryo 48 h after fertilization, now segmented
throughout, pigment on yolk sac and body, otoliths formed;
K. Posterior portion of embryo free from yolk and moves freely
within egg membrane, 72 h after fertilization; L. Newly hatched
fish, actual length 4 mm; M. Larval fish 5 days after hatching,
actual length 5 mm; N. Young fish 9 mm in length; 0. Young fish
12 mm in length (CONTINUED). From Kuntz (1916).
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11.6.15.2.5.2 Under a dissecting microscope, separate and discard abnormal
embryos and unfertilized eggs. While they are checked, the embryos are
maintained in seawater at 25°C. The embryos should be in Stages C-6,
Figure 1.
11.6.15.2.5.3 If the test is prepared with four replicates of 15 larvae at
each of six treatments (five effluent concentrations and a control), and the
combined mortality of eggs and larvae prior to the start of the test is less
than 20%, approximately 400 viable embryos are required at this stage.
11.6.15.2.5.4 Embryos are demersal. They should be aerated and incubated at
25"C, at a salinity of 20-30%o and a 16-h photoperiod. The embryos can be
cultured in either a flow-through or static system, using aquaria or
crystallization dishes. However, if the embryos are cultured in dishes, it is
essential that aeration and daily water changes be provided, and the dishes be
covered to reduce evaporation that may cause increased salinity. One-half to
three-quarters of the seawater from the culture vessels can be poured off and
the incubating embryos retained. Embryos cultured in this manner should hatch
in six or seven days.
11.6.15.2.5.5 At 48 h post-fertilization, embryos are examined under a
microscope to determine development and survival. Embryos should be in Stages
I and J, Figure 1. Discard dead embryos. Approximately 360 viable embryos
are required at this stage.
11.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
11.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
11.8 CALIBRATION AND STANDARDIZATION
11.8.1 See Section 4, Quality Assurance.
11.9 QUALITY CONTROL
11.9.1 See Section 4, Quality Assurance.
11.10 TEST PROCEDURES
11.10.1 TEST SOLUTIONS
11.10.1.1 Receiving Waters
11.10.1.1.1 The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 urn NITEX® filter and compared without
dilution, against a control. Using four replicate chambers per test, each
containing 500-750 ml, and 400 ml for chemical analysis, would require
approximately 2.4-3.4 L or more of sample per test per day.
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11.10.1.2 Effluents
11.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used. A
dilution factor of 0.5 provides precision of ± 100%, and allows for testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25.0%, 50.0%, and 100%). Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used. Therefore, USEPA recommends the
use of the > 0.5 dilution factor. If 100%o HSB is used as a diluent, the
maximum concentration of effluent that can be tested will,be 80% at 20%o
salinity and 70% at 30%o salinity.
11.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first
l-to-2 h of the test, additional dilutions at the lower range of effluent
concentrations should be added.
11.10.1.2.3 The volume of effluent required to initiate the test and for
daily renewal of four replicates (minimum of three) per concentration for five
concentrations of effluent and a control, each containing 750 ml of test
solution, is approximately 5 L. Prepare enough test solution (approximately
3400 ml) at each effluent concentration to provide 400 ml additional volume
for chemical analyses (Table 2).
11.10.1.2.4 The salinity of effluent and receiving water tests for sheepshead
minnows should be between 20%o and 30%o. If concurrent effluent and receiving
water testing occurs, the effluent test salinity should closely approximate
that of the receiving water test. If an effluent is tested alone, select a
salinity between 20%o and 30%o, whichever comes closest to the salinity of the
receiving waters. Table 2 illustrates the quantities of effluent, artificial
sea salts, hypersaline brine, or seawater needed to prepare 3 L of test
solution at each treatment level for tests performed at 20%o salinity.
i
11.10.1.2.5 Just prior to test initiation (approximately 1 h), the
temperature of sufficient quantity of the sample to make the test solutions
should be adjusted to the test temperature (25 ± 1°C) and maintained at that
temperature during the addition of dilution water.
i
11.10.1.2.6 Higher effluent concentrations (i.e., 25%, 50%, and 100%) may
require aeration to maintain adequate dissolved oxygen concentrations.
However, if one solution is aerated, all concentrations must be aerated.
Aerate effluent as it warms and continue to gently aerate test solutions in
the test chambers for the duration of the test.
11.10.1.2.7 Effluent dilutions should be prepared for all replicates in each
treatment in one beaker to minimize variability among the replicates. The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.
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TABLE 2. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20%o, USING 20%o
SALINITY DILUTION WATER PREPARED FROM NATURAL SEAWATER,
HYPERSALINE BRINE, OR ARTIFICIAL SEA SALTS
Effluent
Solution
1
2
3
4
5
Control
Total
Effluent
Cone. (%)
1001'2
50
25
12.5
6.25
0.0
Solutions To
Volume of
Effluent Solution
6800 mL
3400 mL Solution 1
3400 mL Solution 2
3400 mL Solution 3
3400 mL Solution 4
Be Combined
Volume of Diluent
Seawater (20%o)
+ 3400 mL
+ 3400 mL
+ 3400 mL
+ 3400 mL
3400 mL
17000 mL
This illustration assumes: (1) the use of 750 mL of test solution in
each of four replicates and 400 mL for chemical analysis (total of
3,400 mL) for the control and each of five concentrations of effluent
(2) an effluent dilution factor of 0.5, and (3) the effluent lacks
appreciable salinity. A sufficient initial volume (6,800 mL) of
effluent is prepared by adjusting the salinity to the desired level.
In this example, the salinity is adjusted by adding artificial sea
salts to the 100% effluent, and preparing a serial dilution using 20%o
seawater (natural seawater, hypersaline brine, or artificial seawater).
Following addition of salts, the effluent is stirred for 1 h to ensure
that the salts have dissolved. The salinity of the initial 6,800 mL of
100% effluent is adjusted to 20%o by adding 136 g of dry artificial sea
salts (FORTY FATHOMS®). Test concentrations are then made by mixing
appropriate volumes of salinity-adjusted effluent and 20%o salinity
dilution water to provide 6,800 mL of solution for each concentration.
If hypersaline brine alone (100%o) is used to adjust the salinity of
the effluent, the highest concentration of effluent that could be
achieved would be 80% at 20%o salinity. When dry sea salts are used to
adjust the salinity of the effluent, it may be desirable to use a
salinity control prepared under the same conditions and used to
determine survival and growth.
The same procedures would be followed in preparing test concentrations
at other salinities between 20%o and 30%o: (1) the salinity of the bulk
(initial) effluent sample would be adjusted to the appropriate salinity
using artificial sea salts or hypersaline brine, and (2) the remaining
effluent concentrations would be prepared by serial dilution, using a
large batch (17,000 mL) of seawater for dilution water, which had been
prepared at the same salinity as the effluent, using natural seawater,
or hypersaline or artificial sea salts and deionized water.
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11.10.1.3 Dilution Water
11/10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving
water), MSB prepared from natural seawater, or artificial seawater prepared
from FORTY FATHOMS® or 6P2 sea salts (see Table 1 and Section 7, Dilution
Water). Other artificial sea salts may be used for culturing sheepshead
minnows and for the larval survival and growth test if the control criteria
for acceptability of test data are satisfied.
11.10.2 START OF THE TEST
I
11.10.2.1 Tests should begin as soon as possible, preferably within 24 h
after sample collection. The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
tests unless permission is granted by the permitting authority. In no case
should the sample be used in a test more than 72 h after sample collection
(see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and
Sample Preparation for Toxicity Tests).
11.10.2.2 If the embryos have been incubating at 25°C, 30%o salinity, and a
16-h photoperiod, for 5 to 6 days with aeration and daily water renewals,
approximately 24 h prior to hatching, the salinity of the seawater in the
incubation chamber may be reduced from 30%> to the test salinity, if lower
than 30%o. In addition to maintaining good water quality, reducing the
salinity and/or changing the water may also help to initiate hatching over the
next 24 h. A few larvae may hatch 24 h ahead of the majority. Remove these
larvae and reserve them in a separate dish, maintaining the same culture
conditions. It is preferable to use only the larvae that hatch in the 24 h
prior to starting the test. However, if sufficient numbers of larvae do not
hatch within the 24-h period, the larvae that hatch prior to 24 h are added to
the test organisms. The test organisms are then randomly selected for the
test. When eggs or larvae must be shipped to the test site from a remote
location, it may be necessary to use larvae older than 24-h because of the
difficulty in coordinating test organism shipments with field operations.
However, in the latter case, the larvae should not be more than 48-h old at
the start of the test and should all be within 24-h of the same age.
11.10.2.3 Label the test chambers with a marking pen. Use of color coded
tape to identify each treatment and replicate. A minimum of five effluent
concentrations and a control are used for each test. Each treatment
(including controls) should have four (minimum of three) replicates. For
exposure chambers, use 1000 ml beakers, non-toxic disposable plasticware, or
glass chambers with a sump area as illustrated in the inland silverside test
method (see Section 13).
i
11.10.2.4 Prepare the test solutions and add to the test chambers.
11.10.2.5 The test is started by randomly placing larvae from the common pool
into each test chamber until each chamber contains 15 (minimum of 10) larvae,
for a total of 60 larvae (minimum of 30) for each concentration (see
Appendix A). The amount of water added to the chambers when transferring the
larvae should be kept to a minimum to avoid unnecessary dilution of the test
concentrations.
77
-------
11.10.2.6 The chambers may be placed on a light table to facilitate counting
the larvae.
11.10.2.7 Randomize the position of the test chambers at the beginning of the
test (see Appendix A). Maintain the chambers in this configuration throughout
the test. Preparation of a position chart may be helpful.
11.10.3 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE
11.10.3.1 The light quality and intensity should be at ambient laboratory
levels, which is approximately 10-20 ME/m/s, or 50 to 100 foot candles
(ft-c), with a photoperiod of 16 h light and 8 h darkness. The water
temperature in the test chambers should be maintained at 25 ± 1°C. The test
salinity should be in the range of 20 to 30%o to accommodate receiving waters
that may fall within this range. Conduct of this test at salinities less than
20%o may cause an unacceptably low growth response and thereby invalidate the
test. The salinity should vary by no more than ± 2%o among the chambers on a
given day. If effluent and receiving water tests are conducted concurrently,
the salinities of these tests should be similar.
11.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
11.10.4.1 Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain a satisfactory DO. The DO should be
measured on new solutions at the start of the test (Day 0) and before daily
renewal of test solutions on subsequent days. The DO should not fall below
4.0 mg/L (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests). If it is necessary to
aerate, all treatments and the control should be aerated. The aeration rate
should not exceed 100 bubbles/min, using a pipet with a 1-2 mm orifice, such
as a 1-mL KIMAX® serological pipet No. 37033, or equivalent. Care should be
taken to ensure that turbulence resulting from aeration does not cause undue
stress on the fish.
11.10.5 FEEDING
11.10.5.1 Artemia nauplii are prepared as described above.
11.10.5.2 Sheepshead minnow larvae are fed newly-hatched (less than 24-h old)
Artemia nauplii once a day from hatch day 0 through day 6; larvae are not fed
on day 7. Feed 0.10 g nauplii per test chamber on days 0-2, and 0.15 g
nauplii per test chamber on days 3-6. Equal amounts of Artemia nauplii must
be added to each replicate test chamber to minimize the variability of larval
weight. Sufficient numbers of nauplii should be fed to ensure that some
remain alive overnight in the test chambers. An adequate but not excessive
amount should be provided to each replicate on a daily basis. Feeding
excessive amounts of nauplii will result in a depletion in DO to a lower than
acceptable level (below 4.0 mg/L). Siphon as much of the uneaten Artemia
nauplii as possible from each chamber daily to ensure that the larvae
principally eat newly hatched nauplii.
78
-------
11.10.5.3 On days 0-2, weigh 4 g wet weiight or pipette 4 ml of concentrated,
rinsed Artemia nauplii for a test with five treatments and a control.
Resuspend the 4 g Artemia in 80 mL of natural or artificial seawater .in a 100
ml beaker. Aerate or swirl Artemia to maintain a thoroughly mixed suspension
of nauplii. Dispense 2 ml Artemia suspension by pipette or adjustable syringe
to each test chamber. Collect only enough Artemia in the pipette or syringe
for one test chamber or settling of Artemia may occur, resulting in unequal
amounts of Artemia being distributed to the replicate test chambers.
11.10.5.4 On days 3-6, weigh 6 g wet weight or pipette 6 ml Artemia
suspension for a test with five treatments and a control. Resuspend the 6 g
Artemia in 80 ml of natural or artificial seawater in a 100 ml beaker. Aerate
or swirl as 2 ml is dispensed to each test chamber.
i
11.10.5.5 If the survival rate in any test replicate on any day falls below
50%, reduce the volume of Artemia added to that test chamber by one-half
(i.e., from 2 ml to 1 ml) and continue feeding one-half the volume through
day 6. Record the time of feeding on data sheets (Figure 2).
11.10.6 DAILY CLEANING OF TEST CHAMBERS
i
11.10.6.1 Before the daily renewal of test solutions, uneaten and dead
Artemia, dead fish larvae, and other debris are removed from the bottom of the
test chambers with a siphon hose. As much of the uneaten Artemia as possible
should be siphoned from each chamber to ensure that the larvae principally eat
newly hatched nauplii. Alternately, a large pipet (50 mL), fitted with a
safety pipet filler or rubber bulb, can be used. Because of their small size
during the first few days of the tests, larvae are easily drawn into the
siphon tube when cleaning the test chambers. By placing the test chambers on
a light box, inadvertent removal of live larvae can be greatly reduced because
they can be more easily seen. If the water siphoned from the test chambers is
collected in a white plastic tray, the live larvae caught in the siphon can be
retrieved and returned to the appropriate test chamber. Any incidence of
removal of live larvae from the test chambers by the siphon during cleaning,
and subsequent return to the chambers, should be noted in the test records.
11.10.7 OBSERVATIONS DURING THE TEST
11.10.7.1 Routine Chemical and Physical Determinations
I
11.10.7.1.1 DO is measured at the beginning and end of each 24-h exposure
period in one test chamber at each test concentration and in the control.
11.10.7.1.2 Temperature, pH, and salinity are measured at the end of each
24-h exposure period in one test chamber at each test concentration and in the
control. Temperature should also be monitored continuously, observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
vessels at least at the end of the test to determine the temperature variation
in the environmental chamber.
11.10.7.1.3 The pH is measured in the effluent sample each day.
I
79
-------
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81
-------
11.10.7.1.4 Record all the measurements on the data sheet (Figure 2).
11.10.7.2 Routine Biological Observations
11.10.7.2.1 The number of live larvae in each test chamber are recorded daily
(Figure 7), and the dead larvae are discarded.
11.10.7.2.2 Protect the larvae from unnecessary disturbance during the test
by carrying out the daily test observations, solution renewals, and removal of
dead larvae, carefully. Make sure the larvae remain immersed during the
performance of the above operations.
11.10.8 TEST SOLUTION RENEWAL
11.10.8.1 The test solutions are renewed daily using freshly prepared
solution, immediately after cleaning the test chambers. For on-site toxicity
studies, fresh effluent and receiving water samples used in toxicity tests
should be collected daily, and no more than 24 h should elapse between
collection of the sample and use in the test (see Section 8, Effluent and
Receiving Water Sampling, Sample Handling, and Sample Preparation for Toxicity
Tests). For off-site tests, a minimum of three samples must be collected,
preferably on days one, three, and five. Maintain the samples at 4°C until
used.
11.10.8.2 For test solution renewal, the water level in each chamber is
lowered to a depth of 7 to 10 mm, which leaves 15 to 20% of the test solution.
New test solution (750 mL) should be added slowly by pouring down the side of
the test chamber to avoid excessive turbulence and possible injury to the
larvae.
11.10.9 TERMINATION OF THE TEST
11.10.9.1 The test is terminated after 7-d of exposure. At test termination,
dead larvae are removed and discarded. The surviving larvae in each test
chamber (replicate) are counted and immediately prepared as a group for dry
weight determination, or are preserved as a group in 4% formalin or 70%
ethanol. Preserved organisms are dried and weighed within 7 days. For
safety, formalin should be used under a hood.
11.10.9.2 For immediate drying and weighing, siphon or pour live larvae onto
a 500 urn mesh screen in a large beaker to retain the larvae and allow Artemia
and debris to be rinsed away. Rinse the larvae with deionized water to wash
away salts that might contribute to the dry weight. Sacrifice the larvae in
an ice bath of deionized water.
11.10.9.3 Small aluminum weighing pans can be used to dry and weigh the
larvae. Mark for identification an appropriate number of small aluminum
weighing pans (one per replicate). Weigh to the nearest 0.01 rag, and record
the weights (Figure 3).
82
-------
Test Dates:
Species:.
Pan
No.
Cone.
&
Reo.
Initial
Wt.
(mq)
Final
Wt.
(nig)
Diff.
(mg)
No.
.arvae
Av. Wt./
Larvae
(mg)
Figure 3. Data form for the sheepshead minnow, Cyprinodon van'egatus,
larval survival and growth test. Dry weights of larvae (from
USEPA 1987b).
83
-------
11.10.9.4 Immediately prior to drying, rinse the preserved larvae in
distilled water. The rinsed larvae from each test chamber is transferred to a
tared weighing pan 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 until weighed, to prevent the absorption of moisture
from the air. Weigh to the nearest 0.01 mg all weighing pans containing dried
larvae'and subtract the tare weight to determine the dry weight of larvae in
each replicate. Record the weights (Figure 3). For each test chamber, divide
the final dry weight by the number of original larvae in the test chamber to
determine the average individual dry weight, and record (Figure 3). For the
controls, also calculate the mean weight' per surviving fish in the test
chamber to evaluate if weights met test acceptable criteria (see Section 12).
Complete the summary data sheet (Figure 4) after calculating the average
measurements and statistically analyzing the dry weights and percent survival.
Average dry weights should be expressed to the nearest 0.001 mg.
11.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
11.11.1 A summary of test conditions and test acceptability criteria is
listed in Table 3.
11.12 ACCEPTABILITY OF TEST RESULTS
11.12.1 The tests are acceptable if (1) the average survival of control
larvae equals or exceeds 80%, and (2) the average dry weight per surviving
unpreserved control larvae is equal to or greater than 0.60 mg, or (3) the
average dry weight per surviving preserved control larvae is equal to or
greater than 0.50 mg. The above minimum weights presume that the age of the
larvae at the start of the test is less than or equal to 24 h.
11.13 DATA ANALYSIS
11.13.1 GENERAL
11.13.1.1 Tabulate and summarize the data. A sample set of survival and
growth response data is listed in Table 4.
11.13.1.2 The endpoints of toxicity tests using the sheepshead minnow larvae
are based on the adverse effects on survival and growth. The LC50, the IC25,
and the IC50 are calculated using point estimation techniques (see Section 9,
Chronic Toxicity Test Endpoints and Data Analysis). LOEC and NOEC values, for
survival and growth, are obtained using a hypothesis testing approach such f,as
Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel,
1959; Miller, 1981)(see Section 9). Separate analyses are performed for the
estimation of the LOEC and NOEC endpoints and for the estimation of the LC50,
IC25 and IC50. Concentrations at which there is no survival in any of the
test chambers are excluded from the statistical analysis of the NOEC and LOEC
for survival and growth, but included in the estimation of the LC50, IC25 and
IC50. See the Appendices for examples of the manual computations, program
listings, and examples of data input and program output.
84
-------
Test Dates:
Species:
Effluent Tested:
TREATMENT
NO. LIVE
LARVAE
SURVIVAL
(0/\
(/<,)
MEAN DRY VIT/
LARVAE (MG)'
± SD
SI6NIF. DIFF.
FROM CONTROL
(o)
MEAN
TEMPERATURE
(°C)
± SD
MEAN SALINITY
%0
± SD
AVE DISSOLVED
OXYGEN
(MG/L) ± SD
COMMENTS:
Figure 4. Data form for the sheepshead minnow, Cypn'nodon variegatus,
larval survival and growth test. Summary of test results (from
USEPA, 1987b).
85
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL
SURVIVAL AND GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS
1. Test type:
2. Salinity:
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. larvae per test chamber:
12. No. replicate chambers per
concentration:
13. No. larvae per concentration:
14. Source of food:
15. Feeding regime:
16. Cleaning:
17. Aeration:
Static renewal
20%o to 32%o (± 2%o of the selected
test salinity)
25 ± 1'C
Ambient laboratory illumination
10-20 ME/m2/s (50-100 ft-c) (ambient
laboratory levels)
16 h light, 8 h darkness
600 mL - 1 L beakers or equivalent
500-750 mL/replicate (loading and DO
restrictions must be met)
Daily
Newly hatched larvae (less than 24 h
old; 24-h range in age)
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
Newly hatched Artemia nauplii, (less
than 24-h old)
Feed once a day 0.10 g wet weight
Artemia nauplii per replicate on Days
0-2; Feed 0.15 g wet weight Artemia
nauplii per replicate on Days 3-6
Siphon daily, immediately before test
solution renewal and feeding
None, unless DO falls below 4.0 mg/L,
then aerate all chambers. Rate should
be less than 100 bubbles/min
86
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL
SURVIVAL AND GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS
(CONTINUED)
18. Dilution water:
19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sampling requirements:
25. Sample volume required:
Uncontaminated source of natural
seawater; deionized water mixed with
hypersaline brine or artificial sea
salts (HW Marinemix®, FORTY FATHOMS®,
GP2 or equivalent)
Effluent: Minimum of 5 and a control
Effluents: > 0.5
Receiving waters:
7 days
None, or > 0.5
Survival and growth (weight)
80% or greater survival in controls;
average dry weight per surviving
organism in control chambers should be
0.60 mg or greater, if unpreserved, or
0.50 mg or greater after no more than
7 days in 4% formalin or 70% ethanol
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 are
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,
Subsection 8.5.4)
6 L per day
87
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TABLE 4. SUMMARY OF SURVIVAL AND GROWTH DATA FOR SHEEPSHEAD MINNOW,
CYPRINODON,VARIEGATUS, LARVAE EXPOSED TO AN EFFLUENT FOR
SEVEN DAYS1
Effl.
Cone.
(%)
0.0
6.25
12.5
25.0
50.0
100.0
Proportion of
Survival in Replicate
Chambers
A
1.0
1.0
1.0
1.0
0.8
0.0
B
1.0
1.0
1.0
1.0
0.8
0.0
C
1.0
0.9
1.0
1.0
0.7
0.0
D
1.0
1.0
1.0
0.8
0.6
0.0
Mean
Prop.
Surv
1.00
0.98
1.00
0.95
0.73
0.00
Avg Dry Wgt (mg)
1
1
1
1
0
Repl
A
.29
.27
.32
.29
.62
--
icate
B
1.32
1.00
1.37
1.33
0.56
--
In
Chambers
C
1.59
0.97
1.35
1.20
0.46
--
D
1.27
0.97
1.34
0.94
0.46
--
Mean
Dry Wgt
(mg)
1.368
1.053
1.345
1.190
0.525
--
1 Four replicates of 10 larvae each.
11.13.1.3 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. Tests for normality
and homogeneity of variance are included in Appendix B. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
11.13.2 EXAMPLE OF ANALYSIS OF SHEEPHEAD MINNOW, CYPRINODON VARIEGATUS
SURVIVAL DATA
11.13.2.1 Formal statistical analysis of the survival data is outlined in
Figures 5 and 6. The response used in the analysis is the proportion of
animals surviving in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the LC50 endpoint. Concentrations at which there is no survival
in any of the test chambers are excluded from statistical analysis of the NOEC
and LOEC, but included in the estimation of the 1C, EC, and LC endpoint.
11.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
88
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STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL HYPOTHESIS TESTING
SURVIVAL DATA
PROPORTION SURVIVING
ARC SINE
TRANSFORMATION
SHAPIRO-WILICS TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
YES
T-TESTWITH
BONFERRONI
ADJUSTMENT
I YES
EQUAL NUMBER OF
REPLICATES?
NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 5. Flowchart for statistical analysis of the sheepshead minnow,
Cyprinodon variegatus, larval survival data by hypothesis
testing.
89
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STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
\
i
TWO OR MORE
PARTIAL MORTALITIES?
NO
YES
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2 TEST)
NO
YES
ONE OR MORE
PARTIAL MORTALITIES?
i
YES
r
NO
GRAPHICAL METHOD
LC50
PROBIT METHOD
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONC.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
NO
YES
SPEARMAN-KARBER
METHOD
i
r
TRIMMED SPEARMAN-
KARBER METHOD
LC50AND95%
CONFIDENCE
INTERVAL
Figure 6, Flowchart for statistical analysis of the sheepshead minnow,
Cyprinodon van'egatus, larval survival data by point estimation,
90
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Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Milk's
Test, and Bartlett's Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure. j
11.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t-test with the Bonferroni adjustment (see
Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.
11.13.2.4 Probit Analysis (Finney, 1971; see Appendix 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 Analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber
Method, or the Graphical Method may be used (see Appendices H-K).
i
11.13.2.5 Example of Analysis of Survival Data
11.13.2.5.1 This example uses the survival data from the Sheepshead Minnow
Larval Survival and Growth Test. The proportion surviving in each replicate
must first be transformed by the arc sine square root transformation procedure
described in Appendix B. The raw and transformed data, means and variances of
the transformed observations at each effluent concentration arid control are
listed in Table 5. A plot of the survival proportions is provided in
Figure 7. Since there was 100% mortality in all four replicates for the 100%
concentration, it was not included in the statistical analysis and was
considered a qualitative mortality effect.
11.13.2.6 Test for Normality
11.13.2.6.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 6.
11.13.2.6.2 Calculate the denominator, D, of the statistic:
D = £ (X.-X)2
i=l
Where: X1- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
•
91
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TABLE 5. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, SURVIVAL DATA
Repncate
RAW
ARC SINE
TRANSFORMED
Mean (Y,)
s*
i1
A
B
C
D
A
B
C
D
Control
1.0
1.0
1.0
1.0
1.412
1.412
1.412
1.412
1.412
0.0
1
Effluent Concentration (%)
6.25
1.0
1.0
0.9
1.0
1.412
1.412
1.249
1.412
1.371
0.007
2
12.5
1.0
1.0
1.0
1.0
1.412
1.412
1.412
1.412
1.412
0.0
3
25.0
1.0
1.0
1.0
0.8
• 1.412
1.412
1.412
1.107
1.336
0.023
4
50.0
0.8
0.8
0.7
0.6
1.107
1.107
0.991
0.886
1.023
0.011
5
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Effluent Concentration (%)
Replicate Control
6.25
A 0.0 0.041
B 0.0 0.041
C 0.0 -0.122
D 0.0 0.041
11.13.2.6.3 For this set of data,
n = 20
X = 1 (-0
20
D = 0.1236
12.5 25.0
0.0 0.076
0.0 0.076
0.0 0.076
0.0 -0.229
.001) = 0.000
50.0
0.084
0.084
-0.032
-0.137
92
-------
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93
-------
11.13.2.6.4 Order the centered observations from smallest to largest
X<1)sX(2)< ...
where XO) denotes the ith ordered observation. The ordered observations for
this example are listed in Table 7.
11.13.2.6.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a1? a2, ... ak where k is n/2 if n is even and (n-l)/2
if n is odd. For the data in this example, n = 20 and k = 10. The a,- values
are listed in Table 8.
11.13.2.6.6 Compute the test statistic, W, as follows:
W = -[fa, (x(n~i+u -X(i})}2
D ±=i
The differences x i X
-------
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
ai
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
X(n-,>1) _ X(i)
0.313
0.221
0.198
0.108
0.076
0.041
0.041
0.041
0.0
0.0
|X(20>
X!S
X07>
X(16)
x;«;-
fe
xai)
X(D
: X(2>
- x<3)
- x(4)
- x<5)
- x(6)
- x(7)
- x<8>
- x<9)
- x<10)
11.13.2.6.8 Since the data do not meet the assumption of normality, Steel's
Many-one Rank Test will be used to analyze the survival data.
11.13.2.7 Steel's Many-one Rank Test
11.13.2.7.1 For each control and concentration combination, combine the data
and arrange the observations in order of size from smallest to largest.
Assign the ranks (1, 2, ..., 8} to the ordered observations with a rank of 1
assigned to the smallest observation, rank of 2 assigned to the next larger
observation, etc. If ties occur when ranking, assign the average rank to each
tied observation.
11.13.2.7.2 An example of assigning ranks to the combined data for the
control and 6.25% effluent concentration is given in Table 9. This ranking
procedure is repeated for each control/concentration combination. The
complete set of rankings is summarized in Table 10. The ranks are next summed
for each effluent concentration, as shown in Table 11.
95
-------
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 6.25% EFFLUENT
CONCENTRATION FOR STEEL'S MANY-ONE RANK TEST
Rank
Transformed
Proportion
Surviving
Effluent
Concentration
1
5
5
5
5
5
5
5
1.249
1.412
1.412
1.412
1.412
1.412
1.412
1.412
6.25
6.25
6.25
6.25
Control
Control
Control
Control
Repli- Control
TABLE 10. TABLE OF RANKS
Effluent Concentration (%)
6.25
12.5
25.0
50.0
*>"•»
A
B
C
D
2Us
1.412
1.412
1.412
1.412
(5,4.5,5,6
(5,4.5,5,6
(5,4.5,5,6
(5,4.5,5,6
.5)
.5)
.5)
.5)
1.412
. 1.412
1.249
1.412
(5)
(5)
(D
(5)
1.412
1.412
1.412
1.412
(4.5)
(4.5)
(4.5)
(4.5)
1.412
1.412
1.412
1.107
(5)
(5)
(5)
(1)
1.107
1.107
0.991
0.886
(3.5)
(3.5)
(2)
(1)
TABLE 11. RANK SUMS
Effluent Concentration
Rank Sum
6.25
12.5
25.0
50.0
16
18
16
10
96
-------
11.13.2.7.3 For this example, determine if the survival in any of the
effluent concentrations is significantly lower than the survival in the
control. If this occurs, the rank sum at that concentration would be
significantly lower than the rank sum of the control. Thus, compare the rank
sums for the survival at each of the various effluent concentrations with some
"minimum" or critical rank sum, at or below which the survival would be
considered significantly lower than the control. At a significance level of
0.05, the minimum rank sum in a test with four concentrations (excluding the
control) and four replicates is 10 (see Table 5, Appendix E).
11.13.2.7.4 Since the rank sum for the 50% effluent concentration is equal to
the critical value, the proportion surviving in the 50% concentration is
considered significantly less than that in the control. Since no other rank
sums are less than or equal to the critical value, no other concentrations
have a significantly lower proportion surviving than the control. Hence, the
NOEC and the LOEC are the 25% and 50% concentrations, respectively.
11.13.2.8 Calculation of the LC50
11.13.2.8.1 The data used for the calculation of the LC50 is summarized in
Table 12. For estimating the LC50, the data for the 100% effluent
concentration with 100% mortality is included.
11.13.2.8.2 Because there are at least two partial mortalities in this set of
test data, calculation of the LC50 using Probit Analysis i<; recommended. For
this set of data, however, the test for heterogeneity of variance was
significant. Probit Analysis is not appropriate in this case. Inspection of
the data reveals that, once the data is smoothed and adjusted, the proportion
mortality in the lowest effluent concentration will not be zero although the
proportion mortality in the highest effluent concentration will be one.
Therefore, the Spearman-Karber Method is appropriate for this data.
11.13.2.8.3 Before the LC50 can be calculated the data must be smoothed and
adjusted. For the data in this example, because the observed proportion
mortality for the 12.5% effluent concentration is less than the observed
response proportion for the 6.25% effluent concentration, the observed
responses for the control and these two groups must be averaged:
Pcf =PiS =P/ a. 0-00+0-025+0.00 = °-025 =f 0.0083
Where: p^ = the smoothed observed mortality proportion for effluent
concentration i. | -
11.13.2.8.3.1 Because the rest of the responses are monotonic, additional
smoothing is not necessary. The smoothed observed proportion mortalities are
shown in Table 12.
11.13.2.8.4 Because the smoothed observed proportion mortality for the
control is now greater than zero, the data in each effluent concentration must
97
-------
be adjusted using Abbott's formula (Finney, 1971). The adjustment takes the
form: *
Where: p^ - the smoothed observed proportion mortality for the control
P? • (Pi - Po) / (1 - Po)
p? = the smoothed observed proportion mortality for effluent
concentration i
11.13.2.8.4.1 For the data in this example, the data for each effluent
concentration must be adjusted for control mortality using Abbott's formula,
as follows:
Po
a _ _a _ _a _ Pi ~Po _ 0 . 0083-Q . 0083 0.00 n n
) ~ Pi - Pa - — - —r— = —— =0.0
1-0.0083
0.9917
a _ P/-Po _ 0.05-0.0083 _ 0.0417
P3 =
1-0.0083
0.9917
= 0.042
_ a P/-PoS
P4 =
i-PcT
a =
~
0.275-0.0083
1-0.0083
0.2667
0.9917
= 0.269
_ 1.000-0.0083 _ 0.9917 _
1-0.0083 0.9917 ~
_ ± ...
~ '
The smoothed, adjusted response proportions for the effluent concentrations
are shown in Table 12.
TABLE 12. DATA FOR EXAMPLE OF SPEARMAN-KARBER ANALYSIS
Effluent
Concentration
%
Control
6.25
12.5
25.0
50.0
100.0
Number
of Deaths
0
1
0
2
11
40
Number of
Organisms
Exposed
40
40
40
40
40
40
Mortality
Proportion
0.000
0.025
0.000
0.050
0.275
1.000
Smoothed
Mortality
Proportion
0.0083
0.0083
0.0083
0.0500
0.2750
1.0000
Smoothed,
Adjusted
Mortality
Proportion
0.000
0.000
0.000
0.042
0.269
1.000
98
-------
11.13.2.8.5 Calculate the Iog10 of the estimated LC50, m, as follows:
m =
Where: p* = the smoothed adjusted proportion mortality at concentration i
Xf = the Iog10 of concentration i
k = the number of effluent concentrations tested, not including the
control
11.13.2.8.5.1 For this example, the Iog10 of the estimated LC50, m, is
calculated as follows:
m = [(0.000 - 0.000) (0.7959 + 1.0969)]/2 +
[(0.042 - 0.000) (1.0969 + 1.3979)]/2 +
[(0.269 - 0.042) (1.3979 + 1.6990)]/2 +
[(1.000 - 0.269) (1.6990 + 2.0000)]/2
I
= 1.755873
i
11.13.2.8.6 Calculate the estimated variance of m as follows:
( }
Where: X,- = the Iog10 of concentration i
n,- = the number of organisms tested at effluent concentration i
p? = the smoothed adjusted observed proportion mortality at effluent
concentration i
k = the number of effluent concentrations tested, riot including the
control
i
11.13.2.8.6.1 For this example, the estimated variance of m, V(m), is
calculated as follows:
...
V(m) = (0.000)(1.000)(1.3979 - 0.7959)^/4(39) +
(0.042)(0.958)(1.6990 - 1.0969)74(39) +
(0.269)(0.731)(2.0000 - 1.3979)74(39)
= 0.0005505
11.13.2.8.7 Calculate the 95% confidence interval for m: m ± 2.0 7 V(m)
99
-------
11.13.2.8.7.1 For this example, the 95% confidence interval for m is
calculated as follows:
1.755873 ± 2 ^0.0005505 = (1.754772, 1.756974)
11.13.2.8.8 The estimated LC50 and a 95% confidence interval for the
estimated LC50 can be found by taking base10 antilogs of the above values.
11.13.2.8.8.1 For this example, the estimated LC50 is calculated as follows:
LC50 = antilog(m) - antilog(1.755873) = 57.0%.
11.13.2.8.8.2 The limits of the 95% confidence interval for the estimated
LC50 are calculated by taking the antilogs of the upper and lower limits of
the 95% confidence interval for m as follows:
lower limit: antilog(l.754772) = 56.9%
upper limit: antilog(1.756974) = 57.1%
11.13.3 EXAMPLE OF ANALYSIS OF SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS,
GROWTH DATA
11.13.3.1 Formal statistical analysis of the growth data is outlined in
Figure 8. The response used in the statistical analysis is mean weight per
original organism for each replicate. The IC25 and IC50 can be calculated for
the growth data via a point estimation technique (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis). Hypothesis testing can be used to
obtain an NOEC and LOEC for growth. Concentrations above the NOEC for
survival are excluded from the hypothesis test for growth effects.
11.13.3.2 The statistical analysis using hypothesis testing consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test. The underlying assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested. The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the nonparametric
test, Steels' Many-one Rank Test, is used to determine the NOEC and LOEC
endpoints. If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
11.13.3.3 Additionally, if unequal numbers of replicates occur among the
concentration levels tested there are parametric and nonparametric alternative
analyses. The parametric analysis is a t test with the Bonferroni adjustment.
The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric
alternative. For detailed information on the Bonferroni adjustment, see
Appendix D.
11.13.3.4 The data, mean and variance of the observations at each
concentration including the control are listed in Table 13. A plot of the
mean weights for each treatment is provided in Figure 9. Since there is no
100
-------
1
STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
GROWTH
GROWTH DATA
MEAN DRY WEIGHT
POINT ESTIMATION
HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FOR SURVIVAL)
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILK-S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
1
EQUAL NUM!
REPLJCAT
NO
r 1
3EROF
ES?
YES
T-TESTWITH niiMMPTPQ
BONFERRONI DUNNETTS
ADJUSTMENT TEST
i
EQU
R
YES
r
STEEL'S MANY-ONE
RANK TEST
t
ENDPOINT ESTIMATES
NOEC, LOEC
AL NUMBER OF
EPUCATIES?
1 r
NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
1
Figure 8. Flowchart for statistical analysis of the sheepshead minnow,
Cypn'nodon van'egatus, larval growth data.
101
-------
survival in the 100% concentration, it is not considered in the growth
analysis. Additionally, since there is significant mortality in the 50%
effluent concentration, its effect on growth is not considered.
TABLE 13. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, GROWTH DATA
Replicate Control
Effluent Concentration (%)
6.25 12.5 25.0 50.0 100.0
A
B
C
D
MfanCY,.)
sf
1!
1.29
1.32
1.59
1.27
1.368
0.0224
1
1.27
1.00
0.97
0.97
1.053
0.0212
2
1.32
1.37
1.35
1.34
1.345
0.0004
3
1.29
1.33
1.20
0.94
1.190
0.0307 -
4 5
_
-
-
"
-
6
11.13.3.5 Test for Normality
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 ithin a
concentration from each observation in that concentration. The centered
observations are summarized in Table 14.
TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Effluent Concentration (%)
Replicate Control
6.25
12.5
25.0
A
B
C
D
-0.078
-0.048
0.222
-0.098
0.217
-0.053
-0.083
0.083
-0.025
0.025
0.005
-0.005
0.100
0.140
0.010
-0.250
102
-------
to
1 EHU
8
:uj<
5 S>£
<: 3<2 =
tc >f£
1 i|i
z mx±
5 xo u
3 ^w^
gts
*
o
o
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Ill
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o
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c:
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c:
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M- O
o s-
a>
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o -a
(6uj) 1HOI3M AdQ
cr>
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3
cn
103
-------
11.13.3.5.2 Calculate the denominator, D, of the test statistic:
D = £ (Xt-
1=1
Where: X,- = the ith centered observation
X - the overall mean of the centered observations
n = the total number of centered observations.
For this set of data, n = 16
X = _L_(-0.004) = -0.00025 = 0.00
16
D = 0.2245
11.13.3.5.3 Order the centered observations from smallest to largest:
x<1) is the ith ordered observation. These ordered observations are
listed in Table 15.
TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
xci>
-0.250
-0.098
-0.083
-0.083
-0.078
-0.053
-0.048
-0.025
i
9
10
11
12
13
14
15
16
xci>
-0.005
0.005
0.010
0.025
O.'lOO
0.140
0.217
0.222
104
-------
11.13.3.5.4 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a., a
., 2,
(n-l)/2 if n is odd. For'the'data in "this example,'n
values are listed in Table 16.
ak where k is n/2 if n is even and
16 and k = 8. The
TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
- X
(i)
1
2
3
4
5
6
7
8
0.5056
0.3290
0.2521
0.1939
0.1447
0.1005
0.0593
0.0196
0.472
0.315
0.223
0.183
0.103
0.063
0.053
0.020
X(16)
X(15)
X(14>
X(13)
X(12)
xdD
X(10)
x<9)
I
- x<1)
- XC2)
- X<3)
J(4)
- x<5)
- X(6>
- X(7>
v<8)
~ A
11.13.3.5.5 Compute the test statistic, W, as follows:
± (X <*-
The differences x
For this set of data:
W =
(i)
- X are listed in Table 16.
1 (0.4588)2 = 0.938
0.2245
11.13.3.5.6 The decision rule for this test is to compare W 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
example, the critical value at a significance level of 0.01 and 16
observations (n) is 0.876. Since W = 0.938 is greater than the critical
value, the conclusion of the test is that the data are normally distributed.
i
11.13.3.6 Test for Homogeneity of Variance
11.13.3.6.1 The test used to examine whether the variation in mean dry weight
is the same across all effluent concentrations including the control, is
105
-------
Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
f ol1ows:
V,) in
in
B =
Where: V- = degrees of freedom for each effluent concentration and
1 control, V, = (n,- - 1)
n,- = the number of replicates for concentration i
p = number of levels of effluent concentration including the
control
In - loge
i = 1, 2, ..., p where p is the number of concentrations
including the control
-52 _
C = 1 + [3 (p-1) ] -1 [ f 1/V, - ( f V,) -
11.13.3.6.2 For the data in this example (see Table 14), all effluent
concentrations including the control have the same number of replicates
(n, - 4 for all i). Thus, V,- = 3 for all i.
11.13.3.6.3 Bartlett's statistic is therefore:
B = [(12)ln(0.0137) - 3 £ In(Si)]/1.139
[12(-4.290) - 3(-18.960)]/1.139
5.396/1.139
4.737
106
-------
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 11.345. Since B = 4.737 is 'less than the
critical value of 11.345, conclude that the variances are not different.
j
11.13.3.7 Dunnett's Procedure
I
11.13.3.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table :17..
TABLE 17. ANOVA TABLE
Source df
Between p - 1
Within N - p
Total N - 1
Sum of Squares
(SS)
SSB
SSW
SST
1
Mean Square(MS)
(SS/df)
i
S* = SSB/(p-l)
s* = SSW/(N-P)
-
Where: p = number of concentration levels including the control
N = total number of observations n, + n, ... + n
* P
n,- = number of observations in concentration i
!
SSB = f,Tl/n±-G2/N Between Sum of Squares:
SST = £ £ YJ.j-G2/N Total Sum of Squares
2=1:7=1
SSW = SST-SSB
Within Sum of Squares
107
-------
6 - the grand total of all sample observations
, G = E z^
I,- - the total of the replicate measurements for concentration i
Y-5 = the jth observation for concentration i (represents the mean
dry weight of the mysids for concentration i in test
chamber j)
11.13.3.7.2 For the data in this example:
n
N = 16
n - 4
Ti = Y., + Y12 + Y13 + Y14 = 5.47
Tj - Y2 + Y22 + Y23 + Y2t = 4.21
T, - Y,] + Y^ + Y,, + Y,4 = 5.38
T~4 = Y^J + Y42 + Y^ + Y44 = 4.76
G - T, + T2 + T3 + T4 = 19.82
SSB = *tTl/n±-G*/N
_1_(99.247) - (19.82)2 = 0.260
4 16
SST
25.036 - (19. 82)2 = 0.484
16
SSW = SST-SSB
s? = SSB/CP-D
SSW/(N-p)
= 0.484 - 0.260 = 0.224
0.260/(4-l) = 0.087
0.224/(16-4) = 0.019
108
-------
11.13.3.7.3 Summarize these calculations in the ANOVA table (Table 18)
TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source df Sum of Squares Mean Siquare(MS)
(SS) (SS/df)
Between
Within
3
12
0.260
0.224
0.087
0.019
Total 15 0.484
11.13.3.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
ti =
Where: Yf = mean dry weight for effluent concentration i
Y1 = mean dry weight for the control
Sw = square root of the within mean square
n, = number of replicates for the control
n,- = number of replicates for concentration i.
109
-------
11.13.3.7.5 Table 19 includes the calculated t values for each concentration
and control combination. In this example, comparing the 6.25% concentration
with the control, the calculation is as follows:
TABLE 19. CALCULATED T VALUES
Effluent Concentration (%) i t,-
6.25
12.5
25.0
2
3
4
3.228
0.236
1.824
. _ (1.368-1.053)
Co •"*
[0.138^(1/4) + (1/4)]
11.13.3.7.6 Since the purpose of this test is to detect a significant
reduction in mean weight, a one-sided test is appropriate. The critical value
for this one-sided test is found in Table 5, Appendix C. For an overall alpha
level of 0.05, 12 degrees of freedom for error and three concentrations
(excluding the control) the critical value is 2.29. The mean weight for
concentration i is considered significantly less than the mean weight for the
control if tt is greater than the critical value. Since t2 is greater than
2.29, the 6.25% concentration has significantly lower growth than the control.
However, the 12.5% and 25% concentrations do not exhibit this effect. Hence
the NOEC and the LOEC for growth cannot be calculated.
11.13.3.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be. calculated:
MSD = d S^CL/nJ + (1/n)
Where: d = the critical value for Dunnett's Procedure
Sw » the square root of the within mean square
n = the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n, » the number of replicates in the control.
110
-------
11.13.3.7.8 In this example:
MSD = 2.29 (0.10)^(1/4) + (1/4)
= 2.29 (0.138)(0.707)
= 0.223
11.13.3.7.9 Therefore, for this set of data, the minimum
difference that can
be detected as statistically significant is 0.223 mg.
11.13.3.7.10 This represents a 16% reduction in mean weight from the control.
11.13.3.8 Calculation of the ICp
11.13.3.8.1 The growth data from Table 4 are utilized in this example. As
seen from Table 4 and Figure 7, the observed means are not monotonically non-
increasing with respect to concentration (mean response for each higher
concentration is not less than or equal to the mean response for the previous
concentration and the responses between concentrations do not follow a linear
trends). Therefore, the means are smoothed prior to calculating the 1C. In
the following discussion, the observed means are represented by Y,- and the
smoothed means by M,-.
11.13.3.8.2 Starting with the control mean, Y., = 1.368 and Y2 = 1.053, we see
that Y, > Y2. Set M, = Y,. Comparing Y2 to Y3, Y2 < Y3. \ -
11.13.3.8.3 Calculate the smoothed means:
M2 = M3 = (Y2 + Y3)/2 = 1.199
11.13.3.8.4 Since Y, = 0 < Y5 = 0.525 < Y, = 1.190 < Y. - 1.345, set M, =
1.199, M4 = 1.190, M5 = 0.525, and set M6 - 0.
11.13.3.8.5 Table 20 contains the response means and smoothed means and
Figure 10 gives a plot of the smoothed response curve.
TABLE 20. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, MEAN GROWTH
RESPONSE AFTER SMOOTHING
Effluent
Cone. (%)
Control
6.25
12.50
25.00
50.00
100.00
i
1
2
3
4
5
6
Response Means Smoothed Means
(mg) Y, (mg) M;
1.
1.
1.
1.
0.
0.
368
053
345
189
525
0
1.368
1.199
1.199
1.189
0.525
0.0
111
-------
^T^T^O'dddodddOO
c
o
T3
O
c
*^^
I
o
•o
as
0)
Q.
O)
CD
.C
s-
Q)
oo
-!-> O
a} S-
T3 O)
3 •>
a} to
s- a .
o en
-------
11.13.3.8.6 An IC25 and IC50 can be estimated using the Linear Interpolation
Method. A 25% reduction in weight> compared to the controls, would result in
a mean dry weight of 1.026 mg, where M^l-p/lOO) = 1.368(1-25/100). A 50%
reduction in mean dry weight, compared to the controls, would result in a mean
dry weight of 0.684 mg. Examining the smoothed means and their associated
concentrations (Table 4), the response, 1.026 mg, is bracketed by C4 = 25.0%
effluent and C5 = 50.0% effluent. The response (0.684 mg) is bracketed by
C4 = 25.0% effluent and C5 = 50% effluent.
11.13.3.8.7 Using the equation from Section 4.2 of Appendix M, the estimate
of the IC25 is calculated as follows:
ICp =
IC25 = 25.0 + [1.368(1 - 25/100) - 1.189] (50.00 - 25.00)
(0.525 - 1.189)
31.2%.
11.13.3.8.8 Using the equation frontSection 4.2 of Appendix L, the estimate
of the IC50 is calculated as follows:
ICp=Cj+ [ML (l-p/100) -AT,]
(C
IC50 - 50.0 + [1.368(1-50/100) - 0.525] (100.00-50.00)
(0.0 - 0.525)
= 44.0%.
11.13.3.8.9 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 31.1512%. The empirical
95% confidence interval for the true mean was 22.0420% and 36.3613%. The
computer program output for the IC25 for this data set is shown in Figure 11.
11.13.3.8.10 When the ICPIN program was used to analyze this set of data for
the IC50, requesting 80 resamples, the estimate of the IC50 was 44.0230%. The
empirical 95% confidence interval for the true mean was 39.1011% and 49.0679%.
The computer program output is shown in Figure 12.
113
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
1
2
3
4
1
0
1.29
1.32
1.59
1.27
2
6.25
1.27
1
.972
.97
3
12.5
1.32
1.37
1.35
1.34
4
25
. 1.29
1.33
1.2
.936
5
50
.62
.560
.46
.46
6
100
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Test Ending Date:
Test Species: Cyprinodon variegatus
Test Duration: 7-d
DATA FILE: sheep.icp
OUTPUT FILE: sheep.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
4
4
4
4
4
4
Concentration
%
0.000
6.250
12.500
25.000
50.000
100.000
Response
Means
1.368
1.053
1.345
1.189
0.525
0.000
Std. Pooled
Dev. Response Means
0.150
0.145
0.021
0.177
0.079
0.000
1.368
1.199
1.199
1.189
0.525
0.000
The Linear Interpolation Estimate: 31.1512 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 30.6175 Standard Deviation: 2.9490
Original Confidence Limits: Lower: 25.4579 Upper: 34.4075
Expanded Confidence Limits: Lower: 22.0420 Upper: 36.3613
Resampling time in Seconds: 1.70 Random Seed: -2137496326
Figure 11. ICPIN program output for the IC25.
114
-------
Cone. ID
Cone. Tested 0 6.25
Response 1 1.29 1.27
Response 2 1.32 1
Response 3 1.59 .972
Response 4 1.27 .97
12.5 25
1.32 1.29
1.37 1.33
1.35 1.2
1.34 .936
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Test Ending Date:
Test Species: Cyprinodon variegatus
Test Duration: 7-d
DATA FILE: sheep. icp
OUTPUT FILE: sheep. i 50
50 100
.62 0
. 560 0
.46 0
.46 0
Cone
ID
1
2
3
4
5
6
The
Number Concentration
Replicates
4
4
4
4
4
4
Linear Interpolation
%
0.000
6.250
12.500
25.000
50.000
100.000
Estimate:
Response Std.
Means Dev.
Pooled
Response Means
1.368 0.150 1.368
1.053 0.145 1.199
1.345 0.021
1.189 0.177
1.199
1.189
0.525 0.079 0.525
0.000 0.000 0.000
44.0230 Entered P
Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
44.3444 Standard Deviation: 1.7372
Lower: 40.9468 Upper: 47.1760
Lower: 39.1011 Upper: 49.0679
1.70 Random Seed: -156164614
Figure 12. ICPIN program output for the IC50.
115 !
-------
11.14 PRECISION AND ACCURACY
11.14.1 PRECISION
11.14.1.1 Single-Laboratory Precision
11.14.1.1.1 Data on the single-laboratory precision of the sheepshead minnow
larval survival and growth test using FORTY FATHOMS® artificial seawater,
natural seawater, and 6P2 with copper sulfate, sodium dodecyl sulfate, and
hexavalent chromium, as reference toxicants, are given in Tables 21-27. The
IC25, IC50, or LC50 data (coefficient of variation), indicating acceptable
precision for'the reference toxicants (copper, sodium dodecyl sulfate, and
hexavalent chromium), are also listed in these Tables.
11.14.1.2 Multilaboratory Precision
11.14.1.2.1 Data from a study of multilaboratory test precision, involving a
total of seven tests by four participating laboratories, are listed in
Table 27. The laboratories reported very similar results, indicating good
inter!aboratory precision. The coefficient of variation (IC25) was 44.2% and
(IC50) was 56.9%, indicating acceptable precision.
11.14.2 ACCURACY
11.14.2.1 The accuracy of toxicity tests cannot be determined.
116
-------
TABLE 21. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE
FROM FISH MAINTAINED AND SPAWNED IN FORTY FATHOMS® ARTIFICIAL
SEAWATER, JJSING COPPER (CU) SULFATE AS A REFIEREINCE
TOXICANT1'2'3'*'5
Most
Test
Number
1
2
3
4
5
6
7
8
n:
Mean:
CV(%):
NOEC
(M9/L)
507
< 507
< 507
50r
< 507
50
50
50
5
NA
NA
IC25
IC50 Sensitive
(Mg/L) Og/L) Endpoint6
113.3
54.3
41.8
63.2
57.7
48.3
79.6
123.5
p
72.7
41.82
152.3 S
97.5 G
71.4 G
90.8 S
99.8 S
132.5 G
159.7 G
236.4 G
8
130.0
40.87
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by Donald J. Klemm, Bioassessment and Ecotoxicology
Branch, EMSL, Cincinnati, OH.
All tests were performed using Forty Fathoms® synthetic seawater.
Three replicate exposure chambers, each with 15 larvae, were used for
the control and each copper concentration. Copper concentrations
used in Tests 1-6 were: 50, 100, 200, 400, and 800 ng/l. Copper
concentrations in Tests 7-8 were: 25, 50, 100, 200 and 400 A*g/L.
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity test
see Section 4, Quality Assurance.
Endpoints: G=growth; S=survival. i
Lowest concentration tested was 50 /zg/L (NOEC Range;; > 50* - 50
117
-------
TABLE 22. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINIDON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE
FROM FISH MAINTAINED AND SPAWNED IN FORTY FATHOMS® ARTIFICIAL
SEAWATER, USING SODIUM DODECYL SULFATE (SDS) AS A REFERENCE
TOXICANT1'2'3'4'5'6
Test
Number
1
2
3
4
5
6
n:
Mean:
CV(%):
! Data from USEPA
NOEC
(mg/L)
1.0
1.0
1.0
0.5
1.0
0.5
6
NA
NA
(1988a) and USEPA
IC25
(mg/L)
1.2799
1.4087
2.3051
1.9855
1.1901
1.1041
6
1.5456
31.44
(1991a).
Most
IC50 Sensitive
(mg/L) Endpoint
1.5598
1.8835
2.8367
2.6237
1.4267
1.4264
6
1.9595
31.82
+ anrl I7^*n+ftVT/*r
S
S
S
G
S
G
\1 nn\/
Branch, EMSL, Cincinnati, OH.
All tests were performed using Forty Fathoms® synthetic seawater.
Three replicate exposure chambers, each with 15 larvae, were used for
the control and each SDS concentration. SDS concentrations in Tests
1-2 were: 1.0, 1.9, 3.9, 7.7, and 15.5 mg/L. SDS concentrations in
Tests 3-6 were: 0.2, 0.5, 1.0, 1.9, and 3.9 mg/L.
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
NOEC Range: 0.5 -1.0 mg/L (this represents a difference of one
exposure concentration).
Endpoints: G=growth; S=survival
118
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TABLE 23. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINIDON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
PERFORMED IN NATURAL SEAWATER, USING LARVAE FROM FISH
MAINTAINED AND SPAWNED IN NATURAL SEAWATER, USING COPPER (CU)
SULFATE AS A REFERENCE TOXICANT1-2'J'*'5'6 ;
Test
Number
NOEC
(M9/L)
IC25
IC50
Most
Sensitive
Endpoint
1
2
3
4
5
n:
Mean:
CV(%):
1 Data from US EPA
125
31
125
125
125
5
NA
NA
(1988a) and USEPA
320.3
182.3
333.4
228.4'
437.5
5
300.4
33.0
(1991a).
437.5
323.0
484.4
343.8
NC8
4
396.9
19.2
1 r-m hi i ir»i
S
G
S
S
S
5
6
7
8
i v-j 1.0 f/ci i ui llltvj i/jr ucui yc riui IIOUII aiiu L.IIOC IUl C I I U; UIM.ll) Uotrn,
Narragansett, RI.
Three replicate exposure chambers, each with 10-15 larvae, were used
for the control and each copper concentration. Copper concentrations
were: 31, 63, 125, 250, and 500 //g/L.
NOEC Range: 31 - 125 ng/L (this represents a difference of two exposure
concentrations).
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=growth; S=survival.
NC = No linear interpolation estimate could be calculated from the
data, since none of the group response means were less than 50
percent of the control response mean.
119
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TABLE 24. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINIDON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
PERFORMED IN NATURAL SEAWATER, USING LARVAE FROM FISH
MAINTAINED AND SPAWNED IN NATURAL SEAWATER, USING SODIUM
DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT1 f2'3'4'5'6
Test
Number
1
2
3
4
5
n:
Mean:
CV(%) :
\ Data from USEPA
** Tfte*^ o nov»f nv^maH
NOEC
(mg/L)
2.5
1.3
1.3
1.3
1.3
5
NA
NA
(1988a) and USEPA
K\/ £anwia Mnvvi cnr
IC25
(mg/L)
2.9
NCI8
1.9
2.4
1.5
4
2.2
27.6
(1991a).
i anH Fl i CP Tr»V*i
Most
IC50 Sensitive
(mg/L) Endpoint
3.60
NC29
2.4
NC2
1.8
3
2.6
35.3
olln. FRI -N. U!
S
G
S
G
S
SFPA.
5
6
7
8
Narragansett, RI. •
Three replicate exposure chambers, each with 10-15 larvae, were used
for the control and each SDS concentration. SDS concentrations were:
0.3, 0.6, 1.3, 2.5, and 5.0 mg/L.
NOEC Range: 1.3 - 2.5 mg/L (this represents a difference of one
exposure concentration).
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=growth; S=survival.
NCI - No linear interpolation estimate could be calculated from the
data, since none of the group response means were less than 75
percent of the control response mean.
NC2 = No linear interpolation estimate could be calculated from the
data, since none of the group response means were less than 50
percent of the control response mean.
120
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TABLE 25. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND RO TH TEST
PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE
FROM FISH MAINTAINED AND SPAWNED IN FORTY FATHOMS® ARTIFICIAL
SEAWATER, AND HEXAVALENT CHROMIUM AS THE REFERENCE
TOXICANT1'2'3-*'5
Test
Number
1
2
3
4
5
NOEC
(mg/L)
2.0
1.0
4.0
2.0
1.0
IC25
(mg/L)
5.8
2.9
6.9
2.4
3.1
IC50
(mg/L)
11.4
9.9
11.5
9.2
10.8
Most
Sensitive
Endpoint
G
G
G
G
G
n:
Mean:
5
NA
NA
5
4.2
47.6
5
10.6
9.7
Tests performed by Donald Klemm, Bioassessment and Ecotoxicology
Branch, EMSL, Cincinnati, OH. .
All tests were performed using Forty Fathoms® synthetic seawater.
Three replicate exposure chambers, each with 15 larvae, were used for
the control and each hexavalent chromium concentration. Hexavalent
chromium concentrations used in Tests 1-5 were: 1.0, 2.0, 4.0, 8.0,
16.0, and 32.0 mg/L.
NOEC Range: 1.0 - 4.0 mg/L (this represents a difference of four
exposure concentrations)
Adults collected in the field. ; .
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=growth; S=survival.
121
-------
TABLE 26. COMPARISON OF LARVAL SURVIVAL (LC50) AND GROWTH (IC50) VALUES
FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EXPOSED TO
SODIUM DODECYL SULFATE (SDS) AND COPPER (CU) SULFATE IN GP2
ARTIFICIAL SEAWATER MEDIUM OR NATURAL SEAWATER172'*'4
SDS (mg/L)
COPPER
Survival
GP2
NSW
GP2
NSW
455
467
390
412
485
528
Growth
GP2
Mean
CV (%)
7.49
8.70
8.38
8.19
7.7
8.13
8.87
8.85
8.62
4.9
7.39
8.63
8.48
8.17
8.3
8.41
8.51
9.33
8.75
5.8
GP2
341
496
467
NSW
333
529
776
Mean
CV (%)
437
9.4
475
12.3
435
18.9
546
40.7
3
4
Tests performed by George Morrison and Glen Modica, ERL-N, USEPA
Narragansett, RI.
Three replicate exposure chambers, each with 10-15 larvae, were used
for the control and each SDS concentration. SDS concentrations were-
0.3, 0.6, 1.3, 2.5, and 5.0 mg/L.
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
122
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TABLE 27. DATA FROM INTERLABORATORY STUDY OF THE SHEEPSHEAD MINNOW,
CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST USING AN
INDUSTRIAL EFFLUENT AS A REFERENCE TOXICANT1'2'3
Laboratory A
Laboratory B
Laboratory C
Laboratory D
n:
Mean:
Test
Number
1
2
1
2
1
2
Most Sensitive Endooint
NOEC
3.2 (S,G)
3.2 (S,G)
3.2 (S,6)
3.2 (S,G)
1.0 (S)
3.2 (S,G)
1.0 (G)
7
NA
NA
IC25
7.4 (S)
7.6 (S)
5.7 (G)
5.7 (G)
4.7 (S)
7.4 (G)
5.2 (S)
7
5.5
44.2
IC50
7.4 (G)
14.3 (G)
9.7 (G)
8.8 (G)
7.2 (S)
24.7 (G)
7.2 (S)
7
11.3
56.9
1 Data from USEPA (1987b), USEPA (1988a), and USEPA (1991a).
2 Effluent concentrations were: 0.32, 1.0, 3.2, 10.0, and 32.0/».
3 NOEC Range: 1.0 - 3.2% (this represents a difference of one exposure
concentration).
4 Endpoints: G=growth; S=survival.
123
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SECTION 12
TEST METHOD
SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS
EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST
METHOD 1005.0
12.1 SCOPE AND APPLICATION
12.1.1 This method, adapted in part from USEPA (1981) and USEPA (19875)
estimates the chronic toxicity of effluents and receiving waters to the
sneepsnead minnow, Cyprinodon van'egatus, using embryos and larvae in a
nine-day, static renewal test. The effects include the synergistic,
antagonistic, and additive effects of all the chemical, physical, and
biological components which adversely affect the physiological and biochemical
functions of the test organisms. The test is useful in screening for
teratogens because organisms are exposed during embryonic development.
12.1.2 Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LC50s).
12.1.3 Detection limits of the toxicity of an effluent or chemical substance
are organism dependent,
12.1.4 Brief excursions in toxicity may not be detected using 24-h composite
samples. Also, because of the long sample collection period involved in
composite sampling, and because the test chambers are not sealed, highly
volatile and highly degradable toxicants present in the source may not be
detected in the test.
12.1.5 This test is commonly used in one of two forms: (1) a definitive test
consisting of a minimum of five effluent concentrations and a control, and (2)
a receiving water test(s), consisting of one or more receiving water
concentrations and a control.
12.2 SUMMARY OF METHOD
12.2.1 Sheepshead minnow, Cyprinodon van'egatus, embryos and larvae are
exposed in a static renewal system to different concentrations of effluent or
to receiving water starting shortly after fertilization of the eggs through
four days posthatch. Test results are based on the total frequency of both
mortality and gross morphological deformities (terata).
12.3 INTERFERENCES
12.3.1 Toxic substances may be introduced by contaminants in dilution water
glassware, sample hardware, and testing equipment (see Section 5, Facilities
Equipment, and Supplies). '
124
-------
12 3 2 Adverse effects of low dissolved oxygen concentrations (DO), high
concentrations of suspended and/or dissolved solids, and extremes of pH may
mask the effect of toxic substances.
12 3 3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
12 3 4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
12.4 SAFETY
12.4.1 See Section 3, Health and Safety.
12.5 APPARATUS AND EQUIPMENT
12.5.1 Facilities for holding and acclimating test organisms.
i
12.5.2 Sheepshead minnow culture unit -- see Subsection 6.13 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.
12 5 2 1 A test using 15 embryos per test vessel and four replicates per
concentration, will require 360 newly-fertilized embryos at the start of the
test A test with a minimum of 10 embryos per test vessel and three
replicates per concentration, and with five effluent concentrations and a
control, will require a minimum of 180 embryos at the start cif the test.
12 5 3 Brine shrimp, Artemia, culture unit -- for feeding sheepshead minnow
larvae in the continuous culture unit, (see Subsection 6.12! below).
12 5.4 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L, and maintain
sample temperature at 4°C.
12.5.5 Environmental chamber or equivalent facility with!temperature control
(25 ± 1°C).
12.5.6 Water purification system -- Millipore Milli-Q®, deionized water (DI)
or equivalent. .
12 5 7 Balance -- analytical, capable of accurately weighing to 0.00001 g.
Note- An analytical balance is not needed for this test but is needed for
other specified toxicity test methods with growth endpoints.
12 5 8 Reference weights, Class S -- for checking the performance of the
balance. The reference weights should bracket the expected weights of
reagents, and the expected weights of the weighing pans and the weights of the
weighing pans plus larvae. i
125
-------
12.5.9 Air pump -- for oil free air supply.
.12.5.10 Air lines, and air stones -- for aerating water containing embryos,
larvae, or supplying air to test solution with low DO.
12.5.11 Meters, pH and DO -- for routine physical and chemical measurements.
12.5.12 Standard or micro-Winkler apparatus -- for determining DO (optional).
12.5.13 Dissecting microscope -- for examining embryos and larvae.
12.5.14 Light box -- for counting and observing embryos and larvae.
12.5.15 Refractometer -- for determining salinity.
12.5.16 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.
12.5.17 Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.
12.5.18 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
12.5.19 Test chambers -- four (minimum of three), borosilicate glass or
non-toxic plastic labware per test concentration. Care must be taken to avoid
inadvertently removing embryos or larvae when test solutions are decanted from
the chambers. To avoid potential contanimation from the air and excessive
evaporation of test solutions during the test, the chambers should be covered
with safety glass plates or sheet plastic (6 mm thick). The covers are
removed only for observation and removal of dead organisms.
12.5.20 Beakers -- six Class A, borosilicate glass or non-toxic plasticware,
1000 mL for making test solutions.
12.5.21 Wash bottles -- for deionized water, for washing embryos from
substrates and containers, and for rinsing small glassware and instrument
electrodes and probes.
12.5.22 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
12.5.23 Pi pets, volumetric -- Class A, 1-100 ml.
12.5.24 Pipets, automatic -- adjustable, 1-100 ml.
12.5.25 Pipets, serological -- 1-10 ml, graduated.
12.5.26 Pipet bulbs and fillers -- PROPIPET®,- or equivalent.
12.5.27 Droppers and glass tubing with fire polished aperatures, 4 mm ID --
for transferring embryos and larvae.
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12.5.28 Siphon with bulb and clamp -- for cleaning test chambers.
i
r
12.5.29 NITEX® or stainless steel mesh sieves, (< 150 im> 500 /un, and 3 to 5
mm) -- for collecting Artemia nauplii and fish embryos, and for spawning
baskets, respectively (NITEX® is available from Sterling Marine Products, 18
Label Street, Montclair, NJ 07042; 201-783-9800).
• i
12.6 REAGENTS AND CONSUMABLE MATERIALS
12.6.1 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
i
12.6.2 Data sheets (one set per test) -- for data recording (see Figure 1).
i
12.6.3 Tape, colored -- for labelling test chambers.
12.6.4 Markers, waterproof -- for marking containers, etc.
12.6.5 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) -- for standards and calibration check (see USEPA Method 150.1,
USEPA, 1979b).
12.6.6 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA, 1979b), or reagents -- for modified Vfinkier analysis.
12.6.7 Laboratory quality assurance samples and standards --for calibration
of the above methods.
12.6.8 Reference toxicant solutions -- see Section 4, Quality Assurance.
12.6.9 Reagent water -- defined as distilled or deionized water that does not
contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
'
12.6.10 Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
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
%ol among chambers on a given day. If effluent and receiving water tests are
conducted concurrently, the salinities of the water should be similar.
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.
127
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Test Dates:
Type Effluent:
Effluent Tested:
Original pH:
Species:
Field:
Lab:
Test:
Salinity:
D.O.:
CONCENTRATION:
Replicate I
DAY
^Live/Dead
Embrvo-Larvae
Terata
Terno. (°C)
Salinity foot)
D.O. (m/L)
DH
0
1
2
3
4
5
6
7
8
9
CONCENTRATION:
Replicate II;
Comments:
DAY
^Live/Dead
Embrvo-Larvae
Terata
Temo. CO
Salinity (ppt)
D.O. (ma/L)
DH
0
1
2
3
4
5
6
7
8
9
Note: Final endpoint for this test is total mortality (combined total number
of dead embryos, dead larvae, and deformed larvae) (see Subsection 12.10.8 and
12.13).
Figure 1. Data form for sheepshead minnow, Cypn'nodon variegatus,
embryo-larval survival/teratogenicity test. Daily record of
embryo-larval survival/terata and test conditions.
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CONCENTRATION:
Reolicate III:
DAY
#Live/Dead
Embrvo- Larvae
Terata
Temo. (°)
Salinitv foot)
D.O. fma/L)
PH
n
i
2
3
4
5
6
7
8
9
CONCENTRATION:
REPLICATE IV
DAY
#Live/Dead
Embrvo -Larvae
Terata
Temo. (°C)
Salinitv foot)
D.O. (mq/L)
pH
0
1
2
3
4
5
6
7
8
9
Comments:
Note: Final endpoint for this test is total mortality (combined total number
of dead embryos, dead larvae, and deformed larvae) (see Subsection 12.10.8 and
12.13).
Figure 1. Data form for sheepshead minnow, Cyprinodon vsn'egatus,
embryo-larval survival/teratogenicity test. Daily record of
embryo-larval survival/terata and test conditions; (CONTINUED).
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1.3 Hypersaline brine (HSB): HSB has several advantages that make it
fm^}6 f°r +SQ tn tox1city testing. It can be made from any high Sity
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity. HSB derived from natural seawater
rLnnne%Sary trace "^als' biogenic ™"°™s, and some of the
components necessary for adequate growth, survival, and/or
ion or marine and estuarine organisms, and may be stored for •
iicprt ac a rf-i f w?Jnout any apparent degradation. However if 100% HSB is
, • uon dll"ent' tne maximum concentration of effluent that can be tpstpri
using HSB is limited to 80% at 20%. salinity, and 70% at 30%0 salinity
that'(l)3has Ihhiahesu f°ntainer (or makil?9 HSB from natural seawater is one
SKI ,.
immersed directly into the seawater, ensure that the heater mateHals do not
le ^ubstances that would contaminate the br?ne Jne
thermostatica1^ controlled heat exchanger made
" US6d ^ °n - Compressors to
;ln^:? Bef°re addin9 seawater to the brine generator, thoroughly clean
generator, aeration supply tube, heater, and any other materials that w?ll
e^^
contaminaton
-°
incoming tide to minimize the possibility of
Jater^houlri hSeaoSe^Ure °f tje^wwater is increased slowly to 40°C. The
water eSratf™ T£ Jo prevent temperature stratification and to increase
salinity is attained, the HSB should be
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12.6.10.3.7 Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine. For example, if the
salinity of the HSB is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0. The proportion of brine is 1 part in 5 (one part brine
to four parts deionized water). To make 1 L of seawater at 20%o salinity from
a HSB of 100%o, divide 1 L (1000 ml) by 5.0. The result, 200 ml, is the
quantity of HSB needed to make 1 L of sea water. The difference, 800 ml, is
the quantity of deionized water required. j
i
12.6.10.3.8 Table 1 illustrates the composition of test solutions at 20%o if
they are prepared by serial dilution of effluent with 20%o salinity seawater.
12.6.10.4 Artificial sea salts: HW MARINEMIX® brand sea salts (Hawaiian
Marine Imports Inc., 10801 Kempwood, Suite 2, Houston, TX 77043) have been
used successfully at the USEPA, Region 6, Houston laboratory to culture
sheepshead minnows and perform the embryo-larval survival and teratogenicity
test. EMSL-Cincinnati has found FORTY FATHOMS® artificial sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-321-1189), to
be suitable for culturing sheepshead minnows and for performing'the larval
survival and growth test and embryo-larval test. Artificial sea salts may be
used for culturing sheepshead minnows and for the embryo larval test if the
criteria for acceptability of test data are satisfied (see Subsection 12.11).
12.6.10.4.1 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 salts should be mixed in a separate
container -- not the culture tank. The deionized water used in hydration
should be in the temperature range of 21-26°C. Seawater made from artificial
sea salts is conditioned (Spotte, 1973; Spotte et al., 1984; Bower, 1983)
before it is used for culturing or testing. After adding the water, place an
airstone in the container, cover, and aerate the solution mildly for at least
24 h before use. '•
12.6.11 BRINE SHRIMP, ARTEMIA, CULTURE -- for feeding cultures.
12.6.11.1 Newly-hatched Artemia nauplii are used as food in the sheepshead
minnow culture, and a brine shrimp culture unit should be prepared (USEPA,
1993a). Although there are many commercial sources of brine shrimp cysts, the
Brazilian or Colombian strains are currently preferred because the supplies
examined have had low concentrations of chemical residues and produce nauplii
of suitably small size. For commercial sources of brine shrimp, Artemia,
cysts, see Table 2 of Section 5, Facilities, Equipment, and Supplies; and
Section 4, Quality Assurance.
12.6.11.2 Each new batch of Artemia cysts must be evaluated for size
(Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al., 1980) and nutritional
suitability (Leger, et al., 1985; Leger, et al., 1986) against known suitable
reference cysts by performing a side by side larval growth test using the
"new" and "reference" cysts. The "reference" cysts used in the suitability
test may be a previously tested and acceptable batch of cysts, or may be
obtained from the Quality Assurance Research Division, Environmental
j
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TABLE 1. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20%o, USING
20%o NATURAL OR ARTIFICIAL SEAWATER, HYPERSALINE BRINE, OR
ARTIFICIAL SEA SALTS
Solutions To Be Combined
Effluent
Solution
1
2
3
4
5
Control
Total
Ef f 1 uent
Cone.
fo/\
(/o)
1001'2
50
25
12.5
6.25
0.0
Volume of Volume of Diluent
Effluent Seawater (20%o)
Solution
4000 mL
2000 mL Solution 1 + 2000 mL
2000 mL Solution 2 + 2000 mL
2000 mL Solution 3 + 2000 mL
2000 mL Solution 4 + 2000 mL
2000 mL
10000 mL
This illustration assumes: (1) the use of 400 mL of test solution in
each of four replicates and 400 mL for chemical analysis (total of
2000 mL) for the control and five concentrations of effluent (2) an
effluent dilution factor of 0.5, and (3) the effluent lacks appreciable
salinity. A sufficient initial volume (4000 mL) of effluent is prepared
by adjusting the salinity to the desired level. In this example, the
salinity is adjusted by adding artificial sea salts to the 100%
effluent, and preparing a serial dilution using 20%o seawater (natural
seawater, hypersaline brine, or artificial seawater). The salinity of
the initial 4000 mL of 100% effluent is adjusted to 20%o by adding 80 g
of dry artificial sea salts (HW MARINEMIX or FORTY FATHOMS®), and mixing
for 1 h. Test concentrations are then made by mixing appropriate
volumes of salinity-adjusted effluent and 20%> salinity dilution water
to provide 4000 mL of solution for each concentration. If hypersaline
brine alone <100%o) is used to adjust the salinity of the effluent, the
highest concentration of effluent that could be achieved would be 80% at
2 20%o salinity, and 70% at 30%o salinity.
The same procedures would be followed in preparing test concentrations
at other salinities between 20%o and 30%o: (1) The salinity of the bulk
(initial) effluent sample would be adjusted to the appropriate salinity
using artificial sea salts or hypersaline brine, and (2) the remaining
effluent concentrations would be prepared by serial dilution, using a
large batch (10 L) of seawater for dilution water, which had been
prepared at the same salinity as the effluent, using natural seawater,
hypersaline and deionized water.
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Monitoring Systems Laboratory, Cincinnati, OH 45268; 513-569-7325. A sample
of newly hatched Artemia nauplii from each new batch of cysts should be
chemically analyzed. The Artemia cysts should not be used if the
concentration of total organic chlorine pesticides exceeds 0.15 ng/g wet
weight or the total concentration of organochlorine pesticides plus PCBs
exceeds 0.30 pg/g wet weight. (For analytical methods see USEPA, 1982).
12.6.11.3 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or a solution prepared by adding 35.0 g
uniodized salt (NaCl) or artificial sea salts to 1 L of deionized
water, to a 2-L separatory funnel, or equivalent.
2. Add 10 ml Artemia cysts to the separatory funnel and aerate for
24 h at 27°C. (Hatching time varies with incubation temperature
and the geographic strain of Artemia used (see USEPA, 1985d,
USEPA, 1993a; and ASTM, 1993).
3. 'After 24 h, cut off the air supply in the separatory funnel.
Artemia nauplii are phototactic, and will concentrate at the
bottom of the funnel if it is covered for 5-10 minutes. To
prevent mortality, do not leave the concentrated nauplii at the
bottom of the funnel more than 10 min without aeration.
4. Drain the nauplii into a beaker or funnel fitted with a < 150 /M
NITEX® or stainless steel screen, and rinse with seawater or
equivalent before use.
12.6.11.4 Testing Artemia nauplii as food for toxicity te;st organisms.
12.6.11.4.1 The primary criterion for acceptability of each new supply of
brine shrimp cysts is the ability of the nauplii to support good survival and
growth of the sheepshead minnow larvae. The larvae used to evaluate the
suitability of the brine shrimp nauplii must be of the same geographical
origin, species, and stage of development as those used routinely in the
toxicity tests. Sufficient data to detect differences in survival and growth
should be obtained by using three replicate test vessels, each containing a
'minimum of 15 larvae, for each type of food.
i
i
12.6.11.4.2 The feeding rate and frequency, test vessels, volume of control
water, duration of the test, and age of the nauplii at the start of the test,
should be the same as used for the routine toxicity tests.
i
12.6.11.4.3 Results of the brine shrimp nutrition assay, 'where there are only
two treatments, can be evaluated statistically by use of ai t test. The "new"
food is acceptable if there are no statistically significant differences in
the survival and growth of the larvae fed the two sources of nauplii.
12.6.11.4.4 The average seven-day survival of larvae should be 80% or
greater, and (2) the average dry weight of larvae should be 0.60 mg or
greater, if dried and weighed immediately after the test, or (3) the average
dry weight of larvae should be 0.50 mg or greater, if the larvae are preserved
in 4% formalin before drying and weighing. The above minimum weights presume
that the age of the larvae at the start of the test is not; greater than 24 h.
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12.6.12 TEST ORGANISMS, SHEEPSHEAD MINNOWS, CYPRINODON VARIEGATUS
12.6.12.1 Brood Stock
12.6.12.1.1 Adult sheepshead minnows for use as brood stock may be obtained
by seine in Gulf of Mexico and Atlantic coast estuaries, from commercial
sources, or from young fish raised to maturity in the laboratory. Feral brood
stocks and first generation laboratory fish are preferred, to minimize
inbreeding.
12.6.12.1.2 To detect disease and to allow time for acute mortality due to
the stress of capture, field-caught adults are observed in the laboratory a
minimum of two weeks before using as a source of gametes. Injured or diseased
fish are discarded.
12.6.12.1.3 Sheepshead minnows can be continuously cultured in the laboratory
from eggs to adults. The larvae, juvenile, and adult fish should be kept in
appropriate size rearing tanks, maintained at ambient laboratory temperature.
The larvae should be fed sufficient newly hatched Artemia nauplii daily to
assure that live nauplii are always present. Juveniles are fed frozen adult
brine shrimp and a commercial flake food, such as TETRA SM-80®, available from
Tetra Sales (U.S.A), 201 Tabor Road, Morris Plains, NJ 07950; 800-526-0650, or
MARDEL AQUARIAN® Tropical Fish Flakes, available from Mardel Laboratories,
Inc., 1958 Brandon Court, Glendale Heights, IL 60139; 312-351-0606, or
equivalent. Adult fish are fed flake food three or four times daily,
supplemented with frozen adult brine shrimp.
12.6.12.1.3.1 Sheepshead minnows reach sexual maturity in three-to-five
months after hatch, and have an average standard length of approximately 27 mm
for females and 34 mm for males. At this time, the males begin to exhibit
sexual dimorphism and initiate territorial behavior. When the fish reach
sexual maturity and are to be used for natural spawning, the temperature
should be controlled at 18-20°C.
12.6.12.1.4 Adults can be maintained in natural or artificial seawater in a
flow-through or recirculating, aerated system consisting of an all-glass
aquarium, or a "Living Stream" (Figid Unit, Inc., 3214 Sylvania Avenue,
Toledo, OH 43613; 419-474-6971), or equivalent.
12.6.12.1.5 The system is equipped with an undergravel or outside biological
filter-of shells (see Spotte, 1973 or Bower, 1983 for conditioning the
biological filter), or a cartridge filter, such as a MAGNUM® Filter, available
from Carolina Biological Supply Co., Burlington, NC 27215; 800-334-5551, or an
EHEIM® Filter, available from Hawaiian Marine Imports Inc., P.O. Box 218687,
Houston, TX 77218; 713-492-7864, or equivalent, at a salinity of 20-30%o and a
photoperiod of 16 h light/8 h dark.
12.6.12.2 Obtaining Embryos for Toxicity Tests
12.6.12.2.1 Embryos can be shipped to the laboratory from an outside source
or obtained from adults held in the laboratory. Ripe eggs can be obtained
either by natural spawning or by intraperitoneal injection of the females with
134
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human chorionic gonadotrophin (HCG) hormone, available from United States
Biochemical Corporation, Cleveland, OH 44128; 216-765-5000. If the culturing
system for adults is temperature controlled, natural spawning can be induced.
Natural spawning is preferred because repeated spawnings can be obtained from
the same brood stock, whereas with hormone injection, the brood stock is
sacrificed in obtaining gametes.
12.6.12.2.2 It should be emphasized that the injection and hatching schedules
given below are to be used only as guidelines. Response to the hormone varies
from stock to stock and with temperature. Time to hatch and percent hatch
also vary among stocks and among batches of embryos obtained from the same
stock, and are dependent on temperature, DO, and salinity.
.
12.6.12.2.3 Forced Spawning
.
12.6.12.2.3.1 HCG is reconstituted with sterile saline or Ringer's solution
immediately before use. The standard HCG vial contains 1,000 IU to be
reconstituted in 10 ml of saline. Freeze-dried HCG which comes with
premeasured and sterilized saline is the easiest to use. Use of a 50 IU dose
requires injection of 0.05 ml of reconstituted hormone solution.
Reconstituted HCG may be used for several weeks if kept in the refrigerator.
12.6.12.2.3.2 Each female is injected intraperitoneally with 50 IU HCG on two
consecutive days, starting at least 4 days prior to the beginning of a test.
Two days following the second injection, eggs are stripped from the females
and mixed with sperm derived from excised macerated testes. At least ten
females and five males are used per test to ensure that there is a sufficient
number of viable embryos.
12.6.12.2.3.3 HCG is injected into the peritoneal cavity, just below the
skin, using as small a needle as possible. A 50 IU dose is recommended for
females approximately 27 mm in standard length. A larger or smaller dose may
be used for fish which are significantly larger or smaller than 27 mm. With
injections made on days one and two, females which are held at 25°C should be
ready for stripping on Day 4. Ripe females should show pronounced abdominal
swelling, and release at least a few eggs in response to a gentle squeeze.
Injected females should be isolated from males. It may be helpful if fish
that are to be injected are maintained at 20°C before injection, and the
temperature raised to 25°C on the day of the first injection.
12.6.12.2.3.4 Prepare the testes immediately before stripping the eggs from
the females. Remove the testes from three-to-five males. The testes are
paired, dark grey organs along the dorsal mid!ine of the abdominal cavity. . If
the head of the male is cut off and pulled away from the rest of the fish,
most of the internal organs can be pulled out of the body cavity, leaving the
testes behind. The testes are placed in a few ml of seawater until the eggs
are ready.
•
12.6.12.2.3.5 Strip the eggs from the females, into a dish containing 50-100
ml of seawater, by firmly squeezing the abdomen. Sacrifice the females and
remove the ovaries if all the ripe eggs do not flow out freely. Break up any
i
135
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clumps of ripe eggs and remove clumps of ovarian tissue and underripe eggs.
Ripe eggs are spherical, approximately 1 mm in diameter, and almost clear.
12.6.12.2.3.6 While being held over the dish containing the eggs, the testes
are macerated in a fold of NITEX® screen (250-500 urn mesh) dampened with
seawater. The testes are then rinsed with seawater to remove the sperm from
tissue, and the remaining sperm and testes are washed into the dish. Let the
eggs and milt stand together for 10-15 min, swirling occasionally.
12.6.12.2.3.7 Pour the contents of the dish into a beaker, and insert an
airstone. Aerate gently, such that the water moves slowly over the eggs, and
incubate at 25°C for 60-90 min. After incubation, wash the eggs on a NITEX®
screen and resuspend them in clean seawater.
12.6.12.2.4 Natural Spawning
12.6.12.2.4.1 Short-term (Demand) Embryo Production
12.6.12.2.4.1.1 Adult fish should be maintained at 18-20°C in a temperature
controlled system. To obtain embryos for a test, adult fish (generally, at
least eight-to-ten females and three males) are transferred to a spawning
chamber, with a photoperiod of 16 h light/8 h dark and a temperature of 25°C,
two days before the beginning of the test. The spawning chambers are
approximately 20 X 35 X 22 cm high (USEPA, 1978), and consist of a basket of
3-5 mm NITEX® mesh, made to fit into a 57-L (15 gal) aquarium. Spawning
generally will begin within 24 h or less. The embryos will fall through the
bottom of the basket and onto a collecting screen (250-500 /jm mesh) or tray
below the basket. The collecting tray should be checked for embryos the next
morning. The number of eggs produced is highly variable. The number of
spawning units required to provide the embryos needed to perform a toxicity
test is determined by experience. If the trays do not contain sufficient
embryos after the first 24 h, discard the embryos, replace the trays, and
collect the embryos for another 24 h or less. To help keep the embryos clean,
the adults are fed while the screens are removed.
12.6.12.2.4.1.2 The embryos are collected in a tray placed on the bottom of
the tank. The collecting tray consists of ± 150 /^m NITEX® screen attached to
a rigid plastic frame. The collecting trays with newly-spawned, embryos are
removed from the spawning tank, and the embryos are collected from the screens
by washing them with -a wash bottle or removing them with a fine brush. The
embryos from several spawning units may be pooled in a single container to
provide a sufficient number to conduct the test(s). The embryos are
transferred into a petri dish or equivalent, filled with fresh culture water,
and are examined using a dissecting microscope or other suitable magnifying
device. Damaged and infertile eggs are discarded (see Figure 2). It is
strongly recommended that the embryos be obtained from fish cultured in-house,
rather than from outside sources, to eliminate the uncertainty of damage
caused by shipping and handling that may not be observable, but which might
affect the results of the test.
12.6.12.2.4.1.3 After sufficient embryos are collected for the test, the
adult fish are returned to the (18-20°C) culture tanks.
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12.6.12.2.4.2 Sustained Natural Embryo Production
12.6.12.2.4.2.1 Sustained (long-term), daily, embryo production can be
achieved by maintaining mature fish in tanks, such as a (2I85--L or 75-gal)
LIVING STREAM® tank, at a temperature of 23-25°C. Embryos are produced daily,
and when needed, embryo "collectors" are placed on the bottom of the tank on
the afternoon preceding the start of the test. The next morning, the embryo
collectors are removed and the embryos are washed into a shallow glass culture
dish using artificial seawater.
12.6.12.2.4.2.2 Four embryo collectors, approximately 20 cm X 45 cm, will
approximately cover the bottom of the 285-L tank. The collectors are
fabricated from plastic fluorescent light fixture diffusors (grids), with
cells approximately 14 mm deep X 14 mm square. A screen consisting of 500 /*m
mesh is attached to one side (bottom) of the grid with silicone adhesive. The
depth and small size of the grid protects the embryos from predation by the
adult fish.
12.6.12.2.4.2.3 The brood stock is replaced annually with feral stock.
12.6.12.2.5 Test Organisms I
12.6.12.2.5.1 Embryos spawned over a less than 24-h period, are used for the
test. These embryos may be used immediately to start a test or may be placed
in a suitable container and transported for use at a remote location. When
overnight transportation is required, embryos should be obtained when they are
no more than 8-h old. This permits the tests at the remote site to be started
with less than 24-h old embryos. Embryos should be transported or shipped in
clean, insulated containers, in well aerated or oxygenated fresh seawater or
aged artificial sea water of correct salinity, and should be protected from
extremes of temperature and any other stressful conditions during transport.
Instantaneous changes of water temperature when embryos are transferred from
culture unit water to test dilution water, or from transport container water
to on-site test dilution, should be less than 2°C. Instantaneous changes of
pH, dissolved ions, osmotic strength, and DO should also be kept to a minimum.
12.6.12.2.5.2 The number of embryos needed to start the test will depend on
the number of tests to be conducted and the objectives. If the test is
conducted with four replicate test chambers (minimum of three) at each
toxicant concentration and in the control, with 15 embryos; (minimum of 10) in
each test chamber, and the combined mortality of embryos prior to the start of
the test is less than 20%, 400 viable embryos are required for the test.
12.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
12.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
12.8 CALIBRATION AND STANDARDIZATION
12.8.1 See Section 4, Quality Assurance.
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12.9 QUALITY CONTROL
12.9.1 See Section 4, Quality Assurance.
12.10 TEST PROCEDURES
12.10.1 TEST SOLUTIONS
12.10.1.1 Receiving Waters
12.10.1.1.1 The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 urn NIT.EX® filter and compared without
dilution, against a control. Using four replicate chambers per test, each
containing 400-500 mL, and 400 mL for chemical analysis, would require
approximately 2.0-2.5 L or more of sample per test per day.
12.10.1.2 Effluents
12.10.1.2.1 The selection of the effluent test concentration should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used. A
dilution factor of 0.5 provides precision of ± 100%, and allows for testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used. Therefore, USEPA recommends the
use of the z. 0.5 dilution factor. If 100%o salinity MSB is used as a diluent,
the maximum concentration of effluent that can be tested will be 80% at 20%o
and 70% at 30%o salinity.
12.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first
l-to-2 h of the test, additional dilutions at the lower range of effluent
concentrations should be added.
12.10.1.2.3 The volume of effluent required to initiate the test and for
daily renewal of four replicates (minimum of three) per concentration for five
concentrations of effluent and a control, each containing 400 mL of test
solution, is approximately 4 L. Prepare enough test solution (approximately
3000 mL) at each effluent concentration to refill the test chambers and
provide at least 400 mL additional volume for chemical analyses.
12.10.1.2.4 Maintain the effluent at 4°C. Plastic containers such as 8-20 L
cubitainers have proven successful for effluent collection and storage.
12.10.1.2.5 Just prior to test initiation (approximately 1 h), the
temperature of a sufficient quantity of the sample(s) to make the test
solutions should be adjusted to the test temperature (25 ± 1°C) and maintained
at that temperature during the addition of dilution water.
138
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12.10.1.2.6 Higher effluent concentrations (i.e., 25%, 50%, and 100%) may
require aeration to maintain adequate dissolved oxygen concentrations.
However, if one solution is aerated, all concentrations must be.aerated.
Aerate effluent as it warms and continue to gently aerate test solutions in
the test chambers for the duration of the test.
12.10.1.2.7 Effluent dilutions should be prepared for all replicates in each
treatment in one beaker to minimize variability among the replicates. The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.
12.10.1-.3 Dilution Water
12.10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving
water), HSB prepared from natural seawater, or artifical seawater prepared
from FORTY FATHOMS® or GP2 sea salts (see Table 3 and Section 7, Dilution
Water). Other artifical sea salts may be used for culturing sheepshead
minnows if the control criteria for acceptability of test data are satisfied.
12.10.2 START OF THE TEST
I
12.10.2.1 Tests should begin as soon as possible, preferably within 24 h
after sample collection. For on-site toxicity studies, no more than 24 h
should elapse between collection of the effluent and use in an embryo-larval
study. The maximum holding time following retrieval of the sample from the
sampling device should not exceed 36 h for off-site toxicity studies unless
permission is granted by the permitting authority. In no case should the
sample be used in a test more than 72 h after sample collection (see Section
8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests).
12.10.2.2 Label the test chambers with a marking pen. Use color-coded tape
to identify each treatment and replicate. A minimum of five effluent
concentrations and a control are used for each effluent test. Each
concentration (including controls) is to have four replicates (minimum of
three). Use 500 ml beakers, crystallization dishes, nontoxic disposable
plastic labware, or equivalent for test chambers.
12.10.2.3 Prepare the test solutions (see Table 1) and add to the test
chambers.
12.10.2.4 Gently agitate and mix the embryos to be used in the test in a
large container so that eggs from different spawns are evenly dispersed.
12.10.2.5 The test is started by randomly placing embryos from the common
pool, using a small bore (2 mm), fire polished, glass tube calibrated to
contain approximately the desired, number of embryos, into each of four
replicate test chamber, until each chamber contains 15 embryos (minimum of
10), for a total of 60 embryos (minimum of 30) for each concentration (four
replicates recommended, three minimum) (see Appendix A). The amount of water
added to the chambers when transferring the embryos should be kept to a
minimum to avoid unnecessary dilution of the test concentrations.
139
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12.10.2.6 After the embryos have been distributed to each test chamber,
examine and count them. Remove and discard damaged or infertile eggs and
replace with undamaged embryos. It may be more convenient and efficient to
transfer embryos to intermediate containers of dilution water for examination
and counting. After the embryos have been examined and counted in the
intermediate container, assign them to the appropriate test chamber and
transfer them with a minimum of dilution water.
12.10.2.7 Randomize the position of the test chambers at the beginning of the
test (see Appendix A). Maintain the chambers in this configuration throughout
the test. Preparation of a position chart may be helpful.
12.10.3 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE
12.10.3.1 The light quality and intensity should be at ambient laboratory
levels, approximately 10-20 ^E/m/s, or 50 to 100 foot candles (ft-c), with a
photoperiod of 16 h of light and 8 h of darkness. The test water temperature
should be maintained at 25 ± 1°C. The salinity should be 5%o to 32%o ± 2%o to
accommodate receiving waters that may fall within this range. The salinity
should vary no more than ± 2%o among the chambers on a given day. If
effluent and receiving water tests are conducted concurrently, the salinities
of these tests should be similar.
12.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
12.10.4.1 Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain satisfactory DO. The DO should not fall
below 4.0 mg/L (see Section 8, Effluent and Receiving Water Sampling, Sample
Holding, and Sample Preparation for Toxicity Tests). If it is necessary to
aerate, all treatments and the control should be aerated. The aeration rate
should not exceed 100 bubbles/min, using a pipet with a 1-2 mm orifice, such
as a 1-mL KIMAX® Serological Pipet No. 37033, or equivalent. Care should be
taken to ensure that turbulence resulting from the aeration does not cause
undue physical stress to the fish.
12.10.5 FEEDING
12.10.5.1 Feeding is not required.
12.10.6 OBSERVATIONS DURING THE TEST
12.10.6.1 Routine Chemical and Physical Determinations
12.10.6.1.1 DO is measured at the beginning and end of each 24-h exposure
period at each test concentration and in the control.
12.10.6.1.2 Temperature, pH, and salinity are measured at the end of each
24-h exposure period in one test chamber at each test concentration and in the
control. Temperature should also be monitored continuously or observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
140
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chambers at least at the end of. the test to determine the temperature
variation in the environmental chambers.
12.10.6.1.3 The pH is measured in the effluent sample each day before new
test solutions are made.
12.10.6.1.4 Record all measurements on the data sheet (Figure 1).
12.10.6.2 Routine Biological Observations
12.10.6.2.1 At the end of the first 24 h of exposure, before renewing the
test solutions, examine and count the embryos. Remove the dead embryos (milky
colored and opaque) and record the number. If the rate of mortality or fungal
infection exceeds 20% in the control chambers, or if excessive
nonconcentration related mortality occurs, terminate the test and start a new
test with new embryos. If the above mortality conditions do not occur,
continue the test for the full nine days.
12 10 6 2.2 At 25°C, hatching begins on about the sixth day,. After hatching
begins, count the number of dead and live embryos and the .number of hatched,
dead, live, and deformed and/or debilitated larvae, dally (see Figure 2 for
illustrations of morphological development of embryo and larva). Deformed
larvae are those with gross morphological abnormalities such as curved spines,
lack of appendages, lack of fusiform shape (non-distinct mass), a colored
beating heart in an opaque mass, lack of mobility, abnormal swimming, or other
characteristics that preclude survival. Remove dead embryos and dead and
deformed larvae as previously discussed and record the numbers for all test
observations (see Figure 2).
12 10 6 2 3 Protect the embryos and larvae from unnecessary disturbance
during the test by carefully carrying out the daily test observations,
solution renewals, and removal of dead organisms. Make sure the test
organisms remain immersed during the performance of the above operations.
12.10.7 DAILY CLEANING OF TEST CHAMBERS j
12 10 7 1 Since feeding is not required, test chambers are not cleaned daily
unless accumulation of particulate matter at the bottm of the tank causes a
problem.
12.10.8 TEST SOLUTION RENEWAL
12 10 8 1 The test solutions are renewed daily using freshly prepared
solution, immediately after cleaning the test chambers. For on-site toxicity
studies, fresh effluent and receiving water samples used in toxicity tests
should be collected daily, and no more than 24 h should elapse between
collection of the sample and use in the test (see Section 8,. Effluent and
Receiving Water Sampling, Sample Handling, and Sample Preparation for Toxicity
Tests). For off-site tests, a minimum of three samples must be collected,
preferably on days one, three, and five. Maintain the samples at 4°C until
used.
i
141
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Figure 2. Embryonic development of sheepshead minnow, Cypn'nodon variegatus:
A. Mature unfertilized egg, showing attachment filaments and
micropyle, X33; B. Blastodisc fully developed; C,D. Blastodisc, 8
cells; E. Blastoderm, 16 cells; F. Blastoderm, late cleavage
stage; 6. Blastoderm with germ ring formed, embryonic shield
developing; H. Blastoderm covers over 3/4 of yolk, yolk noticeably
constricted; I. Early embryo. From Kuntz (1916).
142
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Figure 2. Embryonic development of sheepshead minnow, Cypnnodonvanegatus:
J Embryo 48 h after fertilization, now segmented throughout,
pigment on yolk sac and body, otoliths formed; K. Posterior
portion of embryo free from yolk and moves freely within egg
membrane, 72 h after fertilization; L. Newly hatched fish, actual
length 4 mm; M. Larval fish 5 days after hatching, actual length 5
mm; N. Young fish 9 mm in length; 0. Young fish 12 mm in length
(CONTINUED). From Kuntz (1916). |
143
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12.10.8.2 The test solutions are adjusted to the correct salinity and renewed
mof waie? £eiS£# -0lJhCte? SfPlf' DuHng the dail* renewal process? 7^0
UL»f« £ 1 r •" th^ chamber to ensure that the embryos and larvae
SXSJ KUbm^d dm;in9 uthe ^newal process. New test solution (400 ml)
pynnldnn%h dKSl°Wly ty Pour1n9 down the side of the test chamber to avoid
exposing the embryos and larvae to excessive turbulence.
12.10.8.3 Prepare test solutions daily, making a minimum of five
concentrates and a control. If concurrent effluent and receiving water
testing occurs, the effluent test salinity should closely approximate that of
the receiving water test. If an effluent is tested alone, selec^a salinity
which approximately matches the salinity of the receiving waters Table 1
!U?S? ?S quantities of effluent, seawater, deionized water, and
artificial sea salts needed to prepare 3 L of test solution at each effluent
concentration for tests conducted at 20%o salinity.' ernuent
12.10.9 TERMINATION OF THE TEST
JnJ^A ThS-*uSt iS terminated after nine days of exposure, or four days
post-hatch, whichever comes first. Count the number of surviving dead and
deformed and/or debilitated larvae, and record the numbers of ewh. The
nJSlTVJT6 ale treated as dead- Keep a separate record of the total
solution med larvae for use in ^porting the teratogenicity of the test
12.11 ACCEPTABILITY OF TEST RESULTS
^ aCC6ptable' SUrvival in the controls must
12.12 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
°f teSt C0nd1tions and test acceptability criteria is
12.13 DATA ANALYSIS
12.13.1 GENERAL
12.13.1.1 Tabulate and summarize the data.
JH?'1;.2 Tue endP°ints of this toxicity test are based on total mortality
combined number of dead embryos, dead larvae, and deformed larvae. The EC
endpoints are calculated using Probit Analysis (Finney, 1971) LOEC and NOFr
values, for total mortality, are obtained Jslng a hypothes s test approach
(Steel'5 ?9^6tS lle^lpsn (°?M^ J955) or'steel's MarJ-owRanTSs*?
^teei, 1959, Miller, 1981). See the Appendices for examples of the manual
computations, program listings, and examples of data input and program Su?put.
144
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TABLE 2 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL
SURVIVAL AND TERATOGENICITY TEST WITH EFFLUENTS AND RECEIVING
WATERS
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photpperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. of embryos per chamber:
12. No. replicate test chambers
per concentration:
13. No. embryos per concentration:
14. Feeding regime:
15. Aeration:
16. Dilution water:
17. Test concentrations:
Static renewal
5%o to 32%o (± 2%o of the selected
test salinity)
25 ± 1°C
Ambient laboratory "light
i
10-20 AiE/m2/s, or 50-100 ft-c (ambient
laboratory levels)
16 h light, 8 h darkness
400-500 mL
250-400 mL per replicate (loading and
DO restrictions must be met)
Daily
less than 24 h old
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
Feeding not required
None unless DO falls below 4.0 mg/L
Uncontaminated source of natural
seawater; deionized water mixed with
hypersaline brine or artificial sea
salts (HW Marinemix®, FORTY FATHOMS®,
GP2, or equivalent)
Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water
or minimum of 5 and a control
145
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TABLE 2.
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL
™^NN?lS{°GENICITV TEST WITH EFFLUENTS AN° ««*'«*
18. Dilution factor:
19. Test duration:
20. Endpoints:
21. Test acceptability criteria:
22. Sampling requirements:
23. Sample volume required:
Effluent: > 0.5
Receiving waters:
9 days
None, or > 0.5
Percent hatch; percent larvae dead or
with debilitating morphological and/or
behavior abnormalities such as: gross
deformities; curved spine;
disoriented, abnormal swimming
behavior; surviving normal larvae from
original embryos
80% or greater survival in controls
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 are 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, Subsection 8.5.4)
5 L per day
146
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12 13 1 3 The statistical tests described here must be used with a knowledge
of'the assumptions upon which the tests are contingent. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
12 13 2 EXAMPLE OF ANALYSIS OF SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS,
EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY DATA
12 13 2.1 Formal statistical analysis of the total mortality data is outlined
in Fiqure 3. The response used in the analysis is the total mortality
proportion in each test or control chamber. Separate analyses are performed
for the estimation of the NOEC and LOEC endpoints and for the estimation of
the EC endpoint. Concentrations at.which there ^ 100% mortality in all of
the test chambers are excluded from the statistical analysis of the NOEC and
LOEC, but included in the estimation of the EC endpoints.
12.13.2.2 For the case of equal numbers of replicates across; all
concentrations and the control, the evaluation of the NOEC arid LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity ot
variance, are formally tested. The test for normality is the Shapiro-Wilk s
Test, and Bartlett's Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.
12 13 2 3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment (see
Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.
12 13 2 4 Probit Analysis (Finney, 1971; see Appendix 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 tit the
Probit Analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber
Method or the Graphical Method may be used (see Appendice H-K).
i
12.13.2.5 Example of Analysis of Survival Data -
12 13.2.5.1 The data for this example are listed in Table 3. Total
mortality, expressed as a proportion (combined total number of dead embryos,
dead larvae and deformed larvae divided by the number of embryos at start of
test) is the response of interest. The total mortality proportion in each
replicate must first be transformed by the arc sine square root transformation
procedure described in Appendix B. The raw and transformed data, means and
variences of the transformed observations at each SDS concentration and
control are listed in Table 3. A plot of the data is provided in Figure 4.
Since there is 100% total mortality in all replicates for the 8.0 mg/L
concentration, it is not included in this statistical analysis and is
considered a qualitative mortality effect.
147
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STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW EMBRYO-LARVAL
SURVIVAL AND TERATOGENICITY TEST
TOTAL MORTALITY
TOTAL NUMBER OF DEAD EMBRYOS,
DEAD LARVAE, AND DEFORMED LARVAE
PROBIT ANALYSIS
ARC SINE
TRANSFORMATION
ENDPOINT ESTIMATE
EC1
SHAPIRO-WILJCSTEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
EQUAL NUMBER OF
REPLICATES?
NO
HETEROGENEOUS
VARIANCE
YES
EQUAL NUMBER OF
REPLICATES?
YES
NO
TWITH
ERRONI
TMENT
DUNNETTS
TEST
STEEL'S MANY-ONE
RANK TEST
r-J
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 3. Flowchart for statistical analysis of sheepshead minnow,
Cyprinodon varlegatus, embryo-larval survival and teratoqenicity
test. Survival and terata data.
148
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TABLE 3. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL
TOTAL MORTALITY DATA
Concentration (mq/L)
Replicate Control 0.5 1.0
2.0
4.0
8.0
RAW
A
B
c
D
0.
0.
0.
0.
1
0
1
0
0
0
0
0
.0
.2
.2
.1
0
0
0
0
.0
.1
.1
.2
0.3
0.1
0.2
0.4
0
0
0
0
.9
.7
.8
.8
1.0
1.0
1.0
1.0
ARC SINE
TRANS-
FORMED
Mean (Y,-)
i
1
A
B
C
D
0.322
0.159
0.322
0.159
0.241
0.009
1
0.159
0.464
0.464
0.322
0.352
0.021
2
0.159
0.322
0.322
0.464
0.317
0.016
3
0.580
0.322
0.464
0.685
0.513
0.024
4
1.249
0.991
1.107
1.107
1.114
0.011
5
12.13.2.6 Test for Normality
12.13.2.6.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from,each observation in that concentration,! The centered
observations are summarized in Table 4.
TABLE 4. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Replicate
Control
0.5
SDS Concentration (mg/L)
1.0
2.0
4.0 8.0
A
B
C
D
0.081
-0.082
0.081
-0.082
-0.193
0.112
0.112
-0.030
-0.158
0.005
0.005
0.147
0.067
-0.191
0.135
-0.123
-0.049 -0.007
0.172
-0.007
.
-
-
-
149
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w z
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150
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12.13.2.6.2 Calculate the denominator, D, of the statistic:
D= £ (X± - X)
1=1
Where: Xj = the ith centered observation
X = the overall mean of the centered observations
n - the total number of centered observations
12.13.2.6.3 For this set of data, n = 20
1
X =
(-0.005) =0.000
20
D = 0.2428
I
I
12.13.2.6.4 Order the centered observations from smallest to largest
(n)
X < X < ... < X
where XC1) denotes the ith ordered observation.
this example are listed in Table 5.
The ordered observations for
TABLE 5. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
X(D
-0.193
-0.191
-0.158
-0.123
-0.082
-0.082
-0.049
-0.030
-0.007
-0.007
•?
X(i>
11 0.005
12 0.005
13 0.067
14
15
16
0.081
0.081
0.112
17 0.112
18 0.135
19 0.147
20
0.172
151
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12.13.2.6.5 From Table 4, Appendix B, for the number of observations, n,
Ar^'f******'* 4* I* M A«HM ^.£>S A J _ ._. J. ... _ _ _-_!___ I • I J* • f+ m
obtain the coefficients a,, a2, ..., ak where k is n/2 if n is even and
(n-l)/2 if n is odd. For the data in this example, n = 20 and k = 10.
values are listed in Table 6.
The a;
TABLE 6. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
ai
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
0.365
0.338
0.293
0.235
0.194
0.163
0.130
0.097
0.012
0.012
X(20)
x<19)
x<18)
X(17)
X(16)
•X(15>
XCU)
x<13)
x<12>
XC11)
- X(1)
- x(2)
- x<3)
- x<4)
- x(5)
- x(6)
- x(7)
- x(8>
- x(9)
- x(10>
12.13.2.6.6 Compute the test statistic, W, as follows:
w=± ~
D
The differences x(n"i+1) - Xci) are listed in Table 6. For the data in this
example,
W
1 (0.4807)2 = 0.952
0.2428
12.13.2.6.7 The decision rule for this test is to compare W as calculated in
Section 13.2.6.6 to a 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 the data in this example, the critical value at a
significance level of 0.01 and n = 20 observations is 0.868. Since W = 0.952
is greater than the critical value, conclude that the data are normally
distributed.
12.13.2.7 Test for Homogeneity of Variance
12.13.2.7.1 The test used to examine whether the variation in mean proportion
mortality is the same across all toxicant concentrations including the
152
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control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic
is as follows:
in
In
B =
Where: V,- =
degrees of freedom for each copper concentration and
control, V,- = (nf - 1)
p = number of concentration levels including the control
In = loge
i = 1, 2, ..., p where p is the number of concentrations
including the control
n- = the number of replicates for concentration i.
s2 =
c =
1=1
1=1
1=1
12.13.2.7.2 Since B is approximately distributed as chi-square with p - 1
degrees of freedom when the variances are equal, the appropriate critical
value is obtained from a table of the chi-square distribution for p - 1
degrees of freedom and a significance level of 0.01. If B is less than the
critical value then the variances are assumed to be equal.i
12.13.2.7.3 For the data in this example, M- = 3, p = 5, F2 = 0.0162, and
C = 1.133. The calculated B value is:
B =
(15) [ln(0.0162)]-3Eln<
1.33
15(-4.1227) - 3(-20.9485)
1.33
= 0.886
153
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12.13.2.7.4 Since B is approximately distributed as chi-square with p - 1
degrees of freedom when the variances are equal, the appropriate critical
value for the test is 13.277 for a significance level of 0.01. Since B =
0.886 is less than the critical value of 13.277, conclude that the variances
are not different.
12.13.2.8 Dunnett's Procedure
12.13.2.8.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 7.
TABLE 7. ANOVA TABLE
Source
df
Sum of Squares
(SS)
Mean Square(MS)
(SS/df)
Between
Within
Total
P
N
N
- 1
- P
- 1
SSB
SSW
SST
SB = SSB/(p-l)
S* = SSW/(N-p)
Where: p -= number of SDS concentration levels including the control
N - total number of observations n1 + n2 ... + n
n{ = number of observations in concentration i
SSB = f,Tl/n±-G2/N Between Sum of Squares
1=1
SST = £ ^Ylj-Gz/N Total Sum of Squares
SSW = SST-SSB
Within Sum of Squares
154
-------
the grand total of all sample observations, G
= £**
Y,
ij
the total of the replicate measurements for concentration i
the jth observation for concentration i (represents the
proportion surviving for toxicant concentration i in test
chamber j)
12.13.2.8.2 For the data in this example:
n1 = n2 = n3 = n4 = n5 = 4
N = 20
'11
Y,/ = 0.962
'21 + Y22 + Y23 + Y24 = 1.409
T3 = Y31 + Y32 + Y33 + Y34 = 1.267
' " + Y,, + Y43 + Y44 = 2.051
+ Y53 + Y54 = 4-454
i:: % +
6=1,
T3 + T4 = 10.143
SSB = f^Tl/ni-G2/N
1 (28.561) - (10.143)2 = 1.996
4 20
SST =
YJ.,-G2/N
7.383 - (10.143)
20
2.239
SSW = SST-SSB = 2.239 - 1.996 = 0.243
S* = SSB/(p-l) = 1.996/(5-l) = 0.499
i
I
SJ; = SSW/(N-p) = 0.243/(20-5) = 0.016
12.13.2.8.3 Summarize these calculations in the ANOVA table (Table 8)
155
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TABLE 8. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
df
Sum of Squares
(SS)
Mean Square(MS)
(SS/df)
Between
Within
Total
4
15
19
1.996
0.243
2.239
0.499
0.016
12.13.2.8.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
fci =
Where: Yf = mean proportion surviving for concentration i
Y! * mean proportion surviving for the control
Sw - square root of the within mean square
n1 = number of replicates for the control
n- = number of replicates for concentration i.
Since we are looking for an increased response in percent of total mortality
over control, the control mean is subtracted from the mean at a concentration.
12.13.2.8.5 Table 9 includes the calculated t values for each concentration
and control combination. In this example, comparing the 0.5 mg/L
concentration with the control the calculation is as follows:
(0.352 - 0.241)
[0.1265^(1/4)+(1/4)]
= 1.241
12.13.2.8.6 Since the purpose of this test is to detect a significant
increase in total mortality, a one-sided test is appropriate. The critical
value for this one-sided test is found in Table 5, Appendix C. For an overall
alpha level of 0.05, 15 degrees of freedom for error and four concentrations
(excluding the control) the critical value is 2.36. The mean proportion of
156
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TABLE 9. CALCULATED T VALUES
SDS Concentration (mg/L)
0.5
1.0
2.0
4.0
i
2
3
4
5
*'
1.241
0.850
3.041
9.760
total mortality for concentration "i" is considered significantly less than
the mean proportion of total mortality for the control if t,- is greater than
the critical value. Therefore, the 2.0 mg/L and the 4.0 mg/L concentrations
have significantly higher mean proportions of total mortality than the
control. Hence the NOEC is 1.0 mg/L and the LOEC is 2.0 mg/L.
12.13.2.8.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
MSD = d SCL/nJ + (l/n)
Where: d = the critical value for Dunnett's procedure
'
Sw = the square root of the within mean square [
n, = the number of replicates in the control. \
n = The common number of replicates at each concentration (this
assumes equal replication at each concentration)
12.13.2.8.8 In this example:
MSD = 2.36 (0.1265) v/d/4) + (1/4)
= 2.36 (0.1265)(0.7071)
= 0.211
12.13.2.8.9 The MSD (0.450) is in transformed units. To determine the MSD in
terms of percent survival, carry out the following conversion.
157 !
-------
1. Add the MSD to the transformed control mean.
0.241 + 0.211 = 0.452
2. Obtain the untransformed values for the control mean and the sum
calculated in 1. -
[ Sine (0.241) ]2 = 0.057
[ Sine (0.452) ]2 = 0.191
3. The untransformed MSD (MSDU) is determined by subtracting the
untransformed values from step 2.
MSDU = 0.191 - 0.057 = 0.134
12.13.2.8.10 Therefore, for this set of data, the minimum difference in mean
proportion of total mortality between the control and any SDS concentration
that can be detected as statistically significant is 0.134.
12.13.2.8.11 This represents a 268% increase in mortality from the control.
12.13.2.9 Calculation of the LC50
12.13.2.9.1 The data used for the Probit Analysis is summarized in Table 10.
To perform the Probit Analysis, run the USEPA Probit Analysis Program.
An example of the program input and output is supplied in Appendix H.
TABLE 10. DATA FOR PROBIT ANALYSIS
SDS Concentration (mq/L)
Number Dead
Number Exposed
Control 0.5
4 4
40 40
1.0 2.0 4.0
28 32
40 40 40
8.0
40
40
12.13.2.9.2 For this example, the chi-square test for heterogeneity was not
significant. Thus Probit Analysis appears appropriate for this set of data.
12.13.2.9.3 Figure 5 shows the output data for the Probit Analysis of the
data from Table 10 using the USEPA Probit Program.
158
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USEPA PROBIT ANALYSIS PROGRAM
USED FOR CALCULATING LC/EC VALUES
Version 1.5
Probit Analysis of Sheepshead Minnow Embryo-Larval Survival and
Teratogenicity Data
Cone.
Control
0.5000
1.0000
2.0000
4.0000
8.0000
Number
Exposed
40
40
40
40
40
40
Number
Resp.
4
4
2
8
32
40
Observed
Proportion
Responding
0.1000
0.1000
0.0500
0.2000
0.8000
1.0000
Proportion
Responding
Adjusted for
Controls
0.
0,
o!
0,
0000
0174
0372
1265
7816
1.0000
Chi - Square for Heterogeneity (calculated) = 0.883
Chi - Square for Heterogeneity (tabular value) = 7.815
Probit Analysis of Sheepshead Minnow Embryo-Larval Survival and
Teratogenicity Data
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
1.346
3.018
Lower Upper
95% Confidence Limits
0.751
2.539
1.776
3.455
Figure 5. Output for USEPA Probit Program, Version 1.5.
159
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12.14 PRECISION AND ACCURACY
12.14.1 PRECISION
12.14.1.1 Single-Laboratory Precision
12.14.1.1.1 Data on the single-laboratory precision of the sheepshead minnow
embryo-larval survival and teratogenicity test are available for eight tests
with copper sulfate and five tests with sodium dodecyl sulfate (USEPA, 1989a).
The data for the first five tests show that the same NOEC and LOEC,
240 ^g Cu/L and 270 /*g Cu/L, respectively, were obtained in all five tests,
which is the maximum level of precision that can be attained. Three
additional tests (6-8) were performed with narrower (20 /KJ) concentration
intervals, to more precisely identify the threshold concentration. The NOEC
and LOEC for these tests are 200 ^g and 220 /*g Cu/L, respectively. For sodium
dodecyl sulfate, the NOEC's and LOEC's for all tests are 2.0 and 4.0 mg/L,
respectively. The precision, expressed as the coefficient of variation (CV%),
is indicated in Tables 11-12. For copper (Cu), the coefficient of variation,
depending on the endpoint used, ranges from 2.5% to 6.1% which indicates
excellent precision. For sodium dodecyl sulfate (SDS), the coefficient of
variation, depending on the endpoint used, ranges from 11.7% to 51.2%,
indicating acceptable precision.
12.14.1.2 Multilaboratory Precision
12.14.1.2.1
available.
Data on the multilaboratory precision of this test are not yet
12.14.2 ACCURACY
12.14.2.1 The accuracy of toxicity tests cannot be determined.
160
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TABLE 11. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINODON VARIEGATUS, EMBRYO-LARVAL SURVIVAL AND
TERATOGENICITY TEST PERFORMED IN HW MARINEMIX® ARTIFICIAL
SEAWATER, USING EMBRYOS FROM FISH MAINTAINED AND SPAWNED IN HW
MARINEMIX® ARTIFICIAL SEAWATER USING COPPER (CU) SULFATE AS
REFERENCE TOXICANT1'2'3'4'5'6'7
Test
Number (ju<
1
2
3
4
5
6
7
8
n:
Mean:
CV(%):
\ Data from USEPA
^ T*-* <"• 4- t+ r\s\\*£f*u*mr*if\
EC1
EC5
EC10 EC50 NOEC
3/L) UgA) (/*g/L) Ug/L) (/*g/L)
173
*
*
182
171
*
*
195
4
180
6.1
(1988a)
r^\/ T«»rtirti
189
*
*
197
187
*
*
203
4
194
3.8
and USEPA
* Us\1 T 4 t*4"r\\f*
198 234 240
*
*
* 240
* 240
206 240 240
197 234 240
*
*
* < 200
* 220
208 226 220
4
4 7
202 233 NA
2.8
(1991a).
A/iiir»4--ir« D-i/\T/\ri-ioH
2.5 NA
LJ/M i o4- f\n
* -
Facility, Environmental Services Division, Region 6, USEPA, Houston,
Texas. i
Cyprinodon variegatus embryos used in the tests were less than 20 h
old when the tests began. Two replicate test chambers were used for
the control and each toxicant concentration. Ten embryos were
randomly added to each test chamber containing 250 ml. of test or
control water. Solutions were renewed daily. The temperature and
salinity of the test solutions were 24 + 1°C and 20%o, respectively.
Copper test concentrations were prepared using copper sulfate.
Copper concentrations for Tests 1-5 were: 180, 210, 240, 270, and 300
Mg/L- Copper concentrations for Test 6 were: 220, 240, 260, 280, and
300 ^g/L. Copper concentrations for Tests 7-8 were: 200., 220, 240,
260, and 280 ^g/L. Tests were conducted over a two-week period.
Adults collected in the field.
NOEC Range: 200 - 240 ng/L (this represents a difference of two
exposure concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Data did not fit the Probit model.
161
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TABLE 12. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
CYPRINODON VARIEGATUS, EMBRYO-LARVAL SURVIVAL AND
TERATOGENICITY TEST PERFORMED IN HW MARINEMIX® ARTIFICIAL
SEAWATER, USING EMBRYOS FROM FISH MAINTAINED AND SPAWNED IN HW
MARINEMIX® ARTIFICIAL SEAWATER USING SODIUM DODECYL SULFATE
/ c*r\c* \ A r* nrrmrfci^r" Trt v T /"* A HIT ' /1— *•* i^ » ^ *& t •
('
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
SDS) AS REFERENC
EC1
(mg/L)
1.7
*
0.4
1.9
1.3
4
1.3
51.2
E TOXICANT'
EC5
(mg/L)
2.0
*
0.7
2.2
1.7
4
1.6
41.6
EC10
(mg/L)
2.2
*
0.9
2.4
1.9
4
1.9
35.0
EC50
(mg/L)
3.1
*
2.5
3.3
3.0
4
2.9
11.7
NOEC
(mg/L)
2.0
4.0
2.0
2.0
2.0
5
NA
NA
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by Terry Hollister, Aquatic Biologist, Houston Facility,
Environmental Services Division, Region 6, USEPA, Houston, Texas.
Cyprinodon van'egatus embryos used in the tests were less than 20 h old
when the tests began. Two replicate test chambers were used for the
control and each toxicant concentration. Ten embryos were randomly
added to each test chamber containing 250 mL of test or control water.
Solutions were renewed daily. The temperature and salinity of the test
solutions were 24 + 1°C and 20%o, respectively.
SDS concentrations for all tests were: 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L.
Tests were conducted over a three-week period.
Adults collected in the field.
NOEC Range: 2.0 - 4.0 mg/L (this represents a difference of two exposure
concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
- Data did not fit the Probit model.
162
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SECTION 13
TEST METHOD
I
INLAND SILVERSIDE, HENIDIA BERYLLINA, LARVAL SURVIVAL AND GROWTH
METHOD 1006.0
13.1 SCOPE AND APPLICATION
13.1.1 This method estimates the chronic toxicity of effluents and receiving
waters to the inland silverside, Mem'dia beryllina, using seven-to-eleven day
old larvae in a seven day, static renewal test. The effects include the
synergistic, antagonistic, and additive effects of all the chemical, physical,
and biological components which adversely affect the physiological and
biochemical functions of the test species.
13.1.2 Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LCBOs).
13.1.3 Detection limits of the toxicity of an effluent or chemical substance
are organism dependent.
13.1.4 Brief excursions in toxicity may not be detected using 24-h composite
samples. Also, because of the long sample collection period involved in
composite sampling, and because the test chambers are not sealed, highly
volatile and highly degradable toxicants present in the source may not be
detected in the test.
I
13.1.5 This test is commonly used in one of two forms: (1) a definitive test,
consisting of a minimum of five effluent concentrations and a control, and (2)
a receiving water test(s), consisting of one or more receiving water
concentrations and a control.
13.2 SUMMARY OF METHOD
13.2.1 Inland silverside, Mem'dia berylTina, 7 to 11-d old larvae are exposed
in a static renewal system for seven days to different concentrations of
effluent or to receiving water. Test results are based on the survival and
growth of the larvae.
13.3 INTERFERENCES
13.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
13.3.2 Adverse effects of low dissolved oxygen (DO) concentrations, high
concentrations of suspended and/or dissolved solids, and extremes of pH, may
mask or confound the effects of toxic substances.
163
-------
13.3.3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
13.3.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
13.3.5 Food added during the test may sequester metals and other toxic
substances and confound test results.
13.4 SAFETY
13.4.1 See Section 3, Health and Safety.
13.5 APPARATUS AND EQUIPMENT
13.5.1 Facilities for holding and acclimating test organisms.
13.5.2 Brine shrimp, Artemia, culture unit -- see Subsection 13.6.16 below
and Section 4, Quality Assurance.
13.5.3 Menidia beryllina culture unit -- see Subsection 13.6.17 below,
Middaugh and Hemmer (1984), Middaugh et al. (1986), USEPA (1987g) and USEPA
(1993a) for detailed culture methods. This test requires from 180 to 360 7 to
11 day-old larvae. It is preferable to obtain the test organisms from an in-
house culture unit. If it is not feasible to culture fish in-house, embryos
or larvae can be obtained from other sources by shipping them in well
oxygenated saline water in insulated containers.
13.5.4 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.
13.5.5 Environmental chamber or equivalent facility with temperature control
(25 ± 1-C).
13.5.6 Water purification system -- Mi Hi pore Milli-Q®, deionized water (DI)
or equivalent.
13.5.7 Balance, analytical -- capable of accurately weighing to 0.00001 g.
13.5.8 Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of the weighing pans and the
expected weights of the weighing pans plus fish.
13.5.9 Drying oven -- 50-105°C range, for drying larvae.
13.5.10 Air pump -- for oil-free air supply.
13.5.11 Air lines, plastic or pasteur pipettes, or air stones -- for gently
aerating water containing the fragile larvae or for supplying air to test
solution with low DO.
164
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13.5.12 Meters, pH and DO -- for routine physical and chemical measurements.
13.5.13 Standard or micro-Winkler apparatus -- for calibrating DO (optional).
13.5.14 Desiccator -- for holding dried larvae.
13.5.15 Light box -- for counting and observing larvae.
13.5.16 Refractometer -- for determining salinity.
13.5.17 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.
13.5.18 Thermometers, bulb-thermograph or electronic chart type -- for
continuously recording temperature.
.
13.5.19 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
13.5.20 Test chambers -- four (minimum of three) chambers per concentration
The chambers should be borosilicate glass or nontoxic disposable plastic
labware. To avoid potential contamination from the air and excessive
evaporation of test solutions during the test, the chambers should be covered
during the test with safety glass plates or sheet plastic (6 mm thick).
13.5.20.1 Each test chamber for the inland silverside should contain a
minimum of 750 ml of test solution. A modified Norberg and Mount (1985)
chamber (Figure 1), constructed of glass and silicone cement, has been used
successfully for this test. This type of chamber holds an adequate column of
test solution and incorporates a sump area from which test solutions can be
siphoned and renewed without disturbing the fragile inland silverside larvae
Modifications for the chamber are as follows: 1) 200 ^m mesh NITEX® screen
instead of stainless steel screen; and 2) thin pieces of glass rods cemented
with silicone to the NITEX® screen to reinforce the bottom and sides to
produce a sump area in one end of the chamber. Avoid excessive use of
silicone, while still ensuring that the chambers do not leak and the larvae
cannot get trapped or escape into the sump area. Once constructed, check the
chambers for leaks and repair if necessary. Soak the chambers overnight in
seawater (preferably in flowing water) to cure the silicone cement before use
Other types of glass test chambers, such as the 1000 mL beakers used in the
short-term sheepshead minnow larval survival and growth test, may be used It
is recommended that each chamber contain a minimum of 50 ml per larvae and
allow adequate depth of test solution (5.0 cm).
Jn™'2,1 ,Beaker? " six class A> borosilicate glass or non-toxic plasticware,
1000 ml for making test solutions.
13.5.22 Mini-Winkler bottles -- for dissolved oxygen calibrations.
13.5.23 Wash bottles -- for deionized water, for washing embryos from
substrates and containers, and for rinsing 'small glassware and instrument
electrodes and probes.
165
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GLASS
REINFORCEMENTS
SUMP
Figure 1. Glass chamber with sump area. Modified from Norberg and Mount
(1985). From USEPA (1987c).
13.5.24 Crystallization dishes, beakers, culture dishes, or equivalent -- for
incubating embryos.
13.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
13.5.26 Separatory funnels, 2-L -- Two-four for culturing Artemia.
13.5.27 Pipets, volumetric -- Class A, 1-100 ml.
13.5.28 Pipets, automatic -- adjustable, 1-100 ml.
13.5.29 Pipets, serological -- 1-10 ml, graduated.
13.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent.
13.5.31 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for
transferring larvae.
13.5.32 Siphon with bulb and clamp -- for cleaning test chambers.
13.5.33 Forceps -- for transferring dead larvae to weighing pans.
166
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13.5.34 NITEX® mesh sieves (< 150 A*m, 500 //m, 3 to 5 mm) -- for collecting
Artemia nauplii and fish larvae. (NITEX® is available from Sterling Marine
Products, 18 Label Street, Montclair, NJ 07042; 201-783-9800.)
13.6 REAGENTS AND CONSUMABLE MATERIALS
13.6.1 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
13.6.2 Data sheets (one set per test) -- for data recording.
13.6.3 Tape, colored -- for labelling test chambers.
13.6.4 Markers, waterproof -- for marking containers, etc.
13.6.5 Vials, marked -- 24/test, containing 4% formalin or 70% ethanol, to
preserve larvae (optional).
i
13.6.6 Weighing pans, aluminum -- 26/test (2 extra).
13.6.7 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method 150.1,
USEPA, 1979b). |
'
13.6.8 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA, 1979b), or reagents -- for modified Winkler analysis.
13.6.9 Laboratory quality assurance samples and standards -- for the above
methods.
13.6.10 Reference toxicant solutions -- see Section 4, Quality Assurance.
13.6.11 Ethanol (70%) or formalin (4%) -- for use as a preservative for the
fish larvae.
13.6.12 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
13.6.13 Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water; and Section 8, Effluent and Surface Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
I
13.6.13.1 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 by no more than
± 2%o among the chambers on a given day. If effluent and receiving water
tests are conducted concurrently, the salinities of these tests should be
similar.
i
13.6.13.2 The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
167
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measurable salts. Exposure of Menidia beryl Una larvae to these effluents
will require adjustments in the salinity of the test solutions. It is
important to maintain a constant salinity across all treatments. In addition,
it may be desirable to match the test salinity with that of the receiving
water. Artificial sea salts or hypersaline brine (100%o) derived from natural
seawater may be used to adjust the salinities.
13.6.13,3 Hypersaline brine (MSB): MSB has several advantages that make it
desirable for use in toxicity testing. It can be made from any high quality,
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity. MSB derived from natural seawater
contains the necessary trace metals, biogenic colloids, and some of the
microbial components necessary for adequate growth, survival, and/or
reproduction of marine and estuarine organisms, and may be stored for
prolonged periods without any apparent degradation. However, if 100% HSB is
used as a diluent, the maximum concentration of effluent that can be tested
will be 70% at 30%o salinity and 80% at 20%o salinity.
13.6.13.3.1 The ideal container for making HSB from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a noncorrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine. If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that would contaminate the brine. One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass. If aeration is used, use only oil free air compressors to
prevent contamination.
13.6.13.3.2 Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be in direct contact with the brine. A good quality biodegradable detergent
should be used, followed by several (at least three) thorough deionized water
rinses.
13.6.13.3.3 High quality (and preferably high salinity) seawater should be
filtered to at least 10 /^m before placing into the brine generator. Water
should be collected on an incoming tide to minimize the possibility of
contamination.
13.6.13.3.4 The temperature of the seawater is increased slowly to 40°C.
The water should be aerated to prevent temperature stratification and to
increase water evaporation. The brine should be checked daily (depending on
volume being generated) to ensure that salinity does not exceed 100%o and that
the temperature does not exceed 40°C. Additional seawater may be added to the
brine to obtain the volume of brine required.
13.6.13.3.5 After the required salinity is attained, the HSB should be
filtered a second time through a 1 urn filter and poured directly into portable
containers (20 L cubitainers or polycarbonate water cooler jugs are suitable).
The containers should be capped and labelled with the date the brine was
168
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generated and its salinity. Containers of HSB should be stored in the dark
and maintained at room temperature until used.
.
13.6.13.3.6 If a source of HSB is available, test solutions can be made by
following the directions below. Thoroughly mix together the deionized water
and brine before mixing in the effluent.
13.6.13.3.7 Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine. For example, if the
salinity of the HSB is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0. The proportion of brine is one part in five (one part
brine to four parts deionized water). To make 1 L of seawater at 20%o
salinity from a HSB of 100%o, divide 1 L (1000 ml) by 5.0. The.result, 200
ml, is the quantity of HSB needed to make 1 L of seawater. The difference,
800 ml, is the quantity of deionized water required.
13.6.13.3.8 Table 1 illustrates the composition of test solutions at 20%o if
they are made by combining effluent (0%o), deionized water arid HSB at 100%o
salinity. The volume (ml) of brine required is determined by using the amount
calculated above. In this case, 200 ml of brine is required for 1 L;
therefore, 600 ml would be required for 3 L of solution. The volumes of HSB
required are constant. The volumes of deionized water are determined by
subtracting the volumes of effluent and brine from the total volume of
solution: 3,000 ml - mL effluent - ml HSB = ml deionized water.
13.6.13.4 Artificial sea salts: A modified GP2 artificial seawater
formulation (Table 2) has been successfully used to perform the inland
silverside survival and growth test. The use of GP2 for holding and culturing
of adults is not recommended at this time.
13.6.13.4.1 The GP2 artificial sea salts (Table 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 gm NaHC03, in 500 ml
deionized water. Add 2.5 ml of this stock solution for each liter of the GP2
artificial seawater. j
13.6.14 ROTIFER CULTURE --for feeding cultures and test organisms
13.6.14.1 At hatching Mem'dia beryl!ina larvae are too small to ingest
Artemia nauplii and must be fed rotifers, Brachionus pliaitilis. The rotifers
can be maintained in continuous culture when fed algae (see Section 6 and
USEPA, 1987g). Rotifers are cultured in 10-15 L Pyrex® carboys (with a drain
spigot near the bottom) at 25-28°C and 25-35%o salinity. Four 12-L culture
carboys should be maintained simultaneously to optimize production. Clean
carboys should be filled with autoclaved seawater. Alternatively, an
immersion heater may be used to heat saline water in the carboy to 70-80°C for
1 h.
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TABLE 1. PREPARATION OF 3 L SALINE WATER FROM DEIONIZED WATER AND A
HYPERSALINE BRINE OF 100%o NEEDED FOR TEST SOLUTIONS AT
20%o SALINITY
Effluent
Concentration
(%)
80
40
20
10
5
Control
Volume of
Ef f 1 uent
(0%o)
(ml)
2400
1200
600
300
150
0
Volume of
Deionized
Water
(ml)
0
1200
1800
2100
2250
2400
Volume of
Hypersaline
Brine
(ml)
600
600
600
600
600
600
Total
Vol ume
(ml)
3000
3000
3000
3000
3000
3000
Total
4,650
9,750
3,600
18,000
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TABLE 2. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF
GP2 ARTIFICIAL SEAWATER FOR THE INLAND SILVERSIDE,
MENIDIA BERYLLINA, TOXICITY TESTU'S
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 • 6 H20
CaCl2 • 2 H20
SrCl2 • 6 H20
NaHC03
\ Modified GP2
Concentration
(9/L)
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
from Spotte et al . (1984)
Amount; (g)
Required for
20 L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
1
(19905). The salinity is 30.89 g/L.
GP2 can be diluted with deionized (DI) water to the desired test
salinity.
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13.6.14.2 When the water has cooled to 25-28°C, aerate and add a start-up
sample of rotifers (50 rotifers/mL) and food (about 1 L of a dense algal
culture). The carboys should be checked daily to ensure that adequate food is
available and that the rotifer density is adequate. If the water appears
clear, drain 1 L of culture water and replace it with algae. Excess water can
be removed through the spigot drain and filtered through a < 60 /j,m mesh
screen. Rotifers collected on the screen should be returned to the culture.
If a more precise measure of the rotifer population is needed, rotifers
collected from a known volume of water can be resuspended in a smaller volume,
killed with formalin and counted in a Sedgwick-Rafter cell. If the density
exceeds 50 rotifers/mL, the amount of food per day should be increased to 2 L
of algae suspension. The optimum density of approximately 300-400 rotifers/mL
may be reached in 7-10 days and is sustainable for 2-3 weeks. At these
densities, the rotifers should be cropped daily. Keeping the carboys away
from light will reduce the amount of algae attached to the carboy walls. When
detritus accumulates, populations of ciliates, nematodes, or harpacticoid
copepods that may have been inadvertently introduced can rapidly take over the
culture. If this occurs, discard the cultures.
13.6.15 ALGAL CULTURES -- for feeding rotifer cultures
13.6.15.1 Tetraselmus suecica or Chlorella sp. (see USEPA, 1987a) can be
cultured in 20-L polycarbonate carboys that are normally used for bottled
drinking water. Filtered seawater is added to the carboys and then autoclaved
(110°C for 30 min). After-cooling to room temperature, the carboys are placed
in a temperature chamber controlled at 18-20°C. One liter of 7. suecica or
Chlorella sp. starter culture and 100 mL of nutrients are added to each
carboy.
13.6.15.2 Formula for algal culture nutrients.
13.6.15.2.1 Add 180 g NaNO
3>
12 g NaH2P04,
and 6.16 g EDTA to 12 L of
deionized water. Mix with a magnetic stirrer until all salts are dissolved
(at least 1 h).
13.6.15.2.2 Add 3.78 g FeCl3 • 6 H20 and stir again. The solution should be
bright yellow.
13.6.15.2.3 The algal culture is vigorously aerated via a pipette inserted
through a foam stopper at the top of the carboy. A dense algal culture should
develop in 7-10 days and should be used by day 14. Thus, start-up of cultures
should be made on a daily or every second day basis. Approximately 6-8
continuous cultures will meet the feeding requirements of four 12-L rotifer
cultures. When emptied, carboys are washed with soap and water and rinsed
thoroughly with deionized water before reuse.
13.6.16 BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test
organisms
13.6.16.1 Newly hatched Artemia nauplii are used as food for inland
silverside larvae in toxicity tests. Although there are many commercial
sources of brine shrimp cysts, the Brazilian or Colombian strains are being
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used because the supplies examined have had low concentrations of chemical
residues and produce nauplii of suitably small size. For commercial sources
of brine shrimp, Artemia, cysts, see Table 2 of Section 5., Facilities,
Equipment, and Supplies and Section 4, Quality Assurance.
13.6.16.2 Each new batch of Artemia cysts must be evaluated for size
(Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al., 1980) and nutritional
suitability (see Leger et. al., 1985; Leger et al., 1986) against known
suitable reference cysts by performing a side by side larval growth test using
the "new" and "reference" cysts. The "reference" cysts used in the
suitability test may be a previously tested and acceptable batch of cysts, or
may be obtained from the Quality Assurance Research Division, Environmental
Monitoring Systems Laboratory, Cincinnati, OH 45268; 513-569-7325. A sample
of newly-hatched Artemia nauplii from each new batch of cysts should be
chemically analyzed. The Artemia cysts should not be used if the
concentration of total organochlorine pesticides exceeds 0.15 /^g/g wet weight
or that the total concentration of organochlorine pesticides plus PCBs does
not exceed 0.30 »g/g wet weight. (For analytical methods, see USEPA 1982).
13.6.16.2.1 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or a solution prepared by adding 35.0 g uniodized
salt (NaCl) or artificial sea salts to 1 L of deioriized water, to a
2-L separatory funnel or equivalent.
2. Add 10 ml Artemia cysts to the separatory funnel arid aerate for 24 h
at 27°C. (Hatching time varies with incubation temperature and the
geographic strain of Artemia used (see USEPA, 1985d; USEPA, 1993a; and
ASTM, 1993.)
3. After 24 h, cut off the air supply in the separatory funnel. Artemia
nauplii are phototactic and will concentrate at the bottom of the
funnel if it is covered for 10-15 minutes to prevent mortality, do not
leave the concentrated nauplii at the bottom of the funnel more than
10 minutes without aeration.
4. Drain the nauplii into a beaker or funnel fitted with < 150 ^m NITEX®
or stainless steel screen, and rinse with seawater or equivalent
before use.
13.6.16.3 Testing Artemia nauplii as food for toxicity test organisms.
13.6.16.3.1 The primary criterion for acceptability of each new supply of
brine shrimp cysts is the ability of the nauplii to support good survival and
growth of the inland silverside larvae (see Subsection 13.11). The larvae
used to evaluate the suitability of the brine shrimp nauplii must be of the
same geographical origin, species, and stage of development as those used
routinely in the toxicity tests. Sufficient data to detect differences in
survival and growth should be obtained by using three replicate test chambers
each containing a minimum of 15 larvae, for each type of food.
13.6.16.3.2 The feeding rate and frequency, test vessels and volume of
control water, duration of the test, and age of the nauplii at the start of
the test, should be the same as used for the routine toxicity tests.
I
i
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13.6.16.3.3 Results of the brine shrimp nutrition assay, where there are only
two treatments, can be evaluated statistically by use of a t test. The "new"
food is acceptable if there are no statistically significant differences in
the survival and growth of the larvae fed the two sources of nauplii.
13.6.16.4 Use of Artemia nauplii as food for inland silverside, Menidia
beryllina, larvae.
13.6.16.4.1 Menidia beryllina larvae begin feeding on newly hatched Artemia
nauplii about five days after hatching, and are fed Artemia nauplii daily
throughout the 7-day larval survival and growth test. Survival of Menidia
beryllina larvae 7-9 days old is improved by feeding newly hatched (< 24 h
old) Artemia nauplii. Equal amounts of Artemia nauplii must be fed to each
replicate test chamber to minimize the variability of larval weight.
Sufficient numbers of nauplii should be fed to ensure that some remain alive
overnight in the test chambers. An adequate but not excessive amount should
be provided to each replicate on a daily basis. Feeding excessive amounts of
nauplii will result in a depletion in DO to below an acceptable level (below
4.0 mg/L). As much of the uneaten Artemia nauplii as possible should be
siphoned from each chamber prior to test solution renewal to ensure that the
larvae principally eat newly hatched nauplii.
13.6.17 TEST ORGANISMS, INLAND SILVERSIDE, MENIDIA BERYLLINA
13.6.17.1 The inland silverside, Menidia beryllina, is one of three species
in the atherinid family that are amenable to laboratory culture; and one of
four atherinid species used for chronic toxicity testing. Several atherinid
species have been utilized successfully for early life stage toxicity tests
using field collected (Goodman et al., 1985) and laboratory reared adults
(Middaugh and Takita, 1983; Middaugh and Hemmer, 1984; and USEPA, 1987g). The
inland silverside, Menidia beryllina, populates a variety of habitats from
Cape Cod, Massachusetts, to Florida and west to Vera Cruz, Mexico (Johnson,
1975). It can tolerate a wide range of temperature, 2.9 - 32.5°C (Tagatz and
Dudley, 1961; Smith, 1971) and salinity, of 0%o - 58%. (Simmons, 1957;
Renfro, 1960), having been reported from the freshwaters of the Mississippi
River drainage basin (Chernoff et al., 1981) to hypersaline lagoons (Simmons,
1957). Ecologically, Menidia spp. are important as major prey for many
prominent commercial species (e.g., bluefish (Pomatomus saltatrix), mackerel
(Scomber scombrus), and striped bass (Morone saxatilis) (Bigelow and
Schroeder, 1953). The inland silverside, Menidia beryllina, is a serial
spawner, and will spawn under controlled laboratory conditions. Spawning can
be induced by diurnal interruption in the circulation of water in the culture
tanks (Middaugh et al., 1986; USEPA, 1987a). The eggs are demersal,
approximately 0.75 mm in diameter (Hildebrand and Schroeder, 1928), and adhere
to vegetation in the wild, or to filter floss in laboratory culture tanks.
The larvae hatch in 6-7 days when incubated at 25°C and maintained in seawater
ranging from 5%o to 30% (USEPA, 1987a). Newly hatched larvae are 3.5-4.0 mm
in total length (Hildebrand, 1922).
13.6.17.2 Inland silverside, Menidia beryllina, adults (see USEPA, 1987g and
USEPA, 1993a for detailed culture methods) may be cultured in the laboratory
or obtained from the Gulf of Mexico or Atlantic coast estuaries throughout the
174
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year (Figure 2). Gravid females can be collected from low salinity waters
along the Atlantic coast during April to July, depending on the latitude.
The most productive and protracted spawning stock can be obtained from adults
brought into the laboratory. Broodstocks, collected from local estuaries
twice each year (in April and October), will become sexually active after
1-2 months and will generally spawn for 4-6 months. j
13.6.17.3 The fish can be collected easily with a beach seine (3-6 mm mesh),
but the seine should not be completely landed onto the beach. Silversides are
very sensitive to handling and should never be removed from the water by net
-- only by beaker or bucket.
i
13.6.17.4 Samples may contain a mixture of inland silverside, Mem'dia
beryllina, and Atlantic silverside, Mem'dia mem'dia, on the Atlantic coast or
inland silverside and tidewater silverside, Mem'dia peninsulae, on the Gulf
Coast (see USEPA, 1987g for additional information on morphological
differences for identification). Johnson (1975) and Chernoff et al. (1981)
have attempted to differentiate these species. In the northeastern United
States, M. beryllina juveniles and adults are usually considerably smaller
than M. mem'dia juveniles and adults (Bengtson, 1984), and can be separated
easily in the field on that basis.
13.6.17.5 Record the water temperature and salinity at each collection site.
Aerate (portable air pump, battery operated) the fish and transport to the
laboratory as quickly as possible after collection. Upon 'arrival at the
laboratory, the fish and the water in which they were collected are
transferred to a tank at least 0.9 m in diameter. A filter system should be
employed to maintain water quality (see USEPA, 1987g). Laboratory water is
added to the tank slowly, and the fish are acclimated at the rate of 2°C per
day, to a final temperature of 25°C, and about 5%o salinity per day, to a
final salinity in the range of 20%o - 32%o. The seawater in each tank should
be brought to a minimum volume of 150 L. A density of about 50 fish/tank is
appropriate. Maintain a photoperiod of 16 h light/8 h dark. Feed the adult
fish flake food or frozen brine shrimp twice daily and Artemia nauplii once
daily. Siphon the detritus from the bottom of the tanks weekly.
13.6.17.6 Larvae for a toxicity test can be obtained from the broodstock by
spawning onto polyester aquarium filter-fiber substrates, 15 cm long X 10 cm
wide X 10 cm thick, which are suspended with a string 8-10 cm below the
surface of the water and in contact with the side of the holding tanks for
24-48 h, 14 days prior to the beginning of a test. The floss should be gently
aerated by placing it above an airstone, and weighted down with a heavy
non-toxic object. The embryos, which are light yellow in color, can be seen
on the floss, and are round and hard to the touch compared to the soft floss.
13.6.17.7 Remove as much floss as possible from the embryos. The floss
should be stretched and teased to prevent the embryos from clumping. The
embryos should be incubated at the test salinity and lightly aerated. At
25°C, the embryos will hatch in about 6-8 days. Larvae arta fed about 500
rotifer larvae/day from hatch through four days post-hatch. On Days 5 and 6,
newly hatched (less than 12-h old) Artemia nauplii are mixed with the
i
175
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A. Adult, ca. 64 mm SL
fcAji?-=*w :-f •*-'-!•-< jiff.'-'. .'.. .11.1.-
r^SS^S^^AvTvwv^^vJSSscsw
F. Larva, 6.7 mm TL
G. Larva, 8.9 mm TL
Figure 2. Inland silverside, Menidia beryllina: A. Adult, ca. 64 mm SL; B.
Egg (diagrammatic), only bases of filaments shown; C. Egg, 2-cell
stage; D. Egg, morula stage; E. Advanced embryo, 2 1/2 days after
fertilization. From Martin and Drewry (1978).
176
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rotifers, to provide a transition period. After Day 7, only nauplii are fed,
and the age range for the nauplii can be increased from U!-h old to 24-h old.
13.6.17.8 Silverside larvae are very sensitive to handling and shipping
during the first week after hatching. For this reason, if organisms must be
shipped to the test laboratory, it may be impractical to use larvae less than
11 days old because the sensitivity of younger organisms may result in
excessive mortality during shipment. If organisms are to be shipped to a test
site, they should be shipped only as (1) early embryos, so that they hatch
after arrival, or (2) after they are known to be feeding viiell on Artemia
nauplii (8-10 days of age). Larvae shipped at 8-10 days of age would be 9 to
11 days old when the test is started. Larvae that are hatched and reared in
the test laboratory can be used at 7 days of age.
13.6.17.9 If four replicates of 15 larvae are used at each effluent
concentration and in the control, 360 larvae will be needed for each test.
13.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
13.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
13.8 CALIBRATION AND STANDARDIZATION
13.8.1 See Section 4, Quality Assurance. !
13.9 QUALITY CONTROL
•
13.9.1 See Section 4, Quality Assurance.
I
13.10 TEST PROCEDURES
13.10.1 TEST SOLUTIONS
i
13.10.1.1 Receiving Waters
13.10.1.1.1 The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 ^m NITEX® filter and compared without
dilution, against a control. Using four replicate chambers per test, each
containing 500-750 mL, and 400 mL for chemical analysis, would require
approximately 2.4-3.4 L or more of sample per day.
13.10.1.2 Effluents
13,10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used.
A dilution factor of 0.5 provides precision of ± 100%, and allows for testing
of concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Test precision shows
177
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little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used. Therefore, USEPA
recommends the use of the > 0.5 dilution factor. If 100% salinity MSB is used
as a diluent, the maximum concentration of effluent that can be tested will be
80% at 20%o salinity, and 70% at 30%o salinity.
13.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first 1
to 2 h of the test, additional dilutions at the lower range of effluent
concentrations should be added.
13.10.1.2.3 The volume of effluent required to initiate the test and for
daily renewal of four replicates per treatment for five concentrations of
effluent and a control, each containing 750 ml of test solution, is
approximately 5 L. Prepare enough test solution at each effluent
concentration to provide 400 ml additional volume for chemical analyses.
13.10.1.2.4 Tests should begin as soon as possible after sample collection,
preferably within 24 h. The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
studies unless permission is granted by the permitting authority. In no case
should the test be started more than 72 h after sample collection (see Section
8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests, subsection 8.5.4).
13.10.1.2.5 Just prior to test initiation (approximately 1 h), the
temperature of a sufficient quantity of the sample to make the test solution
should be adjusted to the test temperature (25 ± 1°C) and maintained at that
temperature during the addition of dilution waters.
13.10.1.2.6 Effluent dilutions should be prepared for all replicates in each
treatment in one beaker to minimize variability among the replicates. The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.
13.10.1.3 Dilution Water
13.10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving
water), HSB prepared from natural seawater, or artificial seawater prepared
from FORTY FATHOMS® or GP2 sea salts (see Table 2 and Section 7, Dilution
Water). Other artificial sea salts may be used for culturing sheepshead
minnows and for the larval survival and growth test if the control criteria
for acceptability of test data are satisfied.
13.10.2 START OF THE TEST
13.10.2.1 Inland silverside larvae 7 to 11 days old can be used to start the
survival and growth test. At this age, the inland silverside feed on
newly-hatched Artemia nauplii. At 25°C, tests with inland silverside larvae
can be performed at salinities ranging from 5%o to 32%o. If the test salinity
ranges from 16%o to 32%o, the salinity for spawning, incubation, and culture
178
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of the embryos and larvae should be maintained within this salinity range. If
the test salinity is in the range of 5%o to 15%o, the embryos may be spawned
at 30%o, but egg incubation and larval rearing should be at the test salinity.
If the specific salinity required for the test differs from the rearing
salinity, adjustments of 5%o daily should be made over the three days prior to
start of test.
13.10.2.2 One Day Prior to Beginning of Test
13.10.2.2.1 Set up the Artemia culture so that newly hatched nauplii will be
available on the day the test begins, (see Section 7).
13.10.2.2.2 Increase the temperature of water bath, room.i or incubator to the
required test temperature (25 ± 1°C).
13.10.2.2.3 Label the test chambers with a marking pen. Use of color coded
tape to identify each concentration and replicate is helpful., A minimum of
five effluent concentrations and a control should be selected for each test.
Glass test chambers, such as crystallization dishes, beakers, or chambers with
a sump area (Figure 1), with a capacity for 500-750 ml of test solution,
should be used.
13.10.2.2.4 Randomize the position of test chambers in the temperature-
controlled water bath, room, or incubator at the beginning of the test, using
a position chart. Assign numbers for the position of each test chamber using
a table of random numbers or similar process (see Appendix A for an example of
randomization). Maintain the chambers in this configuration throughout the
test, using a position chart.
13.10.2.2.5 Because inland silverside larvae are very sensitive to handling,
it is advisable to distribute them to their respective test chambers which
contain control water on the day before the test is to begin„ Each test
chamber should contain 15 larvae (minimum 10) and it is recommended that there
be four replicates (minimum of three) for each concentration and control.
13.10.2.2.6 Seven to 11 day old larvae are active and difficult to capture
and are subject to handling mortality. Carefully remove larvae (2-3-at a
time) by concentrating them in a corner of the aquarium or; culture vessel, and
capture them with a wide-bore pipette, small petri dish, crystallization dish,
3-4 cm in diameter, or small pipette. They are active and will readily escape
from a pipette. Randomly transfer the larvae (2-3 at a time) into each test
chamber until the desired number (15) is attained. See Appendix A for an
example of randomization. After the larvae are dispensed, use a light table
to verify the number in each chamber.
.
13.10.2.3 Before beginning the test remove and replace any dead larvae from
each test chamber. The test is started by removing approximately 90% of the
clean seawater from each test chamber and replacing with the appropriate test
solution.
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13.10.3 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE
13.10.3.1 The light quality and intensity should be at ambient laboratory
levels, which is approximately 10-20 pE/m/s, or 50 to 100 foot candles
(ft-c), with a photoperiod of 16 h of light and 8 h of darkness. The water
temperature in the test chambers should be maintained at 25 ± 1°C. The test
salinity should be in the range of 5%o to 32%o, and the salinity should not
vary by more than ± 2%o among the chambers on a given day. If effluent and
receiving water tests are conducted concurrently, the salinities of these
tests should be similar.
13.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
13.10.4.1 Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain satisfactory DO. The DO should be measured
on new solutions at the start of the test (Day 0) and before daily renewal of
test solutions on subsequent days. The DO should not fall below 4.0 mg/L (see
Section 8, Effluent and Receiving Water Sampling, Sample Handling and Sample
Preparation for Toxicity Tests). If it is necessary to aerate, all
concentrations and the control should be aerated. The aeration rate should
not exceed 100 bubbles/min, using a pi pet with a 1-2 mm orifice such as a 1-mL
KIMAX® serological pi pet No. 37033, or equivalent. Care should be taken to
ensure that turbulence resulting from aeration does not cause undue stress to
the fish.
13.10.5 FEEDING
13.10.5.1 Artemia nauplii are prepared as described above.
13.10.5.2 The test larvae are fed newly-hatched (less than 24-h-old) Artemia
nauplii once a day from Day 0 through Day 6; larvae are not fed on Day 7.
Equal amounts of Artemia nauplii must be fed to each replicate test chamber to
minimize the variability of larval weight.. Sufficient numbers of nauplii
should be fed to ensure that some remain alive overnight in the test chambers.
An adequate, but not excessive amount of Artemia nauplii, should be provided
to each replicate on a daily basis. Feeding excessive amounts of Artemia
nauplii will result in a depletion in DO to below an acceptable level. Siphon
as much of the uneaten Artemia nauplii as possible from each chamber daily to
ensure that the larvae principally eat newly hatched nauplii.
13.10.5.3 On days 0-2, transfer 4 g wet weight or pipette 4 mL of
concentrated, rinsed Artemia nauplii to seawater in a 100 mL beaker, and bring
to a volume of 80 mL. Aerate or swirl the suspension to equally distribute
the nauplii while withdrawing individual 2 mL portions of the Artemia nauplii
suspension by pipette or adjustable syringe to transfer to each replicate test
chamber. Because the nauplii will settle and concentrate at the tip of the
pipette during the transfer, limit the volume of concentrate withdrawn each
time to a 2-mL portion for one test chamber helps ensure an equal distribution
to the replicate chambers. Equal distribution of food to the replicates is
critical for successful tests.
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13.10.5.4 On days 3-6, transfer 6 g wet weight or 6 mL of the Artemia nauplii
concentrate to seawater in a 100 mL beaker. Bring to a volume of 80 mL and
dispense as described above.
I
13.10.5.5 If the larvae survival rate in any replicate on any day falls below
50%, reduce the volume of Artemia nauplii suspension added to that test
chamber by one-half (i.e., reduce from 2 ml to 1 ml) and continue feeding
one-half the volume through day 6. Record the time of feeding on the data
sheets. |
I
13.10.6 DAILY CLEANING OF TEST CHAMBERS
13.10.6.1 Before the daily renewal of test solutions, uneaten and dead
Artemia, and other debris are removed from the bottom of the test chambers
with a siphon hose. Alternately, a large pipet (50 mL), fitted with a safety
pi pet filler or rubber bulb, can be used. If the test chambers illustrated in
Figure 1 are used, remove only as much of the test solution from the chamber
as is necessary to clean, and siphon the remainder of the test solution from
the sump area. Because of their small size during the first few days of the
test, larvae are easily drawn into a siphon tube when cleaning the test
chambers. By placing the test chambers on a light box, inadvertent removal of
larvae can be greatly reduced because they can be more easily seen. If the
water siphoned from the test chambers is collected in a white plastic tray,
the live larvae caught up in the siphon can be retrieved, and returned by
pipette to the appropriate test chamber and noted on data sheet. Any
incidence of removal of live larvae from the test chambers by the siphon
during cleaning, and subsequent return to the chambers should be noted in the
test records.
13.10.7 OBSERVATIONS DURING THE TEST
13.10.7.1 Routine Chemical and Physical Determinations
13.10.7.1.1 DO is measured at the beginning and end of each 24-h exposure
period in one test chamber at all test concentrations and in the control.
13.10.7.1.2 Temperature, pH, and salinity are.measured at the end of each
24-h exposure period in one test chamber at all test concentrations and in the
control. Temperature should also be monitored continuously or observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
chambers at least the end of the test to determine the temperature variation
in the environmental chamber.
13.10.7.1.3 The pH is measured in the effluent sample each day before new
test solutions are made.
13.10.7.1.4 Record all measurements on the data sheet (Figure 3).
13.10.7.2 Routine Biological Observation
181
-------
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183
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13.10.7.2.1 The number of live larvae in each test chamber are recorded daily
(Figure 3), and the dead larvae are discarded.
13.10.7.2.2 Protect the larvae from unnecessary disturbances during the test
by carrying out the daily test observations, solution renewals, and removal of
dead larvae. Make sure the larvae remain immersed at all times during the
performance of the above operations.
13.10.8 TEST SOLUTION RENEWAL
13.10.8.1 The test solutions are renewed daily using freshly prepared
solutions, immediately after cleaning the test chambers. The water level in
each chamber is lowered to .a depth of 7-to-10 mm, leaving 10 to 15% of the
test solution. New test solution is added slowly by refilling each chamber
with the appropriate amount of test solution without excessively disturbing
the larvae. If the modified chamber is used (Figure 1), renewals should be
poured into the sump area using a narrow bore (approximately 9 mm ID) funnel.
13.10.8.2 The effluent or receiving water used in the test is stored in an
incubator or refrigerator at 4°C. Plastic containers such as 8-20 L
cubitainers have proven suitable for effluent collection and storage. For
on-site toxicity studies no more than 24 h should elapse between collection of
the effluent and use in a toxicity test (see Section 8, Effluent and Receiving
Water Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).
13.10.8.3 Approximately 1 h before test initiation, a sufficient quantity of
effluent or receiving water sample is warmed to 25 ± 1°C to prepare the test
solutions. A sufficient quantity of effluent should be warmed to make the
daily test solutions.
13.10.8.3.1 An illustration of the quantities of effluent and seawater needed
to prepare test solution at the appropriate salinity is provided in Table 2.
13.10.9 TERMINATION OF THE TEST
13.10.9.1 The test is terminated after 7 days of exposure. At test
termination dead larvae are removed and discarded. The surviving larvae in
each test chamber (replicate) are counted, and immediately prepared as a group
for dry weight determination, or are preserved in 4% formalin or 70% ethanol.
Preserved organisms are dried and weighed within 7 d. For safety, formalin
should be used under a hood.
13.10.9.2 For immediate drying and weighing, siphon or pour live larvae onto
a 500 /im mesh screen in a large beaker to retain the larvae and allow Artemia
to be rinsed away. Rinse the larvae with deionized water to remove salts that
might contribute to the dry weight. Sacrifice the larvae in an ice bath of
deionized water.
13.10.9.3 Small aluminum weighing pans can be used to dry and weigh larvae.
An appropriate number of aluminum weigh pans (one per replicate) are marked
for identification and weighed to 0.01 mg, and the weights are recorded
(Figure 4) on the data sheets.
184
-------
Test Dates:
Species:.
Pan
No.
Cone.
&
Rep.
Initial
Wt.
Final
Wt.
(mg)
Diff.
(mg)
No.
Larvae
Av. Wt./
Larvae
(mg)
Figure 4. Data form for the inland silverside, Menidia beryllina, larval
survival and growth test. Dry weights of larvae (from USEPA,
1987b).
185
-------
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 13.1). 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.
13.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
13.11.1 A summary of test conditions and test acceptability criteria is
listed in Table 3. '
13.12 ACCEPTABILITY OF TEST RESULTS
13.12.1 Test results are acceptable if (1) the average survival of control
larvae is equal to or greater than 80%, and (2) where the test starts with
7-day old larvae, the average dry weight per surviving control larvae, when
dried immediately after test termination, is equal to or greater than 0.50 mg,
or the average dry weight of the control larvae preserved not more than 7 days
in 4% formalin or 70% ethanol equals or exceeds 0.43 mg.
13.13 DATA ANALYSIS
13.13.1 GENERAL
13.13.1.1 Tabulate and summarize the data.
13.13.1.2 The endpoints of toxicity tests using the inland silverside are
based on the adverse effects on survival and growth. The LC50, the IC25, and
the IC50 are calculated using point estimation techniques (see Section 9,
Chronic Toxicity Test Endpoints and Data Analysis). LOEC and NOEC values, for
survival and growth, are obtained using a hypothesis testing approach such as
Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel,
1959; Miller, 1981) (see Section 9). Separate analyses are performed for the
estimation of the LOEC and NOEC endpoints and for the estimation of the LC50,
IC25, and IC50. Concentrations at which there is no survival in any of the
test chambers are excluded from the statistical analysis of the NOEC and LOEC
for survival and growth but included in the estimation of the LC50, IC25, and
IC50. See the Appendices for examples of the manual computations and examples
of data input and program output.
186
-------
Test Dates:
Species:
Effluent Tested:
TREATMENT
NO. LIVE
LARVAE
SURVIVAL
(%)
MEAN DRY WT/
LARVAE (MG)
± SD
SIGNIF. DIFF.
FROM CONTROL
(o)
MEAN
TEMPERATURE
CO
± SD
MEAN SALINITY
%0
+ SD
AVE. DISSOLVED
OXYGEN
(MG/L) ± SD
COMMENTS:
Figure 5. Data form for the inland silverside, Mem'dia beryllina, larval
survival and growth test. Summary of test results (from USEPA,
1987c).
187
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL AND
GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS
1. Test type:
2. Salinity:
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. larvae per test
chamber:
12. No. replicate chambers
per concentration:
13. No. larvae per concentration:
14. Source of food:
15. Feeding regime:
16. Cleaning:
Static renewal
5%o to 32%o (± 2%o of the selected
test salinity)
25 ± 1°C
Ambient laboratory illumination
10-20 /*E/m2/s (50-100 ft-c) (Ambient
laboratory levels)
16 h light, 8 h darkness
600 mL - 1 L containers
500-750 mL/replicate (loading and DO
restrictions must be met)
Daily
7-11 days post hatch; 24-h range in
age
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
Newly hatched Artemia nauplii
(survival of 7-9 days old Menidia
beryl Una larvae improved by feeding
24 h old Artemia)
Feed 0.10 g wet weight Artemia nauplii
per replicate on days 0-2; Feed 0.15 g
wet weight Artemia nauplii per
replicate on days 3-6
Siphon daily, immediately before test
solution renewal and feeding
188
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL AND
GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sampling requirement:
25. Sample volume required:
None, unless DO concentration falls
below 4.0 mg/L, then aerate all
chambers. Rate should be less than
100 bubbles/min.
;
Uncontaminated source of natural sea
water, artificial seawater; deionized
water mixed with hypersaline brine or
artificial sea salts (HW Marinemix®,
FORTY FATHOMS®, GP2 or equivalent)
Effluent: Minimum of 5 and a control
Receiving Waters: 100% receiving water
or minimum of 5 and a control
Effluents: > 0.5
Receiving waters:
7 days
None, or > 0.5
Survival and growth (weight)
80% or greater survival in controls,
0.50 mg average dry weight of control
larvae where test starts with 7-days
old larvae and dried immediately after
test termination, or 0.43 mg or
greater average dry weight per
surviving control larvae, preserved
not more than 7 days in 4% formalin or
70% ethanol
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 are
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,
Subsection 8.5.4)
6 L per day
189
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13.13.1.3 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
13.13.2 EXAMPLE OF ANALYSIS OF INLAND SILVERSIDE, MENIDIA BERYLLINA,
SURVIVAL DATA
13.13.2.1 Formal statistical analysis of the survival data is outlined in
Figures 6 and 7. The response used in the analysis is the proportion of
animals surviving in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the LC50 endpoint. Concentrations at which there is no survival
in any of the test chambers are excluded from statistical analysis of the NOEC
and LOEC, but included in the estimation of the 1C, EC, and LC endpoint.
13.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Wilk's
Test, and Bartlett's Test is used to test for the homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.
13.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment (see
Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.
13.13.2.4 Probit Analysis (Finney, 1971; see Appendix 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 H-K).
13.13.2.5 Example of Analysis of Survival Data
13.13.2.5.1 This example uses the survival data from the inland silverside
larval survival and growth test. The proportion surviving in each replicate
in this example must first be transformed by the arc sine transformation
procedure described in Appendix B. The raw and transformed data, means and
variances of the transformed observations at each effluent concentration and
control are listed in Table 4. A plot of the data is provided in Figure 8.
Since there is 100% mortality in all three replicates for the 50% and 100%
concentrations, they are not included in this statistical analysis and are
considered a qualitative mortality effect.
190
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STATISTICAL ANALYSIS OF INLAND SILVERSIDE LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL HYPOTHESIS TESTING
SURVIVAL DATA
PROPORTION SURVIVING
ARC SINE
TRANSFORMATION
SHAPIRO-WILKSTEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTS TEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
T-TESTWITH
BONFERRONI
ADJUSTMENT
EQUAL NUMEIER OF
REPLICATES?
YES
NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRON1 ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 6. Flowchart for statistical analysis of the inland silverside,
Menidia beryllina, survival data by hypothesis testing.
I
191
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STATISTICAL ANALYSIS OF INLAND SILVERSIDE LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
TWO OR MORE
PARTIAL MORTALITIES?
NO
YES
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2 TEST)
NO
YES
ONE OR MORE
PARTIAL MORTALITIES?
\
YES
r
NO _
GRAPHICAL METHOD
LC50
PROBIT METHOD
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONG.?
NO
YES
SPEEARMAN-KARBER
METHOD
TRIMMED SPEARMAN-
KARBER METHOD
LC50AND95%
CONFIDENCE
INTERVAL
Figure 7. Flowchart for statistical analysis of the inland silverside,
Mem'dia beryTTina, survival data by point estimation.
192
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193
-------
TABLE 4. INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL DATA
Effluent Concentration^)
Replicate Control
6.25
12.5
25.0
50.0 100.0
RAW
ARC SINE
TRANS-
FORMED
Mean(Y,}
s?
i
A
B
C
A
B
C
0.80
0.87
0.93
1.107
1.202
1.303
1.204
0.010
1
0.73
0.80
0.87
1.024
1.107
1.202
1.111
0.008
2
0.80
0.33
0.60
1.107
0.612
0.886
0.868
0.061
3
0.40 0.0
0.53 0.0
0.07 0.0
0.685
0.815
0.268
0.589
0.082
4
0.0
0.0
0.0
-
-
13.13.2.6 Test for Normality
13.13.2.6.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 5.
TABLE 5. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Replicate
Control
Effluent Concentration (%)
6.25 12.5 25.0
A
B
C
-0.097
-0.002
0.099
-0.087
-0.004
0.091
0.239
-0.256
0.018
0.096
0.226
-0.321
194
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13.13.2.6.2 Calculate the denominator, D, of the statistic:
D = £
i=l
~v\ 2
Where: X,- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
13.13.2.6.3 For this set of data, n =12
X = L(0.002) = 0.0
12 i
D - 0.3214
13.13.2.6.4 Order the centered observations from smallest to largest:
X(1)
0.018
0.091
0.096
0.099
0.226
0.239
13.13.2.6.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a2, ... ak where k is n/2 if n is even and (n-l)/2
if n is odd. For the data in this example, n = 12 and k =| 6. The a,- values
are listed in Table 7.
195
-------
13.13.2.6.6 Compute the test statistic, W, as follows:
. j
D 1=1
The differences x(n"!+1} - X
- x(2>
- x<3)
- x<4)
- x(5>
- XC6)
13.13.2.6.7 The decision rule for this test is to compare W as calculated in
Subsection 13.2.6.6 to a 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 the data in this example, the critical value at a
significance level of 0.01 and n = 12 observations is 0.805. Since W = 0.945
is greater than the critical value, conclude that the data are normally
distributed.
13.13.2.7 Test for Homogeneity of Variance
13.13.2.7.1 The test used to examine whether the variation in survival is the
same across all effluent concentrations including the control, is Bartlett's
Test (Snedecor and Cochran, 1980). The test statistic is as follows:
B =
- Ev^ in
Where: V,
P
In
degrees of freedom for each effluent concentration and
control, V,- = (nf - 1)
number of levels of effluent concentration including the
control
196
-------
i = 1, 2, ..., p where p is the number of concentrations
including the control
n,- = the number of replicates for concentration i.
sr =
t
C = l+[3(p-l)]-1[El/IA-(
2=1 2=1 ~
13.13.2.7.2 For the data in this example (See Table 4), all effluent
concentrations including the control have the same number of replicates
(n, = 3 for all i). Thus, Vf = 2 for all i.
13.13.2.7.3 Bartlett's statistic is therefore:
B = [(8)1^2(0.0402) -2 £ In (S*)] /1.2083
2=1
= [8(-3.21391) - 2(-14.731)]/1.2083
= 3.7508/1.2083
= 3.104
13.13.2.7.4 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 11.345. Since B = 3.104 is less than the
critical value of 11.345, conclude that the variances are not different.
13.13.2.8 Dunnett's Procedure
13.13.2.8.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 8.
197
-------
TABLE 8. ANOVA TABLE
Source
Between
Within
Total
df Sum of Squares
(SS.)
p - 1 SSB
N - p SSW
N - 1 SST
Mean Square(MS)
(SS/df)
SB i SSB/(p-l)
Sy = SSW/(N-p)
Where: p - number of SDS concentration levels including the control
N = total number of observations n, + n2 ... + np
nf = number of observations in concentration i
SSB = ~ETi/ni-G2/N Between Sum of Squares
SST =
Total Sum of Squares
SSW = SST-SSB
Within Sum of Squares
G = the grand total of all sample observations, G = £ T±
Tf = the total of the replicate measurements for
concentration i
u = the jth observation for concentration i (represents the
proportion surviving for toxicant concentration i in test
chamber j)
198
-------
13.13.2.8.2 For the data in this example:
= n2 = n3 = n4 = 3
N = 12
T1 = Yn + Yia + Y13 = 3.612
T, = Y21 + Y22 + Y23 = 3.333
T3 = Y31 + Y32 + Y33 - 2-605
T4 = Y41 + Y42 + Y43 =1.768
G = T, + T2 + T3 + T4 - 11.318
SSB = f^Tl/ni-G2/N
SST =
1 (34.067) - fll.318)z = 0.681
3 12
1.002
i-lj'-l
= 11.677 - (11. 318V
12
SSW = SST-SSB
= 1.002 - 0.681 = 0.321
SB = SSB/(p-l) = 0.681/(4-l) = 0.227
Sy = SSW/(N-p) = 0.321/(12-4) = 0.040
13.13.2.8.3 Summarize these calculations in the ANOVA table (Table 9)
TABLE 9. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
df
3
8
Sum of Squares
(SS)
0.681
0.321
Mean Square(MS)
(SS/df)
i
0.227
0.040
Total
11
1.002
199
-------
13.13.2.8.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where: Y? = mean proportion surviving for effluent concentration i
Y,, « mean proportion surviving for the control
SM » square root of the within mean square
n., - number of replicates for the control
nf = number of replicates for concentration i.
13.13.2.8.5 Table 10 includes the calculated t values for each concentration
and control combination. In this example, comparing the 1.0% concentration
with the control the calculation is as follows:
(1.204 - 1.111)
[0.020
= 0.570
TABLE 10. CALCULATED T VALUES
Effluent Concentration(%)
6.25
12.5
25.0
2
3
4.
0.570
2.058
3.766
13.13.2.8.6 Since the purpose of this test is to detect a significant
reduction in survival, a one-sided test is appropriate. The critical value
for this one-sided test is found in Table 5, Appendix C. For an overall alpha
level of 0.05, eight degrees of freedom for error and three concentrations
(excluding the control) the critical value is 2.42. The mean proportion
surviving for concentration i is considered significantly less than the mean
proportion surviving for the control if t,- is greater than the critical value.
Therefore, only the 25.0% concentration has a significantly lower mean
proportion surviving than the control. Hence the NOEC is 12.5% and the LOEC
is 25.0%.
200
-------
13.13.2.8.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
MSD = d Sw
\
Where: d = the critical value for Dunnett's Procedure
Su = the square root of the within mean square
n = the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n1 = the number of replicates in the control.
13.13.2.8.8 In this example:
MSD = 2.42 (0.20)^(1/3)+(1/3)
= 2.42 (0.20)(0.817)
= 0.395
13.13.2.8.9 The MSD (0.395) is in transformed units. To determine the MSD in
terms of percent survival, carry out the following conversion.
1. Subtract the MSD from the transformed control mean
1.204 - 0.395 = 0.809
2. Obtain the untransformed values for the control mean and the
difference calculated in step 1.
[ Sine (1.204) ]* = 0.871
[ Sine (0.809) ]2 = 0.524
3. The untransformed MSD (MSDJ is determined by subtracting the
untransformed values from step 2. I
MSDU = 0.871 - 0.524 = 0.347
13.13.2.8.10 Therefore, for this set of data, the minimum difference in mean
proportion surviving between the control and any effluent concentration that
can be detected as statistically significant is 0.347. >
j
13.13.2.8.11 This represents a 40% decrease in survival from the control.
201
-------
13.13.2.9 Calculation of the LC50
13.13.2.9.1 The data used for the Probit Analysis is summarized in Table 11,
To perform the Probit Analysis, run the USEPA Probit Analysis Program. An
example of the program input and output is supplied in Appendix H.
TABLE 11. DATA FOR-PROBIT ANALYSIS
Eff1uent Concentration (%)
Control 6.25 12.5 25.0 50.0 100.0
Number Dead
Number Exposed
6
45
9
45
19
45
30
45
45
45
45
45
13.13.2.9.2 For this example, the chi-square test for heterogeneity was not
significant. Thus Probit Analysis appears to be appropriate for this set of
data.
13.13.2.9.3 Figure 9 shows the output data for the Probit Analysis of the
data from Table 11 using the USEPA Probit Program.
13.13.3 ANALYSIS OF INLAND SILVERSIDE, MENEDIA BERYLLINA, GROWTH DATA
13.13.3.1 Formal statistical analysis of the growth data is outlined in
Figure 10. The response used in the statistical analysis is mean weight per
original organism for each replicate. The IC25 and IC50 can be calculated for
the growth data via a point estimation technique (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis). Hypothesis testing can be used to
obtain an NOEC and LOEC for growth. Concentrations above the NOEC for
survival are excluded from the hypothesis test for growth effects.
13.13.3.2 The statistical analysis using hypothesis tests consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test. The underlying assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested. The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance. If either of these test fails, the nonparametric
test, Steel's Many-one Rank Test, is used to determine the NOEC and LOEC
endpoints. If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
13.13.3.3 Additionally, if unequal numbers of replicates occur among the
concentration levels tested there are parametric and nonparametric alternative
analyses. The parametric analysis is a t test with the Bonferroni adjustment.
The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric
alternative. For detailed information on the Bonferroni adjustment, see
Appendix D.
202
-------
Probit Analysis of Inland Silverside Larval Survival Data
Cone.
Control
6
12
25
50
100
Chi
Chi
.2500
.5000
.0000
.0000
.0000
- Square
- Square
Number
Exposed
45
45
45
45
45
45
for Heterogeneity
for Heterogeneity
Observed
Number Proportion
Resp. Responding
6
9
19
30
45
45
(calculated)
(tabular value)
0
0
0
0
1
1
.1333
.2000
.4222
.6667
.0000
.0000
Proportion
Responding
Adjusted for
Controls
0
0
0
0
1
1
-
.0000
.0488
.3130
.6037
.0000
.0000
4.149
7.815
Probit Analysis of Inland Silverside Larval Survival Data
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
4.980
18.302
Lower Upper
95% Confidence Limits
2
13
.023
.886
7.789
22.175
Figure 9. Output for USEPA Probit Analysis Program,, Version 1.5.
203
-------
STATISTICAL ANALYSIS OF INLAND SILVERSIDE LARVAL
SURVIVAL AND GROWTH TEST
GROWTH
GROWTH DATA
MEAN DRY WEIGHT
1
r
POINT ESTIMATION
HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FOR SURVIVAL)
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILICS TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
T-TESTWITH
BONFERRONI
ADJUSTMENT
EQUAL NUMBER OF
REPLICATES?
YES
NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 10. Flowchart for statistical analysis of the inland silverside,
Menidia beryl Una, growth data.
204
-------
13.13.3.4 The data, mean and variance of the growth observations at each
concentration including the control are listed in Table 12. A plot of the
data is provided in Figure 11. Since there was no survival in the 50% and
100% concentrations, these are not considered in the growth analysis.
Additionally, since there is significant mortality in the 25% effluent
concentration, its effect on growth is not considered. i
TABLE 12. INLAND SILVERSIDE, MENIDIA BERYLLINA, GROWTH DATA
Effluent Concentration (%)
Replicate Control 6.25 12.5 25.0 50.0 100.0
Me
Si
i
A
B
C
an (Yf)
0
0
0
0
0
1
.751
.849
.907
.836
.0062
0
0
0
0
0
2
.737
.922
.927
.859
.0130
0
0
0
0
0
3
.722
.285
.718
.575
.063
0.
0.
1.
0.
0.
4
196
312
079
196
0136
-
.
-
5 6
13.13.3.5 Test for Normality
i
13.13.3.5.1 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 13.
TABLE 13. CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Effluent Concentration (%)
Replicate Control 6.25 12.5
A -0.085 0.147
B 0.013 -0.290
0.00
0.166
C 0.071 0.143 -0.117
205
-------
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206
-------
13.13.3.5.2 Calculate the denominator, D, of the test statistic:
i=l
Where: X,- = the ith centered observation
X = the overal1 mean of the centered observations
n = the total number of centered observations.
•i
For this set of data, n = 9
X = _J_(-0.002) = 0.000
9 I
D = 0.167
13.13.3.5.3 Order the centered observations from smallest to largest:
y(1) y(2) < v(n)
I
Where XO) is the ith ordered observation. These ordered observations are
listed in Table 14.
TABLE 14. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WALK'S EXAMPLE
1
1
2
3
4
5
X<0
-0.290
-0.117
-0.085
0.000
0.013
i
6
7
8
9
X(i>
0.071
0.116
0.143
0.147
i
13.13.3.5.4 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a?, ..., a, where k is n/2 if n is even and (n-
l)/2 if n is odd. For the data in this example, n = 9 and k = 4. The a,-
values are listed in Table 15.
207
-------
13.13.3.5.5 Compute the test statistic, W, as follows:
W=
D
The differences x(n"i+1) - Xci> are listed in Table 15. For this set of data:
W =
1 (0.3997)2 = 0.964
0.1657
TABLE 15. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1
2
3
4
0.5888
0.3244
0.1976
0.0947
0.437
0.260
0.201
0.071
x<9)
x<8)
XC7)
X(6>
- XC1)
- x<2)
- x<3)
- x<4>
13.13.3.5.6 The decision rule for this test is to compare W 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
example, the critical value at a significance level of 0.01 and nine
observations (n) is 0.764. Since W = 0.964 is greater than the critical
value, the conclusion of the test is that the data are normally distributed.
13.13.3.6 Test for Homogeneity of Variance
13.13.3.6.1 The test used to examine whether the variation in mean dry weight
is the same across all effluent concentrations including the control, is
Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
follows:
B =
In
Where: Vf = degrees of freedom for each effluent concentration and
control, V,- = (nf - 1)
p = number of levels of effluent concentration including the
control
208
-------
i = 1,2, ..., p where p is the number of concentrations including
the control
In = loge
n,- = number of replicates for concentration i
v±sl)
1=1
C =
13.13.3.6.2 For the data in this example, (See Table 13)
concentrations including the control have the same number
3 for all i). Thus, V5 = 2 for all i.
13.13.3.6.3 Bartlett's statistic is therefore:
B = [(6)ln(0.274) -2
all effluent
of replicates (n,-
1/1.25
= [6(-3.5972)-2(ln(0.0062)+ln(0.013())+ln(0.0631))]/1.25
= [-26.583 - (-24.378)]/1.25
= 2.236
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 = 2.236 is less than the critical
value of 9.210, conclude that the variances are not different.
209
-------
13.13.3.7 Dunnett's Procedure
13.13.3.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 16.
TABLE 16. ANOVA TABLE
Source df
Between p - 1
Within N - p
Total N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
SB = SSB/(p-l)
Sy = SSW/(N-p)
Where: p « number of effluent concentrations including the control
N « total number of observations n, + n2 ... + n
nf » number of observations in concentration i
SSB = £ Tl/n± - G2/N Between Sum of Squares
SST -
i-U-i
Total Sum of Squares
SSW » SST-SSB
Within Sum of Squares
the grand total of all sample observations,
i=1
the total of the replicate measurements for concentration i
the jth observation for concentration i (represents the mean
dry weight of the fish for toxicant concentration i in test
chamber j)
210
-------
13.13.3.7.2 For the data in this example:
n, = n2 = n3 = 3
N = 9
T, - Y,. + Y1P + Y13 = 0.751 + 0.849 + 0.907 = 2.507
T, = Yp + Y~ + Yp3 = 0.727 + 0.922 + 0.927 =2.576
T3 " Y3i + Y32 + Y33 = 0.722 + 0.285 + 0,718 = 1.725
G = T, + T2 + T3 = 6.808
SSB =
_1_(15.896) - (6.80S)2 = 0.1488
SST
5.463 - (6. 80S)2 = 0.3131
SSW = SST-SSB = 0.3131 - 0.1488 == 0.1643
i
S2 = SSB/(p-l) = 0.1488/(3-l) = 0.0744
Sj = SSW/(N-p) = 0.1643/(9-3) = 0.0274
13.13.3.7.3 Summarize these calculations in the ANOVA table (Table 17)
TABLE 17. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source df Sum of Squares Mean Square(MS)
(SS) - (SS/df)
Between
Within
2
6
0.1488
0.1643
p. 0744
0.0274
Total 8 0.3131
211
-------
13.13.3.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration and control combination as follows:
Where: Yf = mean dry weight for effluent concentration i
Y., - mean dry weight for the control
Sw - square root of the within mean square
n, - number of replicates for the control
n,- = number of replicates for concentration i.
13.13.3.7.5 Table 18 includes the calculated t values for each concentration
and control combination. In this example, comparing the 6.25% concentration
with the control the calculation is as follows:
(0.836 - 0.859)
[0.1655^(1/3)+(1/3)]
= -0.120
TABLE 18. CALCULATED T VALUES
Effluent Concentration (%)
6.25
12.5
2
3
-0.170
1.931
13.13.3.7.6 Since the purpose of this test is to detect a significant
reduction in mean weight, a one-sided test is appropriate. The critical value
for this one-sided test is found in Table 5, Appendix C. For an overall alpha
level of 0.05, six degrees of freedom for error and two concentrations
(excluding the control) the critical value is 2.34. The mean weight for
concentration i is considered significantly less than mean weight for the
control if tf is greater than the critical value. Therefore, all effluent
concentrations in this example do not have significantly lower mean weights
than the control. Hence the NOEC and the LOEC for growth cannot be
calculated.
13.13.3.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
212
-------
MSD = d Sw ^(1/1^)+(1/12)
Where: d = the critical value for Dunnett's Procedure
Sw = the square root of the within mean square
i
n = the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n1 = the "number of replicates in the control.
13.13.3.7.8 In this example:
MSD = 2.34 (0.1655)V(1/3) + (1/3)
= 2.34 (0.1655)(0.8165)
= 0.316
13.13.3.7.9 Therefore, for this set of data, the minimum difference that can
be detected as statistically significant is 0.316 mg.
13.13.3.7.10 This represents a 37.8% reduction in mean weight from the
control.
!
13.13.3.8 Calculation of the ICp
13.13.3.8.1 The growth data from Tables 4 and 12 are utilized in this
example. As seen in Table 19 and Figure 11, the observed means are not
monotonically non-increasing with respect to concentration (the mean response
for each higher concentration is not less than or equal to the mean response
for the previous concentration, and the reponses between concentrations do not
follow a linear trend). Therefore, the means are smoothed prior to
calculating the 1C. In the following discussion, the observed means are
represented by Y,- and the smoothed means by M,-.
.
13.13.3.8.2 Starting with the control mean, Y., = 0.836 and Y2 = 0.859, we see
that Y, < Y2. Set M,- = Y,-.
j
13.13.3.8.3 Calculate the smoothed means:
i
M1 - M2 = (Y1 + Y2)/2 = °'847
13.13.3.8.4 Since Y5 = 0 < Y4 = 0.196 < Y3 = 0.575 < M2, set M3 = 0.575, M4 =
0.196, and M5 = 0.
213
-------
13.13.3.8.5 Table 19 contains the response means and the smoothed means and
Figure 12 gives a plot of the smoothed response curve.
TABLE 19. INLAND SILVERSIDE MEAN GROWTH RESPONSE AFTER SMOOTHING
Ef f 1 uent
Cone.
(%)
Control
6.25
12.50
25.00
50.00
i
1
2
3
4
5
Response
Means,
Yj
(mg)
0.836
0.859
0.575
0.196
0.00
Smoothed
Means,
M-
(mg)
0.847
0.847
0.575
0.196
0.0
13.13.3.8.6 An IC25 and IC50 can be estimated using the Linear Interpolation
Method. A 25% reduction in weight, compared to the controls, would result in
a mean dry weight of 0.627 mg, where M^l-p/100) = 1.847(1-25/100). A 50%
reduction in mean dry weight, compared to the controls, would result in a mean
weight of 0.418 mg. Examining the smoothed means and their associated
concentrations (Table 20), the response, 0.627 mg, is bracketed by C, = 6.25%
effluent and C3 = 25.0% effluent. The response (0.418) is bracketed by C, =
12.5% and by C4 - 25% effluent. 3
13.13.3.8.7 Using the equation from Section 4.2 of Appendix L, the estimate
of the IC25 is calculated as follows:
ICp =
IC25 = 6.25 + [0.847(1 - 25/100) - 0.847](12.50 - 6.25)
= 11.1%.
(0.575 - 0.847)
214
-------
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215
-------
13.13.3.8.8 Using the equation from Section 4.2 of Appendix L, the estimate
of the IC50 is calculated as follows: ,
ICp -Cj
IC50 = 6.25 + [0.847(1 - 50/100) - 0.847] (12.50 - 6.25)
= 17.5%.
(0.575 - 0.847)
13.13.3.8.9 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 11.1136%. The empirical
95% confidence interval for the true mean was 5.7119% to 19.2112%. The
computer program output for the IC25 for this data set is shown in Figure 13.
13.13.3.8.10 When the ICPIN program was used to analyze this set of data for
the IC50, requesting 80 resamples, the estimate of the IC50 was 17.4896%. The
empirical 95% confidence interval for the true mean was 6.4891% to 22.4754%
effluent. The computer program output is shown in Figure 14.
13.14 PRECISION AND ACCURACY
13.14.1 PRECISION
13.14.1.1 Single-Laboratory Precision
13.14.1.1.1 Data on the single-laboratory precision of the inland silverside
larval survival and growth test using copper (CU) sulfate and sodium dodecyl
sulfate (SDS) as reference toxicants, in natural seawater and GP2 are provided
in Tables 20-22. In Tables 20-21, the coefficient of variation for copper
based on the IC25 is 43.2% and for SDS is 43.2% indicating acceptable
precision. In the five tests with each reference toxicant, the NOEC's varied
by only one concentration interval, indicating good precision. The
coefficient of variation for all reference toxicants based on the IC50 in two
types of seawater (GP2 and natural) ranges from 1.8% to 50.7% indicating
acceptable precision. Data in Table 22 show no detectable differences between
tests conducted in natural and artificial seawaters.
13.14.1.2 Multilaboratory Precision
13.14.1.2.1 Data on the multilaboratory precision of the inland silverside
larval survival and growth test are not yet available.
13.14.2 ACCURACY
13.14.2.1 The accuracy of toxicity tests cannot be determined.
216
-------
Cone. ID
Cone. Tested
Response
Response
Response
1
2
3
1
0
.751
.849
.907
2
6.25
.727
.922
.927
3
12.5
.722
.285
.718
4
25
.196
.312
.079
5
50
0
0
0
6
100
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Test Ending Date:
Test Species: Menidia beryl!ina
Test Duration: 7-d
DATA FILE: silver.icp
OUTPUT FILE: silver.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
%
0.000
6.250
12.500
25.000
50.000
100.000
Response Std.
Means Dev.
0.836 0.079
0.859 0.114
0.575 0.251
0.196 0.117
0.000 0.000
0.000 0.000
Pooled
Response Means
0.847
0.847
0.575
0.196
0.000
0.000
The Linear Interpolation Estimate: 11.1136 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 11.5341 Standard Deviation: 2.1155
Original Confidence Limits: Lower: 8.5413 Upper: 14.9696
Expanded Confidence Limits: Lower: 5.7119 Upper: 19.2112
Resampling time in Seconds: 1.43 Random Seed: -1912403737
Figure 13. ICPIN program output for the IC25.
217
-------
Cone. ID
Cone. Tested
Response
Response
Response
1
2
3
1
0
.751
.849
.907
2
6.25
.727
.922
.927
3
12.5
.722
.285
.718
4
25
.196
.312
.079
5
50
0
0
0
6
100
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Test Ending Date:
Test Species: Menidia beryllina
Test Duration: 7-d
DATA FILE: silver.icp
OUTPUT FILE: silver.i50
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
%
0.000
6.250
12.500
25.000
50.000
100.000
Response
Means
0.836
0.859
0.575
0.196
0.000
0.000
Std. Pooled
Dev. Response Means
0.079
0.114
0.251
0.117
0.000
0.000
0.847
0.847
0.575
0.196
0.000
0.000
The Linear Interpolation Estimate: 17.4896 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 16.9032 Standard Deviation: 2.4973
Original Confidence Limits: Lower: 12.2513 Upper: 19.8638
Expanded Confidence Limits: Lower: 6.4891 Upper: 22.4754
Resampling time in Seconds: 1.43 Random Seed: -1440337465
Figure 14. ICPIN program output for the IC50.
218
-------
TABLE 20. SINGLE-LABORATORY PRECISION OF THE INLAND SILVERSIDE, MENIDIA
BERYLLINA, SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL
SEAWATER, USING LARVAE FROM FISH MAINTAINED AND SPAWNED IN
NATURAL StAWATER, AND COPPER (CU) AS A REFERENCE
172'*'4'^6'7
Most
Test
Number
n:
Mean:
CV(%)
1
2
3
4
5
•
\ Data from USEPA
NOEC
Ug/L)
63
125
63
125
31
5
NA
NA
(1988a) and USEPA
IC25
U9/L)
96.2
207.2
218.9
177.5
350.1
5
209.9
43.7
(1991a)
IC50 , Sensitive
(jug/L) Endpoint
I
148.6
NC8
493.4
241.4
479.8
i 4
340.8
50.7
i
S
s
G
S
G
4
5
i —^ —. . j — ..Vii iuvii \AIIVI i_ i i o i* i w i *^ i i vs } L.IX i_ ii * UOi_rr\5
Narragansett, RI.
Three replicate exposure chambers with 10-15 larvae were used for the
control and each copper concentration. Copper concentrations were: 31,
63, 125, 250, and 500 Mg/L.
Adults collected in the field.
S = Survival effects. G = Growth data at these toxicant concentrations
were disregarded because there was a significant reduction in survival.
NOEC Range: 31 - 125 /j.g/1 (this represents a difference of two exposure
concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
NC = No linear interpolation estimate could be calculated from the ata,
since none of the group response means were less than 50 percent of the
control response mean.
219
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TABLE 21. SINGLE-LABORATORY PRECISION OF THE INLAND SILVERSIDE, MENIDIA
BERYLLINA, SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL
SEAWATER, USING LARVAE FROM FISH MAINTAINED AND SPAWNED IN
NATURAL SEAWATER, AND SODIUM DODECYL SULFATE (SDS) AS A
REFERENCE TOXICANT1'2'3'4'5'6'7
Test
Number
NOEC
(mg/L)
IC25
(mg/L)
IC50
(mg/L)
Most
Sensitive
Endpoint
1
2
3
4
5
1.3
•1.3
1.3
1.3
1.3
0.3
1.6
1.5
1.5
1.6
1.7
1.9
1.9
1.9
2.2
S
S
S
S
S
n:
Mean:
CV(X):
5
NA
NA
5
1.3
43.2
5
1.9
9.4
I Data from USEPA (1988a) and USEPA (1991a)
Tests performed by George Morrison and Elise Torello, ERL-N, USEPA,
Narragansett, RI.
Three replicate exposure chambers with 10-15 larvae were used for the
control and each SDS concentration. SDS concentrations were: 0.3, 0.6,
1.3, 2.5, and 5.0 mg/L.
Adults collected in the field.
S » Survival Effects. Growth data at these toxicant concentrations
were disregarded because there was a significant reduction in survival.
6 NOEC Range 1.3 mg/L.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
220
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TABLE 22. COMPARISON OF THE SINGLE-LABORATORY PRECISION OF THE INLAND
SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL (LC50) AND
GROWTH (IC50) VALUES EXPOSED TO SODIUM DODECYL SULFATE (SDS)
OR COPPER (CU) SULFATE IN GP2 ARTIFICIAL SEAWATER MEDIUM OR
NATURAL SEAWATER (NSW)1'2'3'4
SDS (mg/L)
Survival
GP2
NSW
Growth
GP2
NSW
3.59
4.87
5.95
Mean 4.81
CV (%) 24.6
3.69 3.60
4.29 5.54
8.05
6.70
5.34 . 5.28
44.2
29.6
3.55
5.27
8.53
5.79
43.8
Copper
GP2
247
215
268
NSW
256
211
240
GP2
260
236C
NC5
NSW
277
223
238
Mean
CV (%)
243
10.9
236
9.8
248
6.9
246
11.2
Tests performed by George Morrison and Glen Modica, ERL-N, USEPA,
Narragansett, RI.
Three replicate exposure chambers with 10-15 larvae per treatment.
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance. !
NC= No linear interpolation estimate could be calculated from the data,
since none of the group response means were less than 50 percent of the
control response mean.
221
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SECTION 14
TEST METHOD
MYSID, HYSIDOPSIS BAHIA, SURVIVAL,
GROWTH, AND FECUNDITY TEST
METHOD 1007.0
14.1 SCOPE AND APPLICATION
14.1.1 This method, adapted in part from USEPA (1987d), estimates the chronic
toxicity of effluents and receiving waters to the mysid, Mysidopsis bahia,
during a seven-day, static renewal exposure. The effects include the
synergistic, antagonistic, and additive effects of all the chemical, physical,
and additive components which adversely affect the physiological and
biochemical functions of the test organisms.
14.1.2 Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LCSOs).
14.1.3 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
14.1.4 Brief excursions in toxicity may not be detected using 24-h composite
samples. Also, because of the long sample collection period involved in
composite sampling and because the test chambers are not sealed, highly
volatile and highly degradable toxicants present in the source may not be
detected in the test.
14.1.5 This test is commonly used in one of two forms: (1) a definitive
test, consisting of a minimum of five effluent concentrations and a control,
and (2) a receiving water test(s), consisting of one or more receiving water
concentrations and a control.
14.2 SUMMARY OF METHOD
14.2.1 Mysidopsis bahia 7-day old juveniles are exposed to different
concentrations of effluent, or to receiving water in a static system, during
the period of egg development. The test endpoints are survival, growth
(measured as dry weight), and fecundity (measured as the percentage of females
with eggs in the oviduct and/or brood pouch).
14.3 INTERFERENCES
14.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
14.3.2 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
222
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14.3.3 The test results can be confounded by (1) the presence of pathogenic
and/or predatory organisms in the dilution water, effluent, and receiving
water, (2) the condition of the brood stock from which the test animals were
taken, (3) the amount and type of natural food in the effluent, receiving
water, or dilution water, (4) nutritional value of the brine shrimp, Artemia
nauplii, fed during the test, and (5) the quantity of brine shrimp, Artemia
nauplii, or other food added during the test, which may sequester metals and
other toxic substances, and lower the DO.
14.4 SAFETY \
14.4.1 See Section 3, Health and Safety.
14.5 APPARATUS AND EQUIPMENT I
14.5.1 Facilities for holding and acclimating test organisms.
14.5.2 Brine shrimp, Artemia, culture unit -- see Subsection 14.6.12 below
and Section 4, Quality Assurance. ;
14.5.3 Mysid, Mysidopsis bahia, culture unit -- see Subsection 6 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.
14.5.4 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.
14.5.5 Environmental chamber or equivalent facility with temperature control
(26 ± 1°C).
I
I
14.5.6 Water purification system -- Millipore Milli-Q®, doionized water or
equivalent.
i
14.5.7 Balance -- Analytical, capable of accurately weighing to 0.00001 g.
14.5.8 Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of the weighing pans and weighing
pans plus organisms.
14.5.9 Drying oven -- 50-105°C range, for drying organisms.
14.5.10 Desiccator -- for holding dried organisms.
14.5.11 Air pump -- for oil-free air supply.
14.5.12 Air lines, and air stones -- for aerating cultures, brood chambers,
and holding tanks, and supplying air to test solutions with low DO.
14.5.13 Meters, pH and DO -- for routine physical and chemical measurements.
-
1
223
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14.5.14 Tray -- for test vessels; approximately 90 X 48 cm to hold 56
vessels.
14.5.15 Standard or micro-Winkler apparatus -- for determining DO and
checking DO meters.
14.5.16 Dissecting microscope (350-400X magnification) -- for examining
organisms in the test vessels to determine their sex and to check for the
presence of eggs in the oviducts of the females.
14.5.17 Light box -- for illuminating organisms during examination.
14.5.18 Refractometer or other method -- for determining salinity.
14.5.19 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.
14.5.20 Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.
14.5.21 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
14.5.22 Test chambers -- 200 ml borosilicate glass beakers or non-toxic 8 oz
disposable plastic cups (manufactured by Falcon Division of Becton, Dickinson
Co., 1950 Williams Dr., Oxnard, CA 93030) or other similar containers.
Forty-eight (48) test vessels are required for each test (eight replicates at
each of five effluent concentrations and a control). To avoid potential
contamination from the air and excessive evaporation of test solutions during
the test, the chambers should be covered with safety glass plates or sheet
plastic (6 mm thick).
14.5.23 Beakers or flasks -- six, borosilicate glass or non-toxic
plasticware, 2000 mL for making test solutions.
14.5.24 Wash bottles -- for deionized water, for washing organisms from
containers and for rinsing small glassware and instrument electrodes and
probes.
14.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 50-2000 ml for making test solutions.
14.5.26 Separatory funnels, 2-1 -- Two-four for culturing Artemia.
14.5.27 Pipets, volumetric -- Class A, 1-100 ml.
14.5.28 Pipets, automatic -- adjustable, 1-100 ml.
14.5.29 Pipets, serological -- 1-10 ml, graduated.
14.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent.
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14.5.31 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for
transferring organisms.
14.5.32 Forceps -- for transferring organisms to weighing pans.
,
14.5.33 NITEX® or stainless steel mesh sieves (< 150 //m, 500-1000 /j.m, 3-5 mm)
-- for concentrating organisms. (NITEX® is available from Sterling Marine
Products, 18 Label Street, Montclair, NJ 07042; 201-783-9800).
14.5.34 Depression glass slides or depression spot plates -- two, for
observing organisms.
14.6 REAGENTS AND CONSUMABLE MATERIALS
14.6.1 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
14.6.2 Data sheets (one set per test) -- for data recording (Figures 14, 15,
and 16).
14.6.3 Tape, colored -- for labelling test chambers. !
14.6.4 Markers, waterproof -- for marking containers, etc,
j
14.6.5 Weighing pans, aluminum -- to determine the dry weight of organisms.
14.6.6 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) -- for standards and calibration check (see USEPA Method 150 1
USEPA, 1979b).
14.6.7 Membranes and filling solutions -- for dissolved oxygen probe (see
USEPA Method 360.1, USEPA, 1979b), or reagents for modified Winkler analysis.
14.6.8 Laboratory quality assurance samples and standards -- for the above
methods.
14.6.9 Reference toxicant solutions -- see Section 4, Quality Assurance.
14.6.10 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies). j
14.6.11' Effluent, receiving water, and dilution water --see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests. Dilution water
containing organisms that might prey upon or otherwise interfere with the test
organisms should be filtered through a fine mesh net (with 150 u.m or smaller
openings).
14.6.11.1 Saline test and dilution water -- The salinity oif the test water
must be in the range of 20% to 30%o. The salinity should vary by no more
225
-------
than ± 2%o among the chambers on a given day. If effluent and receiving water
tests are conducted concurrently, the salinities of these tests should be
similar.
14.6.11.2 The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts. Exposure of mysids to these effluents will require
adjustments in the salinity of the test solutions. It is important to
maintain a constant salinity across all treatments. 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 (HSB)
derived from natural seawater or artificial sea salts.
14 6.11.3 HSB has several advantages that make it desirable for use in
toxicity testing. It can be made from any high quality, filtered seawater by
evaporation, and can be added to the effluent or to deionized water to
increase the salinity. Brine derived from natural seawater contains the
necessary trace metals, biogenic colloids, and some of the microbial
components necessary for adequate growth, survival, and/or reproduction of
marine and estuarine organisms, and may be stored for prolonged periods
without any apparent degradation. However, if 100%o HSB is used as a diluent,
the maximum concentration of effluent that can be tested is 80% effluent at
30%o salinity and 70% effluent at 30% salinity.
14 6 11 3 1 The ideal container for making brine from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a non-corrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine. If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that would contaminate the brine. One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass. If aeration is used, only oil-free air compressors should be
used to prevent contamination.
14.6.11.3.2 Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be in direct contact with the brine. A good quality biodegradable detergent
should be used, followed by several (at least three) thorough deionized water
rinses.
14.6.11.3.3 High quality (and preferably high salinity) seawater should be
filtered to at least 10 ^m before placing into the brine generator. Water
should be collected on an incoming tide to minimize the possibility of
contamination.
14.6.11.3.4 The temperature of the seawater is increased slowly to 40°C. The
water should be aerated to prevent temperature stratification and to increase
water evaporation. The brine should be checked daily (depending on the volume
being enerated) to ensure that the salinity does not exceed 100%o and that.the
temperature does not exceed 40°C. Additional seawater may be added to the
brine to obtain the volume of brine required.
226
-------
14.6.11.3.5 After the required salinity is attained, the HSB should be
filtered a second time through a 1 /im filter and poured directly into portable
containers (20-L cubitainers or polycarbonate water cooler jugs are suitable).
The containers should be capped and labelled with the date the brine was
generated and its salinity. Containers of HSB should be s;tored in the dark
and maintained under room temperature until used.
14.6.11.3.6 If a source of HSB is available, test solutions can be made by
following the directions below. Thoroughly mix together the deionized water
and HSB before mixing in the effluent.
I
14.6.11.3.7 Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine. For example, if the
salinity of the brine is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0. The proportion of brine is 1 part in 5 (one part brine
to four parts deionized water). To make 1 L of seawater at 20%o salinity from
a HSB of 100%o, 200 ml of brine and 800 ml of deionized water are required.
14.6.11.3.8 Table 2 illustrates the composition of 1800 ml test solutions at
20%o if they are made by combining effluent (0%o), deioni2f.ed water and HSB of
100%o (only). The volume (ml) of brine required is determined by using the
amount calculated above. In this case, 200 ml of brine is; required for 1 L;
therefore, 360 ml would be required for 1.8 L of solution. The volumes of HSB
required are constant. The volumes of deionized water are determined by
subtracting the volumes of effluent and brine from the total volume of
solution: 1800 ml - ml effluent - ml brine = ml deionized water.
14.6.11.4 Artifical sea salts: FORTY FATHOMS® brand sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-321-1189) have
been used successfully to culture and perform life cycle tests with mysids
(Home, et al., 1983; ASTM, 1993) (see Section 7, Dilution Water). HW
Marinemix® (Hawaiian Marine Imports, Inc., P.O. Box 218687, Houston, TX 77218;
713-492-7864 sea salts have been used successfully to culture mysids and
perform the mysid toxicity test (USEPA Region 6 Houston Laboratory; EMSL-
Cincinnati). In addition, a slightly modified version of the GP2 medium
(Spotte et al., 1984) has been successfully used to perform the mysid
survival, growth, and fecundity test (Table 1).
i
14.6.11.4.1 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 hydation
should be in the temperature range of 21-26°C. Seawater made from artificial
sea salts is conditioned (Spotte, 1973; Spotte, et al., 1984; Bower, 1983)
before it is used for culturing or testing. After adding the water, place an
airstone in the container, cover, and aerate the solution mildly for 24 h
before use.
14.6.11.4.2 The GP2 reagent grade chemicals (Table 1) 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
i
227
-------
TABLE 1. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
ARTIFICIAL SEAWATER FOR THE MYSID, MYSIDOPSIS BAHIA, TOXICITY
TEST1'*'*
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 • 6 H20
CaCl2 • 2 H20
SrCl2 • 6 H20
NaHC03
1 Modified GP2 from Spotte
™ Tl* s* >«nvif>t4**i4*ii«>\M4* <•»••» T 4« <•» ** *^f
Concentration
(9/L)
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
et al. (1984).
•1 y'ttf'tMSt/tM^XM'N^-vrVMf* I.I
Amount (g)
Required for
20L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
j-ivtsN 4- •* \ff\v\ <£%A/tm 1 1C CD A
(1990b). The salinity is 30.89 g/L.
GP2 can be diluted with deionized (DI) water to the desired test
salinity.
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 NaHC03 in 500 mL of
deionized water. Add 2.5 mL of this stock solution for each liter of the GP2
artificial seawater.
14.6.12 BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test
organisms.
14.6.12.1 Newly hatched Artemia nauplii are used for food for the stock
cultures and test organisms. Although there are many commercial sources of
brine shrimp cysts, the Brazilian or Colombian strains are preferred because
the supplies examined have had low concentrations of chemical residues and
228
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TABLE 2. QUANTITIES OF EFFLUENT, DEIONIZED WATER, AND HYPERSALINE BRINE
(100%o) NEEDED TO PREPARE 1800 ML VOLUMES OF TEST SOLUTION
WITH A SALINITY OF 20%o
Effluent Volume of Volume of Volume of
Concentration Effluent Deionized Hypersaline Total Volume
(%) (0%o) Water Brine (mL)
(mL) (ml) (mL)
80 1440 0 360 1800
40 720 720 360 1800
20 360 1080 360 i 1800
10 180 1260 360 1800
5 90 1350 360 i 1800
Control 0 1440 360 i, 1800
i
I
Total 2790 5850 2160 t 10800
produce nauplii of suitably small size. For commercial sources of brine
shrimp, Artemia, cysts, see Table 2 of Section 5, Facilities, Equipment, and
Supplies); and Section 4, Quality Assurance.
14.6.12.2 Each new batch of Artemia cysts must be evaluated for size
(Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al., 1980) and nutritional
suitability (Leger, et al., 1985, Leger, et al., 1986) against known suitable
reference cysts by performing a side-by-side larval growth test using the
"new" and "reference" cysts. The "reference" cysts used in the suitability
test may be a previously tested and acceptable batch of cysts, or may be
obtained from the Quality Assurance Research Division, EMSL, Cincinnati, OH
45268, 513-569-7325. A sample of newly-hatched Artemia nauplii from each new
batch of cysts should be chemically analyzed. The Artemis cycts should not be
used if the concentration of total organic chlorine exceeds 0.15 ^g/g wet
229
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weight or the total concentration of organochlorine pesticides plus PCBs
exceeds 0.30 /*g/g wet weight (For analytical methods see USEPA, 1982).
14.6.12.2.1 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or an aqueous uniodized salt (NaCl) solution
prepared with 35 g salt or artificial sea salts to 1 L of deionized
water, to a 2-L separatory funnel, or equivalent.
2. Add 10 ml Artemia cysts to the separatory funnel and aerate for 24 h
at 27°C. Hatching time varies with incubation temperature and the
geographic strain of Artemia used (see USEPA, 1985a; USEPA, 1993a;
ASTM, 1993).
3. After 24 h, cut off the air supply in the separatory funnel.
Artemia nauplii are phototactic, and will concentrate at the bottom
of the funnel if it is covered for 5-10 minutes. To prevent
mortality, do not leave the concentrated nauplii at the bottom of the
funnel more than 10 min without aeration.
4. Drain the nauplii into a beaker or funnel fitted with a < 150 ^m
NITEX® or stainless steel screen, and rinse with seawater or
equivalent before use.
14.6.12.3 Testing Artemia nauplii as food for toxicity test organisms.
14.6.12.3.1 The primary criteria for acceptability of each new supply of
brine shrimp, cysts is adequate survival, growth, and reproduction of the
mysids. The mysids used to evaluate the acceptability of the brine shrimp
nauplii must be of the same geographical origin and stage of development (7
days old) as those used routinely in the toxicity tests. Sufficient data to
detect differences in survival and growth should be obtained by using eight
replicate test chambers, each containing 5 mysids, for each type of food.
14.6.12.3.2 The feeding rate and frequency, test vessels, volume of control
water, duration of the test, and age of the Artemia nauplii at the start of
the test, should be the same as used for the routine toxicity tests.
14.6.12.3.3 Results of the brine shrimp, Artemia, nauplii nutrition assay,
where there are only two treatments, can be evaluated statistically by use of
a t test. The "new" food is acceptable if there are no statistically
significant differences in the survival, growth, and reproduction of the
mysids fed the two sources of nauplii.
14.6.13 TEST ORGANISMS, Mysidopsis bahia (see Rodgers et al., 1986 and USEPA,
1993a for information on mysid ecology).
14.6.13.1 Brood Stock
14.6.13.1.1 To provide an adequate supply of juveniles for a test, mysid,
Mysidopsis bahia, cultures should be started at least four weeks before the
test animals are needed. At least 200 mysids, Mysidopsis bahia, should be
placed in each culture tank to ensure that 1500 to 2000 animals will be
available by the time preparations for a test are initiated.
230
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14.6.13.1.2 Mysids, Mysidopsis bahia, may be shipped or otherwise transported
in polyethylene bottles or CUBITAINERS®. Place 50 animals in 700 ml of
seawater in a 1-L shipping container. To control bacterial growth and prevent
DO depletion during shipment, do not add food. Before closing the shipping
container, oxygenate the water for 10 min. The mysids, Mysidopsis bahia, will
starve if not fed within 36 h, therefore, they should be shipped so that they
are not in transit more than 24 h.
14.6.13.1.3 The identification of the Mysidopsis bahia stock culture should
be verified using the key from Heard (1982), Price (1978), Price, (1982),
Stuck et al. (1979a), and Stuck et al.-(1979b). Records of the verification
should be retained along with a few of the preserved specimens.
14.6.13.1.4 Glass aquaria (120- to 200-L) are recommended for cultures.
Other types of culture chambers may also be convenient. Three or more
separate cultures should be maintained to protect against loss of the entire
culture stock in case of accident, low DO, or high nitrite levels, and to
provide sufficient numbers of juvenile mysids, Mysidopsis bahia, for toxicity
tests. Fill the aquaria about three-fourths full of seawater. A flow-through
system is recommended if sufficient natural seawater is available, but a
closed, recirculating or static renewal system may be used.if proper water
conditioning is provided and care is exercised to keep the pH above 7.8 and
nitrite levels below 0.05 mg/L.
14.6.13.1.5 Standard aquarium undergravel filters should be used with either
the flow-through or recirculating system to provide aeration and a current
conducive to feeding (Gentile et al., 1983). The undergravel filter is covered
with a prewashed, coarse (2-5 mm) dolomite substrate, 2.5 cm deep for
flow-through cultures or 10 cm deep for recirculating cultures.
14.6.13.1.6 The recirculating culture system is conditioned as follows:
1. After the dolomite has been added, the filter is attached to the air
supply and operated for 24 h.
2. Approximately 4 L of seed water obtained from a successfully
operating culture is added to the culture chamber,
3. The nitrite level is checked daily with an aquarium test kit or with
EPA Method 354.1 (USEPA, 1979b).
4. Add about 30 ml of concentrated Artemia nauplii every other day until
the nitrite level reaches at least 2.0 mg/L. The nitrite will
continue to rise for several days without adding more Artemia nauplii
and will then slowly decrease to less than 0.05 mg/L,,
5. After the nitrite level falls below 0.05 mg/L, add another 30 mL of
Artemia nauplii concentrate and check the nitrite concentration every
day.
6. Continue this cycle until the addition of Artemia nauplii does not
cause a rise in the nitrite concentration. The culture chamber is
then conditioned and is ready to receive mysids.
7. Add only a few (5-20) mysids at first, to determine if conditions are
favorable. If these mysids are still doing well after a week, several
hundred more can be added.
231
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14.6.13.1.7 It is important to add enough food to keep the adult animals from
cannibalizing the young, but not so much that the DO is depleted or that there
is a buildup of toxic concentrations of ammonia and nitrite. Just enough
newly-hatched Artemia nauplii are fed twice a day so that each feeding is
consumed before the next feeding.
14.6.13.1.8 Natural seawater is recommended as the culture medium, but HSB
may be used to make up the culture water if natural seawater is not available.
EMSL-Cincinnati has successfully used FORTY FATHOMS® artificial sea salts for
culturing and toxicity tests of mysids, and USEPA, Region 6 has used HW
MARINEMIX® artificial sea salts.
14.6.13.1.9 Mysids, Mysidopsis bahia, should be cultured at a temperature of
26 ± 1°C. No water temperature control equipment is needed if the ambient
laboratory temperature remains in the recommended range, and if there are no
frequent, rapid, large temperature excursions in the culture room.
14.6.13.1.10 The salinity should be maintained at 30 ± 2%o, or at a lower
salinity (but not less than 20%o) if most of the tests will be conducted at a
lower salinity.
14.6.13.1.11 Day/n'ight cycles prevailing in most laboratories will provide
adequate illumination for normal growth and reproduction. A 16-h/8-h day/night
cycle in which the light is gradually increased and decreased to simulate dawn
and dusk conditions, is recommended.
14.6.13.1.12 Mysid, Mysidopsis bahia, culture may suffer if DOs fall below 5
mg/L for extended periods. The undergravel filter will usually provide
sufficient DO. If the DO drops below 5 mg/L at 25°C and 30%o, additional
aeration should be provided. Measure the DO in the cultures daily the first
week and then at least weekly thereafter.
14.6.13.1.13 Suspend a clear glass or plastic panel over the cultures, or use
some other means of excluding dust and dirt, but leave enough space between
the covers and culture tanks to allow circulation of air over the cultures.
14.6.13.1.14 If hydroids or worms appear in the cultures, remove the mysids
and clean the chambers thoroughly, using soap and hot water. Rinse once with
acid (10% HC1) and three times with distilled or deionized water. Mysids with
attached hydroids should be discarded. Those without hydroids should be
transferred by hand pipetting into three changes of clean seawater before
returning them to the cleaned culture chamber. To guard against predators,
natural seawater should be filtered through a net with 30 /j,m mesh openings
before entering the culture vessels.
14.6.13.1.15 Mysids, Mysidopsis bahia, are very sensitive to low pH and
sudden changes in temperature. Care should be taken to maintain the pH at 8.0
± 0.3, and to limit rapid changes in water temperature to less than 3°C.
14.6.13.1.1(5 Mysids, Mysidopsis bahia, should be handled carefully and as
little as possible so that they are not unnecessarily stressed or injured.
They should be transferred between culture chambers with long handled cups
232
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with netted bottoms. Animals should be transferred to the test vessels with a
large bore pipette (4-mm), taking care to release the animals under the
surface of the water. Discard any mysids that are injured during handling.
14.6.13.1.17 Culture Maintenance (Also See USEPA, 1993a)
14.6.13.1.17.1 Cultures in closed, recirculating systems are fed twice a day.
If no nauplii are present in the culture chamber after four hours, the amount
of food should be increased slightly. In flow-through systems, excess food
can be a problem by promoting bacterial growth and low dissolved oxygen.
14.6.13.1.17.2 Careful culture maintenance is essential. The organisms
should not be allowed to become too crowded. The cultures should be cropped
as often as necessary to maintain a density of about 20 mysids per liter. At
this density, at least 70% of the females should have eggs in their brood
pouch. If they do not, the cultures are probably under stress, and the cause
should be found and corrected. If the cause cannot be found, it may be
necessary to restart the cultures with a clean culture chamber, a new batch of
culture water, and clean gravel.
14.6.13.1.17.3 In closed, recirculating systems, about one third of the
culture water should be replaced with newly prepared seawater every week.
Before siphoning the old media from the culture, it is recommended that the
sides of the vessel be scraped and the gravel carefully turned over to prevent
excessive buildup of algal growth. Twice a year the mysids should be removed
from the recirculating cultures, the gravel rinsed in clean seawater, the
sides of the chamber washed with clean seawater, and the gravel and animals
returned to the culture vessel with newly conditioned seawater. No detergent
should be used, and care should b.e taken not to rinse all the bacteria from
the gravel.
14.6.13.2 Test Organisms
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 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.
14.6.13.2.2 Eight days before the test is to start, sufficient gravid females
are placed in brood chambers. Assuming that 240 juveniles are needed for each
test, approximately half this number (120) of gravid females should be
transferred to brood chambers. The mysids are removed from the culture tank
with a net or netted cup and placed in 20-cm diameter finger bowls. The
gravid females are transferred from the finger bowls to the brood chambers
with a large-bore pipette or, alternatively, are transferred by pouring the
contents of the finger bowls into the water in the brood chambers.
14.6.13.2.3 The mysid juveniles may be collected for the toxicity tests by.
transferring gravid females from the stock cultures to netted (1000 /^m)
flow-through containers (Figure 1) held within 4-1 glass, wide-mouth
233
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INFLOW
OUTFLOW
NETTED
CHAMBER
SEPARATORY
FUNNEL
NETTED
CHAMBER
CULTURE DISH
Figure 1. Apparatus (brood chamber) for collection of juvenile mysids,
Nysidopsis bahia. From USEPA (1987d).
separatory funnels. Newly released juveniles.can pass through the netting,
whereas the females are retained. The gravid females are fed newly hatched
Artemia nauplii, and are held overnight to permit the release of young. The
juvenile mysids are collected by opening the stopcock on the funnel and
collecting them in another container from which they are transferred to
holding tanks using a wide bore (4 mm ID) pipette. The brood chambers usually
require aeration to maintain sufficient DO and to keep the food in suspension.
14.6.13.2.4 The temperature in the brood chamber should be maintained at the
upper acceptable culture limit (26 - 27°C), or 1°C higher than the cultures,
to encourage faster brood release. At this temperature, sufficient juveniles
should be produced for the test.
14.6.13.2.5 The newly released juveniles (age = 0 days) are transferred to
20-L glass aquaria (holding vessels) which are gently aerated. Smaller
holding vessels may be used, but the density of organisms should not exceed 10
mysids per liter. The test animals are held in the holding vessel for six
days prior to initiation of the test. The holding medium is renewed every
other day.
14.6.13.2.6 During the holding period, the mysids are acclimated to the
salinity at which the test will be conducted, unless already at that salinity.
The salinity should be changed no more than 2%o per 24 h to minimize stress on
the juveniles.
234
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14.6.13.2.7 The temperature during the holding period is critical to mysid
development, and must be maintained at 26 ± 1°C. If the temperature cannot be
maintained in this range, it is advisable to hold the juveniles an additional
day before beginning the test.
14.6.13.2.8 During the holding period, just enough newly-hatched Artemia
nauplii are fed twice a day (a total of at least 150 nauplii per mysid per
day) so that some food is constantly present.
i
14.6.13.2.9 If the test is to be performed in the field, the juvenile mysids,
Mysidopsis bahia, should be gently siphoned into 1-L polyethylene wide-mouth
jars with screw-cap lids filled two-thirds full with clean seawater from the
holding tank. The water in these jars is aerated for 10 min, and the jars are
capped and packed in insulated boxes for shipment to the test site. Food
should not be added to the jars to prevent the development of excessive
bacterial growth and a reduction in DO.
.
14.6.13.2.10 Upon arrival at the test site (in less than 24 h) the mysids,
Mysidopsis bahia, are gently poured from the jars into 20-cm diameter glass
culture dishes. The jars are rinsed with salt water to dislodge any mysids
that may adhere to the sides. If the water appears milky, siphon off half of
it with a netted funnel (to avoid siphoning the mysids) and replace with clean
salt water of the same salinity and temperature. If no Artemia nauplii are
present in the dishes, feed about 150 Artemia nauplii per mysid.
14.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
14.7.1 See Section 8, Effluent and Receiving Water Sampling., Sample Handling,
and Sample Preparation for Toxicity Tests.
14.8 CALIBRATION AND STANDARDIZATION
14.8.1 See Section 4, Quality Assurance.
14.9 QUALITY CONTROL
I •
14.9.1 See Section 4, Quality Assurance.
14.9.2 The reference toxicant recommended for use with the mysid 7-day test
is copper sulfate or sodium dodecyl sulfate.
14.10 TEST PROCEDURES
l
I
14.10.1 TEST DESIGN
'
14.10.1.1 The test consists of at least five effluent concentrations plus a
site water control and a reference water treatment (natural seawater or
seawater made up from hypersaline brine, or equivalent).
14.10.1.2 Effluent concentrations are expressed as percent effluent.
235
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14.10.1.3 Eight replicate test vessels, each containing 5 to 7 day old
animals, are used per effluent concentration and control.
14.10.2 TEST SOLUTIONS
14.10.2.1 Receiving waters
14.10.2.1.1 The sampling point(s) is determined by the objectives of the
test. At estuarine and marine sites, samples are usually collected at mid-
depth. Receiving water toxicity is determined with samples used directly as
collected or with samples passed through a 60 urn NITEX® filter and compared
without dilution, against a control. Using eight replicate chambers per test,
each containing 150 ml, and 400 ml for chemical analysis, would require
approximately 1.6 L or more of sample per test per day.
14.10.2.2 Effluents
14.10.2.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used. A
dilution factor of 0.5 provides precision of ± 100%, and testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used. Therefore, USEPA recommends the
use of the ^ 0.5 dilution factor. If 100%o MSB is used as a diluent, the
maximum concentration of effluent that can be tested will be 80% at 20%o and
70% at 30%o salinity.
14.10.2.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If high mortality is observed during the first l-to-2 h of
the test, additional dilutions at the lower range of effluent concentrations
should be added.
14.10.2.2.3 The volume of effluent required for daily renewal of eight
replicates per concentration for five concentrations of effluent and a
control, each containing 150 ml of test solution, is approximately 1200 ml.
Prepare enough test solution (approximately 1600 ml) at each effluent
concentration to provide 400 ml additional volume for chemical analyses.
14.10.2.2.4 Just prior to test initiation (approximately 1 h), the
temperature of a sufficient quantity of the sample to make the test solutions
should be adjusted to the test temperature (26 ± 1°C) and maintained at that
temperature during the addition of dilution water.
14.10.2.2.5 Higher effluent concentrations (i.e., 25%, 50%, and 100%) may
require aeration to maintain adequate dissolved oxygen concentrations.
However, if one solution is aerated, all concentrations must be aerated.
Aerate effluent as it warms and continue to gently aerate test solutions in
the test chambers for the duration of the test.
236
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14.10.2.2.6 Effluent dilutions should be prepared for all replicates in each
treatment in one flask to minimize variability among the replicates. The test
chambers (cups) are labelled with the test concentration and replicate number.
Dispense 150 ml of the appropriate effluent dilution to each test chamber.
14.10.2.3 Dilution Water
14.10.2.3.1 Dilution water may be uncontaminated natural seawater (receiving
water), HSB prepared from natural seawater, or artifical seawater prepared
from FORTY FATHOMS® or GP2 sea salts (see Table 1 and Section 7, Dilution
Water). Other artifical sea salts may be used for culturing mysid and for the
survival, growth, and fecundity test if the control criteria for acceptability
of test data are satisfied.
14.10.3 START OF THE TEST \
14.10.3.1 The test should begin as soon as possible, preferably within 24 h
after sample collection. The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
tests unless permission is granted by the permitting authority. In no case
should the test be started more than 72 h after sample collection (see
Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests).
j
14.10.3.2 Begin the test by randomly placing five animals (one at a time) in
each test cup of each treatment using a large bore (4 mm ID) pipette (see
Appendix A for an example of randomization). It is easier to capture the
animals if the volume of water in the dish is reduced and the dish is placed
on a light table. It is recommended that the transfer pipette be rinsed
frequently because mysids tend to adhere to the inside surface.
14.10.4 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
14.10.4.1 The light quality and intensity under ambient laboratory conditions
are generally adequate. Light intensity of 10-20 /iE/m/si or 50 to 100 foot
candles (ft-c), with a photoperiod of 16 h light and 8 h darkness. It is
critical that the test water temperature be maintained at 26 ± 1°C. It is
recommended that the test water temperature be continuously recorded. The
salinity should vary no more than ± 2%o among chambers on a given day. If
effluent and receiving water tests are conducted concurrently, the salinities
of these tests should be similar.
I
14.10.4.1.1 If a water bath is used to maintain the test temperature, the
water depth surrounding the test cups should be at least 2.5 cm deep.
14.10.4.1.2 Rooms or incubators with high volume ventilation should be used
with caution because the volatilization of the test solutions and evaporation
of dilution water may cause wide fluctuations in salinity. Covering the test
cups with clear polyethylene plastic may help prevent volatilization and
evaporation of the test solutions.
237
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14.10.5 DISSOLVED OXYGEN (DO) CONCENTRATION
14.10.5.1 Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain a satisfactory DO. The DO should be
measured on new solutions at the start of the test (Day 0) and before daily
renewal of test solutions on subsequent days. The DO should not fall below
4.0 mg/L (see Section.8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests). If it is necessary to
aerate, all treatments and the control should be aerated. The aeration rate
should not exceed 100 bubbles/minute, using a pipet with a 1-2 mm orifice,
such as a 1-mL KIMAX® serological pipet No. 37033, or equivalent. Care should
be taken to ensure that turbulence resulting from aeration does not cause
undue stress on the mysid.
14.10.6 FEEDING
14.10.6.1 Artemia nauplii are prepared as described above.
14.10.6.2 During the test, the mysids in each test chamber should be fed
Artemia nauplii, (less than 24-h old), at the rate of 150 nauplii per mysid
per day. Adding the entire daily ration at a single feeding immediately after
test solution renewal may result in a significant DO depression. Therefore,
it is preferable to feed half of the daily ration immediately after test
solution renewal, and the second half 8 - 12 h later. Increase the feeding if
the nauplii are consumed in less than 4 h. It is important that the nauplii
be washed before introduction to the test chamber.
14.10.7 DAILY CLEANING OF TEST CHAMBERS
14.10.7.1 Before the daily renewal of test solutions, uneaten and dead
Artemia, dead mysids and other debris are removed from the bottom of the test
chambers with a pipette. As much of the uneaten Artemia as possible should be
removed from each chamber to ensure that the mysids principally eat new
hatched nauplii. By placing the test chambers on a light box, inadvertent
removal of live mysids can be greatly reduced because they can be more easily
seen. Any incidence of removal of live mysids from the test chambers during
cleaning, and subsequent return to the chambers should be noted in the test
records.
14.10.8 OBSERVATIONS DURING THE TEST
14.10.8.1 Routine Chemical and Physical Determinations
14.10.8.1.1 DO is measured at the beginning and end of each 24-h exposure
period in one test chamber at. each test concentration and in the control.
14.10.8.1.2 Temperature, pH, and salinity are measured at the end of each
24-h exposure period in one test chamber at each concentration and in the
control. Temperature should also be monitored continuously observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
238
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chambers at least at the end of the test to determine temperature variation in
environmental chamber.
14.10.8.1.3 The pH is measured in the effluent sample each day before new
test solutions are made.
14.10.8.2 Routine Biological Observations
14.10.8.2.1 The number of live mysids are counted and recorded each day when
the test solutions are renewed (Figure 2). Dead animals and excess food
should be removed with a pipette before test solutions are renewed.
14.10.8.2.2 Protect the mysids from unnecessary distrubance during the test
by carrying out the daily test observations, solution renewals, and removal of
the dead mysids, carefully. Make sure the mysids remain immersed during the
performance of the above operations.
14.10.9 TEST SOLUTION RENEWAL
•
14.10.9.1 Before the daily renewal of test solutions, slowly pour off all but
10 mL of the old test medium into a 20 cm diameter culture dish on a light
table. Be sure to check for animals that may have adhered to the sides of the
test chamber. Rinse them back into the test cups. Add 150 mL of new test
solution slowly to each cup. Check the culture dish for animals that may have
been poured out with the old media, and return them to the test chamber.
14.10.10 TERMINATION OF THE TEST
14.10.10.1 After measuring the DO, pH, temperature, and salinity and
recording survival, terminate the test by pouring off the test solution in all
the cups to a one cm depth and refilling the cups with clean seawater. This
will keep the animals alive, but not exposed to the toxicant, while waiting to
be examined for sex and the presence of eggs.
14.10.10.2 The live animals must be examined for eggs and the sexes
determined within 12 h of the termination of the test. If the test was
conducted in the field, and the animals cannot be examined on site, the live
animals should be shipped back to the laboratory for processing. Pour each
replicate into a labelled 100 mL plastic screw capped jar, arid send to the
laboratory immediately.
14.10.10.3 If the test was conducted in the laboratory, or when the test
animals arrive in the laboratory from the field test site, the test organisms
must be processed immediately while still alive as follows:
14.10.10.3.1 Examine each replicate under a stereomicroscope (240X) to
determine the number of immature.animals, the sex of the mature animals, and
the presence or absence of eggs in the oviducts or brood sacs of the females
(see Figures 3-6). This must be done while the mysids are alive because they
turn opaque upon dying. This step should not be attempted! by a person who has
not had specialized training in the determination of sex and presence of eggs
i
239
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TEST:
START DATE:.
SALINITY:
DAY1
DAY 2
DAYS
DAY 4
DAY 6
DAY 6
DAY 7
DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
DAY 0
DAY?
TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
TEMP
TEMP
SALINITY
SALINITY
D.O.
D.O.
PH
pH
TRTMT
TRTMT
TEMP
TEMP
SALINITY
SALINITY
D.O.
D.O
pH
pH
Figure 2. Data form for the mysid, Mysidopsis bahia, water quality
measurements. From USEPA (1987d).
240
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MATURE FEMALE, EGGS IN OVIDUCTS
. eyestalk
antennule
statocyst
telson
developing brood sac
oviducts with developing ova
Figure 3. Mature female, mysid, Mysidopsis bahia, with eggs in oviducts. From
USEPA (1987d).
in the oviduct. NOTE: Adult females without eggs in the oviduct or brood sac
look like immature mysids (see Figure 6).
1 " I
14.10.10.3.2 Record the number of immatures, males, females with eggs and
females without eggs on data sheets (Figure 7).
14.10.10.3.3 Rinse the mysids by pipetting them into a small netted cup and
dipping the cup into a dish containing deionized water. Using forceps, place
the mysids from each replicate cup on tared weighing boats and dry at 60°C for
24 h or at 105°C for at least 6 h.
i
14.10.10.3.4 Immediately upon removal from the drying oven, the weighing pahs
were placed in a dessicator until weighed, to prevent absorption of moisture
from the,air. Weigh to the nearest mg. Record weighing pans and subtract the
241
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MATURE FEMALE, EGGS IN BROOD SAC
. eyestalk
carapace
antennule
antenna
statocyst
telson
brood sac with
developing embroyos
uropod
brood sac with
developing embryos
oviducts with developing ova
telson
uropods
Figure 4. Mature female mysid, Mysidopsis bahia, with eggs in oviducts and
developing embryos in the brood sac. Above: lateral view. Below-
dorsal view. From USEPA (1987d).
tare weight to determine the dry weight of the mysid in each replicate.
Record the weights (Figure 8). For each test chamber, divide the first dry
?£&,*,! V5e numker °f ordinal mys1ds per repiicate to determine the average
individual dry weight and record data. For the controls also calculate the
tSt SiSnJaPi •JUrVl^ng."or?1d 1n Jhe test Chamber to evaluate if weights met
test acceptability criteria (see Subsection 14.2).
Jf;l°;?;3A5 PiSCSS °! alumi™m foil d-cm square) or small aluminum weighing
10 mg in weight Y W619 analyses' The weighing pans should not exceed
Num5er ea
-------
MATURE MALE
eyestalk
antennule
K^^^mtm^
antenna
gonad.
t
pleopods
uropod "
statocyst
telson
Figure 5. Mature male mysid, Mysidopsis bahia. From USEPA (1987d).
14.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
14.11.1 A summary of test conditions and test acceptability criteria is
listed in Table 3.
14.12 ACCEPTABILITY OF TEST RESULTS
!
14.12.1 The minimum requirements for an acceptable test are 80% survival and
an average weight of at least 0.20 mg/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.
14.13 DATA ANALYSIS
14.13.1 GENERAL
I
14.13.1.1 Tabulate and summarize the data. Table 4 presents a sample set of
survival, growth, and fecundity data.
243
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IMMATURE
, eyestalk
carapace
antennule
antenna
statocyst
telson
Figure 6. Immature mysid, Mysidopsis bahia, (A) lateral view, (B) dorsal view.
From USEPA (1987d).
14.13.1.2 The endpoints of the mysid 7-day chronic test are based on the
adverse effects on survival, growth, and egg development. The LC50, the IC25,
and the IC50 are calculated using point estimation techniques (see Section 9,
Chronic Toxicity Test Endpoints and Data Analysis). LOEC and NOEC values for
survival, growth, and fecundity are obtained using a hypothesis testing
approach such as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank
Test (Steel, 1959; Miller, 1981) (see Section 9). Separate analyses are
performed for the estimation of the LOEC and NOEC endpoints and for the
estimation of the LC50, IC25, and IC50. Concentrations at which there is no
survival in any of the test chambers are excluded from the statistical
analysis of the NOEC and LOEC for survival, growth, and fecundity, but
included in the estimation of the LC50, IC25, .and IC50. See the Appendices
for examples of the manual computations, and examples of data input and
program output.
14.13.1.3 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
244
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TEST:
.START DATE:
SAI TNITY:
TREATMENT/
REPLICATE
2
8
2
3
5
7
8
2
*
B
6
7
8
DAY 1
# ALIVE
DAY 2
* ALIVE
DAY 3
* ALIVE
DAY 4
* ALIVE
DAY 5
* ALIVE
DAY 6
* ALIVE
DAY 7
* ALIVE
FEMALES
W/EGGS
FEMALES
NO EGGS
MALES
IMMATURES
Figure 7. Data form for the mysid, Mysidopsis bahia, survival and fecundity
data. From USEPA (1987d).
245
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TEST:
START DATE:.
SALINITY:
TMATMINT/
ItlrUCATC
1
2
I
«
f
a
7
•
t
2
3
4
S
9
7
I
1
2
3
4
t
a
7
t
DAYI
»AUVE
DAY 2
»AUVE
DAY 3
»AUVE
DAY 4
* ALIVE
DAY 5
* ALIVE
DAY 8
* ALIVE
DAY 7
* ALIVE
FEMALES
W/ECGS
FEMALES
NO EGGS
MALES
IMMATURES
Figure 7. Data form for the mysid, Mysidopsis bahia, survival and fecundity
data (CONTINUED). From USEPA (1987d).
246
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TEST:
START DATE:.
SALINITY:
TREATMENT*
REPLICATE
1
2
3
4
C
5
6
7
8
1
2
3
4
1
6
7
8
1
2
3
4
2
5
6
7
8
PAN #
TARE
WT.
TOTAL
WT.
ANIMAL
WT.
t
#OF
ANIMALS
.'
1 .
I
!
1
!
X WT./
ANIMAL
Figure 8. Data
From
form for the mysid, Mysidopsis bahia, dry weight measurements,
USEPA (1987d).
247
-------
TEST:
START DATE:.
SALINITY:
Figures. Data^fo™ for «. n^id, (g»**ps/s -Mi,, dry weight neasurercents
248
-------
TABLE 3 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE MYSID, MYSIDOPSIS BAHIA, SEVEN DAY SURVIVAL, GROWTH, AND
FECUNDITY TEST WITH EFFLUENTS AND RECEIVING WATERS
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber:
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. larvae per concentration:
14. Source of food:
15. Feeding regime:
16. Cleaning:
Static renewal
20%o to 30%o (± 2%o of the selected
test salinity)
26 ± 1°C
Ambient laboratory illumination
10-20 ME/m2/s (50-100 ft-c.)
(ambient laboratory "levels)
16 h light, 8 h darkness, with phase
in/out period
8 oz plastic disposable cups, or 400
mL glass beakers
150 mL per replicate
Daily i
7 days
5 (minimum)
8 (minimum)
40 (minimum)
Newly hatched Artemia nauplii (less
than 24 h old)
Feed 150 24 h old nauplii per mysid
daily, half after test solution
renewal and half after 8-12 h.
Pipette excess food from cups daily
immediately before test solution
renewal and feeding,,
249
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE MYSID, HYSIDOPSIS BAHIA, SEVEN DAY SURVIVAL, GROWTH, AND
FECUNDITY TEST WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sampling requirements:
25. Sample volume required:
None unless DO falls below 4.0 mg/L,
then gently aerate in all cups
Uncontaminated source of natural
seawater, deionized water mixed with
hypersaline brine or artificial sea
salts (HW Marinemix®, FORTY FATHOMS®,
GP2 or equivalent)
Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water
or minimum of 5 and a control
Effluents: > 0.5 series
Receiving waters: None, or > 0.5
7 days
Survival, growth, and egg development
80% or greater survival, average dry
weight 0.20 mg or greater in controls;
fecundity may be used if 50% or more
of females in controls produce eggs
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 are
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,
Subsection 8.5.4)
3 L per day
250
-------
TABLE 4. DATA FOR MYSIDOPSIS BAHIA 7-DAY SURVIVAL, GROWTH, AND FECUNDITY
TEST1
Treatment
Control
50 ppb
r r
100 ppb
210 ppb
450 ppb
Replicate
Chamber
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Total
Mysids
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
No. Total
Al i ve Femal es
4 1
4 2
5 3
5 1
5 2
5 5
5 2
4 3
4 2
5 3
4 3
4 0
5 5
5 2
4 4
5 3
3 3
5 2
5 1
5 2
5 3
3 1
4 4
4 0
5 1
4 2
1 1
4 3
3 1
4 2
4 1
4 3
0 0
1 0
0 0
1 0
0 0
0 0
0 0
2 1
Femal es Mean
w/Eggs Weight
1 0.146
2 0.118
2 0.216
1 0.199
2 0.176
4 0.243
2 0.213
3 0.144
1 0.154
1 0.193
2 0.190
0 0.190
2 0.256
1 0.191
1 0.122
1 0.177
1 0.114
1 0.172
0 0.160
1 0.199
2 0.165
0 0.145
1 0.207
0 0.186
0 0.153
0 0.094
0 0.017
0 0.122
0 0.052
0 0.154
0 0.110
0 0.103
0 - -
0 0.012
0 - -
0 0.002
0 - -
0 - -
0 - -
0 0.081
Dat.a provided by Lussier, Kuhn and Sewall, Environment
Laboratory, U.S. Environmental Protection Agency,
251
al Research
N^arreigansett, RI.
-------
14.13.2 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA, SURVIVAL DATA
14.13.2.1 Formal statistical analysis of the survival data is outlined in
Figures 9 and 10. The response used in the analysis is the proportion of
animals surviving in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endppints and for the
estimation of the LC50 endpoint. Concentrations at which there is no survival
in any of the test chambers are excluded from statistical analysis of the NOEC
and LOEC, but included in the estimation of the LC, EC, and 1C endpoints.
14.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Milk's
Test, and Bartlett's. Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.
14.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t-test with the Bonferroni adjustment (see
Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.
14.13.2.4 Probit Analysis (Finney, 1971; see Appendix G) 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 H-K).
14.13.2.5 The proportion of survival in each replicate must first be
transformed by the arc sine transformation procedure described in Appendix B.
The raw and transformed data, means and variances of the transformed
observations at each concentration including the control are listed in
Table 5. A plot of the survival data is provided in Figure 11.
252
-------
STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST
i
SURVIVAL HYPOTHESIS TESTING
SURVIVAL DATA
PROPORTION SURVIVING
ARC SINE
TRANSFORMATION
SHAPIRO-WILK-S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTS TEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
EQUAL NUMBER OF
REPLICATES?
NO
YES
T-TESTWITH
BONFERRONI
ADJUSTMENT
YES
NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFIERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 9. Flowchart for statistical analysis of mysid, Mysidopsis
survival data by hypothesis testing.
253
-------
STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
1
r
TWO OR MORE
PARTIAL MORTALITIES?
YES
IS PROBIT MODEL '
APPROPRIATE?
(SIGNIFICANT X2 TEST)
YES
NO
NO
ONE OR MORE
PARTIAL MORTALITIES?
i
YES
r
NO
GRAPHICAL METHOD
LC50
PROBIT METHOD L
Af
H
- hi
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
NO
YES
SPEARMAN-KARBER
METHOD
L.C50AND95%
CONFIDENCE
INTERVAL
« '
TRIMMED SPEARMAN-
KARBER METHOD
Figure 10. Flowchart for statistical analysis of mysid, Mysidopsis bahia,
survival data by point estimation.
254
-------
o
o
ec
o
u.
Ul
UJ
CQ
a.
a.
UJ
o
8
-8
O)
>
d)
(XI
CD
Jd
O
(O
O)
to
•r~
•c:
to
-Q
§•
•fj
o
o.
o
a.
in
O
+->
o
cZ
O O> 00
^ 0 CD
r». o!o d
NOlldOdOdd IVAIAdnS
CD
255
-------
TABLE 5. MYSID, MYSIDOPSIS BAHIA, SURVIVAL DATA
Concentration (ODD)
Repl
RAW
ARC 'SINE
TRANS-
FORMED
Mean(Y,)
si
1
i
icate
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Control
0.80
0.80
1.00
1.00
1.00
1.00
1.00
0.80
1.107
1.107
1.345
1.345
1.345
1.345
1.345
1.107
1.256
0.015
1
50.0
0.80
1.00
0.80
0.80
1.00
1.00
0.80
1.00
1.107
1.345
1.107
1.107
1.345
1.345
1.107
1.345
1.226
0.016
2
100.0
0.60
1.00
1.00
1.00
1.00
0.60
0.80
0.80
0.886
1.345
1.345
1.345
1.345
0.886
1.107
1.107
1.171
0.042
3
210.0
1.00
0.80
0.20
0.80
0.60
0.80
0.80
0.80
1.345
1.107
0.464
1.107
0.886
1.107
1.107
1.107
1.029
0.067
4
450.0
0.00
0.20
0.00
0.20
0.00
0.00
0.00
0.40
0.225
0.464
0.225
0.464
0.225
0.225
0.225
0.685
0.342
0.031
5
14.13.2.6 Test for Normality
14.13.2.6.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The centered
observations are listed in Table 6.
14.13.2.6.2 Calculate the denominator, D, of the test statistic:
D = £ (x±-x)2
Where: X,- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations.
256
-------
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Concentration
Replicate
1
2
3
4
5
6
7
8
Control
(Site Water)
-0.149
-0.149
0.089
0.089
0.089
0.089
0.089
-0.149
50.0
-0.119
0.119
-0.119
-0.119
0.119
0.119
-0.119
0.119
100.0
-0.285
0.174
0.174
0.174
0.174
-0.285
-0.064
-0.064
210.0
1
1
0.316
0.078
-0.565
0.078
-0.142
0.078
0.078
0.078
450.0
-0.117
0.121
-0.117
0.121
-0.117
-0.117
-0.117
0.342
14.13.2.6.3 For this set of data, n = 40
X = _L(-0.006)
40
0.0
D = 1.197
14.13.2.6.4 Order the centered observations from smallest to largest:
X(1)
The differences x
example:
- X(1) are listed in Table 7. For this data in this
257
-------
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
i X<0. i X(f)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
-0.565
-0.285
-0.285
-0.149
-0.149
-0.149
-0.143
-0.119
-0.119
-0.119
-0.119
-0.117
-0.117
-0.117
-0.117
-0.117
-0.064
-0.064
0.078
0.078
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.078
0.078
0.078
0.089
0.089
0.089
0.089
0.089
0.119
0.119
0.119
0.119
0.121
0.121
0.174
0.174
0.174
0.174
0.316
0.342
W = 1 (1.0475)2 = 0.9167
1.197
14.13.2.6.7 The decision rule for this test is to compare W as calculated in
Subsection 14.13.2.6.5 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.
14.13.2.6.8 Since the data do not meet the assumption of normality, Steel's
Many-one Rank Test will be used to analyze the survival data.
14.13.2.7 Steel's Many-one Rank Test
14.13.2.7.1 For each control and concentration combination, combine the data
and arrange the observations in order of size from smallest to largest.
Assign the ranks (1, 2, ... , 16) to the ordered observations with a rank of 1
assigned to the smallest observation, rank of 2 assigned to the next larger
258
-------
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
observation, etc.
tied observation.
ai
0.3964
0.2737
0.2368
0.2098
0.1878
0.1691
0.1526
0.1376
0.1237
0.1108
0.0986
0.0870
0.0759
0.0651
0.0546
0.0444
0.0343
0.0244
0.0146
0.0049
If ties
win-i-nj _ wti
0.907
0.601
0.459
0.323
0.323
0.323
0.264
0.240
0.238
0.238
0.238
0.236
0.206
0.206
0.206
0.206
0.153
0.142
0.0
0.0
occur when ranking,
j
: xc4o> -
x«9> .
x<38) -
X(37) _
X(36) _
x<35) .
x(34) -
Y<33)
A
; Y<32>
' i A ~
x<31) -
X(30) .
x<29> _
Y(28)
A
,-• x(27> -
X(26) _
v(25)
A ""
XC24> _
Y<23>
! A ~
Y<22>
A ~
X(21> -
i •
assign the average
xd)
x<2)
X(3)
X(4)
X(5)
X(6)
x<7)
X(8)
x<9)
X(10)
XC11)
XC12)
X(13)
X(H>
x<15>
x<16>
X(17)
x<18)
X(19)
Y<20)
A
rank to each
14.13.2.7.2 An example of assigning ranks to the combined data for the
control and 50.0 ppb concentration is given in Table 9. This ranking
procedure is repeated for each control/concentration combination. The
complete set of rankings is summarized in Table 10. The ranks are then summed
for each concentration level, as shown in Table 11.
i
14.13.2.7.3 For this example, determine if the survival in any of the
concentrations is significantly lower than the survival in the control. If
this occurs, the rank sum at that concentration would be significantly lower
than the rank sum of the control. Thus compare the rank sums for the survival
at each of the various concentration levels with some "minimum" or critical
rank sum, at or below which the survival would be considered significantly
lower than the control. At a significance level of 0.05, the minimum rank sum
in a test with four concentrations (excluding the control) and eight
replicates is 47 (See Table 5, Appendix E). i
i1
14.13.2.7.4 Since the rank sum for the 450 ppb concentration level is less
than the critical value, the proportion surviving in that concentration is
considered significantly less than that in the control. Since no other rank
i
259 !
-------
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 50 PPB CONCENTRATION LEVEL
FOR STEEL'S MANY-ONE RANK TEST
Transformed Proportion
Rank of Total Mortality Concentration
4
4
4
4
4
4
4
12
12
12
12
12
12
12
12
12
1.107
1.107
1.107
1.107
1.107
1.107
1.107
1.571
1.571
1.571
1.571
1.571
1.571
1.571
1.571
1.571
Control
Control
Control
50 ppb
50 ppb
50 ppb
50 ppb
Control
Control
Control
Control
Control
50 ppb
50 ppb
50 ppb
50 ppb
sums are less than or equal to the critical value, no other concentrations
have a significantly lower proportion surviving than the control. Hence, the
NOEC and the LOEC are assumed to be 210.0 ppb and 450.0 ppb, respectively.
14.13.2.8 Calculation of the LC50
14.13.2.8.1 The data used for the Probit Analysis is summarized in Table 12.
For the Probit Analysis, run the USEPA Probit Analysis Program. An example of
the program output is provided in Figure 12.
14.13.2.8.2 For this example, the chi-square test for heterogeneity was not
significant. Thus Probit Analysis appears to be appropriate for this set of
data.
14.13.3 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA GROWTH DATA
14.13.3.1 Formal statistical, analysis of the growth data is outlined in
Figure 13. The response used in the statistical analysis is mean weight per
original of males and females combined per replicate. The IC25 and IC50 can
be calculated for the growth data via a point estimation technique (see
Section 9, Chronic Toxicity Test Endpoints and Data Analysis). Hypothesis
testing can be used to obtain an NOEC and LOEC for growth. Concentrations
above the NOEC for survival are excluded from the hypothesis test for growth effect
260
-------
TABLE 10. TABLE OF RANKS
1
Concentration (ppb)
Repli-
cate
1
2.
3
4
5
6
7
8
1
1
1
1
1
1
1
1
Control
.107(4,
.107(4,
.345(12
.345(12
.345(12
.345(12
.345(12
.107(4,
5,6.
5,6.
,12,
,12,
,12,
,12,
,12,
5,6.
5,10)
5,10)
13.5,14)
13.5,14)
13.5,14)
13.5,14)
13.5,14)
5,10)
1
1
1
1
1
1
1
1
50
.107(4)
.345(12)
.107(4)
.107(4)
.345(12)
.345(12)
.107(4)
.345(12)
0
1
1
1
1
0
1
1
100
.886(1.5)
.345(12)
.345(12)
.345(12)
.345(12)
.886(1.5)
.107(5)
.107(5)
210
1.345(13.5)
1.107(6.5)
0.464(1)
1.107(6.5)
0.886(2)
1.107(6.5)
1.107(6.5)
1.107(6.5)
0
0
0
0
0
0
0
0
450
.225(3)
.464(6.5)
.225(3)
.464(6.5)
.225(3)
.225(3)
.225(3)
.685(8)
Control ranks are given in the order of the concentration with which
they were ranked.
TABLE 11. RANK SUMS
Concentration
Rank Sum
50
100
210
450
64
61
49
36
14.13.3.2 The statistical analysis using hypothesis tests; consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test. The underlying, assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested. The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the nonparametric
test, Steel's Many-one Rank Test, is used to determine the NOEC and LOEC
endpoints. If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
I
14.13.3.3 Additionally, if unequal numbers of replicates occur among the
concentration levels tested, there are parametric and nonparametric
alternative analyses. The parametric analysis is a t test with the Bonferroni
adjustment. The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
261
-------
Probit Analysis of Mysidopsis bshia Survival Data
Cone.
Control
50.0000
100.0000
210.0000
450.0000
Number
Exposed
40
40
40
40
40
Number
Resp.
3
4
6
11
36
Observed
Proportion
Responding
Proportion Adjusted for
Responding
0.0750
0.1000
0.1500
0.2750
0.9000
Controls
0.0000
-.0080
0.0480
0.1880
0.8880
Chi - Square for Heterogeneity (calculated) = 0.725
Chi - Square for Heterogeneity (tabular value) = 5.991
Probit Analysis of Mysidopsis bahia Survival Data
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
123.112
288.873
Lower Upper
95% Confidence Limits
65.283
239.559
165.552
335.983
Figure 12. Output for USEPA Probit Analysis Program, version 1.5.
262
-------
TABLE 12. DATA FOR PROBIT ANALYSIS
Control
50,0
Concentration (ppb)
100.0
210.0
450.0
No Dead
No Exposed
3
40
4
40'
6
40
'
11
40 i
36
40
nonparametric alternative. For detailed information on the Bonferroni
adjustment, see Appendix D.
14.13.3.4 The data, mean and variance of the observations at each
concentration including the control for this example are listed in Table 13.
A plot of the data is provided in Figure 14. Since there is significant
mortality in the 450 ppb concentration, its effect on growth is not
considered.
i
TABLE 13. MYSID, MYSIDOPSIS BAHIA, GROWTH DATA
Replicate Control
Concentration (»pb)
50.0
100.0
210.0
450.0
Mean(Y,)
ST
i
1
2
3
4
.5
6
7
8
0.146
0.118
0.216
0.199
0.176
0.243
0.213
0.144
0.182
0.00186
1
0.157
0.193
0.190
0.190
0.256
0.191
0.122
0.177
0.184
0.00145
2
0.114
0.172
0.160
0.199
0.165
0.145
0.207
0.186
0.168
0.00091
3
0.1153
0.071
0.017
0.112
0.052
0.154
0.110
0.103
0.101
0.00222
4
0.012
-
0.002
-
-
-
0.081
_
-
5
14.13.3.5 Test for Normality
, j
14.13.3.5.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
263
-------
STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST
GROWTH
POINT ESTIMATION
ENDPOINT ESTIMATE
IC25, IC50
GROWTH DATA
MEAN DRY WEIGHT
HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FOR SURVIVAL
SHAPIRO-WILICS TEST
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
NON-NORMAL DISTRIBUTION
HETEROGENEOUS
VARIANCE
1
NO
r
EQUAL NUMBER OF
REPLICATES?
T-TESTWITH
BONFERRONI
ADJUSTMENT
i
YES
DUNNETTS
TEST
}
YES
r
EQUAL NUMBER OF
REPLICATES?
1 NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 13. Flowchart for statistical analysis of mysid, Mysidopsis bahia,
growth data.
264
-------
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265
-------
concentration from each observation in that concentration. The centered
observations are listed in Table 14.
TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Concentration
Replicate
1
2
3
4
5
6
7
8
Control
-0.036
-0.064
0.034
0.017
-0.006
0.061
0.031
-0.038
50.0
-0.030
0.009
0.006
0.006
0.072
0.007
-0.062
-0.007
100.0
-0.054
0.004
-0.008
0.031
-0.003
-0.023
0.039
0.018
(DDb)
210.0
0.052
-0.007
-0.084
0.021
-0.049
0.053
0.009
0.002
14.13.3.5.2 Calculate the denominator, D, of the statistic:
D = £ (X^X)2 .
Where: X,- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
14.13.3.5.3 For this set of data, n = 32
X = _i_ (0.007) = 0.000
32
D = 0.0451
14.13.3.5.4 Order the centered observations from smallest to largest
v<1) < v(2) < < w(n)
where XCl) denotes the ith ordered observation. The ordered observations for
this example are listed in Table 15.
266
-------
TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-0.084
-0.064
-0.062
-0.054
-0.049
-0.038
-0.036
-0.030
-0.023
-0.008
-0.007
-0.007
-0.006
-0.003
0.002
0.004
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
0.006
0.006
0.007
0.009
0.009
0.017
0.018
0.021
0.031
0.031
0.034
0.039
0.052
0.053
0.061
0.072
14.13.3.5.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a2, ... ak where k is n/2 if n is even and (n-l)/2
if n is odd. For the data in this example, n = 32 and k == 16. The a,- values
are listed in Table 16.
14.13.3.5.6 Compute the test statistic, W, as follows:
W =
D
The differences x
-------
TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
- X
en
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.4188
0.2898
0.2462
0.2141
0.1878
0.1651
0.1449
0.1265
0.1093
0.0931
0.0777
0.0629
0.0485
0.0344
0.0206
0.0068
0.156
0.125
0.115
0.106
0.088
0.072
0.067
0.061
0.044
0.026
0.024
0.016
0.015
0.010
0.004
0.002
xc»>
X(S1>
X 0>
y(29)
,,(28)
x(27>
v C^o/
* ^
x(2)
XC4>
X(23)
x( >
y(21)
x<20)
v(19)
X(18)
X(17)
- x(l>
- x<2)
- X
- x<4)
- x(5)
- x(6)
- x^'
X\oJ
- x<9)
- x<10>
- x<11>
w(12)
- x^^'
- x<^
- x !
- x(16>
14.13.3.6 Test for Homogeneity of Variance
14.13.3.6.1 The test used to examine whether the variation in mean weight of
the mysids is the same across all concentration levels including the control,
is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
fol1ows:
B =
Where: V,
p s
In =
ni =
degrees of freedom for each copper concentration and
control, V,- = (n,- - 1)
number of concentration levels including the control
loge
1, 2, ..., p where p is the number of concentrations
including the control
the number of replicates for concentration i.
268
-------
, c =
i-i
14.13.3.6.2 For the data in this example (See Table 13), all concentrations
including the control have the same number of replicates (n- = 8 for all i).
Thus, V,- = 7 for all i.
14.13.3.6.3 Bartlett's statistic is therefore:
B- [(28)ln(0.0016) -
= [28(-6.4315) - 7(-25.9357)]/1.06
= [-180.082 - (-181.5499)]/1.06
= 1.385
i
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. Since B = 1.385 is less than the critical
value of 9.210, conclude that the variances are not different.
14.13.3.7 Dunnett's Procedure
14.13.3.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 17.,
TABLE 17. ANOVA TABLE
Source
Between
Within
Total
df
P - 1
N - p
N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
S* = SSB/(p-l)
S* = SSW/(N-p)
269
-------
Where: p
N
n
number of concentration levels including the control
total number of observations n., + n2 ... + np
number of observations in concentration i
SSB
= 2^,
Tl/n±-G2/N
Between Sum of Squares
SST =
YJ.j-G2/N
Total Sum of Squares
SSW = SST-SSB
Within Sum of Squares
G = the grand total of all sample observations, G ±
= £ T
I,. = the total of the replicate measurements for concentration i
Y,-j - the jth observation for concentration i (represents the mean
dry weight of the mysids for concentration i in test
chamber j)
14.13.3.7.2 For the data in this example:
n, = n2 = n3 = n4 = 8
N = 32
T - Y -i- V
J.1 ~ .'.11 ..12
^4 - Y41 + Y42
Y18 = 1.455
Y28 = 1.473
Y38 = 1.348
Y,. = 0.805
T1 + T2 + T3 + T4 = 5-081
SSB = f,Tl/n±-G2/N
1_(6.752) - (5.08i
8 32
= 0.0372
SST =
0.889 - (5.08ir = 0.0822
32
270
-------
SSW = SST-SSB = 0.0822 - 0.0372 - 0.0450
S* = SSB/(p-l) = 0.0372/(4-l) = 0.0124
S* = SSW/(N-p) = 0.0450/(32-4) = 0.0016
14.13.3.7.3 Summarize these calculations in the ANOVA table (Table 18)
TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
3
28
31
Sum of Squares
(SS)
0.0372
0.0450
0.0822
Mean Square (MS)
(SS/df)
0.0127
0.0016
14 13.3.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where: Y, = mean dry weight for concentration i
Y, = mean dry weight for the control
Su = square root of the within mean square
n, = number of replicates for the control i
n- = number of replicates for concentration i i
14 13 3 7 5 Table 19 includes the calculated t values for each concentration
and control combination. In this example, comparing the 50.0 ppb
concentration with the control the calculation is as follows:
271
-------
(0.182-0.184)
[0 . 04CVU/8) +(1/8) ]
-0.100
TABLE 19. CALCULATED T VALUES
Concentration (ppb)
50.0
100.0
210.0
2
3
4
-0.150
0.700
4.050
14.13.3.7.6 Since the purpose of this test is to detect a significant
reduction in mean weight, a one-sided test is appropriate. The critical value
for this one-sided test is found in Table 5, Appendix C. For an overall alpha
level of 0.05, 28 degrees of freedom for error and three concentrations
(excluding the control) the approximate critical value is 2.15. The mean
weight for concentration "i" is considered significantly less than the mean
!S195r« « tte contro1 1f *! is grater than the critical value. Therefore,
the 210.0 ppb concentration has significantly lower mean weight than the
control. Hence the NOEC and the LOEC for growth are 100.0 ppb and 210.0 ppb
respectively. KK '
14.13.3.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
Where: d
S
n
= d
the critical value for Dunnett's Procedure
>w - the square root of the within mean square
the common number of replicates at each concentration
(this assumes equal replication at each concentration)
r\i = the number of replicates in the control.
14.13.3.7.8 In this example:
MSD = 2.15(0.04)^(1/8)+(1/8)
= 2.15 (0.04)(0.5)
= 0.043
272
-------
14 13 3.7.9 Therefore, for this set of data, the minimum idifference that can
be detected as statistically significant is 0.043 mg.
14.13.3.7.10 This represents a 23.6% reduction in mean weight from the
control.
14.13.3.8 Calculation of the ICp
14 13 3 8 1 The growth data from Table 13 are utilized in this example. As
seen in', the observed means are not monotonically non-increasing with respect
to concentration. Therefore, it is necessary to smooth the means prior to
calculating the 1C. In the following discussion, the observed means are
represented by Y,- and the smoothed means by Mr
14.13.3.8.2 Starting with the control mean, Y, = 0.182 arid Y2 = 0.184, we see
that Y1 < Y2
Calculate the smoothed means:
M, = M2 = (Y, + Y2)/2 = 0.183
14 13 3 8.3 Since Y5 = 0.025 < Y, - 0.101 < Y3 = 0.168 < M2, set M3 = 0.168
and M =0.101, and M5 = 0.025. Table 20 contains the smoothed means and
Figure 15 gives a plot of the smoothed response curve.
TABLE 20. MYSID, MYSIDOPSIS BAHIA,
MEAN
GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(ppb)
Control
50.0
100.0
210.0
450.0
i
1
2
3
4
5
Response
Means
Y1 (mg)
0.182
0.184
0.168
0.101
0.012
Smoothed
Mean
M,- (mg)
0.183
0.183
0.168
0.101
0.012!
14 13 3 8 4 An IC25 and IC50 can be estimated using the Linear Interpolation
Method." A 25% reduction in weight, compared to the controls, would result in
a mean weight of 0.136 mg, where M^l-p/100) = 0.183(1-25/100). A 50%
reduction in mean dry weight, compared to the controls, would result in a mean
weight of 0.091 mg. Examining the smoothed means and their associated
concentrations (Table 20), the response, 0.136 mg, is bracketed by C3 = 100
ppb and C, = 210 ppb. The response, 0.091 mg, is bracketed by C4 = 210 ppb
and C5 = 450 ppb.
273
-------
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274
-------
14.13.3.8.5 Using the equation in Section 4.2 from Appendix L, the estimate
of the IC25 is calculated as follows: i
icp = Cj
IC25 = 100 + [0.183(1 - 25/100) - 0.166] (210 - 100)
= 151 ppb.
14.13.3.8.6 Using Equation 1 from Appendix L, the estimai
calculated as follows:
ICp = C^ [M, (l-p/100) -Afj] \C(j^ ~_l
(0.101 - 0.168)
e of the IC50 is
IC50 = 210 + [0.183(1 - 50/100) - 0.101] (450 - 210)
(0.012 - 0.101)
= 236 ppb.
14.13.3.8.7 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 147.170 ppb. The
empirical 95.0% confidence interval for the true mean was 97.0905 ppb and
186.6383 ppb. The computer program output for the IC25 for this data set is
shown in Figure 16.
14.13.3.8.8 When the ICPIN program was used to analyze this set of data for
the IC50, requesting 80 resamples, the estimate of the IC50 was 230.755 ppb.
The empirical 95.7% confidence interval for the true mean was (183.84 ppb to
277.9211 ppb). The computer program output for the IC50 for this data set is
shown in Figure 17.
14.13.4 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA, FECUNDITY DATA
14.13.4.1 Formal statistical analysis of the fecundity data is outlined in
Figure 18. The response used in the statistical analysis is the proportion of
females with eggs in each test or control chamber. If no females were present
in a replicate, a response of zero should not be used. Instead there are no
data available for that replicate and the number of replicates for that level
of concentration or the control should be reduced by one. Separate analyses
are performed for the estimation.of the NOEC and LOEC endpoints, and for the
estimation of the EC, LC, and 1C endpoints. The data for a concentration are
excluded from the statistical analysis of the NOEC and LOEC endpoints if no
eggs were produced in all of the replicates in which females existed.
However, all data are included in the estimation of the IC25 and IC50.
275
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
.146
.118
.216
.199
.176
.243
.213
.144
2
50
.154
.19
.193
.190
.190
.191
.122
.177
3
100
.114
.172
.160
.199
.165
.145
.207
.186
4
210
.153
.094
.017
.122
.052
.154
.110
.103
5
450
0
.012
0
.002
0
0
0
.081
*** Inhibition Concentration Percentage Estimate ***
Toxi cant/Eff1uent:
Test Start Date: Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.i25
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
M9/1
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Std. Pooled
Dev. Response Means
0.043
0.038
0.030
0.047
0.028
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate: 150.6446 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 147.1702 Standard Deviation: 23.7984
Original Confidence Limits: Lower: 97.0905 Upper: 186.6383
Resampling time in Seconds: 0.11 Random Seed: -1623038650
Figure 16. ICPIN program output for the IC25.
276
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
.146
.118
.216
.199
.176
.243
.213
.144
2
30
.154
.193
.190
.190
.256
.191
.122
.177
3
100
.114
.172
.160
.199
.165
.145
.207
.186
4
210
.153
.094
.017
.122
.052
.154
.110
.103
5
450
0
.012
0
.002
0
0
0
.081
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date: Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.i50
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
ug/1
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Std. Pooled
Dev. : iResponse Means
0.043
0.0313
0.030
0.047
0.028
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate: 234.6761 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Resampling time in Seconds:
230.7551 Standard Deviation: 30.6781
Lower: 183.8197 Upper: 277.9211
0.16 Random Seed: -628896314
Figure 17. ICPIN program output for the IC50.
277
-------
STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST
FECUNDITY
FECUNDITY DATA
PROPORTION OF FEMALES WITH EGGS
POINT ESTIMATION
HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FOR SURVIVAL)
ENDPO1NT ESTIMATE
IC25, IC50
ARC SINE TRANSFORMATION
SHAPIRO-WIIJCS TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
YES
T-TESTWITH
BONFERRONI
ADJUSTMENT
I
EQUAL NUMBER OF
REPLICATES?
YES
NO
STEEL'S MANY-ONE
RANKTEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 18. Flowchart for statistical analysis of mysid, Mysidopsis bahia,
fecundity data.
278
-------
14 13.4.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Under!yinq assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Wilk s
Test and Bartlett's Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure. |
14 13 4 3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment
(Appendix D). The Wilcoxon Rank Sum Test with the Bonferrom adjustment is
the nonparametric alternative.
14 13 4 4 The proportion of female mysids, Mysidopsis bahia, with eggs in
each replicate must first be transformed by the arc sine square root
transformation procedure described in Appendix B. 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. The raw and
transformed data, means and variances of the transformed observations at each
test concentration including the control are listed in Table 21. Since there
is significant mortality in the 450 ppb concentration, its effect on
reproduction is not considered. Additionally, since no eggs were produced by
females in any of the replicates for the 210 ppb concentration, it is not
included in this statistical analysis and is considered a qualitative
reproductive effect. A plot of the mean proportion of female mysids with eggs
is illustrated in Figure 19.
14.13.4.5 Test for Normality
i
14.13.4.5.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The .centered
observations are listed in Table 22.
14.13.4.5.2 Calculate the denominator, D, of the statistic:
D = t(X,-X)2 . !
Where: Xs = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
279
-------
*r§
* *
o>
o
I I I I I I I I I I I I I I I I I I I I I I I I I I I
iq^;cOCM!i-'o
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CO
a.
^
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8
UJ
O
CO
01
O1
CD
CO
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t
CO
•o
I"
00
cu
(T3
I
O
s-
o .
Q.
O
a.
SOO3 HUM S3-|V1N3=I NOIiyOdOdd
5-
13
O>
280
-------
TABLE 21. MYSID, MYSIDOPSIS BAHIA, FECUNDITY DATA: PERCENT FEMALES WITH
EGGS
Test Concentration (oob)
Repl
RAW
ARC SINE
TRANS-
FORMED
MeanCY,-)
?T
i
icate
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Control
1.00
1.00
0.67
1.00
1.00
0.80
1.00
1.00
1.57
1.57
0.96
1.57
1.57
1.12
1.57
1.57
1.44
0.064
1
50.0
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
100.0 210.0
0.33 0.0
0.50 0.0
0.00 0.0
0.50 0.0
0.67
0.00
0.25
-
0.0
0.0
0.0
0.0
0.61
0.78
0.00
0.78
0.96
0.00
0.52
-
0.52
0.147
3
..
..
„
-
4
281
-------
TABLE 22. CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Test Concentration (ODD)
Replicate
1
2
3
4
5
6
7
8
Control
0.13
0.13
-0.48
0.13
0.13
-0.32
0.13
0.13
50.0
0.07
-0.10
0.25
-
-0.03
0.07
-0.19
-0.10
100.0
0.09
0.26
-0.52
0.26
0.44
-0.52
0.00
14.13.4.5.3 For this set of data, n = 22
(0.000) = 0.000
22
' D = 1.4412
14.13.4.5.4 Order the centered observations from smallest to largest:
where Xci> denotes the ith ordered observation. The ordered observations for
this example are listed in Table 23.
14.13.4.5.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients an, a2, ... ak where k is n/2 if n is even and (n-l)/2
if n is odd. For the data in this example, n = 22 and k = 11. The a, values
are listed in Table 24.
14.13.4.5.6 Compute the test statistic, W, as follows:
1 Ji Z
fir — -1- r > => I v(tt-i+l) _ y (i) \ ~\
W — — L " "i {•"• •"• I J
The differences xcn"i+1> - Xci> are listed in Table 24. For the data in this
example:
W = 1 (1.1389)2 = 0.900
1.4412
282
-------
TABLE 23. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
x
0.74 XQ:J^
0.57 X
0.32 X(!,}
0.23
0.23
0.16
0.13
0.06
0.02
X
X\ 16)
._
x<15)
X
v(13)
x<12>
- x(1)
- X
- x^
- x<4)
- x(5)
- x<6)
X(7j
„
- x<8)
- X
- xc;;>
- x<11>
14.13.4.5.7 The decision rule for this test is to compare M as calculated in
Subsection 14.13.4.5.6 to a critical value found in Table 6, Appendix B. If
the computed M 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 = 22 observations is 0.878. Since M = 0.900
is greater than the critical value, conclude that the data are normally
distributed.
283
-------
14.13.4.6 Test for Homogeneity of Variance
14.13.4.6.1 The test used to examine whether the variation in proportion of
female mysids with eggs is the same across all concentration levels including
the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test
statistic is as follows:
[(
£ V InS2 - £ v.
B =
Where: V
p
In
f
1 =
degrees of freedom for each copper concentration and
control, Vf = (n,. - 1)
number of concentration levels including the control
loge
1, 2, ..., p where p is the number of concentrations
including the control
n,- - the number of replicates for concentration i.
c =
i-i
14.13.4.6.2 For the data in this example (see Table 21), n. = 8, n? = 7 and
n3 - 7. Thus, the respective degrees of freedom are 7, 6 and 6.
14.13.4.6.3 Bartlett's statistic is therefore:
B - [(19)ln(0.077) - (7 ln(0.064) + 6 ln(0.021) + 6 ln(0.147))]/1.07
- [19(-2.564) - (-53.925)]/1.07
- [-48.716 - (-53.925)1/1.07
- 4.868
284
-------
14.13.4.6.4 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
two degrees of freedom, is 9.210. Since B = 4.868 is less than the critical
value of 9.210, conclude that the variances are not different.
i
14.13.4.7 T test with the Bonferroni Adjustment
14.13.4.7.1 A t test with the Bonferroni adjustment is used as an alternative
to Dunnett's Procedure when, as in this set of data, the number of replicates
is not the same for all concentrations. Like Dunnett's Procedure, it uses a
pooled, estimate of the variance, which is equal to the error value calculated
in an analysis of variance. To obtain an estimate of the pooled variance,
construct an ANOVA table as described in Table 25.
TABLE 25. ANOVA TABLE
Source
Total
df
Sum of Squares
(SS)
- 1
SST
Mean Square(MS)
(SS/df).
Between
Within
P
N
- 1
- P
SSB
SSW
2
SB =
2
s«-
SSB/(p-l)
SSW/(N-p)
Where: p = number concentration levels including the control
N = total number of observations n1 + n2 ... +nf[
nf = number of observations in concentration i
SSB - Y,T\/ni-Gz/N Between Sum of Squares'
i-l
SST =
:-G2/N
SSW = SST-SSB
Total Sum of Squares
Within Sum of Squares
285
-------
6 = the grand total of all sample observations, G = £ T±
TJ = the total of the replicate measurements for
concentration i
Y,-j - the -jth observation for concentration i (represents the
proportion of females with eggs for concentration
i in test chamber j)
14.13.4.7.2 For the data in this example:
n, = 8 n2 = 7 n3 = 7
N = 22
J2: Y2': v22
I3 - Y31 + Y32
Y18 = 11.5
Y27 = 4.94
Y37 = 3.65
G = T, + T2 + T3 = 20.09
SSB =
132.25 + 24.40 + 13.32
877
403.61 = 3,57
22
SST =
= 23.396 - 403.61 = 5.05
22
SSW = SST-SSB. = 5.05 - 3.57 = 1.48
SB - SSB/(p-l) = 3.57/(3-l) = 1.785
Sy = SSW/(N-p) = 1.48/(22-3) = 0.078
286
-------
14.13.4.7.3 Summarize these calculations in the ANOVA table (Table 26).
TABLE 26. ANOVA TABLE FOR THE T TEST WITH BONFERRONI'S ADJUSTMENT EXAMPLE
Source df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between
Within
Total
2
19
21
3.57
1.48
5.05
1.785
0.078
1
1
14.13.4.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where: Y,- = mean proportion of females with eggs for. concentration i
Y! = mean proportion of females with eggs for the control
SH = square root of the within mean square
n,, = number of replicates for the control
n,- = number of replicates for concentration i.
14.13.4.7.5 Table 27 includes the calculated t values for each concentration
and control combination. In this example, comparing the 50.0 ppb
concentration with the control the calculation is as follows:
,_ _ (1.44 - 0.52)
[0.279
= 5.05
287
-------
TABLE 27. CALCULATED T VALUES
Test Concentration (ppb)
50.0
100.0
2
3
5.05
6.37
14.13.4.7.6 Since the purpose of this test is to detect a significant
reduction in mean proportion of females with eggs, a one-sided test is
appropriate. The critical value for this one-sided test is found in Table 5,
Appendix D, Critical Values for the t test with Bonferroni's adjustment.
For an overall alpha level of 0.05, 19 degrees of freedom for error and two
concentrations (excluding the control) the approximate critical value is
2.094. The mean proportion for concentration "i" is considered significantly
less than the mean proportion for the control if t, is greater than the
critical value. Therefore, the 50.0 ppb and the 100.0 ppb concentrations have
significantly lower mean proportion of females with eggs than the control.
Hence the LOEC for fecundity is 50.0 ppb.
14.13.4.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
MSD = t SrfJCL/Ili) + (1/72)
Where: t = the critical value for the t test with Bonferroni's adjustment
Sw = the square root of the within mean square
n » the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n, = the number of replicates in the control.
14.13.4.7.8 In this example:
MSD = 2.094 (0.279)^(1/8)+(1/7)
= 2.094 (0.279)(0.518)
= 0.303
14.13.4.7.9 • Therefore, for this set of data, the minimum difference that can
be detected as statistically significant is 0.30.
14.13.4.7.10 The MSD (0.30) is in transformed units. To determine the MSD in
terms of percent of females with eggs, carry out the following conversion.
288
-------
1. Subtract the MSD from the transformed control mean.
1.44 - 0.30 = 1.14
I
i
2. Obtain the untransformed values for the control mean and the
difference calculated in 4.10.1.
[ Sine (1.44) J2 = 0.983
'
[ Sine (1.14) ]2 = 0.823
3. The untransformed MSD (MSD ) is determined by subtracting the
untransformed values from 14.4.8.10.2.
MSDU = 0.983 - 0.823 = 0.16
14.13.4.7.11 Therefore, for this set of data, the minimum difference in mean
proportion of females with eggs between the control and any copper
concentration that can be detected as statistically significant is 0.16.
.
14.13.4.7.12 This represents a 17% decrease in proportion of females with
eggs from the control.
14.13.4.8 Calculation of the ICp
14.13.4.8.1 The fecundity data in Table 4 are utilized in this example.
Table 28 contains the mean proportion of females with eggs for each toxicant
concentration. As can be seen, the observed means are monotonically
nonincreasing with respect to concentration. Therefore, it is not necessary
to smooth the means prior to calculating the 1C. Figure 20 gives a plot of
the response curve. !
TABLE 28. MYSID,. MYSIDOPSIS BAHIA, MEAN PROPORTION
OF FEMALES WITH EGGS |
Toxicant
Cone.
(ppb)
Control
50.0
100.0
210.0
450.0
i
1
2
3
4
5
Response Si
Means, Y1 M<
noothed
jans, M.-
(proportion) (proportion)
0.934
0.426
0.934
0.426
0.317 0.317
0.000 0.000
0.010 0.000
14.13.4.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation
Method. A 25% reduction in mean proportion of females with eggs, compared to
289
-------
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*
"IO
QL
Q.
O
\-
CC
O
O
I-
1
1
i • • • • i • ' • • i ' • • • i • ' • • i ' ' ' • i '^^^ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
oii-oeocoN-toio^coevii-o
»••••••••••••
T-1-T-OOOOOOOOOO
SDD3
dO NOIldOcJOtld NV3W
CD
cn
o>
3
OS
to
-Q
a.
•§
T3
•r-
to
O)
r™-
0*
O)
C
O
o
O.
O
Q.
OJ
ou
O
'SI
O
CM
O)
=5
O5
290
-------
the controls, would result in a mean proportion of 0.701, where M^(l-p/100) =
0.934(1-25/100). A 50% reduction in mean proportion of females with eggs,
compared to the controls, would result in a mean proportion of 0.467.
Examining the means and their associated concentrations (Table 28), the
response, 0.701, is bracketed by C., = 0 ppb and C2 = 50 ppb. The response,
0.467, is bracketed by C, = 0 ppb and C2 = 50 ppb. ;
14.13.4.8.3 Using the equation in Section 4.2 from Appendix L, the estimate
of the IC25 is calculated as follows:
icp - Cj
IC25 = 0 + [0.934(1 - 25/100) - 0.934] (50 - 0)
(0.426 - 0.934)
= 23 ppb.
14.13.4.8.4 Using the equation in Section 4.2 from Appendix L, the estimate
of the IC50 is calculated as follows:
ICp =
w+1)
IC50 = 0 + [0.934(1 - 50/100) - 0.934] (50 - 0)
(0.426 - 0.934)
= 46 ppb. I
14.13.4.8.5 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 22.9745 ppb.
The empirical 95.0% confidence interval for the true mean was 20.0499 ppb to
30.5675 ppb. The computer program output for the IC25 for this data set is
shown in Figure 21. This value is extrapolated below the lowest test
concentration and data should be used cautiously.
I
14.13.4.8.6 When the ICPIN program was used to analyze this set of data for
the IC50, requesting 80 resamples, the estimate of the IC50 v/as 45.9490 ppb.
The empirical 95.0% confidence interval for the true mean was 40.1467 ppb to
63.0931 ppb. The computer program output for the IC50 for this data set is
shown in Figure 22.
291
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
1
1
.67
1
1
.8
1
1
2
50
.5
.33
.67
.4
.5
.25
.33
3
100
.3
.5
0
.5
.67
0
.25
4
210
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date: Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: fecundity
DATA FILE: mysidfe.icp
OUTPUT FILE: mysidfe.i25
Cone.
ID
1
2
3
4
Number
Replicates
8
7
7
8
Concentration
ug/l
0.000
50.000
100.000
210.000
Response
Means
0.934
0.426
0.317
0.000
Std. Pooled
Dev. Response Means
0.127
0.142
0.257
0.000
0.934
0.426
0.317
0.000
flt
The Linear Interpolation Estimate: 22.9745 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 23.8871 Standard Deviation: 3.0663
Original Confidence Limits: Lower: 20.0499 Upper: 30.5765
Resampling time in Seconds: 1.37 Random Seed: 1918482350
Figure 21. ICPIN program output for the IC25.
292
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
1
1
.67
1
1
.8
1
1
2
50
.5
.33
.67
.4
.5
.25
.33
3
100
.3
.5
0
.5
.67
0
.25
4
210
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxi cant/Eff1uent:
Test Start Date: Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: fecundity
DATA FILE: mysidfe.icp
OUTPUT FILE: mysidfe.i50
Cone.
ID
1
2
3
4
Number
Replicates
8
7
7
8
Concentration
ug/i
0.000
50.000
100.000
210.000
Response
Means
0.934
0.426
0.317
0.000
Std. Pooled
Dev. Response Means
0.127
0.142
0.257
0.000
0.934
0.426
0.317
0.000
The Linear Interpolation Estimate: 45.9490 Entered P Value: 50
'-
Number of Resamplings: 80
The Bootstrap Estimates Mean: 47.8720 Standard Deviation: 8 2908
Original Confidence Limits: Lower: 40.1467 Upper: 63 0931
Resampling time in Seconds: 1.32 Random Seed: -391064242
Figure 22. ICPIN program output for the IC50.
293
-------
14.14 PRECISION AND ACCURACY
14.14.1 PRECISION
14.14.1.1 Single-Laboratory Precision
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 same in
four of the six tests with Cu and SDS. Growth and fecundity were generally
not acceptable endpoints in either 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.
14.14.1.2 Multilaboratory Precision
14.14.1.2.1 The multilaboratory precision of the test has not yet been
determined.
14.14.2 ACCURACY .
14.14.2.1 The accuracy of toxicity tests cannot be determined.
294
-------
TABLE 29. SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST PERFORMED IN NATURAL
SEAWATER, USING JUVENILES FROM MYSIDS CULTURED AND SPAWNED IN
NATURAL §|AWATER, AND COPPER (CU) SULFATE AS ,A REFERENCE
TOXICANT 'f'4'5'6
Test
Number
1
2
3
4
5
NOEC
(M9/L)
63
125
125
125
125
IC25
96.1
,138.3
156.3
143.0
157.7
. IC50
NC8
175.5
187.5
179.9
200.3
Most
Sensitive
Endpoint
S
S
S
S
S
n:
Mean:
CV(%):
5
NA
NA
5
138.3
18.0
4
185.8
5.8
1
2
3
7
8
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by Randy Camel eo, ERL-N, USEPA, Narragansett, RI.
Eight replicate exposure chambers, each with five juveniles, were used
for the control and each toxicant concentration. The temperature of
the test solutions was maintained at 26 + 1°C.
Copper concentrations in Tests 1-2 were: 8, 16, 31, 63, and 125
Copper concentrations in Tests 3-6 were, 16, 31, 63, ]25, and 250
NOEC Range: 63 - 125 /zg/L (this represents a difference of two
exposure concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=Growth; S=Survival.
NC = No linear interpolation estimate could be calculated from the
data, since none of the group response means were less than 50 percent
of the control concentrations.
295
-------
TABLE 30. SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSIS BAHIA,
SURVIVAL, GROWTH, AND FECUNDITY TEST PERFORMED IN NATURAL
SEAWATER USING JUVENILES FROM MYSIDS CULTURED AND SPAWNED IN
NATURAL SEAWATER, AND SODIUM DODECYL SULFATE (SDS) AS A
REFERENCE TOXICANT1'2'3'4'5'6
Test
Number
NOEC
(mg/L)
IC25
(mg/L)
IC50
(mg/L)
Most
Sensitive
Endpoint
1
2
3
4
5
6
2.5
< 0.3
< 0.6
5.0
2.5
5.0
4.5
NC
NC8
7.8
3.6
7.0
NC9
NC
NC
NC9
•4.6
9.3
S
S
S
S
S
S
n:
Mean:
4
NA
NA
4
5.7
35.0
2
6.9
47.8
1
2
3
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by Randy Cameleo, ERL-N, USEPA, Narragansett, RI.
Eight replicate exposure chambers, each with five juveniles, were used
for the control and each toxicant concentration. The temperature of
the test solutions was maintained at 26 ± 1°C.
SDS concentrations in Tests 1-2 were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L.
in Tests 3-4 were: 0.6, 1.3, 2.5, 5.0 and 10.0 mg/L.
1.3, 2.5, 5.0, 10.0, and
SDS concentrations
SDS concentrations in Tests 5-6 were:
20.0 mg/L.
NOEC Range:
7
8
< 0.3 - 5.0 mg/L (this represents a difference of four
exposure concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=Growth; S=Survival.
NC - No linear interpolation estimate could be calculated from the
data, since none of the group response means were less than 75 percent
of the control response mean.
NC = No linear interpolation estimate could be calculated from the
data, since none of the group response means were less than 50 percent
of the control response mean.
296
-------
TABLE 31. COMPARISON OF SURVIVAL (LC50)1, GROWTH AND FECUNDITY (IC50)1
RESULTS FROM 7-DAY TESTS WITH THE MYSID, MYSIDOPSIS BAHIA,
USING NATURAL SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS
DILUTION WATER AND SODIUM DODECYL SULFATE (SDS) AS A
REFERENCE TOXICANT
Test
Survival LC50
NSW
GP2
Growth IC50
Fecundity IC50
NSW
GP2
NSW
GP2
1
2
3
16.2
20.5
2
16.3
19.2
21.9
16.8
24.2
2
16.3
23.3
24.4
12.0
20.1
2
10.9
18.5
21.7
All LC50/IC50 values in mg/L.
No test performed.
297
-------
TABLE 32. COMPARISON OF SURVIVAL (LC50)1, GROWTH AND FECUNDITY (IC50)1
RESULTS FROM 7-DAY TESTS WITH THE MYSID, NYSIDOPSIS BAHIA,
USING NATURAL SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS
DILUTION WATER AND COPPER (CU) SULFATE AS A REFERENCE
TOXICANT
Test
GP2
1
125
2
142
3
186
Survival LC50
NSW GP2
177 182
--2 173
190 174
Growth
NSW
208
2
195
IC50
GP2
186
210
179
Fecundity IC50
NSW
177
_-2
168
I All LC50/IC50 values in
No test performed.
298
-------
TABLE 33. CONTROL RESULTS FROM 7-DAY SURVIVAL, GROWTH, AND FECUNDITY
TESTS WITH THE MYSID, MYSIDOPSIS BAHIA, USING NATURAL SEAWATER
AND ARTIFICIAL SEAWATER (6P2) AS A DILUTION WATER
Test
1
2
3
4
5
6
Survival (%)
NSW GP2
98 93
80 90
--2 95
94 84
--2 94
80 75
Control 1
Growth (mq)
NSW GP2
0.32 0.32
0.40 0.43
--
2 0.40
0.34 0.37
--
2 0.36
0.40 0.41
Fecundity (%)
NSW GP2
73 77
100 95
--2 100
89 83
83
79 93
Survival as percent of mysids alive after 7 days; growth as mean
individual dry weight; fecundity as percent females With eggs.
No test performed.
299
-------
SECTION 15
TEST METHOD
SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST
METHOD 1008.0
15.1 SCOPE AND APPLICATION
15.1.1 This method, adapted in part from USEPA (1987e), measures the toxicity
of effluents and receiving water to the gametes of the sea urchin, Arbacia
punctulata, during a 1 h and 20 min exposure. The purpose of the sperm cell
toxicity test is to determine the concentration of a test substance that
reduces fertilization of exposed gametes relative to that of the control.
15.1.2 Detection limits of the toxicity of an effluent or chemical substance
are organism dependent.
15.1.3 Brief excursions in toxicity may not be detected using 24-h composite
samples. Also, because of the long sample collection period involved in
composite sampling and because the test chambers are not sealed, highly
volatile and highly degradable toxicants in the source may not be detected in
the test.
15.1.4 This test is commonly used in one of two forms: (1) a definitive test,
consisting of a minimum of five effluent concentrations and a control, and (2)
a receiving water test(s), consisting of one or more receiving water
concentrations and a control.
15.2 SUMMARY OF METHOD
15.2.1 The method consists of exposing dilute sperm suspensions to effluents
or receiving waters for 1 h. Eggs are then added to the sperm suspensions.
Twenty minutes after the eggs are added, the test is terminated by the
addition of preservative. The percent fertilization is determined by
microscopic examination of an aliquot from each treatment. The test results
are reported as the concentration of the test substance which causes a
statistically significant reduction in fertilization.
15.3 INTERFERENCES
15.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
15.3.2 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
300
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15.4 SAFETY
15.4.1 See Section 3, Health and Safety.
15.5. APPARATUS AND EQUIPMENT
!
15.5.1 Facilities for holding and acclimating test organisms.
15.5.2 Laboratory sea urchins, Arbacia punctulata, culture unit -- See
Subsection 6.17, culturing methods below and Section 4, Quality Assurance. To
test effluent or receiving water toxicity, sufficient eggs and sperm must be
available.
15.5.3 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.
15.5.4 Environmental chamber or equivalent facility with temperature control
(20 ± 1°C).
15.5.5 Water purification system -- Millipore Milli-Q®, deionized water (DI)
or equivalent.
15.5.6 Balance -- Analytical, capable of accurately weighing to 0.00001 g.
15.5.7 Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of materials to be weighed.
15.5.8 Air pump -- for oil-free air supply.
i
15.5.9 Air lines, and air stones -- for aerating water containing adults, or
for supplying air to test solutions with low DO.
15.5.10 Vacuum suction device -- for washing eggs.
15.5.11 Meters, pH and DO -- for routine physical and chemical measurements.
15.5.12 Standard or micro-Winkler apparatus -- for determining DO (optional).
15.5.13 Transformer, 10-12 Volt, with steel electrodes -- for stimulating
release of eggs and sperm.
"
15.5.14 Centrifuge, bench-top, slant-head, variable speed -- for washinq
eggs.
•
15.5.15 Fume hood -- to protect the analyst from formaldehyde fumes.
15.5.16 Dissecting microscope -- for counting diluted egg stock.
15.5.17 Compound microscope --for examining and counting sperm cells and
fertilized eggs.
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15.5.18 Sedgwick-Rafter counting chamber -- for counting egg stock and
examining fertilized eggs.
15.5.19 Hemacytometer, Neubauer -- for counting sperm.
15.5.20 Count register, 2-place --for recording sperm and egg counts.
15.5.21 Refractometer -- for determining salinity.
15.5.22 Thermometers, glass or electronic, laboratory grade --for measuring
water temperatures.
15.5.23 Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.
15.5.24 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
15.5.25 Ice bucket, covered -- for maintaining live sperm.
15.5.26 Centrifuge tubes, conical -- for washing eggs.
15.5.27 Cylindrical glass vessel, 8-cm diameter -- for maintaining dispersed
egg suspension.
15.5.28 Beakers -- six Class A, borosilicate glass or non-toxic plasticware,
1000 ml for making test solutions.
15.5.29 Glass dishes, flat bottomed, 20-cm diameter -- for holding urchins
during gamete collection.
15.5.30 Wash bottles -- for deionized water, for rinsing small glassware and
instrument electrodes and probes.
15.5.31 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
15.5.32 Syringes, 1-mL, and 10-mL, with 18 gauge, blunt-tipped needles (tips
cut off) -- for collecting sperm and eggs.
15.5.33 Pipets, volumetric -- Class A, 1-100 ml.
15.5.34 Pipets, automatic -- adjustable 1-100 ml.
15.5.35 Pipets, serological -- 1-10 ml, graduated.
15.6.36 Pipet bulbs and fillers -- PROPIPET®, or equivalent.
15.6 REAGENTS AND CONSUMABLE MATERIALS
15.6.1 Sea Urchins, Arbacia punctuTata minimum 12 of each sex.
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15.6.2 Food -- kelp, Laminaria sp., or romaine lettuce for the sea urchin,
Arbacia punctulata.
15.6.3 Standard salt water aquarium or Instant Ocean Aquarium (capable of
maintaining seawater at 15°C) -- with appropriate filtration and aeration
system.
I
15.6.4 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
15.6.5 Scintillation vials, 20 ml, disposable -- to prepare test
concentrations.
15.6.6 Tape, colored -- for labelling tubes.
15.6.7 Markers, waterproof -- for marking containers, etc.
15.6.8 Parafilm --to cover tubes and vessels containing test materials.
15.6.9 Gloves, disposable; labcoat and protective eyewear -- for personal
protection from contamination.
15.6.10 Data sheets (one set per test) -- for data recording (see Figures 1,
2, and 3).
15.6.11 Acetic acid, 10%, reagent grade, in seawater -- for preparing killed
sperm dilutions. ;
i
15.6.12 Formalin, 1%, in 2 ml of seawater -- for preserving eggs (see
Subsection 10.7 Termination of the Test).
15.6.13 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) -- for standards and calibration check (see USIEPA Method 150.1,
USEPA, 1979b). I
15.6.14 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA, 1979b), or reagents -- for modified Winkler analysis.
15.6.15 Laboratory quality assurance samples and standards •-- for the above
methods.
15.6.16 Reference toxicant solutions -- see Section 4, Quality Assurance.
15.6.17 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms.
15.6.18 Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
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TEST DATE:
SAMPLE:
COMPLEX EFFLUENT SAMPLE:
COLLECTION DATE:
SALINITY/ADJUSTMENT:
PH/ADJUSTMENT REQUIRED: _
PHYSICAL CHARACTERISTICS:
STORAGE:
COMMENTS:
SINGLE COMPOUND:
SOLVENT (CONC):
TEST CONCENTRATIONS:
DILUTION WATER:
CONTROL WATER:
TEST TEMPERATURE:
TEST SALINITY: _
COMMENTS:
Figure 1. Data form for (1) fertilization test using sea urchin, Arbacia
punctulata.
304
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TEST DATE:
SAMPLE: _
SPERM DILUTIONS:
HEMACYTOMETER COUNT, E:
x 104 = SPM SOLUTION E =
SPERM CONCENTRATIONS: SOLUTION E x 40 = SOLUTION A =
SOLUTION E x 20 = SOLUTION B =
SOLUTION E x 5 = SOLUTION D =
SOLUTION SELECTED FOR TEST (
DILUTION: SPM/(5 x 107)
= 5 x 107 SPM):
DF
[(DF) x 10) - 10 =
FINAL SPERM COUNTS =
+ SW., ml.
EGG DILUTIONS:
INITIAL EGG COUNT
ORIGINAL EGG STOCK CONCENTRATION = 10X (INITIAL
EGG COUNT)
VOLUME OF SW TO ADD TO DILUTE EGG STOCK TO 2000/mL:
(EGG COUNT) - 200
CONTROL WATER TO ADD EGG STOCK, mL
FINAL EGG COUNT
,1
TEST TIMES:
SPERM COLLECTED: :
EGGS COLLECTED:
SPERM ADDED:
EGGS ADDED: .
FIXATIVE ADDED:
SAMPLES READ:
SPM
SPM
SPM
Figure 2.
Data form (2) for fertilization test using sea urchin, Arbacia
punctulata.
305
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DATE TESTED:
SAMPLE: .
TOTAL AND UNFERTILIZED EGG COUNT AT END OF TEST:
EFFLUENT
CONC (%)
1
REPLICATE VIAL
TOTAL-UNFERT TOTAL-UNFERT TOTAL-UNFERT TOTAL-UNFERT
STATISTICAL ANALYSIS:
ANALYSIS OF VARIANCE:
CONTROL:
DIFFERENT FROM CONTROL (P)
COMMENTS:
Figure 3. Data form (3) for fertilization test using sea urchin, Arbacia
punctulata.
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15.6.18.1 Saline test and dilution water -- the salinity of the test water
must be 30%o. The salinity should vary by no more than ± 2%o among the
replicates. If effluent and receiving water tests are conducted concurrently,
the salinities of these tests should be similar.
|
15.6.18.2 The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts. Exposure of sea urchin eggs and sperm to these effluents
will require adjustments in the salinity of the test solutions. It is
important to maintain a constant salinity across all treatments. In addition
it may be desireable to match the test salinity with that of the receiving
water. Two methods are available to adjust salinities --hypersaline brine
(MSB) derived from natural seawater or artificial sea salts.
15.6.18.3 Hypersaline brine (MSB): MSB has several advantages that make it
desirable for use in toxicity testing. It can be made from any high quality,
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity. MSB derived from natural seawater
contains the necessary trace metals, biogenic colloids, and some of the
microbial components necessary for adequate growth, survival, and/or
reproduction of marine and estuarine organisms, and may be stored for
prolonged periods without any apparent degradation. However, if 100%o HSB is
used as a diluent, the maximum concentration of effluent that can be tested
will be 80% at 20%o salinity and 70% at 30%o salinity.
15.6.18.3.1 The ideal container for making HSB from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a noncorrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine. If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that woul.d contaminate the brine. One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass. If aeration is utilized, use only oil-free air compressors
to prevent contamination. j
15.6.18.3.2 Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be in direct contact with the brine. A good quality biodegradable detergent
should be used, followed by several (at least three) thorough deionized water
rinses. !
.
15.6.18.3.3 High quality (and preferably high salinity) seawater should be
filtered to at least 10 /j.m before placing into the brine generator. Water
should be collected on an incoming tide to minimize the possibility of
contamination.
15.6.18.3.4 The temperature of the seawater is increased slowly to 40°C. The
water should be aerated to prevent temperature stratification and to increase
water evaporation. The brine should be checked daily (depending on the volume
being generated) to ensure that the salinity does not exceed 100%o and that
.
307
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the temperature does not exceed 40°C. Additional seawater may be added to the
brine to obtain the volume of brine required.
15.6.18.3.5 After the required salinity is attained, the MSB should be
filtered a second time through a 1 f*m filter and poured directly into portable
containers, (20 L cubitainers or polycarbonate water cooler jugs are
suitable). The containers should be capped and labelled with the date the
brine was generated and its salinity. Containers of HSB should be stored in
the dark and maintained under room temperature until used.
15.6.18.3.6 If a source of HSB is available, test solutions can be made by
following the directions below. Thoroughly mix together the deionized water
and brine before mixing in the effluent.
15.6.18.3.7 Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine. For example, if the
salinity of the brine is 100%o and the test is to be conducted at 30%o, 100%o
divided by 30%o = 3.3. The proportion of brine is 1 part in 3.3 (one part
brine to 2.3 parts deionized water). To make 1 L of seawater at 30%o salinity
from a HSB of 100%o, 300 ml of brine and 700 ml of deionized water are
required.
15.6.16.3.8 Table 1 illustrates the preparation of test solutions at 30%o if
they are made by combining effluent (0%o), deionized water and HSB (100%o), or
FORTY FATHOMS® sea salts.
15.6.16.4 Artificial sea salts: FORTY FATHOMS® brand sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-3211189) 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.
15.6.16.4.1 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, et al., 1984; Bower, 1983).-
15.6.16.4.2 The 6P2 reagent grade chemicals (Table 1) 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 NaHC03 in 500 ml of
deionized water. Add 2.5 mL of this stock solution for each liter of the GP2
artifical seawater.
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TABLE 1. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 30%o USING
NATURAL SEAWATER, HYPERSALINE BRINE, OR ARTIFICIAL SEA SALTS
Solutions To Be Combined
Eff1uent
Effluent Concentration
Solution
Volume of
Effluent
Solution
(mL)
Volume of Diluent
Seawater (30%o)
(mL)
1
2
3
4
5
Control
1001 840
50 420 Solution 1 + 420
25 420 Solution 2 + 420
12.5 420 Solutions
6.25 420 Solution 4
0.0
Total
+ 420
+ 420
420
2080
This illustration assumes: (1) the use of 5 mL of test solution in each
of four replicates (total of 20 mL) for the control and five
concentrations of effluent, (2) an effluent dilution factor of 0.5, (3)
the effluent lacks appreciable salinity, and (4) 400 mL of each test
concentration is used for chemical analysis. A sufficient initial
volume (840 mL) of effluent is prepared by adjusting the salinity to
30%o. In this example, the salinity is adjusted by adding artificial
sea salts to the 100% effluent, and preparing a serial dilution using
30%o seawater (natural seawater, hypersaline brine, or artificial
seawater). Stir solutions 1 h to ensure that the salts dissolve. The
salinity of the initial 840 mL of 100% effluent is adjusted to 30%o by
adding 25.2 g of dry artificial sea salts (FORTY FATHOMS®). Test
concentrations are then made by mixing appropriate volumes of salinity
adjusted effluent and 30%o salinity dilution water to provide 840 mL of
solution for each concentration. If hypersaline brine alone (100%o) is
used to adjust the salinity of the effluent, the highest concentration,
of effluent that could be tested would be 70% at 30%o salinity.
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TABLE 2. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
ARTIFICIAL SEAWATER FOR THE SEA URCHIN, ARBACIA PUNCTULATA,
TOXICITY TEST1'2'3
Compound
Concentration
(9/L)
Amount (g)
Required for
20 L
NaCl
Na2S04
KC1
KBr
Na2B407
MgCl2 .
CaCl2 .
SrCl2 .
NaHC03
. 10 H20
6 H20
2 H20
6 H20
] Modified GP2 from Spotte
The constituent salts and
(1990b). The salinity is
3 f\r\n *»••«* L* n ^J*!*lii4>n^J • • * 4» L* *\
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
et al. (1984).
concentrations
30.89 g/L.
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
were taken from USEP/
salinity.
15.6.17 TEST ORGANISMS, SEA URCHINS, ARBACIA PUNCTULATA
15.6.17.1 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. 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.
15.6.17.2 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.
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15.6.17.3 The culture unit should be maintained at 15 ± 3°C, with a water
temperature control device. ;
15.6.17.4 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.
15.6.17.5 Natural or artificial seawater with a salinity of 30% is used to
maintain the adult animals, for all washing and dilution steps, and as the
control water in the tests (see Subsection 15.6.16). i
15.6.17.6 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.
15.6.17.7 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.
15.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
15.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sampling Preparation for Toxicity Tests.
i
15.8 CALIBRATION AND STANDARDIZATION
i
15.8.1 See Section 4, Quality Assurance.
15.9 QUALITY CONTROL
I
15.9.1 See Section 4, Quality Assurance. \
15.10 TEST PROCEDURES
15.10.1 TEST SOLUTIONS
15.10.1.1 Receiving Waters
-
15.10.1.1.1 The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 yum NITEX® filter and compared without
dilution against a control. Using four replicate chambers per test, each
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containing 5 ml, and 400 mL for chemical analysis, would require approximately
420 ml or more of sample per test.
15.10.1.2 Effluents
15.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used.
A dilution factor of 0.5 provides precision of ± 100%, and testing of
concentrations between 6.25% .and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used. Therefore, USEPA recommends the
use of the ;> 0.5 dilution factor. If 100%o HSB is used as a diluent, the
maximum concentration of effluent that can be tested will be 80% at 20%o and
70% at 30%o salinity.
15.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).
15.10.1.2.3 Just prior to test initiation (approximately 1 h), a sufficient
quantity of the sample to make the test solutions should be adjusted to the
test temperature (20 ± 1°C) and maintained at that temperature during the
addition of dilution water.
15.10.1.2.4 The test should begin as soon as possible, preferably within 24 h
of sample collection. The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
tests unless permission is granted by the permitting authority. In no case
should the sample be used in a test more than 72 h after sample collection
(see section 8 Effluent and Receiving Water Sampling, Sample Handling, and
Sample Preparation for Toxicity Test).
15.10.1.2.5 Effluent dilutions should be prepared for all replicates in each
treatment in one beaker to minimize variability among the replicates. The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.
15.10.1.3 Dilution Water
15,10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving
water), HSB prepared from natural seawater, or artifical seawater FORTY
FATHOMS® or GP2 sea salts (see Table 2 and Section 7, Dilution Water).
Prepare 3 L of control water at 30%o using HSB or artificial sea salts (see
Table 1). This water is used in all washing and diluting steps and as control
water in the test. Natural seawater and local waters may be used as
additional controls.
15.10.2 COLLECTION OF GAMETES FOR THE TEST
15.10.2.1 Select four females and place in shallow bowls, barely covering the
shell with seawater. Stimulate the release of eggs by touching the shell with
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steel electrodes connected to a 10-12 volt transformer (about 30 seconds each
time). Collect the eggs from each female using a 10 ml disposable syringe
fitted with an 18-gauge, blunt-tipped needle (tip cut off). Remove the needle
from the syringe before adding the eggs to a conical centrifuge tube. Pool
the eggs. The egg stock may be held at room temperature for several hours
before use. Note: Eggs should be collected first to eliminate possibility of
pre-fertilization.
15.10.2.2 Select four males and place in shallow bowls, biirely covering the
animals with seawater. Stimulate the release of sperm as described above.
Collect the sperm (about 0.25 ml) from each male, using a 1-3 ml disposable
syringe fitted with an 18-gauge, blunt-tipped needle. Pool the sperm.
Maintain the pooled sperm sample on ice. The sperm must be used in a toxicity
test within 1 h of collection. !
15.10.3 PREPARATION OF SPERM DILUTION FOR USE IN THE TEST
I
15.10.3.1 Using control water, dilute the pooled sperm sample to a
concentration of about 5 X 10 sperm/mL (SPM). Estimate the sperm
concentration as described below:
1. Make a sperm dilutions of 1:50, 1:100, 1:200, and 1:400, using 30%o
seawater, as follows:
i
a. Add 400 //L of collected sperm to 20 ml of seawater in Vial A. Mix by
gentle pipetting using a 5-mL pipettor, or by inversion;
b. Add 10 ml of sperm suspension from Vial A to 10 ml of seawater in
Vial B. Mix by gentle pipetting using a 5-mL pipettor, or by
inversion;
c. Add 10 mL of sperm suspension from Vial B to 10 mL of seawater in
Vial C. Mix by gentle pipetting using a 5-mL pipettor, or by
inversion;
d. Add 10 mL of sperm suspension from Vial C to 10 mL of seawater in
Vial D. Mix by gentle pipetting using a 5-mL pipettor, or by
inversion;
e. Discard 10 mL from Vial D. (The volume of all suspensions is 10 mL).
2. Make a 1:2000 killed sperm suspension and determine the SPM.
a. Add 10 mL 10% acetic acid in seawater to Vial C. Cap Vial C and mix
by inversion.
b. Add 1 mL of killed sperm from Vial C to 4 mL of seawater in Vial E.
Mix by gentle pipetting with a 4-mL pipettor.
c. Add sperm from Vial E to both sides of the Neubauer hemacytometer.
Let the sperm settle 15 min.
d. Count the number of sperm in the central 400 squares on both sides of
the hemacytometer using a compound microscope (IGiOX). Average the
counts from the two sides. \
e. SPM in Vial E = 10 x average count. ;
3. Calculate the SPM in all other suspensions using the SPM in Vial E
above:
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SPM in Vial A = 40 x SPM in Vial E
SPM in Vial B = 20 x SPM in Vial E
SPM in Vial D = 5 x SPM in Vial E
SPM in original sperm sample = 2000 x SPM in Vial E
4. Dilute the sperm suspension with a SPM greater than 5 x 107 SPM to 5 x
107 SPM.
Actual SPM/(5 x 107) = dilution factor (DF)
[(DF) x 10] - 10 = mL of seawater to add to vial.
5. Confirm the sperm count by sampling from the test stock. Add 0.1 ml
of test stock to 9.9 ml of 10% acetic acid in seawater, and count with
the hemacytometer. The count should average 50 ± 5.
15.10.4 PREPARATION OF EGG SUSPENSION FOR USE IN THE TEST Note: The egg
suspension may be prepared during the 1-h sperm exposure.
15.10.4.1 Wash the pooled eggs three times using control water with gentle
centrifugation (500xg for 3 minutes using a tabletop centrifuge). If the wash
water becomes red, the eggs have lysed and must be discarded.
15.10.4.2 Dilute the egg stock, using control water, to about 2000 eggs/mL.
1. Transfer the eggs to a glass beaker containing 200 ml of control water
("egg stock").
2. Mix the egg stock using an air-bubbling device. Using a wide-mouth
pi pet tip, transfer 1 ml of eggs from the egg stock to a vial
containing 9 ml of control water. (This vial contains an egg suspension
diluted 1:10 from egg stock).
3. Mix the contents of the vial by inversion. Using a wide-mouth pipet
tip, transfer 1 ml of eggs from the vial to a Sedgwick-Rafter counting
chamber. Count all eggs in the chamber using a dissecting microscope at
24X "egg count".
4. Calculate the concentration of eggs in the stock. Eggs/mL = 10X (egg
count). Dilute the egg stock to 2000 eggs/mL by the formula below.
a. If the egg count is equal to or greater than 200:
(egg count) - 200 = volume (mL) of control water to add to egg stock.
b. If the egg count is less than 200, allow the eggs to settle and
remove enough control water to concentrate the eggs to greater
than 200, repeat the count, and dilute the egg stock as in a.
above. NOTE: It requires 24 mL of a egg stock solution for each
test with a control and five exposure concentrations.
c. Transfer 1 mL of the diluted egg stock to a vial containing 9 mL of
control water. Mix well, then transfer 1 mL from the vial to a
Sedgwick-Rafter counting chamber. Count all eggs using a
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dissecting microscope. Confirm that the final egg count =
2000/mL (± 200). i
15.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
I
15.10.5.1 The light quality and intensity should be at ambient laboratory
levels 10-20 /iE/m /s (50-100 ft-c) with a photoperiod of 16 h light and 8 h
darkness. The water temperature in the test chambers should be maintained at
20 ± 1°C. ' The test salinity should be in the range of 28 to 32%o. The
salinity should vary by no more that ± 2%o among the chambers on a given day.
If effluent and receiving water tests are conducted concurrently, the
salinities of these tests should be similar.
15.10.6 DISSOLVED OXYGEN (DO) CONCENTRATION
15.10.6.1 Aeration may affect the toxicity of effluent and should be used
only as a last resort to maintain a satisfactory DO. The DO concentrations
should be measured on new solutions at the start of the test (Day 0). The DO
should not fall below 4.0 mg/L (see Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample Preparation for Toxicity Tests). If it
is necessary to aerate, all treatments and the control should be aerated. The
aeration rate should not exceed 100 bubbles/minute, using a pipet with a 1-2
mm orifice, such as a 1 mL KIMAX® serological pipet No. 37033, or equivalent.
15.10.7 OBSERVATIONS DURING THE TEST
i
15.10.7.1 Routine Chemical and Physical Observations
15.10.7.1.1 DO is measured at the beginning of the exposure period in one
test chamber at each test concentration and in the control.
i-
15.10.7.1.2 Temperature, pH, and salinity are measured at the beginning of
the exposure period in one test chamber at each concentration and in the
control. Temperature should also be monitored continuously observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
chambers at least at the end of the test to determine temperature variation in
environmental chamber.
15.10.7.1.3 The pH is measured in the effluent sample each day before new
test solutions are made. i
i
15.10.7.1.4 Record all the measurements on the data sheet.
15.10.7.2 Routine Biological Observations
15.10.7.2.1 Fertilization will be determined by the presence of a
fertilization membrane surrounding the egg.
315
-------
15.10.8 START OF THE TEST
15.10.8.1 Effluent/deceiving water samples are adjusted to salinity of 30%o.
Four replicates (minimum of three) are prepared for each test concentration,
using 5 ml of solution in disposable liquid scintillation vials. A 50% (0.5)
concentration series can be prepared by serially diluting test concentrations
with control water. Sufficient test solution is prepared at each effluent
concentration to provide additional volume for chemical analyses, at the high,
medium, and low test concentrations.
15.10.8.2 All test samples are equilibrated at 20°C ± 1°C before addition of
sperm.
15.10.8.3 Within 1 h of collection add 100 pL of appropriately diluted sperm
to each test vial. Record the time of sperm addition.
15.10.8.4 Incubate all test vials at 20 ± 1°C for 1 h.
15.10.8.5 Mix the diluted egg suspension (2000 eggs/mL), using gentle
bubbling. Add 1 ml of diluted egg suspension to each test vial using a wide
mouth pi pet tip. Incubate 20 min at 20 ± 1°C.
15.10.9 TERMINATION OF THE TEST
15.10.9.1 Terminate the test and preserve the samples by adding 2 ml of 1%
formalin in seawater to each vial.
15.10.9.2 Vials should be evaluated within 48 hours.
15.10.9.3 To determine fertilization, transfer about 1 ml eggs from the
bottom of a test vial to a Sedgwick-Rafter counting chamber. Observe the eggs
using a compound microscope (100X). Count between 100 and 200 eggs/sample.
Record the number counted and the number unfertilized. Fertilization is
indicated by the presence of a fertilization membrane surrounding the egg.
Note: adjustment of the microscope to obtain proper contrast may be required
to observe the fertilization membrane. Because samples are fixed in formalin,
a ventilation hood is set up surrounding the microscope to protect the analyst
from prolonged exposure to formaldehyde fumes.
15.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
15.11.1 A summary of test conditions and test acceptability criteria is
listed in Table 3.
15.12 ACCEPTABILITY OF TEST RESULTS
15.12.1 The spermregg ratio routinely employed should result in fertilization
of 70%-90% of the eggs in the control chambers.
316
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT
AND RECEIVING WATERS
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Test chamber size:
7. Test solution volume:
8. No. of sea urchins:
9. No. egg and sp.erm cells
per chamber:
10. No. replicate chambers
per concentration:
11. Dilution water:
12. Effluent concentrations:
Static
I
30%o (± 2%o of the selected test
salinity)
i
20 ± 1°C
Ambient laboratory light during test
preparation j
10-20 ME/m2/s, or 50-100 ft-c (Ambient
laboratory levels)
Disposable (glass) liquid
scintillation vials (20 mL capacity),
presoaked in control water
5 mL
Pooled sperm from four males and
pooled eggs from four females are
used per test
About 2000 eggs and 5, 000,000 sperm
cells per vial
4 (minimum of 3)
Uncontaminated source of natural
seawater; deionized water mixed with
hypersaline brine or artificial sea
salts (HW Marinemix®, FORTY FATHOMS®,
6P2, or equivalent) !
Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving
water or minimum of 5 and a control
317
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT
AND RECEIVING WATERS (CONTINUED)
13. Test dilution factor:
14. Test duration:
15. Endpoint:
16. Test acceptability
criteria:
17. Sampling requirements:
18. Sample volume required:
Effluents: > 0.5
Receiving waters: None or > 0.5
1 h and 20 min
Fertilization of sea urchin eggs
70% - 90% egg fertilization in
controls
One sample collected at test
initiation, and preferably used
within 24 h of the time it is removed
from the sampling device (see Section
8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests,
Subsection 8.5.4)
1 L per test
318
-------
15.13 DATA ANALYSIS
i
15.13.1 GENERAL I
15.13.1.1 Tabulate and summarize the data. Calculate the proportion of
fertilized eggs for each replicate. A sample set of test data is listed in
Table 4.
15.13.1.2 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. The assistance of a
statistician is recommended for analysts who are not proficient in statistics,
'
TABLE 4. DATA FROM SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST1
Copper
Concentration
No. of Eggs No. of Eggs Proportion
Replicate Counted Fertilized Fertilized
Control
A
B
C
100
100
100
85
78
87
0.85
0.78
0.87
2.5
5.0
10.0
20.0
40.0
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
81
65
71
63
74
78
63
66
51
41
41
37
12
30
26
0.81
0.65
0.71
0.63
0.74
0.78
0.63
0.66
0.51
0.41
0.41
0.37
0.12
0.30
0.26
Tests performed by Dennis M. McMullen, Technology Applications, Inc.,
EMSL, Cincinnati, OH.
15.13.1.3 The endpoints of toxicity tests using the sea urchin are based on
the reduction in proportion of eggs fertilized. The IC25 and the IC50 are
calculated using the Linear Interpolation Method (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis). LOEC and NOEC values for
fecundity are obtained using a hypothesis testing approach such as Dunnett's
Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel, 1959; Miller,
319
-------
1981) (see Section 9). Separate analyses are performed for the estimation of
the LOEC and NOEC endpoints and for the estimation of IC25 and IC50. See the
Appendices for examples of the manual computations, and examples of data input
and program output.
15.13.2 EXAMPLE OF ANALYSIS OF SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION
DATA
15.13.2.1 Formal statistical analysis of the fertilization data is outlined
in Figure 4. The response used in the analysis is the proportion of
fertilized eggs in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the IC25 and IC50 endpoints. Concentrations at which there are
no eggs fertilized in any of the test chambers are excluded from statistical
analysis of the NOEC and LOEC, but included in the estimation of the IC25 and
IC50.
15.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Wilk's
Test, and Bartlett's Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.
15.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested, there are. parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment (see
Appendix D). The Wilcoxon Rank Sum test with the Bonferroni adjustment is the
nonparametric alternative.
15.13.2.4 Example of Analysis of Fecundity Data
15.13.2.4.1 This example uses toxicity data from a sea urchin, Arbacia
punctulata, fertilization test performed with copper. The response of
interest is the proportion of fertilized eggs, thus each replicate must first
be transformed by the arc sine square root transformation procedure described
in Appendix B. The raw and transformed data, means and variances of the
transformed observations at each copper concentration and control are listed
in Table 5. The data are plotted in Figure 5.
15.13.2.5 Test for Normality
15.13.2.5.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 6.
320
-------
STATISTICAL ANALYSIS OF SEA URCHIN FERTILIZATION TEST
FERTILIZATION DATA
PROPORTION OF FERTILIZED EGGS
POINT ESTIMATION
ARC SINE
TRANSFORMATION
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILK-S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTS TEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
EQUAL NUMBER OF
REPLICATES?
NO
YES
T-TESTWITH
BONFERRONI
ADJUSTMENT
I YES
NO
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 4. Flowchart for statistical analysis of sea urchin, Arbacia
punctulata, by point estimation.
321
-------
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322
-------
TABLE 5. SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA
Copper Concentration (//g/L)
Repl
RAW
ARC SINE
TRANSFORMED
Mean (YJ
?T
i
TABLE 6
icate Control
A 0.
B 0.
C 0.
A 1.
B 1.
C 1.
85
78
87
173
083
202
1.153
0.004
1
. CENTERED
2.5
0.
0.
0.
1.
0.
1.
1.
0.
2
81
65
71
120
938
002
020
009
OBSERVATIONS
5.0
0.63
0.74
0.78
0.917
1.036
1.083
1.012
0.007
3
10.0
0.63
0.66
0.51
0.917
0.948
0.795
0.887
0.007
4
FOR SHAPIRO-MILK'S
20.0
0.41
0.41
0.3.7
0.695
0.695
0.654
0.681
0.001
!>
EXAMPLE
40.0
0.12
0.30
0.26
0.354
0.580
0.535
0.490
0.014
6
Copper Concentration (//g/L)
Replicate
A
B
C
Control
0.020
-0.070
0.049
0
-0
-0
2.5
.100
.082
.018
5.0
-0.095
0.024
0.071
10.0
0.030
0.061
-0.092
20.0
0.014
0.014
-0.027
40.0
-0.136
0.090
0.045
15.13.2.5.2 Calculate the denominator, D, of the statistic:
D = £ (x,-x)2
i-l
Where: X,- = the ith centered observation ,
X = the overall mean of the centered observations
n = the total number of centered observations
15.13.2.5.3 For this set of data, n = 18 !
X = _L_ (0) = 0
18
D = 0.0822
323
-------
15.13.2.5.4 Order the centered observations from smallest to largest
< ...
1
2
3
4
5
6
7
8
9
-0.136
-0.095
-0.092
-0.082
-0.070
-0.027
-0.018
0.014
0.014
10
11
12
13
14
15
16
17
18
0.020
0.024
0.030
0.045
0.049
0.061
0.071
0.090
0.100
15.13.2.5.5 From Table'4, Appendix B, for the number of observations, n,
obtain the coefficients a15 a2, ... ak where k is n/2 if n is even and (n-l)/2 *
if n is odd. For the data in this example, n = 18 and k = 9. The a,- values \\
are listed in Table 8.
15.13.2.5.6 Compute the test statistic, W, as follows:
W =
D i=i
The differences, x(n"i+1) - X(i), are listed in Table 7. For the data in this
example:
W = 1 (0.2782)2 = 0.942
0.0822
15.13.2.5.7 The decision rule for this test is to compare W as calculated in
2.6 to a 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 the data in this example, the critical value at a
significance level of 0.01 and n = 18 observations is 0.858. Since W = 0.942
is greater than the critical value, conclude that the data are normally
distributed.
324
-------
1
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
ai
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
« _
XC12) .
X(11) .
X(10) _
XC1)
X(2)
X(3)
X(4>
X(5)
X(6)
J(7)
x<8>
X(9,
15.13.2.6 Test for Homogeneity of Variance
15.13.2.6.1 The test used to examine whether the variation in the proportion
of fertilized eggs is the same across all copper concentrations including the
control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic
is as follows:
[(
in
S2 - £
B =
v, in Sf]
Where: V
, = degrees of freedom for each copper concentration and control,
V,- = (n,- - 1)
p = number of levels of copper concentration including the control
n
In
, = the number of replicates for concentration 1.
= loge
i = 1,2, ..., p where p is the number of concentrations including
the control
C =
i=l
1=1
325
-------
15.13.2.6.2 For the data in this example (see Table 5), all copper
concentrations including the control have the same number of replicates (nf
3 for all i). Thus, V,- = 2 for all i .
15.13.2.6.3 Bartlett's statistic is, therefore:
B = [ (12) Irz (0.0007) -2
1/1.194
= [12(-4.962) - 2(-31. 332)]/1.194
= 3.122/1.194
= 3.615
15 13.2.6.4 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 5 degrees
of freedom, is 15.09. Since B = 2.615 is less than the critical value of
15.09, conclude that the variances are not different.
15.13.2.7 Dunnett's Procedure
15.13.2.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 9.
TABLE 9. ANOVA TABLE
Source
Between
Within
Total
df
P - 1
N - p
N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square(MS)
(SS/df)
SB = SSB/ (p-1)
Sy = SSW/(N-p)
Where: p = number of copper concentrations including the control
N = total number of observations n, + n2 ... + np
n,- - number of observations in concentration i
326
-------
SSB = 2^TJ/n±-G2/N Between Sum of Squares
i-l
SST = E EFy-GV-W Total Sum of Squares
SSW = SST-SSB Within Sum of Squares
I
6 = the grand total of all sample observations, G = £ r,
i-i
T,- = the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the
proportion of fertilized eggs for copper concentration i in
test chamber j)
15.13.2.7.2 For the data in this example:
n1 = n2 = n3 = n4 = n5 = n6 = 3
N = 18
T, = ¥„ + Y12 + Y13 = 3.458 |
T2 = Y21 + Y22 + Y23 = 3.060
T3 = Y31 + Y32 + Y33 = 3'036
T4 = Y41 + Y42 + Y43 = 2.660 ;
fc: fe t fc: fc:!:«
G = T,- + T2 + T3 + T4 + T5 + T6 = 15.727
SSB =
i=l
2
= (43.950)/3 - (15.727)/18 = 0.909
SST =
14.732 - (15.727)2/18 - 0.991
SSW = SST-SSB = 0.991 - 0.909 = 0.082
327
-------
SB = SSB/(p-l) = 0.909/(6-l) = 0.182
$„ = SSW/(N-p) = 0.082/(18-6) = 0.007
15.13.2.7.3 Summarize these calculations in the ANOVA table (Table 10)
TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
5
12
17
Sum of Squares
(SS)
0.909
0.082
0.991
Mean Square(MS)
(SS/df)
0.182
0.007
15.13.2.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where: Y? - mean proportion fertilized eggs for copper concentration i
Y, = mean proportion fertilized eggs for the control
Sw » square root of the within mean square
n., - number of replicates for the control
n,- = number of replicates for concentration i.
Since we are looking for a decreased response from the control in the
proportion of fertilized eggs, the concentration mean is subtracted from the
control mean.
328
-------
15.13.2.7.5 Table 11 includes the calculated t values for each concentration
and control combination. In this example, comparing the £.5
concentration with the control the calculation is as follows:
(1.153-1.020)
[0 . 084v/(l/3) + (1/3)]
=1.939
TABLE 11. CALCULATED T VALUES
Copper Concentration (M9/L)
2.5
5.0
10.0
20.0
40.0
2
3
4
5
6
1.939
2.056
3.878
6.882
9.667
15.13.2.7.6 Since the purpose of this test is to detect a significant
decrease in the proportion of fertilized eggs, a one-sided test is
appropriate. The critical value for this one-sided test is found in Table 5,
Appendix D. For an overall alpha level of 0.05, 12 degrees of freedom for
error and five concentrations (excluding the control) the critical value is
2.50. The mean proportion of fertilized eggs for concentration i is
considered significantly less than the mean proportion of fertilized eggs for
the control if t5 is greater than the critical value. Therefore, the 10.0
//g/L, 20.0 /jg/L and 40.0 /Kj/L concentrations have a significantly lower mean
proportion of fertilized eggs than the control. Hence the NOEC is 5.0 //g/L
and the LOEC is 10.0 /^g/L.
15.13.2.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be calculated:
MSD = d
Where: d = the critical value for Dunnett's Procedure
Sw = the square root of the within mean square
n = the common number of replicates at each concentration (this
assumes equal replication at each concentration)
n1 = the number of replicates in the control.
15.13.2.7.8 In this example,
MSD = 2.50 (0.084)^(1/3) + (1/3)
= 2.50 (0.084)(0.816)
= 0.171
329
-------
15.13.2.7.9 The MSD (0.171) is in transformed units. To determine the MSD in
terms of proportion of fertilized eggs, carry out the following conversion.
1. Subtract the MSD from the transformed control mean.
1.153 - 0.171 = 0.982
2. Obtain the untransformed values for the control mean and the difference
calculated in step 1 of 13.2.7.9.
[ Sine (1.153) ]2 = 0.835
[ Sine (0.982) ]2 = 0.692
3. The untransformed MSD (MSDJ is determined by subtracting the
untransformed values from step 2 in 14.2.7.9.
MSDU = 0.835 - 0.692 = 0.143
15.13.2.7.10 Therefore, for this set of data, the minimum difference in mean
proportion of fertilized eggs between the control and any copper concentration
that can be detected as statistically significant is 0.143.
15.13.2.7.11 This represents a 17% decrease in the proportion of fertilized
eggs from the control.
15.13.2.8 Calculation of the ICp
15.13.2.8.1 The fertilization data in Table 4 are utilized in this example.
Table 12 contains the mean proportion of fertilized eggs for each toxicant
concentration. As can be seen, the observed means are monotonically non-
increasing with respect to concentration. Therefore, it is not necessary to
smooth the means prior to calculating the ICp; (see Figure 5 for a plot of the
response curve).
15.13.2.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation
Method. A 25% reduction in mean proportion of fertilized eggs, compared to
the controls, would result in a mean proportion of 0.625, where M/l-p/100) =
0.833(1-25/100). A 50% reduction in mean proportion of fertilized eggs,
compared to the controls, would result in a mean proportion of 0.417.
Examining the means and their associated concentrations (Table 12), the
response, 0.625, is bracketed by C3 = 5.0 ^g/L copper and C4 = 10.0 fj.g/1
copper. The response, 0.417, is bracketed by C4 = 10.0 /j.g/1 copper and C5 =
20.0 /ig/L copper.
330
-------
TABLE 12. SEA URCHIN, ARBACIA PUNCTULAJA, MEAN PROPORTION
OF FERTILIZED EGGS
Copper
Cone.
(M9/L)
Control
2.5
5.0
10.0
20.0
40.0
i
1
2
3
4
5
6
Response_
Means, Y^
(proportion)
0.833
0.723
0.717
0.600
0.397
0.227
Smoothed
Means, Mn-
(proportion)
0.833
0.723
0.717
0.600
0.397
0.227
i
15.13.2.8.3 Using the equation from Section 4.2 in Appendix L, the estimate
of the IC25 is calculated as follows:
IC25 = 5.0 + [0.833(1 - 25/100) - 0.717] (10.0 - 5.0)
= 8.9
1(0.600 1- 0.717)
15.13.2.8.4 Using the equation from Section 4.2 in Appendix L, the estimate
of the IC50 is calculated as follows:
IC50 = 10.0 + [0.833(1 - 50/100) - 0.600] (20.0 - 10.0)
19.0
(0.397 - 0.600)
15.13.2.8.5 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 8.9286 fj.g/1. The
empirical 95.0% confidence interval for the true mean was 3.3036 /^g/L to
14.6025 Mg/L. The computer program output for the IC25 for this data set is
shown in Figure 6.
,
15.13.2.8.6 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC50 was 19.0418 /^g/L.
i
331
-------
Cone. ID
Cone. Tested
Response
Response
Response
1
2
3
1
0
.85
.78
.87
2
2.5
.81
.65
.71
3
5.0
.63
.74
.78
4
10.0
.63
.66
.51
5
20.0
.41
.41
.37
6
40.0
.12
.3
.26
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Copper
Test Start Date: Test Ending Date:
Test Species: SEA URCHIN, Arbacia punctulata
Test Duration:
DATA FILE: urchin.icp
OUTPUT FILE: urchin.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
ug/1
0.000
2.500
5.000
10.000
20.000
40.000
Response
Means
0.833
0.723
0.717
0.600
0.397
0.227
Std. Pooled
Dev. Response Means
0.047
0.081
0.078
0.079
0.023
0.095
0.833
0.723
0.717
0.600
0.397
0.227
The Linear Interpolation Estimate: 8.9286 Entered P Value: 25
— .•••,... — — — — ___ — ____________________ __„_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .. _ _.
Number of Resamplings: 80
The Bootstrap Estimates Mean: 8.7092 Standard Deviation: 1.5324
Original Confidence Limits: Lower: 6.2500 Upper: 11.6304
Expanded Confidence Limits: Lower: 3.3036 Upper: 14.6025
Resampling time in Seconds: 1.59 Random Seed: 1834854321
Figure 6. ICPIN program output for the IC25.
332
-------
The empirical 95.0% confidence interval for the true mean was; 16.1083 ng/l to
23.6429 M9/L- The computer program output for the IC50 for this data set is
shown in Figure 7.
15.14 PRECISION AND ACCURACY
15.14.1 PRECISION I
15.14.1.1 Single-Laboratory Precision
15 14.1.1.1 Single-laboratory precision data for the reference toxicants,
copper (Cu) and sodium dodecyl sulfate (SDS), tested in FORTY FATHOMS®
artificial seawater, GP2 artificial seawater, and natural seawater are
provided in Tables 13-18. The test results were similar in the three types of
seawater. The IC25 and IC50 for the reference toxicants (copper and sodium
dodecyl sulfate) are reported in Tables 13-16. The coefficient of variation,
based on the IC25, is 28.7% to 54.6% for natural and FORTY FATHOMS® seawater,
indicating acceptable precision. The IC50 ranges from 23:3% to 48.2%, showing
acceptable precision.
15.14.1.2 Multilaboratory Precision
15.14.1.2.1 No data are available on the multilaboratory precision of the
test.
15.14.2 ACCURACY
15.14.2.1 The accuracy of toxicity tests cannot be determined.
333
-------
Cone. ID
Cone. Tested
Response
Response
Response
1
2
3
1
0
.85
.78
.87
2
2.5
.81
.65
.71
3
5.0
.63
.74
.78
4
10.0
.63
.66
.51
5
20.0
.41
.41
.37
6
40.0
.12
.3
.26
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Copper
Test Start Date: Test Ending Date:
Test Species: SEA URCHIN, Arbacia punctulata
Test Duration:
DATA FILE: urchin.icp
OUTPUT FILE: urchin.i50
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
ug/1
0.000
2.500
5.000
10.000
20.000
40.000
Response
Means
0.833
0.723
0.717
0.600
0.397
0.227
Std. Pooled
Dev. Response Means
0.047
0.081
0.078
0.079
0.023
0.095
0.833
0.723
0.717
0.600
0.397
0.227
The Linear Interpolation Estimate: 19.0164 Entered P Value: 50
------------- .
Number of Resamplings: 80
The Bootstrap Estimates Mean: 19.0013 Standard Deviation: 0.8973
Original Confidence Limits: Lower: 17.6316 Upper: 21.2195
Expanded Confidence Limits: Lower: 16.1083 Upper: 23.6429
Resampling time in Seconds: 1.65 Random Seed: -823775279
Figure 7. ICPIN program output for the IC50.
334
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TABLE 13. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
PUNCTULATA, FERTILIZATION TEST PERFORMED IN FORTY FATHOMS®
ARTIFICIAL SEAWATER, USING GAMETES FROM ADULTS MAINTAINED IN
FORTY FATHOMS® ARTIFICIAL SEAWATER, OR OBTAINED DIRECTLY FROM
NATURAL SOURCES, AND COPPER (CU) SULFATE AS A REFERENCE
TOXICANT1'2'3'4'5
Test
Number
LOEC
IC25
(M9/L)
IC50
(M9/L)
1 5.0 8.92
2 12.5 26.35
3 < 6.2 11.30
4 6.2 34.28
5 12.5 36.67
n: 4 5
Mean: NA 23.51
CV(%): NA • 54.60
1
2
2
3
4
5
Data from USEPA (1991a)
Tests performed by Dennis McMullen, Technology Applic
EMSL, Cincinnati, OH.
All tests were performed using FORTY FATHOMS® synthet
29.07
38.96
23.93
61.75
75.14
5
45.77
47.87
atiions, Inc.,
ic seawater.
Copper test solutions were prepared with copper sulfcite» Copper
concentrations in Test 1 were: 2.5, 5.0, 10.0, 20.0,
Copper concentrations in Tests 2-5 were: 6.25, 12.5,
100.0 fj,g/l.
and 40.0 jug/L.
25,0, 50.0, and
NOEC Range: < 5.0 - 12.5 nq/l (this represents a difference of one
exposure concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
335
-------
TABLE 14. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
PUNCTULATA, FERTILIZATION TEST PERFORMED IN FORTY FATHOMS®
ARTIFICIAL SEAWATER, USING GAMETES FROM ADULTS MAINTAINED IN
FORTY FATHOMS® ARTIFICIAL SEAWATER, OR OBTAINED DIRECTLY FROM
NATURAL SOURCES, AND SODIUM DODECYL SULFATE (SDS) AS A
REFERENCE TOXICANT1'2'3'4'5-6
Test
Number
NOEC
(mg/L)
IC25
(mg/L)
IC50
(mg/L)
1
2
3
4
5
0.9
0.9
1.8
0.9
1.8
1.11
1.27
2.26
1.90
2.11
1.76
1.79
2.87
2.69
2.78
n:
Mean:
CV(%):
4
NA
NA
5
1.73
29.7
5
2.38
23.3
Data from USEPA ,(1991a)
Tests performed by Dennis M. McMullen, Technology Applications, Inc.,
EMSL, Cincinnati, OH.
All tests were performed using FORTY FATHOMS® synthetic seawater.
NOEC Range: 1.2 -3.3 mg/L (this represents a difference of one exposure
5
6
0.9, 1.8, 3.6, 7.2, and
concentration).
SDS concentrations for all tests were:
14.4 mg/L.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance. '
336
-------
TABLE 15. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
PUNCTULATA, FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER,
USING GAMETES FROM ADULTS MAINTAINED IN NATURAL SEAWATER AND
COPPER (CU) SULFATE AS A REFERENCE TOXICANT 1'^'*':3'6
Test
Number
NOEC
IC25
IC50
(M9/L)
1
2
3
4
5
12.2
12.2
24.4
< 6.1
6.1
14.2
32.4
30.3
26.2
11.2
18.4
50.8
46.
34.
.3
,1
17.2
n:
Mean :
CV(%) :
4
NA
NA
5
22.8
41.9 !
5
29.9
48.2
1
2
3
4
5
6
Data from USEPA (1991a)
Tests performed by Ray Walsh and Wendy Greene, ERL-N, USEPA,
Narragansett, RI.
Copper concentrations were: 6.1, 12.2, 24.4, 48.7, and 97.4 /^g/L.
NOEC Range: < 6.1 - 24.4 /^g/L (this represents a difference of two
exposure concentrations).
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
337
-------
TABLE 16. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
PUNCTULATA, FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER,
USING GAMETES FROM ADULTS MAINTAINED IN NATURAL SEAWATER AND
SODIUM DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT ^•3>^-6
1
2
3
A
5
6
Test NOEC
Number (mg/L)
1 1.8
2 1.8
3 1.8
4 0.9
5 1.8
n: 5
Mean : NA
CV(%): NA
Data from USEPA (1991a).
Tests performed by Ray Walsh and
Narragansett, RI.
SDS concentrations were: 0.9, 1
NOEC Range: 0.9 - 1.8 mg/L (this
exposure concentration).
Adults collected in the field.
For a discussion of the precision
see Section 4, Quality Assurance.
IC25
(mg/L)
2.3
3.9
2.3
2.1
2.3
5
2.58
28.7
IC50
(mg/L)
2.7
5.1
2.9
2.6
2.7
5
3.2
33.3
Wendy Greene, ERL-N, USEPA,
.8, 3.6, 7.3, and 14.5 mg/L.
represents a difference of one
of data from chronic toxicity tests
338
-------
TABLE 17. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
PUNCTULATA, FERTILIZATION TEST PERFORMED IN GP2, USING GAMETES
FROM ADULTS MAINTAINED IN GP2 ARTIFICIAL SEAWATER AND
COPPER (CU) SULFATE AND SODIUM DODECYL SULFATE (SDS) AS
REFERENCE TOXICANTS1'2'3'4'5
Test
1
2
3
4
5
Mean
SD
CV
LC50
29.1
47.6
32.7
78.4
45.6
46.7
19.5
41.8
27
44
29
73
41
Cu
CI
.3-31
.6-50
.8-35
.3-83
.0-50
luQ/L) SDS (ma/L)
.1
.8
.8
.9
.7
NOEC
6.3
25.0
6.3
50.0
12.5
LOEC
12.5
50.0
12.5
100.0
25.0
LC50
2
1
2
2
1
2
0
10
.1
.8
.2
.3
.8
.0
.2
.0
CI
2.0-2.1
1.8-1.9
2.1-2.2
2.2-2.4
1.7-2.8
'
NOEC
1.3
1.3
1.3
1.3
1.3
LOEC
2.5
2.5
2.5
2.5
2.5
Tests performed by Pamela Corneleo, Science Application International
Corp., ERL-N, USEPA, Narragansett, RI.
All tests were performed using GP2 artificial seawater.
Copper concentrations were: 6.25, 12,5, 25.0, 50.0 and 100 ng/L.
SDS concentrations were: 0.6, 1.25, 2.5, 5.0, and 10.0 mg/L. SDS stock
(14.645 mg/mL) provided by EMSL, USEPA, Cincinnati, OH.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance. ,
339
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TABLE 18. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
PUNCTULATA, FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER,
USING GAMETES FROM ADULTS MAINTAINED IN NATURAL SEAWATER AND
COPPER (CU) SULFATE.ANP SODIUM DODECYL SULFATE (SDS) AS
REFERENCE TOXICANTS1'2'"'*
Cu (ua/l)
Test
1
2
3
4
5
LC50
28.6
13.0
67.8
36.7
35.6
CI
26.7-30.6
11.9-14.2
63.2-72.6
33.9-39.8
33.6-37.7
NOEC
6.3
6.3
6.3
< 6.3
< 6.3
LOEC
12.5
12.5
12.5
6.3
6.3
LC50
2.2
1.9
2.2
3.3
2.9
SDS
CI
2.1-2.2
1.9-2.0
2.1-2.3
3.1-3.4
2.8-3.1
(ma/L)
NOEC
1.3
1.3
1.3
< 0.6
< 0.6
LOEC
2.5
2.5
2.5
0.6
0.6
Mean
SD
CV
36.3
20.0
55.1
2.5
0.58
23.2
1 Tests performed by Anne Kuhn-Hines, Catherine Sheehan, Glen Modica, and
Pamela Comeleo, Science Application International Corp., ERL-N, USEPA,
Narragansett, RI.
Copper concentrations were prepared with copper sulfate. Concentrations
were 6.25, 12.5, 25.0, 50.0, and 100 /*g/L.
SDS concentrations were: 0.6, 1.25, 2.5, 5.0, and 10.0 mg/L. SDS stock
(14.64 mg/mL) provided by EMSL, USEPA, Cincinnati, OH.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
340
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1
SECTION 16
TEST METHOD
RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUC1
METHOD 1009.0
ION TEST
16.1 SCOPE AND APPLICATION
16.1.1 This method, adapted in part from USEPA (1987f) measures the effects
of toxic substances in effluents and receiving water on the sexual
reproduction of the marine red macroalga, Champia parvula. The method
consists of exposing male and female plants to test substances for two days,
followed by a 5-7 day recovery period in control medium, during which the
cystocarps mature.
16.1.2 Detection limits of the toxicity of an effluent or chemical substance
are organism dependent.
16.1.3 Brief excursions in toxicity may not be detected us;ing 24-h composite
samples. Also, because of the long sample collection period involved in
composite sampling, highly volatile and highly degradable toxicants present in
the source may not be detected in the test.
16.1.4 This test is commonly used in one of two forms: (1) a definitive test,
consisting of a minimum of five effluent concentrations and a control, and (2)
a receiving water test(s), consisting of one or more receiving water
concentrations and a control.
16.2 SUMMARY OF METHOD
16.2.1 Sexually mature male and female branches of the red macroalga, Champia
parvula, are exposed in a static system for 2 d to different concentrations of
effluent, or to receiving water, followed by a 5 to 7 day recovery period in
control medium. The recovery period allows time for the development of
cystocarps resulting from fertilization during the exposure; period. The test
results are reported as the concentration of the test substance which causes a
statistically significant reduction in the number of cystocarps formed.
16.3 INTERFERENCES
16.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
I
16.3.2 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
16.3.3 Adverse effects of high concentrations of suspended and/or dissolved
solids, and extremes of pH, may mask the presence of toxic substances.
341
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16.3.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
16.4 SAFETY
16.4.1 See Section 3, Safety and Health.
16.5 APPARATUS AND EQUIPMENT
16.5.1 Facilities for holding and acclimating test organisms.
16.5.2 Laboratory red macroalga, Champia parvula, culture unit -- see
culturing methods below. To test effluent or receiving water toxicity,
sufficient numbers of sexually mature male and female plants must be
available.
16.5.3 Samplers -- automatic samplers, preferably with sample cooling
capability, that can collect a 24-h composite sample of 1 L.
16.5.4 Environmental chamber or equivalent facility with temperature control
(23 ± PC).
16.5.5 Water purification system -- Mi 11ipore Milli-Q®, deionized water (DI)
or equivalent.
16.5.6 Air pump -- for oil-free air supply.
16.5.7 Air lines, and air stones -- for aerating cultures.
16.5.8 Balance -- Analytical, capable of accurately weighing to 0.00001 g.
16.5.9 Reference weights, Class S -- for checking performance of balance.
16.5.10 Meter, pH -- for routine physical and chemical measurements.
16.5.11 Dissecting (stereoscope) microscope -- for counting cystocarps.
16.5.12 Compound microscope -- for examining the condition of plants.
16.5.13 Count register, 2-place -- for recording cystocarp counts.
16.5.14 Rotary shaker -- for incubating exposure chambers (hand-swirling
twice a day can be substituted).
16.5.15 Drying oven --to dry glassware.
16.5.16 Filtering apparatus -- for use with membrane filters (47 mm).
16.5.17 Refractometer -- for determining salinity.
16.5.18 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.
342
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16.5.19 Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.
16.5.20 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
16.5.21 Beakers -- Class A, borosilicate glass or non-toxic plasticware, 1000
ml for making test solutions. ;
i
16.5.22 Erlenmeyer flasks, 250 ml, or 200 ml disposable polystyrene cups,
with covers -- for use as exposure chambers.
16.5.23 Bottles -- borosilicate glass or disposable polystyrene cups (200-400
ml) for use as recovery vessels. |
16.5.24 Wash bottles -- for deionized water, for rinsing small glassware and
instrument electrodes and probes.
16.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
16.5.26 Micropipettors, digital, 200 and 1000 nl -- to make dilutions.
16.5.27 Pipets, volumetric -- Class A, 1-100 ml.
16.5.28 Pipettor, automatic -- adjustable, 1-100 ml.
16.5.29 Pipets, serological -- 1-10 mL, graduated. |
i
16.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent.
I
16.5.31 Forceps, fine-point, stainless steel -- for cutting and handling
branch tips.
16.6 REAGENTS AND CONSUMABLE MATERIALS
16.6.1 Mature red macroalga, Champia parvuTa, plants -- see Subsection
16.6.14 below.
16.6.2 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
16.6.3 Petri dishes, polystyrene -- to hold plants for cystocarp counts and
to cut branch tips. Other suitable containers may be used.
.
16.6.4 Disposable tips for micropipettors. |
16.6.5 Aluminum foil, foam stoppers, or other closures -|- to cover culture
and test flasks.
I
343
-------
16.6.6 Tape, colored -- for labelling test chambers.
16.6.7 Markers, waterproof -- for marking containers, etc.
16.6.8 Data sheets (one set per test) -- for data recording.
16.6.9 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method 150.1,
USEPA, 1979b).
16.6.10 Laboratory quality assurance samples and standards for the above
methods.
16.6.11 Reference toxicant solutions see Section 4, Quality Assurance.
16.6.12 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
16.6.13 Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water; and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
16.6.13.1 Saline test and dilution water -- the use of natural seawater is
recommended for this test. A recipe for the nutrients that must be added to
the natural seawater is given in Table 1. The salinity of the test water must
be 30%p, and vary no more than ± 2%o among the replicates. If effluent and
receiving water tests are conducted concurrently, the salinity of these tests
should be similar.
16.6.13.2 The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts. Therefore, exposure of the red macroalga, Champia parvula,
to effluents will usually require adjustments in the salinity of the test
solutions. Although the red macroalga, Champia parvuTa, cannot be cultured in
100% artificial seawater, 100% artificial seawater can be used during the two
day exposure period. This allows 100% effluent to be tested. It is important
to maintain a constant salinity across all treatments. The salinity of the
effluent can be adjusted by adding hypersaline brine (MSB) prepared from
natural seawater (100%o), concentrated (triple strength) salt solution (GP2
described in Table 2), or dry GP2 salts (Table 2), to the effluent to provide
a salinity of 30%o. Control solutions should be prepared with the same
percentage of. natural seawater and at the same salinity (using deionized water
adjusted with dry salts, or brine) as used for the effluent dilutions.
16.6.13.3 Artificial seawater -- A slightly modified version of the GP2
medium (Spotte, et al, 1984) has been used successfully to perform the red
macroalga sexual reproduction test. The preparation of artificial seawater
(GP2) is described in Table 2.
344
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TABLE 1. NUTRIENTS TO BE ADDED TO NATURAL SEAWATER AND TO ARTIFICIAL
SEAWATER (GP2) DESCRIBED IN TABLE 2. THE CONCENTRATED
NUTRIENT STOCK SOLUTION IS AUTOCLAVED FOR 15 MIN (VITAMINS ARE
AUTOCLAVED SEPARATELY FOR 2 MIN AND ADDED AFTER THE NUTRIENT
STOCK SOLUTION IS AUTOCLAVED). THE pH OF THE SOLUTION IS
ADJUSTED TO APPROXIMATELY pH 2 BEFORE AUTOCLAVIN6 TO MINIMIZE
THE POSSIBILITY OF PRECIPITATION
Amount of Reagent Per Liter of Concentrated
Nutrient Stock Solution
Stock Solution For Stock Solution For
Culture Medium Test Medium
Nutrient Stock Solution1
i
NaN03 6.35 g 1.58 g
NaH2P04 . H20 0.64 g 0.16 g
Na2EDTA . 2 H20 133 mg
Na3C6H507 . 2 H20 51 mg 12.8 mg
Iron2 9.75 mL 2.4 mL
Vitamins3 10 mL ' 2.5 mL
Add 10 mL of appropriate nutrient stock solution per liter of culture
or test medium.
A stock solution of iron is made that contains 1 mg iiron/mL. Ferrous
or ferric chloride can be used.
A vitamin stock solution is made by dissolving 4.88 g thiamine HC1,
2.5 mg biotin, and 2.5 mg B12 in 500 mL deionized water. Adjust to
approximately pH 4 before autoclaving 2 min. It is convenient to
subdivide the vitamin stock into 10 mL volumes in test tubes prior to
autoclaving.
345
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TABLE 2. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
ARTIFICIAL SEAWATER FOR USE IN CONJUNCTION WITH NATURAL
SEAWATER FOR THE RED MACROALGA, CHAMPIA PARVULA, CULTURING AND
TOXICITY TESTING1'2'3'4'5'6'^
Compound
Concentration
(9/L)
Amount (g)
Required for
20 L
1
2
3
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 ' 6 H20
CaCl2 ' 2 H20
SrCl2 • 6 H20
NaHC03
Modified GP2 from Spotte
The constituent salts and
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
et al. (1984).
concentrations were taken
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
from USEPA (19
salts separately to avoid precipitation. However, if the sodium
bicarbonate is autoclaved separately (dry), all of the other salts can
be autoclaved together. Since no nutrients are added until needed,
autoclaving is not critical for effluent testing. To minimize
microalgal contamination, the artificial seawater should be autoclaved
when used for stock cultures. Autoclaving (120°C) should be for a
least 10 min for 1-L volumes, and 20 min for 10-to-20 L volumes.
Prepare in 10-L to 20-L batches.
A stock solution of 68 mg/mL sodium bicarbonate is prepared by
autoclaving it as a dry powder, and then dissolving it in sterile
deionized water. For each liter of GP2, use 2.5 mL of this stock
solution.
Effluent salinity adjustment to 30%o can be made by adding the
appropriate amount of dry salts from this formulation, by using a
triple-strength brine prepared from this formulation, or by using a
100%o salinity brine prepared from natural seawater.
Nutrients listed in Table 1 should be added to the artificial seawater
in the same concentration described for natural seawater.
346
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16.6.14 TEST ORGANISMS RED MACROALGA, CHAHPIA PARVULA
16.6.14.1 Cultures
16.6.14.1.1 Mature plants are illustrated in Figure 1. The adult plant body
(thai 1 us) is hollow, septate, and highly branched. New cultures can be
propagated asexually from excised branches, making it possible to maintain
clonal material indefinitely.
tetrasporangia ——
spermatia
fertilization
TETRASPOROPHYTE
— cystocarp
5mm
Figure 1. Life history of the red macroalga, Champia parvula. Upper left:
Size and degree of branching in female branch tips used for toxicity
tests. From USEPA (1987f).
16.6.14.1.2 Unialgal stock cultures of both males and females are maintained
in separate, aerated 1000 ml Erlenmeyer flasks containing 800 ml of the
culture medium. All culture glass must be acid-stripped in 15% HC1 and rinsed
in deionized water after washing. This is necessary since some detergents can
leave a residue that is toxic to the red macroalga, Champia parvula.
Periodically (at least every 6 months) culture glassware should be baked in a
muffle furnace to remove organic material that may build up on its surface.
Alternately, a few ml of concentrated sulfuric acid can be rolled around the
inside of wet glassware. Caution: the addition of acid to the wet glassware
generates heat.
i
347
-------
16.6.14.1.3 The culture medium is made from natural seawater to which
additional nutrients are added. The nutrients added are listed in Table 1.
Almost any nutrient recipe can be used for the red macroalga, Champia parvula,
cultured in either natural seawater or a 50-50 mixture of natural and
artificial seawaters. Healthy, actively growing plants are the goal, not a
standard nutrient recipe for cultures.
16.6.14.1.4 Several cultures of both males and females should be maintained
simultaneously to keep a constant supply of plant material available. To
maintain vigorous growth, initial stock cultures should be started
periodically with about twenty 0.5 to 1.0 cm branch tips. Cultures are gently
aerated through sterile, cotton-plugged, disposable, polystyrene 1 ml
pipettes. Cultures are capped with foam plugs and aluminum foil and
illuminated with ca. 75 pE/m/s (500 ft-c) of cool-white fluorescent light on
a 16:8 light:dark cycle. Depending on the type of culture chamber or room
used, i.e., the degree of reflected light, the light levels may have to be
adjusted downward. The temperature is 22 to 24°C and the salinity 28-30%o.
Media are changed once a week.
16.6.14.1.5 Prior to use in toxicity tests, stock cultures should be examined
to determine their condition. Females can be checked by examining a few
branch tips under a compound microscope (100 X or greater). Several
trichogynes (reproductive hairs to which the spermatia attach) should be
easily seen near the apex (Figure 2).
16.6.14.1.6 Male plants should be visibly producing spermatia. This can be
checked by placing some male tissue in a petri dish, holding it against a dark
background and looking for the presence of spermatial sori. Mature sori can
also be easily identified by looking along the edge of the thallus under a
compound microscope (Figures 3 and 4).
16.6.14.1.7 A final, quick way to determine the relative "health" of the male
stock culture is to place a portion of a female plant into some of the water
from the male culture for a few seconds. Under a compound microscope numerous
spermatia should be seen attached to both the sterile hairs and the
trichogynes (Figure 5).
16.6.14.2 Culture medium prepared from natural seawater is preferred
(Table 1). However, as much as 50% of the natural seawater may be replaced by
the artificial seawater (GP2) described in Table 2.
16.6.14.2.1 Seawater for cultures is filtered at least to 0.45 pm to remove
most particulates and then autoclaved for 30 minute at 15 psi (120°C). Carbon
stripping the seawater may be necessary before autoclaving to enhance its
water quality (USEPA, 1990b). This is done by adding 2 g activated carbon per
liter of seawater and stirring on a stir plate for 2 h. After stirring filter
through a Whatman number 2 filter, then through a 0.45 membrane filter. The
culture flasks are capped with aluminum foil and autoclaved dry, for 10
minute. Culture medium is made up by dispensing seawater into sterile flasks
and adding the appropriate nutrients from a sterile stock solution.
348
-------
sterile hairs
^Mrichogynes
1 mm
Figure 2. Apex of branch of female plant, showing sterile hairs and
reproductive hairs (trichogynes). Sterile hairs are wider and
generally much longer than trichogynes, and appear hollow except at
the tip. Both types of hairs occur on the entire circumference of
the thai!us, but are seen easiest at the '[edges." Receptive
trichogynes occur only near the branch tips. From USEPA (1987f).
1 cm
spemnatial sorus
Figure 3. A portion of the male thai!us showing spermatial sori. The sorus
areas are generally slightly thicker and somewhat lighter in color.
From USEPA (1987f).
349
-------
cuticle
100 urn
0 O
^spermatia
f\f
\
thallus surface
Figure 4. A magnified portion of a spermatial sorus. Note the rows of cells
that protrude from the thallus surface. From USEPA (1987f).
spermatia
Figure 5. Apex of a branch on a mature female plant that was exposed to
spermatia from a male plant. The sterile hairs and trichogynes are
covered with spermatia. Note that few or no spermatia are attached
to the older hairs (those more than 1 mm from the apex). From USEPA
(1987f).
16.6.14.2.2 Alternately, 1-L flasks containing seawater can be autoclaved.
Sterilization is used to prevent microalgal contamination, and not to keep
cultures bacteria free.
16.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
16.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
350
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16.8 CALIBRATION AND STANDARDIZATION
16.8.1 See Section 4, Quality Assurance.
16.9 QUALITY CONTROL
16.9.1 See Section 4, Quality Assurance.
16.10 TEST PROCEDURES
16.10.1 TEST SOLUTIONS
16.10.1.1 Receiving Waters '
16.10.1.1.1 The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 urn NITEX® filter and compared without
dilution, against a control. Using four replicate chambers per test, each
containing 100 ml, and 400 mL for chemical analysis, would require
approximately 800 mL or more of sample per test.
16.10.1.2 Effluents [
16.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used. A
dilution factor of 0.5 provides precision of ± 100%, and allows for testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used. Therefore, USEPA recommends the
use of the > 0.5 dilution factor. |
16.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).
16.10.1.2.3 The volume of effluent required for the test using a 0.5 dilution
series is approximately 1800 mL. Prepare enough test solution at each
effluent concentration (approximately 800 mL) to provide 100 mL of test
solution for each of four (minimum of three) replicate test chambers and
400 mL for chemical analyses and record data (Figure 6).
16.10.1.2.4 Effluents can be tested at 100%. A 100% concentration of
effluent can be achieved if the salinity of the effluent is adjusted to 30%o
by adding the GP2 dry salt formulation described in Table 2.
i
16.10.1.2.5 Just prior to test initiation (approximately 1 h), the
temperature of sufficient quantity of the sample to make the test solutions
should be adjusted to the test temperature (25 ± 1°C) and maintained at the
temperature during the addition of dilution water.
351
-------
SITE.
COLLECTION DATE
TEST DATE
LOCATION
INITIAL
SALINITY
FINAL
SALINITY
SOURCE OF SALTS FOR1
SALINITY ADJUSTMENT
1Natural seawater, GP2 brine, GP2 salts, etc. (include some indication of amount)
COMMENTS:
Figure 6. Data form for the red macroalga, Champia parvula, sexual
reproduction test. Receiving water summary sheet. From USEPA
(1987f).
352
-------
16.10.1.2.6 Effluent dilutions should be prepared for all replicated in each
treatment in one beaker to minimize variability among the replicates. The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.
16.10.1.3 Dilution Water
16.10.1.3.1. The formula for the enrichment for natural seawater is listed in
Table 1. Both EDTA and trace metals have been omitted. This formula should
be used for the 2-day exposure period, but it is not critical for the recovery
period. Since natural seawater quality can vary among laboratories, a more
complete nutrient medium (e.g., the addition of EDTA) may result in faster
growth (and therefore faster cystocarp development) during the recovery
period.
16.10.2 PREPARATION OF PLANTS FOR TEST
I
16.10.2.1 Once cultures are determined to be usable for toxicity testing
(have trichogynes and sori with spermatia), plant cuttings should be prepared
for the test, using fine-point forceps, with the plants in a little seawater
in a petri dish. For female plants, five cuttings, severed 7-10 mm from the
ends of the branch, should be prepared for each treatment chamber. Try to be
consistent in the number of branch tips on each cutting. For male plants, one
cutting, severed 2.0 to 3.0 cm from the end of the branch, is prepared for
each test chamber. Prepare the female cuttings first, to minimize the chances
of contaminating them with water containing spermatia from the male stock
cultures.
16.10.3. START OF TEST
16.10.3.1 Tests should begin as soon as possible after sample collection,
preferably within 24 h. The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
tests unless permission is granted by the permitting authority. In no case
should the sample be used in a test more than 72 h after sample collection
(see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and
Sample Preparation for Toxicity Test, Subsection 8.5.4). i
16.10.3.2 Just prior to test initiation (approximately 1 h), the temperature
of a sufficient quantity of the sample.to make the test solution should be
adjusted to the test temperature (23 ± 1°C) and maintained at that temperature
during the addition of dilution water.
i
16.10.3.3 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 controls) should have four (minimum of three) replicates.
16.10.3.4 Randomize the position of test chambers at the beginning of the
test.
16.10.3.5 Prepare test solutions and add to the test chambers.
353 i
-------
16.10.3.6 Add five female branches and one male branch to each test chamber.
The toxicant must be present before the male plant is added. I
16.10.3.7 Gently hand swirl the chambers twice a day, or shake continuously
at 100 rpm on a rotary shaker.
16.10.3.8 If desired, the media can be changed after 24 h.
16.10.4 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE
16.10.4.1 The light quality and intensity should be at 75 ^l/m2/s, or 500
foot candles (ft-c) with a photoperiod of 16 h light and 8 h darkness. The
water temperature in the test chambers should be maintained at 23 ± 1°C. The
test salinity should be in the range of 28 to 32%o. The salinity should vary
by no more than ± 2%o among the chambers on a given day. If effluent and
receiving water tests are conducted concurrently, the salinities of these
tests should be similar.
16.10.5 DISSOLVED OXYGEN (DO) CONCENTRATION
16 10.5.1 Aeration may affect the toxicity of effluent and should be used
only as a last resort to maintain a satisfactory DO. The DO concentrations
should be measured on new solutions at the start of the test (Day 0) and
should be measured before renewal of the test solution after 24 h. The DO
should not fall below 4.0 mg/L (see Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample Preparation for Toxicity Tests) If it
is necessary to aerate, all treatments and the control should be aerated. I he
aeration rate should not exceed 100 bubbles/minute, using a pi pet with a
1-2 mm orifice, such as a ImL KIMAX® serological pipet No. 37033, or
equivalent. Care should be taken to ensure that turbulence resulting from the
aeration does not occur.
16.10.6 OBSERVATIONS DURING THE TEST
16.10.6.1 Routine Chemical and Physical Observations
16.10.6.1.1 DO is measured at the beginning and end of each 24-h exposure
period in one test chamber at each concentration and in the control.
16.10.6.1.2 Temperature, pH, and salinity are measured at the end of each
24-h exposure period in one test chamber at each concentration and in the
control. Temperature should also be monitored continuously, observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
chambers at least at the end of the test to determine temperature variation in
environmental chamber.
16.10.6.1.3 The pH is measured in the effluent sample each day before new
test solutions are made.
16.10.6.1.4 Record all the measurements on the data sheet.
354
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16.10.6.2 Routine Biological Observations
16.10.6.2.1 Protect the red macroalga from unnecessary disturbance during the
test by carrying out the daily test observations and solution renewals
carefully.
I
i
16.10.7 TRANSFER OF PLANTS TO CONTROL WATER AFTER 48 H
16.10.7.1 Label the recovery vessels. These vessels can t>e almost any type
of container or flask containing 100 to 200 mL of seawater and nutrients (see
Tables 1 and 2). Smaller volumes can be used, but should be checked to make
sure that adequate growth will occur without having to change the medium.
16.10.7.2 With forceps, gently remove the female branches from test chambers
and place into recovery bottles. Add aeration tubes and foam stoppers.
16.10.7.3 Place the vessels under cool white light (at the same irradiance as
the stock cultures) and aerate for the 5-7 day recovery period. If a shaker
is used, do not aerate the solutions (this will enhance the water motion).
16.10.8 TERMINATION OF THE TEST
16.10.8.1 At the end of the recovery period, count the number of cystocarps
(Figures 7, 8, and 9) per female and record the data (Figure 10). Cystocarps
may be counted by placing females between the inverted halves of a polystyrene
petri dish or other suitable containers with a small amount of seawater (to
hold the entire plant in one focal plane). Cystocarps can be easily counted
under a stereomicroscope, and are distinguished from young branches because
they possess an apical opening for spore release (ostiole) and darkly
pigmented spores.
1 mm
Figure 7. A mature cystocarp. In the controls and lower effluent
concentrations, cystocarps often occur in clusters of 10 or 12.
From USEPA (1987f).
16.10.8.2 One advantage of this test procedure is that if there is
uncertainty about the identification of an immature cystocarp, it is necessary
only to aerate the plants a little longer in the recovery bottles. Within 24
355
-------
young branch
.cells
immature
cystocarp
1 mm
Figure 8. Comparison of a very young branch and an immature cystocarp. Both
can have sterile hairs. Trichogynes might or might not be present
, on a young branch, but are never present on an immature cystocarp.
Young branches are more pointed at the apex and are made up of
larger cells than immature cystocarps, and never have ostioles.
From USEPA (1987f).
Figure 9. An aborted cystocarp. A new branch will eventually develop at the
apex. From USEPA (1987f).
to 48 h, the presumed cystocarp will either look more like a mature cystocarp
or a young branch, or will have changed very little, if at all (i.e., an
aborted cystocarp). No new cystocarps will form since the males have been
removed, and the plants will only get larger. Occasionally, cystocarps will
abort, and these should not be included in the counts. Aborted cystocarps are
easily identified by their dark pigmentation and, often, by the formation of a
new branch at the apex.
16.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
16.11.1 A summary of test conditions and test acceptability criteria is
listed in Table 3.
356
-------
COLLECTION DATE
EXPOSURE BEGAN (date).
EFFLUENT OR TOXICANT
.RECOVERY BEGAN (date).
COUNTED (date)
TREATMENT (% EFFLUENT, /iG/L, or RECEIVING WATER SITES)
1 RFPI TCATFS
CONTROI
A 1
7
3
4
MFAN
R 1
3
4
MFAN
f. 1
2
3
4
MFAN
ft\/ F R A 1 1
MF AN
Temperature
Salinity
Liaht
Source of Dilution Water
Figure 10. Data form for the red macroalga, Champia parvula, sexual
reproduction test. Cystocarp data sheet. From USEPA (1987f)
357
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST
WITH EFFLUENTS AND RECEIVING WATERS
1. Test type:
2. Salinity:
3. Temperature:
4. Photoperiod:
5. Light intensity:
6. Light source:
7. Test chamber size:
8. Test solution volume:
9. No. organisms
per test chamber:
10. No. replicate chambers
per concentration:
11. No. organisms per
concentrations:
12. Dilution water:
13. Test concentrations:
14. Test dilution factor:
Static, non-renewal
30%o (± 2 of the selected test
salinity)
23 ± 1°C
16 h light, 8 h darkness
75 /*E/m2/s (500 ft-c)
Cool-white fluorescent lights
200 mL polystyrene cups, or 250 mL
Erlenmeyer flasks
100 mL (minimum)
5 female branch tips and 1 male plant
4 (minimum of 3)
24 (minimum of 18)
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)
Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water
or minimum of 5 and a control
Effluents: > 0.5
Receiving waters: None or > 0.5
358
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST
WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
15. Test duration:
16. Endpoints:
17. Test acceptability
criteria:
18. Sampling requirements:
19. Sample volume required:
2.day exposure to effluent, followed
by 5 to 7-day recovery period in
control medium for cyctocarp
development i
i
Reduction in cystocarp production
compared to controls
80% or greater survival, and an
average of 10 cystocarps per plant in
controls
One sample collected at test
initiation, and preferably used within
24 h of the time it is removed from
the sampling device (see Section 8,
Effluent and Receiving Water Sampling,
Sampling Handling, and Sample
Preparation for Toxicity Tests,
Subsection 8.5.4).
2 L per test
359
-------
16.12 ACCEPTABILITY OF TEST RESULTS
16.12.1 The test is acceptable if (1) control survival equals or exceeds 80%
and (2) control plants average 10 or more cystocarps per plant.
16.12.2 If plants fragment in the controls or lower exposure concentrations,
it may be an indication that they are under stress.
16.13 DATA ANALYSIS
16.13.1 GENERAL
16.13.1.1 Tabulate and summarize the data.
is listed in Table 4.
A sample set of reproduction data
16.13.1.2 The endpoints of the red macroalga, Champia parvula, toxicity test
are based on the adverse effects on sexual reproduction as the mean number of
cystocarps. The LC50, the IC25, and the IC50 are calculated using point
estimation techniques (see Section 9, Chronic Toxicity Test Endpoints and Data
Analysis). NOEC and LOEC values are obtained using a hypothesis testing
approach, such as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank
Test (Steel, 1959; Miller, 1981) (see Section 9). Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the IC25
and IC50. See the Appendices for examples of the manual computations, program
listing, and example of data input and program output.
16.13.1.3 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
16.13.2 EXAMPLE OF ANALYSIS OF THE RED MACROALGA, CHAMPIA PARVULA,
REPRODUCTION DATA
16.13.2.1 Formal statistical analysis of the data is outlined in Figure 11.
The response used in the analysis is the mean number of cystocarps per
replicate chamber. Separate analyses are performed for the estimation of the
NOEC and LOEC endpoints and for the estimation of the IC25 endpoint and the
IC50 endpoint. Concentrations that have exhibited no sexual reproduction
(less than 5% of controls) are excluded from the statistical analysis of the
NOEC and LOEC, but included in the estimation of the 1C endpoints.
16.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test. The assumptions of Dunnett's Procedures,
normality and homogeneity of variance are formally tested. The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance. Tests for normality and homogeneity of variance are
included in Appendix B. If either of these tests fails, the nonparametric
test, Steel's Many-one Rank Test is used to determine the NOEC and LOEC
endpoints. If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
360
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TABLE 4. DATA FROM THE RED MACROALGA, CHANPIA PARVULA, EFFLUENT
TOXICITY TEST. CYSTOCARP COUNTS FOR INDIVIDUAL PLANTS AND
MEAN COUNT PER TEST CHAMBER FOR EACH EFFLUENT CONCENTRATION1
Effluent
Concentration
(%)
Control
0.8
1.3
2.2
3.6
6.0
10.0
Replicate
Test
Chamber
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
1
19
19
17
10
12
12
10
6
4
1
7
3
2
3
0
1
1
0
0
1
2
2
20
12
25
16
10
9
0
4
4
2
9
2
1
4
4
0
2
4
0
0
1
Plant
3
24
21
18
11
6
9
3
4
2
5
9
2
1
6
3
0
1
3
0
0
0
4 5
7 18
11 23
20 16
12 11
9 10
13 8
5 4
8 4
6 4
4 0
4 6
0 0
5 0
4 2
1 3
0 0
0 0
1 3
0
0 0
0 0
Mean
Cystocarp
Count
17.60
17.20
19.20
12.00
9.40
10.20
4.40
5.20
4.00
2.40
7.00
1.40
1.80
3.80
2.20
0.20
0.80
2.20
0.00
0.20
0.60
Data provided by the ERL-N, USEPA, Narragansett, RI,
361
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STATISTICAL ANALYSIS OF CHAMPIA PARVULA
SEXUAL REPRODUCTION TEST
REPRODUCTION DATA
MEAN CYSTOCARP COUNT
POINT ESTIMATION
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILK-S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTS TEST
HETEROGENEOUS
VARIANCE
1
NO
r
EQUAL NUMBER OF
REPLICATES?
TWITH
•RRONI
TMENT
1 YES
DUNNETTS
TEST
i
YES
'
EQUAL NUMBER OF
REPLICATES?
1 N°
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 11. Flowchart for statistical analysis of the red macroalga, Champia
parvula, data.
362
-------
16.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested there are parametric and nonparametric alternative analyses
The parametric analysis is a t test with the Bonferroni adjustment
(Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is
the nonparametric alternative.
i
16.13.2.4 Example of Analysis of Reproduction Data
16.13.2.4.1 In this example, the data, mean and standard deviation of the
observatfons at each concentration including the control are listed in
Table 5. The data are plotted in Figure 12. As can be seen from the data in
the table, mean reproduction per chamber in the 10% effluent concentration is
less than 5% of the control. Therefore the 10% effluent concentration is not
included in the subsequent analysis. I
TABLE 5. RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION DATA
Effluent Concentration (%)
Replicate Control
0.8
1.3
2.2 3.6
6.0 10.0
A
B
C
Mean(Y,.)
S,-
i
17
17
19
18
1
1
.60
.20
.20
.00
12
12.
9.
10.
10.
1.
2
00
40
20
53
77
4.40
5.20
4.00
4.53
0.37
3
2.40
7.00
1.40
3.60
8.92
4
1
3
2
2
1
5
.80
.80
.20
.60
.12
0.20
0.80
2.20
1.07
1.05
6
0
0
0
0
0
7
.00
.20
.60
.27
.09
16.13.2.5 Test for Normality
16.13.2.5.1 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 6.
363
-------
S-'
O)
O-
co
Q-
S-
«
0
O
S-
U)
CD
O
51
O)
s-
3
O)
CM O
I— T- T-
SdyVOOlSAO dO 'ON
364
-------
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Ef f 1 uent
Replicate
A
B
C
Control
-0.40
-0.80
1.20
0.8
1.47
-1.13
-0.33
1.3
-0.13
0.67
-0.53
Concentration^)
2.2
-1.20
3.40
-2.20
3.6 1
-0.80
1.20
-0.40
6.0
-0.87
-0.27
1.13
16.13.2.5.2 Calculate the denominator, D, of the test statistic:
D = £ (X±-X)2
i=l
Where: X,- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations.
16.13.2.5.3 For this set of data, n = 18
X = J^(O.Ol) = 0.00
18 j
D = 28.7201 |
16.13.2.5.4 Order the centered observations from smallest to largest
X(1)
Where X
-------
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
i Xcn i X(0
1
2
3
4
5
6
7
8
9
-2.20
-1.20
-1.13
-0.87
-0.80
-0.80
-0.53
-0.40
-0.40
10
11
, 12
13
14
15
16
17
18
-0.33
-0.27
-0.13
0.67
1.13
1.20
1.20
1.47
3.40
16.13.2.5.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a.,, a,, ..., a^ where k is n/2 if n is even and (n-
l)/2 if n is odd. For the data in this example, n = 18 and k = 9. The a,-
values are listed in Table 8.
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
af Xn'l - Xci)
1
2
3
4
5
6
7
8
9
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
5.60
2.67
2.33
2.07
1.93
1.47
0.40
0.13
0.07
X(8)
x<7>
XC17)
XC15)
Y<14>
A
X(13)
X(12)
X(11)
X(10)
- x< >
- X?
- xc*>
- XC4>
- x<5)
- x(6>
- x(7>
- X(8)
- X(9)
16.13.2.5.6 Compute the test statistic, W, as follows:
W=
D
366
-------
The differences x(n"i+1) - X
-------
16.13.2.6.3 Bartlett's statistic is therefore:
B = [(12)ln(2.3917) -2 f In (Sf) ]/1.194
i=l
- [12(0.8720) - 2(ln(1.12)+ln(1.77)+...+ln(1.05))]/1.1944
= (10.4640 - 4.0809)/1.1944
= 5.34
16.13.2.6.4 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
five degrees of freedom, is 15.09. Since B = 5.34 is less than the critical
value of 15.09, conclude that the variances are not different.
16.13.2.7 Dunnett's Procedure
16.13.2.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 9.
TABLE 9. ANOVA TABLE
Source df Sum of Squares
(SS)
Between p - 1 SSB
Within N - p SSW
Mean Square(MS)
(SS/df)
SB = SSB/(p - 1)
$1 = SSW/(N - p)
Total N - 1 SST
Where: p = number effluent concentrations including the control
N = total number of observations n1 + n2 ... + np
n- - number of observations in concentration i
368
-------
SSB = &Tl/ni-G2/N Between Sum of Squares
= Y2.-G2/N Total Sum of Squares
SST = 2, 2,
.j
SSW = SST-SSB
Within Sum of Squares
G =
the grand total of all sample observations, G = S 2^
i
T- = the total of the replicate measurements for concentration i
the jth observation for concentration i (represents the
mean (across plants) number of cystocarps for effluent
concentration i in test chamber j)
16.13.2.7.2 For the data in this example:
n« = n, = n,
n5 = n6 =3
N
T!
= 18
- Y
+ Y12 + Y13
+ Y22 + '23
: ?» : >
+ Y52 +
?3 =
*6S
G =
SSB =
17.6 + 17.2 + 19.2
12.0 + 9.4 + 10.2
4.4 + 5.2 + 4.0
2.4 + 7.0 + 1.4
1.8 + 3.8 + 2.2
0.2 + 0.8 + 2.2
T5 + T6 = 121.0
1 (4287.24) - (121.O)2 = 615.69
T" 18
54
31.6
13.6
10.8
7.8
3.2
SST =
= 1457.8 - (121. O)2 = 644.41
18
SSW = SST-SSB
= 644.41 - 615.69 = 28.72
369
-------
SB = SSB/(p-l) = 615.69/(6-l) = 123.14
Sy = SSW/(N-p) = 28.72/(18-6) = 2.39
16.13.2.7.3 Summarize these calculations in the ANOVA table (Table 10)
TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
5
12
17
Sum of Squares
(SS)
615.69
28.72
644.41
Mean Square (MS)
(SS/df)
123.14
2.39
16.13.2.7.4 To perform the individual comparisons, calculate the t
statistic for each concentration, and control combination as follows:
Where: Y, = mean number of cystocarps for effluent concentration i
Y1 = mean number of cystocarps for the control
SM = square root of the within mean square
n1 - number of replicates for the control
rif « number of replicates for concentration i
16.13.2.7.5 Table 11 includes the calculated t values for each concentration
and control combination. In this example, comparing the 0.8% concentration
with the control the calculation is as follows:
(18-10.53)
[1. 55v/(l/3) + (1/3)]
= 5.90
16.13.2.7.6 Since the purpose of this test is to detect a significant
reduction in cystocarp production, a one-sided test is appropriate. The
critical value for this one-sided test is found in Table 5, Appendix C. For
an overall alpha level of 0.05, 12 degrees of freedom for error and five
370
-------
TABLE 11. CALCULATED T VALUES
Effluent
Concentration(%)
0.8
1.3
2.2
3.6
6.0
i
2
3
4
5
6
*i
i . .
5.90
10.64
11.38
12.17
13.38
concentrations (excluding the control) the critical value is 2.50. Mean
cystocarp production for concentration i is considered significantly less
control if t, is greater than the critical value. Therefore, mean cystocarp
productions for all effluent concentrations in this example have significantly
lower cystocarp production than the control. Hence the NOEC is 0.8% and the
LOEC is 0.8%.
16.13.2.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be calculated:
MSD = d S^fCL/nJ + (l/n)
Where: d = the critical value for Dunnett's Procedure
I
SH = the square root of the within mean square
i
n = the common number of replicates at each concentration
(this assumes equal .replication at each concentration)
n., = the number of replicates in the control.
16.13.2.7.8 In this example,
MSD = 2.50(1.55)/(l/3) + (1/3)
= 2.50 (1.55)(.8165)
= 3.16
,
16.13.2.7.9 Therefore, for this set of data, the minimum difference that can
be detected as statistically significant is 3.16 cystocarps.
16.13.2.7.10 This represents a 17.6% reduction in cystocarp production from
the control. •
371
-------
16.13.2.8 Calculation of the ICp
16.13.2.8.1 The sexual reproduction data in Table 5 are utilized in this
example. Table 12 contains the mean number of cystocarps for each effluent
concentration. As can be seen, the observed means are monotonically non-
increasing with respect to concentration. Therefore, it is not necessary to
smooth the means prior to calculating the ICp. Refer to Figure 10 for a plot
of the response curve.
TABLE 12. RED MACROALGA, CHAMPIA PARVULA,
MEAN NUMBER OF CYSTOCARPS
Effluent
Cone.
(%)
Control
0.8
1.3
2.2
3.6
6.0
10.0
i
1
2
3
4
5
6
7
Response
Means
Y,- (mg)
18.00
10.53
4.53
3.60
2.60
1.07
0.27
Smoothed
Means
M, (mg)
18.00
10.53
4.53
3.60
2.60
1.07
0.27
16.13.2.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation
Method. A 25% reduction in mean number of cystocarps, compared to the
controls, would result in a mean number of 13.50 cystocarps, where M^l-p/100)
= 18.00(1-25/100). A 50% reduction in mean number of cystocarps, compared to
the controls, would result in a mean number of 9.00 cystocarps. Examining the
means and their associated concentrations (Table 12), the response, 13.50, is
bracketed by C., = 0.0% effluent and C, = 0.8% effluent. The response, 9.00,
is bracketed by C2 - 0.8% effluent and C3 - 1.3% effluent.
16.13.2.8.3 Using the equation from Section 4.2 in Appendix L, the estimate
of the IC25 is calculated as follows:
ICp = Cj.
IC25 = 0.0 + [18.00(1 - 25/100) - 18.00] (0.8 - 0.0)
= 0.5%.
(10.53 - 18.00)
16.13.2.8.4 Using the equation from Section 4.2 from Appendix L, the estimate
of the IC50 is calculated as follows:
372
-------
IC50 = 0.8 + [18.00(1 - 50/100) - 10.53] (1.3 - 0.8)
(4.53 - 10.53)
= 0.9 %.
16 13.2.8.5 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 0.4821%. The empirical
95.0% confidence interval for the true mean was 0.4013% to 0.6075%. The
computer program output for the IC25 for this data set is shown in Figure 13.
16 13.2.8.6 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC50 was 0.9278%. The empirical
95.0% confidence interval for the true mean was 0.7893% and 1.0576%. The
computer program output for the IC50 for this data set is shown in Figure 14.
16.14 PRECISION AND ACCURACY !
16.14.1 PRECISION
I
16.14.1.1 Single-Laboratory Precision
i
16.14.1.1.1 The single-laboratory precision data from six tests with copper
sulfate (Cu) and six tests with sodium dodecyl sulfate (SDS) are listed in
Tables 13-16. The NOECs with Cu differed by only one concentration interval
(factor of two), showing good precision. The precision of the first four
tests with SDS was somewhat obscured by the choice of toxicant concentrations,
but appeared similar to that of Cu in the last two tests. The IC25 and IC50
are indicated in Tables 13-16. The coefficient of variation, based on the
IC25 for these two reference toxicants in natural seawater and a mixture of
natural seawater and GP2, ranged from 59.6% to 69.0%, and for the IC50, ranged
from 22.9% to 43.7%.
I
16.14.1.2 Multilaboratory Precision
16.14.1.2.1 The multilaboratory precision of the test has not yet been
determined.
16.14.2 ACCURACY \
16.14.2.1 The accuracy of toxicity tests cannot be determined.
373
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1
0
19
20
24
7
18
19
12
21
11
23
17
25
18
20
16
2
.8
10
16
11
12
11
12
10
6
9
10
12
9
9
13
8
3
1.3
10
0
3
5
4
6
4
4
8
4
4
4
2
6
4
4
2.2
1
2
5
4
0
7
9
9
4
6
3
2
2
0
0
5
3.6
2
1
1
5
0
3
4
6
4
2
0
4
3
1
3
6
6
1
0
0
0
0
1
2
1
0
0
0
4
3
1
3
7
10
0
0
0
0
1
0
0
0
0
2
1
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: effluent
Test Start Date: Test Ending Date:
Test Species: RED MACROALGA, Champia parvula
Test Duration:
DATA FILE: champia.icp
OUTPUT FILE: champ1 a.i25
Cone.
ID
1
2
3
4
5
6
7
Number Concentration
Replicates
15
15
15
15
15
15
15
The Linear Interpolation
%
0.000
0.800
1.300
2.200
3.600
6.000
10.000
Estimate:
Response
Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
0.4821
Std.
Dev.
4.928
2.356
2.356
3.066
1.805
1.335
0.594
Pooled
Response Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Resampling time in Seconds:
0.4947 Standard Deviation: 0.0616
Lower: 0.4013 Upper: 0.6075
3.68 Random Seed: 703617166
Figure 13. ICPIN program output for the IC25.
374
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1
0
19
20
24
7
18
19
12
21
11
23
17
25
18
20
16
2
.8
10
16
11
12
11
12
10
6
9
10.
12
9
9
13
8
3
1.3
10
0
3
5
4
6
4
4
8
4
4
4
2
6
4
4
2.2
1
2
5
4
0
7
9
9
4
6
3
2
2
0
0
5
3.6
2
1
1
5
0
3
4
6
4
2
0
4
3
1
3
6
6
1
0
0
0
0
1
2
1
0
0
0
4
3
1
3
7
10
0
0
0
0
1
0
0
0
0
2
1
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: effluent
Test Start Date: Test Ending Date:
Test Species: RED MACROALGA, Champia parvula
Test Duration:
DATA FILE: champia.icp
OUTPUT FILE: champia.iSO
Cone.
ID
1
2
3
4
5
6
7
Number
Replicates
15
15
15
15
15
15
15
Concentration
%
0.000
0.800
1.300
2.200
3.600
6.000
10.000
Response
Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
Std.
Dev. F
4.928
2.356
2.356
3.066
1.805
1.335
0.594
Pooled
Response Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
The Linear Interpolation Estimate:
0.9278 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Resampling time in Seconds:
0.9263 Standard Deviation: 0.0745
Lower: 0.7893 Upper: 1.0576
3.63 Random Seed: -1255453122
Figure 14. ICPIN program output for the IC50.
375
-------
TABLE 13. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA
PARVULA, REPRODUCTION TEST PERFORMED IN A 50/50 MIXTURE OF
NATURAL SEAWATER AND GP2 ARTIFICIAL SEAWATER, USING GAMETES
FROM ADULTS CULTURED IN NATURAL SEAWATER,, THE REFERENCE
TOXICANT USED WAS COPPER (CU) SULFATE1'2'3'4'5
Test
Number
1
2
3
4
5
6
n:
Mean:
CV(%):
1 Data from USEPA
NOEC
UgA)
1.0
1.0
1.0
1.0
0.5
0.5
6
NA
NA
(1991a).
IC25
(/•fl/L)
1.67
1.50
0.69
0.98
0.38
0.38
6
0.93
59.6
IC50
(M9/L)
2.37
1.99
1.53
1.78
0.76
0.75
6
1.5
43.7
M IICCDA
3
4
Narragansett, RI. Tests were conducted at 22°C, in 50/50 GP2 and
natural seawater at a salinity of 30%o.
Copper concentrations were: 0.5, 1.0, 2.5, 5.0, 7.5, and 1.0 ^g/L.
NOEC Range: 0.5 - 1.0 /*g/L (this represents a difference of one
exposure concentration).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
376
-------
TABLE 14. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA
PARVULA, REPRODUCTION TEST PERFORMED IN A 50/50 MIXTURE OF
NATURAL SEAWATER AND GP2 ARTIFICIAL SEAWATER, USING GAMETES
FROM ADULTS CULTURED IN NATURAL SEAWATER. THE REFERENCE
TOXICANT USED WAS SODIUM DODECYL SULFATE (-SDS)1""3'*'5
Test
Number
1
2
3
4
5
6
7
8
9
n:
Mean:
CV(%):
1 Data
2 TQC-+<
NOEC
(mg/L)
< 0.80
0.48
< 0.48
< 0.48
0.26
0.09
0.16
0.09
< 0.29
5
NA
NA
from USEPA (1991 a).
- navFnvmaH hu Cl on ThllV^hv ;
IC25
(mg/L)
1
0.6
0.7
0.4
0.2
0.2
0.1
0.2
0.1 ;
0.3
9
0.31
69.0
snH Mark Taaliabue.
IC50
(mg/L)
0.3
0.6
0.2
0.4
0.5
0.3
0.3
0.2
0.4
9
0.36
37.0
ERL-N. USEPA,
Narragansett, RI. Tests were conducted at 22°C, in 50/50 GP2 and
natural seawater at a salinity of 30%o.
SDS concentrations for Test 1 were: 0.8, 1.3, 2.2, 3.6, 6.0, and
10.0 mg/L. SDS concentrations for Tests 2, 3, and 4 were: 0.48,
0.8, 1.3, 2.2, 3.6, and 6.0 mg/L. SDS concentrations for Tests 5
and 6 were: 0.09, 0.16, 2.26, 0.43, 0.72, and 1.2 mg/L.
NOEC Range: 0.09 - 0.48 mg/L (this represents a difference of two
exposure concentrations).
For a discussion of the precision of data from chronic toxicity
tests see Section 4, Quality Assurance. j
377
-------
TABLE 15. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAHPIA
PARVULA, REPRODUCTION TEST IN NATURAL SEAWATER (30%o
SALINITY),.. THE REFERENCE TOXICANT USED WAS COPPER (CU)
SULFATE1/2'3
Test NOEC
1 1.00
2 0.50
3 0.50
4 0.50
n: 4
Mean : NA
CV(%): NA
Cu (UQ/L)
IC25
2.62
0.71
2.83
0.99
4
1.79
61.09
IC50
4.02
1.66
3.55
4.15
4
3.35
34.45
Data from USEPA (1991a).
Copper concentrations were 0.5, 1.0, 2.5, 5.0, 7.5, and 10 M9/L.
Concentrations of Cu were made from a 100 pg/mL CuSO, standard
obtained from Inorganic Ventures, Inc., Brick, NJ.
Prepared by Steven Ward and Glen Thursby, Environmental Research
Laboratory, USEPA, Narragansett, RI.
378
-------
TABLE 16. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA
PARVULA, REPRODUCTION TEST IN NATURAL SEAWATER (30%o
SALINITY). THE REFERENCE TOXICANT USED WAS SODIUM DODECYL
SULFATE (SDS)1'2'3
Test
1
2
3
4
n:
Mean:
CV(%):
1 Data from USEPA
NOEC
0.60
0.60
0.30
0.15
4
NA
NA
(1991a).
t«*f* i,ifsi*st A AO 7C f\ i
SDS (mq/L)
IC25
0.05
0.48
0.69
0.60
4
0.46
62.29
\tz. ft i c ft ft^ ft en a
IC50
0.50
0.81
0.89
0.81
4
0.75
22.92
nrl 1 Oft mn/l
Concentrations of SDS were made from a 44.64 ± 3.33 mg/mL standard
obtained from the EMSL-USEPA, Cincinnati, OH.
Prepared by Steven Ward and Glen Thursby, Environmental Research
Laboratory, USEPA, Narragansett, RI.
379
-------
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400
411
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APPENDICES
Page
A. Independence, Randomization, and Outliers 403
1. Statistical Independence 403
2. Randomization 403
3. Outliers i, 408
B. Validating Normality and Homogeneity of Variance Assumptions . . . 409
1. Introduction -. 409
2. Tests for Normal Distribution of Data 409
3. Test for Homogeneity of Variance 417
4. Transformations of the Data I 417
C. Dunnett's Procedure 419
1. Manual Calculations 419
2. Computer Calculations I 426
D. T test with the Bonferroni Adjustment ': 432
E. Steel's Many-one Rank Test I 438
F. Wilcoxon Rank Sum Test with the Bonferroni Adjustment 443
G. Single Concentration Toxicity Test - Comparison of Control with
100% Effluent or Receiving Water 450
H. Probit Analysis '•. 454
I. Spearman-Karber Method 457
J. Trimmed Spearman-Karber Method 462
K. Graphical Method [.. 467
L. Linear Interpolation Method 471
!
1. General Procedure 471
401
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APPENDICES (CONTINUED)
Page
2. Data Summary and Plots 471
3. Monotonicity 471
4. Linear Interpolation Method 472
5. Confidence Intervals 473
6. Manual Calculations . 473
7. Computer Calculations 477
Cited References 482
402
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APPENDIX A
INDEPENDENCE, RANDOMIZATION, AND OUTLIERS
1. STATISTICAL INDEPENDENCE
1.1 Dunnett's Procedure and the t test with Bonferroni's adjustment are
parametric procedures based on the assumptions that (1) the observations
within treatments are independent and normally distributed,, and (2) that the
variance of the observations is homogeneous across all toxicant concentrations
and the control. Of the three possible departures from the assumptions,
non-normality, heterogeneity of variance, and lack of independence, those
caused by lack of independence are the most difficult to resolve (see Scheffe,
1959). For toxicity data, statistical independence means that given knowledge
of the true mean for a given concentration or control, knowledge of the error
in any one actual observation would provide no information about the error in
any other observation. Lack of independence is difficult to assess and
difficult to test for statistically. It may also have serious effects on the
true alpha or beta level. Therefore, it is of utmost importance to be aware
of the need for statistical independence between observations and to be
constantly vigilant in avoiding any patterned experimental procedure that
might compromise independence. One of the best ways to help insure
independence is to follow proper randomization procedures throughout the test.
2. RANDOMIZATION '
2.1 Randomization of the distribution of test organisms among test chambers,
and the arrangement of treatments and replicate chambers is an important part
of conducting a valid test. The purpose of randomization is to avoid
situations where test organisms are placed serially into test chambers, or
where all replicates for a test concentration are located adjacent to one
another, which could introduce bias into the test results.
.
2.2 An example of randomization of the distribution of test organisms among
test chambers, and an example of randomization of arrangement of treatments
and replicate chambers are described using the Sheepshead Minnow Larval
Survival and Growth test. For the purpose of the example, the test design is
as follows: Five effluent concentrations are tested in addition to the
control. The effluent concentrations are as follows: 6.25%, 12.5%, 25.0%,
50.0%, and 100.0%. There are four replicate chambers per treatment. Each
replicate chamber contains ten fish. j
2.3 RANDOMIZATION OF FISH TO REPLICATE CHAMBERS EXAMPLE
I
2.3.1 Consider first the random assignment of the fish to the replicate
chambers. The first step is to label each of the replicate chambers with the
control or effluent concentration and the replicate number. The next step is
to assign each replicate chamber four double-digit numbers:. An example of
this assignment is provided in Table A.I. Note that the double digits 00 and
97 through 99 were not used.
403
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TABLE A.I. RANDOM ASSIGNMENT OF FISH TO REPLICATE CHAMBERS EXAMPLE
ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER
Assigned
01,
02,
03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
Numbers
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
68,
69,
70,
71,
72,
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
6.25%
6.25%
6.25%
6.25%
12.5%
12.5%
12.5%
12.5%
25.0%
25.0%
25.0%
25.0%
50.0%
50.0%
50.0%
50.0%
Replicate Chamber
Control ,
Control ,
Control ,
Control ,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
2.3.2 The random numbers used to carry out the random assignment of fish to
replicate chambers are provided in Table A.2. The third step is to choose a
starting position in Table A.2, and read the first double digit number. The
first number read identifies the replicate chamber for the first fish taken
from the tank. For the example, the first entry in row 2 was chosen as the
starting position. The first number in this row is 37. According to Table
A.I, this number corresponds to replicate chamber 1 of the 25.0% effluent
concentration. Thus, the first fish taken from the tank is to be placed in
replicate chamber 1 of the 25.0% effluent concentration.
2.3.3 The next step is to read the double digit number to the right of the
first one. The second number identifies the replicate chamber for the second
fish taken from the tank. Continuing the example, the second number read in
row 2 of Table A.2 is 54. According to Table A.I, this number corresponds to
replicate chamber 2 of the 6.25% effluent concentration. Thus, the second
fish taken from the tank is to be placed in replicate chamber 2 of the 6.25%
effluent concentration.
404
-------
TABLE A.2. TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983)
10 09 73
37 54 20
08 42 26
99 01 90
12 80 79
66 06 57
31 06 01
85 26 97
63 57 33
73 79 64
98 52 01
11 80 50
83 45 29
88 68 54
99 59 46
65 48 11
80 12 43
74 35 09
69 91 62
09 89 32
91 49 91
80 33 69
44 10 48
12 55 07
63 60 64
61 19 69
15 47 44
94 55 72
42 48 11
23 52 37
04 49 35
00 54 99
35 96 31
59 80 80
46 05 88
32 17 90
69 23 46
19 56 54
45 15 51
94 86 43
98 08 62
33 18 51
80 95 10
79 75 24
18 63 33
74 02 94
54 17 84
11 66 44
48 32 47
69 07 49
25 33
48 05
89 53
25 29
99 70
47 17
08 05
76 02
21 35
57 53
77 67
54 31
96 34
02 00
73 48
76 74
56 35
98 17
68 03
05 05
45 23
45 98
19 49
37 42
93 29
04 46
52 66
85 73
62 13
83 17
24 94
76 54
53 07
83 91
52 36
05 97
14 06
14 30
49 38
19 94
48 26
62 32 •
04 06
91 40
25 37
39 02
56 11
98 83
79 28
41 38
76 52 01 35 86
64 89 47 42 96
19 64 50 93 03
09 37 67 07 15
80 15 73 61 47
34 07 27 68 50
45 57 18 24 06
02 05 16 56 92
05 32 54 70 48
03 52 96 47 78
14 90 56 86 07
39 80 82 77 32
06 28 89 80 83
86 50 75 84 01
87 51 76 49 69
17 46 85 09 50
17 72 70 80 15
77 40 27 72 14
66 25 22 91 48
14 22 56 85 14
68 47 92 76 86
26 94 03 68 58
85 15 74 79 54
11 10 00 20 40
16 50 53 44 84
26 45 74 77 74
95 27 07 99 53
67 89 75 43 87
97 34 40 87 21
73 20 88 98 37
75 24 63 38 24
64 05 18 81 59
26 89 80 93 45
45 42 72 68 42
01 39 09 22 86
87 37 92 52 41
20 11 74 52 04
01 75 87 53 79
19 47 60 72 46
36 16 81 08 51
45 24 02 84 04
41 94 15 09 49
96 38 27 07 74
71 96 12 82 96
98 14 50 65 71
77 55 73 22 70
80 99 33 71 43
52 07 98 48 27
31 24 96 47 10
87 63 79 19 76
34
24
23
38
64
36
35
68
90
35
22
50
13
36
91
58
45
43
36
46
46
70
32
12
40
51
59
54
16
68
45
96
33
83
77
05
15
40
43
34
44
89
20
69
31
97
05
59
02
35
67 35
80 52
20 90
31 13
03 23
69 73
30 34
66 57
55 35
80 83
10 94
72 56
74 67
76 66
82 60
04 77
31 82
23 60
93 68
42 75
16 28
29 73
97 92
86 07
21 95
92 43
36 78
62 24
86 84
93 59
86 25
11 96
35 13
60 94
28 14
56 70
95 66
41 92
66 79
88 88
99 90
43 54
15 12
86 10
01 02
79 01
33 51
38 17
29 53
58 40
43 76
40 37
25 60
11 65
66 53
61 70
26 14
48 18
75 48
42 82
05 58
82 48
00 78
79 51
89 28
69 74
23 74
02 10
72 03
67 88
35 54
41 35
65 75
46 97
25 63
37 29
38 48
44 31
87 67
14 16
10 25
38 96
54 62
97 00
40 77
70 07
00 00
15 85
45 43
15 53
88 '96
85 81
33 87
25 91
46 74
71 19
29 69
15 39
68 70
44 01
80 95
20 63
15 95
88 67
98 95
65 81
86 79
73 05
28 46
60 93
60 97
29 40
18 47
90 36
93 78
73 03
21 11
45 52
76 62
96 29
94 75
53 14
57 60
96 64
43 65
65 39
82 39
91 19
03 07
26 25
61 96
54 69
77 97
13 02
93 91
86 74
18 74
66 67
59 04
01 54
39 09
88 69
25 01
74 85
05 45
52 52
56 12
09 97
32 30
10 51
90 91
61 04
33 47
67 43
11 68
33 98
90 74
38 52
82 87
52 03
09 34
52 42
54 06
47 64
56 13
95 71
57 82
16 42
11 39
77 88
08 99
03 33
04 08
48 94
17 70
45 95
61 01
04 25
11 20
22 96
27 93
28 23
45 00
12 48
08 36
31 71
39 24
43 68
79 00
03 54
47 34
54 19
62 52
22 05
56 14
75 80
71 92
33 34
75 75
82 16
17
02
64
97
77
85
39
47
09
44
33
01
10
93
68
86
53
37
90
22
23
40
81
39
82
93
18
92
59
63
35
91
24
92
47
57
23
06
33
56
07
94
98
39
27
21
55
40
46
15
39
00
35
04
12
11
23
18
83
35
50
52
68
29
23
40
14
96
94
54
37
42
22
28
07
42
33
92
25
05
65
23
90
78
70
85
97
84
20
05
35
37
94
00
77
80
36
88
15
O'l
29 27
82 29
08 03
43 62
27 17
19 92
40 30
62 38
49 12
27 38
50 07
77 56
71 17
60 91
47 83
21 81
38 55
28 60
40 05
38 21
08 92
05 08
22 20
70 72
20 73
58 26
21 15
92 74
70 14
52 28
33 71
28 72
10 33
56 52
61 74
39 41
11 89
96 28
82 66
01 45
44 13
54 87
62 46
38 75
93 89
81 45
04 09
46 12
02 00
84 87
49 45
16 65
36 06
76 59
68 33
91 70
97 32
85 79
56 24
84 35
39 98
78 51
78 17
10 62
41 13
65 44
37 63
26 55
64 18
45 98
00 48
23 41
64 13
58 15
17 90
05 27
94 66
59 73
66 70
25 62
24 72
95 29
93 33
01 06
29 41
18 38
63 38
52 07
95 41
11 76
18 80
30 43
11 71
95 79
19 36
17 48
03 24
33 56
99 94
69 38
405
-------
2.3.4 Continue in this fashion until all the fish have been randomly assigned
to a replicate chamber. In order to fill each replicate chamber with ten
fish, the assigned numbers will be used more than once. If a number is read
from the table that was not assigned to a replicate chamber, then ignore it
and continue to the next number. If a replicate chamber becomes filled and a
number is read from the table that corresponds to it, then ignore that value
and continue to the next number. The first ten random assignments of fish to
replicate chambers for the example are summarized in Table A.3.
TABLE A.3. EXAMPLE OF RANDOM ASSIGNMENT OF FIRST TEN FISH TO
REPLICATE CHAMBERS
Fish
First
Second
Third
Fourth
Fifth
Sixth
Seventh
Eighth
Ninth
Tenth
fish
fish
fish
fish
fish
fish
fish
fish
fish
fish
taken
taken
taken
taken
taken
taken
taken
taken
taken
taken
from
from
from
from
from
from
from
from
from
from
tank
tank
tank
tank
tank
tank
tank
tank
tank
tank
Assignment
25.0% effluent,
6.25% effluent,
50.0% effluent
100.0% effluent,
6.25% effluent,
25.0% effluent,
50.0% effluent,
100.0% effluent,
50.0% effluent,
100.0% effluent,
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
1
2
4
4
1
4
1
3
2
4
2.3.5 Four double-digit numbers were assigned to each replicate chamber
(instead of one, two, or three double-digit numbers) in order to make
efficient use of the random number table (Table A.2). To illustrate, consider
the assignment of only one double-digit number to each replicate chamber: the
first column of assigned numbers in Table A.I. Whenever the numbers 00 and 25
through 99 are read from Table A.2, they will be disregarded and the next
number will be read.
2.4 RANDOMIZATION OF REPLICATE CHAMBERS TO POSITIONS EXAMPLE
2.4.1 Next consider the random assignment of the 24 replicate chambers to
positions within the water bath (or equivalent). Assume that the replicate
chambers are to be positioned in a four row by six column rectangular array.
The first step is to label the positions in the water bath. Table A.4
provides an example layout.
2.4.2 The second step is to assign each of the 24 positions four double-digit
numbers. An example of this assignment is provided in Table A.5. Note that
the double digits 00 and 97 through 99 were not used.
2.4.3 The random numbers used to carry out the random assignment of replicate
chambers to positions are provided in Table A.2. The third step is to choose
a starting position in Table A.2, and read the first double-digit number. The
first number read identifies the position for the first replicate chamber of
the control. For the example, the first entry.in row 10 of Table A.2 was
406
-------
TABLE A.4. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
LABELLING THE POSITIONS WITHIN THE WATER BATH
TABLE A.5. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
ASSIGNED NUMBERS FOR EACH POSITION
EXAMPLE
1
7
13
19
2
8
14
20
3
9
15
21
4
10
16
22
i5 •
11
17
2.3
6
12
18
24
EXAMPLE
Assigned
01,
02,
03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
Numbers Position
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
68,
69,
70,
71,
72,
73 1
74
2
75 3
76 4
77 5
78
79
80
81 •
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
6
7
8
9
10
11 ,
12
13
14
15
16
17
18
19
20
21
22
23
24
chosen as the starting position. The first number in this row was 73.
According to Table A.5, this number corresponds to position 1. Thus, the
first replicate chamber for the control will be placed in position 1.
407
-------
2.4.4 The next step is to read the double-digit number to the right of the
first one. The second number identifies the position for the second replicate
chamber of the control. Continuing the example, the second number read in row
10 of Table A.2 is 79. According to Table A.5, this number corresponds to
position 7. Thus, the second replicate chamber for the control will be placed
in position 7.
2.4.5 Continue in this fashion until all the replicate chambers have been
assigned to a position. The first four numbers read will identify the
positions for the control replicate chambers, the second four numbers read
will identify the positions for the lowest effluent concentration replicate
chambers, and so on. If a number is read from the table that was not assigned
to a position, then ignore that value and continue to the next number. If a
number is repeated in Table A.2, then ignore the repeats and continue to the
next number. The complete randomization of replicate chambers to positions
for the example is displayed in Table A.6.
TABLE A.6. EXAMPLE OF RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO
POSITIONS: ASSIGNMENT OF ALL 24 POSITIONS
Control
Control
100.0%
50.0%
100.0%
12.5%
50.0%
50.0%
6.25%
Control
100.0%
25.0%
6.25%
25.0%
Control
50.0%
6.25%
12.5%
100.0%
12.5%
12.5%
25.0%
25.0%
6.25%
2.4.6 Four double-digit numbers were assigned to each position (instead of
one, two, or three) in order to make efficient use of the random number table
(Table A.2). To illustrate, consider the assignment of only one double-digit
number to each position: the first column of assigned numbers in Table A.5.
Whenever the numbers 00 and 25 through 99 are read from Table A.2, they will
be disregarded and the next number will be read.
3. OUTLIERS
3.1 An outlier is an inconsistent or questionable data point that appears
unrepresentative of the general trend exhibited by the majority of the data.
Outliers may be detected by tabulation of the data, plotting, and by an
analysis of the residuals. An explanation should be sought for any
questionable data points. Without an explanation, data points should be
discarded only with extreme caution. If there is no explanation, the analysis
should be performed both with and without the outlier, and the results of both
analyses should be reported.
3.2 Gentleman-Wilk's A statistic gives a test for the condition that the
extreme observation may be considered an outlier. For a discussion of this,
and other techniques for evaluating outliers, see Draper and John (1981).
408
-------
APPENDIX B
VALIDATING NORMALITY AND HOMOGENEITY OF VARIANCE ASSUMPTIONS
1. INTRODUCTION
i
1.1 Dunnett's Procedure and the t test with Bonferroni's adjustment are
parametric procedures based on the assumptions that the observations within
treatments are independent and normally distributed, and that the variance of
the observations is homogeneous across all toxicant concentrations and the
control. These assumptions should be checked prior to using these tests, to
determine if they have been met. Tests for validating the assumptions are
provided in the following discussion. If the tests fail (if the data do not
meet the assumptions), a nonparametric procedure such as Steel's Many-one Rank
Test may be more appropriate. However, the decision on whether to use
parametric or nonparametric tests may be a judgement call, and a statistician
should be consulted in selecting the analysis.
2. TEST FOR NORMAL DISTRIBUTION OF DATA
2.1 SHAPIRO-WILK'S TEST
i
2'.1.1 One formal test for normality is the Shapiro-Wilk's Test (Conover,
1980). The test statistic is obtained by dividing the square of an
appropriate linear combination of the sample order statistics by the usual
symmetric estimate of variance. The calculated W must be greater than zero
and less than or equal to one. This test is recommended for a sample size of
50 or less. If the sample size is greater than 50, the Kollmogorov "D"
statistic (Stephens, 1974) is recommended. An example of the Shapiro-Wilk's
test is provided below.
2.2 The example uses growth data from the Sheepshead Minnow Larval Survival
and Growth Test. The same data are used in the discussion of the homogeneity
of variance determination in Paragraph 3 and Dunnett's Procedure in Appendix
C. The data, the mean and variance of the observations at each concentration,
including the control, are listed in Table B.I.
2.3 The first step of the test for normality is to center the observations by
subtracting the mean of all observations within a concentration from each
observation in that concentration. The centered observations are listed in
Table B.2.
2.4 Calculate the denominator, D, of the test statistic:
•A . ^
r\ — \^ / v v\ 2
U — Z~t \J\.j~~'4\)
Where: X,- = the centered observations, X is the overall mean of the
centered observations, and n is the total number of the
centered observations. For this set of data, X = 0,
and D = 0.1589.
409
-------
TABLE B.I. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH
DATA (WEIGHT IN MG) FOR THE SHAPIRO-WILK'S TEST
Effluent Concentration (%)
Replicate
Control
6.25
12.5
25.0
50.0
1
2
3
Mean(Yf)
?i
i
1.017
0.745
0.862
0.875
0.019
1
1.157
0.914
0.992
1.021
0.015
2
0.998
0.793
1.021
0.937
0.016
3
0.837
0.935
0.839
0.882
0.0031
4
0.715
0.907
1.044
0.889
0.027
5
TABLE B.2. EXAMPLE OF SHAPIRO-WILK'S TEST: CENTERED OBSERVATIONS
Effluent Concentration (%)
Replicate
Control
6.25
12.5
25.0
50.0
1
2
3
0.142
- 0.130
- 0.013
0.136
- 0.107
- 0.029
0.061
- 0.144
0.084
- 0.009
0.053
- 0.043
- 0.174
0.018
0.155
2.4.1 For this set of data,
n = 15
_
15
D = 0.1589
(.0) = 0.0
410
-------
2.5 Order the centered observations from smallest to largest,
X(1) v(2) ., ., vWt
< A < . . . < A I
where X(1> denote the ith order statistic. The ordered observations are
listed in Table B.3.
TABLE B.3. EXAMPLE OF THE SHAPIRO-WILK'S TEST: ORDERED OBSERVATIONS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
- 0.174
- 0.144
- 0.130
- 0.107
- 0.043
- 0.029
- 0.013
- 0.009
0.018
0.053
0.061
0.084
0.136
0.142
0.155
2.6 From Table B.4, for the number of observations, n, obtain the
coefficients a,, a2, ..., ak, where k is n/2 if n is even, and (n-l)/2 if n is
odd. For the data in this example, n = 15, k = 7, and the a,- values are
listed in Table B.5.
2.7 Compute the test statistic, W, as follows:
WD
The differences, X
Cn-i+1)
- x(f),
are listed in Table B.5.
2.8 The decision rule for this test is to compare the critical value from
Table B.6 to the computed W. If the computed value is less than the critical
value, conclude that the data are not normally distributed. For this example,
the critical value at a significance level of 0.01 and 15 observations (n) is
0.835. The calculated value, 0.9516, is not less than the critical value.
Therefore conclude that the data are normally distributed.
411
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-MILK'S TEST (Conover, 1980)
n
1
2
3
4
5
2
0.
•
-
-
•
7071
3
0.7071
0.0000
-
-
•
4
0.6872
0.1667
-
-
•
5
0.6646
0.2413
0.0000
-
•
Number of
6
0.6431
0.2806
0.0875
-
-
Observations
7
0.6233
0.3031
0.1401
0.0000
-
8
0.6052
0.3164
0.1743
0.0561
-
9
0.5888
0.3244
0.1976
0.0947
0.0000
10
0.5739
0.3291
0.2141
0.1224
0.0399
n
1
2
3
4
5
6
7
8
9
10
11
0.
0.
0.
0.
0.
0.
-
-
-
5601
3315
2260
1429
0695
0000
12
0.5475
0.3325
0.2347
0.1586
0.0922
0.0303
-
-
-
13
0.5359
0.3325
0.2412
0.1707
0.1099
0.0539
0.0000
-
-
14
0.5251
0.3318
0.2460
0.1802
0.1240
0.0727
0.0240
-
-
Number of
15
0.5150
0.3306
0.2495
0.1878
0.1353
0.0880
0.0433
0.0000
-
Observations
16
0.5056
0.3290
0.2521
0.1939
0.1447
0.1005
0.0593
0.0196
-
17
0.4968
0.3273
0.2540
0.1988
0.1524
0.1109
0.0725
0.0359
0.0000
18
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
19
0.4808
0.3232
0.2561
0.2059
0.1641
0.1271
0.0932
0.0612
0.0303
0.0000
20
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
21
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
-
-
-
-
4643
3185
2578
2119
1736
1399
1092
0804
0530
0263
0000
22
0.4590
0.3156
0.2571
0.2131
0.1764
0.1443
0.1150
0.0878
0.0618
0.0368
0.0122
-
-
-.
-
23
0.4542
0.3126
0.2563
0.2139
0.1787
0.1480
0.1201
0.0941
0.0696
0.0459
0.0228
0.0000
-
-
-
24
0.4493
0.3098
0.2554
0.2145
0.1807
0.1512
0.1245
0.0997
0.0764
0.0539
0.0321
0.0107
-
-
-
Number of
25
0.4450
0.3069
0.2543
0.2148
0.1822
0.1539
0.1283
0.1046
0.0823
0.0610
0.0403
0.0200
0.0000
-
-
Observations
26
0.4407
0.3043
0.2533
0.2151
0.1836
0.1563
0.1316
0.1089
0.0876
0.0672
0.0476
0.0284
0.0094
-
-
27
0.4366
0.3018
0.2522
0.2152
0.1848
0.1584
0.1346
0.1128
0.0923
0.0728
0.0540
0.0358
0.0178
0.0000
-
28
0.4328
0.2992
0.2510
0.2151
0.1857
0.1601
0.1372
0.1162
0.0965
0.0778
0.0598
0.0424
0.0253
0.0084
-
29
0.4291
0.2968
0.2499
0.2150
0.1864
0.1616
0.1395
0.1192
0.1002
0.0822
0.0650
0.0483
0.0320
0.0159
0.0000
30
0.4254
0.2944
0.2487
0.2148
0.1870
0.1630
0.1415
0.1219
0.1036
0.0862
0.0697
0.0537
0.0381
0.0227
0.0076
412
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED)
\
A-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
31
0.4220
0.2921
0.2475
0.2145
0.1874
0.1641
0.1433
0.1243
0.1066
0.0899
0.0739
0.0585
0.0435
0.0289
0.0144
0.0000
-
-
.
-
32
0.4188
0.2898
0.2462
0.2141
0.1878
0.1651
0.1449
0.1265
0.1093
0.0931
0.0777
0.0629
0.0485
0.0344
0.0206
0.0068
-
-
-
-
33
0.4156
0.2876
0.2451
0.2137
0.1880
0.1660
0.1463
0.1284
0.1118
0.0961
0.0812
0.0669
0.0530
0.0395
0.0262
0.0131
0.0000
-
-
-
34
0.4127
0.2854
0.2439
0.2132
0.1882
0.1667
0.1475
0.1301
0.1140
0.0988
0.0844
0.0706
0.0572
0.0441
0.0314
0.0187
0.0062
-
-
-
Number of
35
0.4096
0.2834
0.2427
0.2127
0.1883
0.1673
0.1487
0.1317
0.1160
0.1013
0.0873
0.0739
0.0610
0.0484
0.0361
0.0239
0.0119
0.0000
-
•
Observations
36
0.4068
0.2813
0.2415
0.2121
0.1883
0.1678
0.1496
0.1331
0.1179
0.1036
0.0900
0.0770
0.0645
0.0523
0.0404
0.0287
0.0172
0.0057
-
•
37
0.4040
0.2794
0.2403
0.2116
0.1883
0.1683
0.1505
0.1344
0.1196
0.1056
0.0924
0.0798
0.0677
0.0559
0.0444
0.0331
0.0220
0.0110
0.0000
"
38
0.4015
0.2774
0.2391
0.2110
0.1881
0.1686
0.1513
0.1356
0.1211
0.1075
0.0947
0.0824
0.0706
0.0592
0.0481
0.0372
0.0264
0.0158
0.0053
•
39
0.3989
0.2755
0.2380
0.2104
0.1880
0.1689
0.1520
0.1366
0.1225
0.1092
0.0967
0.0848
O.CI733
0.0622
O.CI515
O.CI409
O.CI305
0.0203
0.0101
0.0000
40
0.3964
0.2737
0.2368
0.2098
0.1878
0.1691
0.1526
0.1376
0.1237
0.1108
0.0986
0.0870
0.0759
0.0651
0.0546
0.0444
0.0343
0.0244
0.0146
0.0049
\
A-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
41
0.3940
0.2719
0.2357
0.2091
0.1876
0.1693
0.1531
0.1384
0.1249
0.1123
0.1004
0.0891
0.0782
0.0677
0.0575
0.0476
0.0379
0.0283
0.0188
0.0094
0.0000
-
-
-
•
42
0.3917
0.2701
0.2345
0.2085
0.1874
0.1694
0.1535
0.1392
0.1259
0.1136
0.1020
0.0909
0.0804
0.0701
0.0602
0.0506
0.0411
0.0318
0.0227
0.0136
0.0045
-
-
-
"
43
0.3894
0.2684
0.2334
0.2078
0.1871
0.1695
0.1539
0.1398
0.1269
0.1149
0.1035
0.0927
0.0824
0.0724
0.0628
0.0534
0.0442
0.0352
0.0263
0.0175
0.0087
0.0000
-
-
~
44
0.3872
0.2667
0.2323
0.2072
0.1868
0.1695
0.1542
0.1405
0.1278
0.1160
0.1049
0.0943
0.0842
0.0745
0.0651
0.0560
0.0471
0.0383
0.0296
0.0211
0.0126
0.0042
-
-
~
Number of
45
0.3850
0.2651
0.2313
0.2065
0.1865
0.1695
0.1545
0.1410
0.1286
0.1170
0.1062
0.0959
0.0860
0.0765
0.0673
0.0584
0.0497
0.0412
0.0328
0.0245
0.0163
0.0081
0.0000
-
"
Observations
46
0.3830
0.2635
0.2302
0.2058
0.1862
0.1695
0.1548
0.1415
0.1293
0.1180
0.1073
0.0972
0.0876
0.0783
0.0694
0.0607
0.0522
0.0439
0.0357
0.0277
0.0197
0.0118
0.0039
-
*
47
0.3808
0.2620
0.2291
0.2052
0.1859
0.1695
0.1550
0.1420
0.1300
0.1189
0.1085
0.0986
0.0892
0.0801
0.0713
0.0628
0.0546
0.0465
0.0385
0.0307
0.0229
0.0153
0.0076
0.0000
"
48
0.3789
0.2604
0.2281
0.2045
0.1855
0.1693
0.1551
0.1423
0.1306
0.1197
0.1095
0.0998
0.0906
0.0817
0.0731
0.0648
0.0568
0.0489
0.0411
0.0335
0.0259
0.0185
0.0111
0.0037
"
49
0.3770
0.2589
0.2271
0.2038
0.1851
0.1692
0.1553
0.1427
0.1312
0.1205
0.1105
0.1010
0.0919
0.0832
0.0748
0.0667
0.0588
0.0511
0.0436
0.0361
0.0288
0.0215
0.0143
0.0071
0.0000
50
0.3751
0.2574
0.2260
0.2032
0.1847
0.1691
0.1554
0.1430
0.1317
0.1212
0.1113
0.1020
0.0932
0.0846
0.0764
0.0685
0.0608
0.0532
0.0459
0.0386
0.0314
0.0244
0.0174
0.0104
0.0035
413
-------
TABLE B.5. EXAMPLE OF THE SHAPIRO-WILK'S TEST:
TABLE OF COEFFICIENTS AND DIFFERENCES
_ w
1
2
3
4
5
6
7
0.5150
0.3306
0.2495
0.1878
0.1353
0.0880
0.0433
0.329
0.286
0.266
0.191
0.104
0.082
0.031
X(H)
X(3)
*'
* >
X(10)
X(09>
X<08)
- x< >
- x«}
- *?
- x<*>
- *T
- x(«
- x(7)
2.9 In general, if the data fail the test for normality, a transformation
such as to log values may normalize the data. After transforming the data,
repeat the Shapiro Milk's Test for normality.
3. TEST FOR HOMOGENEITY OF VARIANCE
3.1 For Dunnett's Procedure and the t test with Bonferroni's adjustment, the
variances of the data obtained from each toxicant concentration and the
control are assumed to be equal. Bartlett's Test is a formal test of this
assumption. In using this test, it is assumed that the data are normally
distributed.
3.2 The data used in this example are growth data from a Sheepshead Minnow
Larval Survival and Growth Test, and are the same data used in Appendices C
and D. These data are listed in Table B.7, together with the calculated
variance for the control and each toxicant concentration.
3.3 The test statistic for Bartlett's Test (Snedecor and Cochran, 1980) is as
fol1ows:
In
In
B =
Where: Mi = degrees of freedom for each effluent concentration and
control, (Vf = nf - 1)
p = number of levels of toxicant concentration
including the control
In = loge
414
-------
TABLE B. 6,
n 0.01
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0.753
0.687
0.686
0.713
0.730
0.749
0.764
0.781
0.792
0.805
0.814
0.825
0.835
0.844
0.851
0.858
0.863
0.868
0.873
0.878
0.881
0.884
0.888
0.891
0.894
0.896
0.898
0.900
0.902
0.904
0.906
0.908
0.910
0.912
0.914
0.916
0.917
0.919
0.920
0.922
0.923
0.924
0.926
0.927
0.928
0.929
0.929
0.930
. QUANTILES OF THE SHAPIRO MILK'S TEST STATISTIC (Conover,
0.02 0.05 0.10 0.50 0.90 0.95 0.98 0.99
0.756
0.707
0.715
0.743
0.760
0.778
0.791
0.806
0.817
0.828
0.837
0.846
0.855
0.863
0.869
0.874
0.879
0.884
0.888
0.892
0.895
0.898
0.901
0.904
0.906
0.908
0.910
0.912
0.914
0.915
0.917
0.919
0.920
0.922
0.924
0.925
0.927
0.928
0.929
0.930
0.932
0.933
0.934
0.935
0.936
0.937
0.937
0.938
0.767
0.748
0.762
0.788
0.803
0.818
0.829
0.842
0.850
0.859
0.866
0.874
0.881
0.887
0.892
0.897
0.901
0.905
0.908
0.911
0.914
0.916
0.918
0.920
0.923
0.924
0.926
0.927
0.929
0.930
0.931
0.933
0.934
0.935
0.936
0.938
0.939
0.940
0.941
0.942
0.943
0.944
0.945
0.945
0.946
0.947
0.947
0.947
0.789
0.792
0.806
0.826
0.838
0.851
0.859
0.869
0.876
0.883
0.889
0.895
0.901
0.906
0.910
0.914
0.917
0.920
0.923
•0.926
0.928
0.930
0.931
0.933
0.935
0.936
0.937
0.939
0.940
0.941
0.942
0.943
0.944
0.945
0.946
0.947
0.948
0.949
0.950
0.951
0.951
0.952
0.953
0.953
0.954
0.954
0.955
0.955
0.959
0.935
0.927
0.927
0.928
0.932
0.935
0.938
0.940
0.943
0.945
0.947
0.950
0.952
' 0.954
0.956
0.957
0.959
0.960
0.961
0.962
0.963
0.964
0.965
0.965
0.966
0.966
0.967
0.967
0.968
0.968
0.969
0.969
0.970
0.970
0.971
0.971
0.972
0.972
0.972
0.973
0.973
0.973
0.974
0.974
0.974
0.974
0.974
0.998
0.987
0.979
0.974
0.972
0.972
0.972
0.972
0.973
0.973
0.974
0.975
0.975
0.976
0.977
0.978
0.978
0.979
0.980
0.980
0.981
0.981
0.981
0.982
0.982
0.982
0.982
0.983
0.983
0.983
0.983
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.999
0.992
0.986
0.981
0.979
0.978
0.978
0.978
0.979
0.979
0.979
0.980
0.980
0.981
0.981
0.982
0.982
0.983
0.983
0.984
0.984
0.984
0.985
0.985
0.985
0.985
0.985
0.985
0.986
0.986
0.986
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.987
0.987
0.987
0.987
0.988
0.988
0.988
0.988
0.988
0.988
i.ooo
0.996
0.991
0.986
0.985
0.984
0.984
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0,990
0,.990
0.990
0.990
0..990
0..990
0,,990
0..990
1.000
0.997
0.993
0.989
0.988
0.987
0.986
0.986
0.986
0.986
0.986
0.986
0.987
0.987
0.987
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
415
-------
i « 1, 2, ..., p where p is the number of concentrations
including the control
n,. » the number of replicates for concentration i.
;_ i-1
C=l+ [3 (p-1) ] -1 [ I/ V± - (
TABLE B.7. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH DATA
(WEIGHT IN MG) USED FOR BARTLETT'S TEST FOR HOMOGENEITY OF
VARIANCE
Replicate
1
2
3
Mean
Si
i1
Control
1.017
0.745
0.862
0.875
0.019
1
Effl
6.25
1.157
0.914
0.992
1.021
0.015
2
uent Concentration
12.5
0.998
0.793
1.021
0.937
0.016
3
25.0
0.837
0.935
0.839
0.882
0.0031
4
m
50.0
0.715
0.907
1.044
0.889
0.027
5
3.4 Since B is approximately distributed as chi-square with p-1 degrees of
freedom when the variances are equal, the appropriate critical value is
obtained from a table of the chi-square distribution for p-1 degrees of
freedom and a significance level of 0.01. If B is less than the critical
value then the variances are assumed to be equal.
3.5 For the data in this example, V, = 2, p = 5, S2 = 0.0158, and C = 1.2.
The calculated B value is:
2 [5 (In 0.0.158) -
B =
1.2
2 [5(- 4.1477) - (- 22.1247)]
1.2
2.3103
416
-------
3.6 Since B is approximately distributed as chi-square with p - 1 degrees of
freedom when the variances are equal, the appropriate critical value for the
test is 13.3 for a significance level of 0.01. Since B is less than 13.3, the
conclusion is that the variances are not different.
4. TRANSFORMATIONS OF THE DATA j
4.1 When the assumptions of normality and/or homogeneity of variance are not
met, transformations of the data may remedy the problem, so that the data can
be analyzed by parametric procedures, rather than nonparametric technique such
as Steel's Many-one Rank Test or Wilcoxon's Rank Sum Test. Examples of
transformations include log, square root, arc sine square root, and
reciprocals. After the data have been transformed, the Shapiro-Milk's and
Bartlett's tests should be performed on the transformed observations to
determine whether the assumptions of normality and/or homogeneity of variance
are met.
i
4.2 ARC SINE SQUARE ROOT TRANSFORMATION (USEPA, 1993).
4.2.1 For data consisting of proportions from a binomial (response/no
response; live/dead) response variable, the variance within the ith treatment
is proportional to Pi (1 - P.), where ?. is the expected proportion for the
treatment. This clearly violates the homogeneity of variance assumption
required by parametric procedures such as Dunnett's Procedure or the t test
with Bonferroni's adjustment, since the existence of a treatment effect
implies different values of Pf for different treatments, i. Also, when the
observed proportions are based on small samples, or when P,- is close to zero
or one, the normality assumption may be invalid. The arc sine square root
(arc sine J p ) transformation is commonly used for such data to stabilize
the variance and satisfy the normality requirement.
4.2.2 Arc sine transformation consists of determining the angle (in radians)
represented by a sine value. In the case of arc sine square root
transformation of mortality data, the proportion of dead (or affected)
organisms is taken as the sine value, the square root of the sine value is
determined, and the angle (in radians) for the square root of the sine value
is determined. Whenever the proportion dead is 0 or 1, a special modification
of the arc sine square root transformation must be used (Bartlett, 1937). An
explanation of the arc sine square root transformation and the modification is
provided below.
I
4.2.3 Calculate the response proportion (RP) at each effluent concentration,
where:
! .
RP = (number of surviving or unaffected organisms)/(number exposed).
Example: If 12 of 20 animals in a given treatment replicate survive:
RP = 12/20
= 0.60
417
-------
4.2.4 Transform each RP to its arc sine square root, as follows:
4.2.4.1 For RPs greater than zero or less than one:
Angle (radians) = ^
Example: If RP = 0.60:
Angle = arc sine
/0.60
= arc sine 0.7746
= 0.8861 radians
4.2.4.2 Modification of the arc sine square root when RP = 0.
Angle (in radians) = arc sine
Where: N = Number of animals/treatment replicate
Example: If 20 animals are used:
a/so
Angle = arc sine
- arc sine 0.1118
« 0.1120 radians
4.2.4.3 Modification of the arc sine square root when RP = 0
Angle - 1.5708 radians - (radians for RP = 0)
Example: Using above value:
Angle = 1.5708 - 0.1120
= 1.4588 radians
418
-------
APPENDIX C
DUNNETT'S PROCEDURE
1. MANUAL CALCULATIONS
1.1 Dimnett's Procedure (Dunnett, 1955; Dunnett, 1964) is; used to compare
each concentration mean with the control mean to decide if any of the
concentrations differ from the control. This test has an overall error rate
of alpha, which accounts for the multiple comparisons with the control. It is
based on the assumptions that the observations are independent and normally
distributed and that the variance of the observations is homogeneous across
all concentrations and control. (See Appendix B for a discussion on
validating the assumptions). Dunnett's Procedure uses a pooled estimate of
the variance, which is equal to the error value calculated in an analysis of
variance. Dunnett's Procedure can only be used when the same number of
replicate test vessels have been used at each concentration and the control.
When this condition is not met, the t test with Bonferroni's adjustment is
used (see Appendix D).
I
1.2 The data used in this example are growth data from a Sheepshead Minnow
Larval Survival and Growth Test, and are the same data used in Appendices B
and D. These data are listed in Table C.I.
TABLE C.I. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL
GROWTH DATA (WEIGHT IN MG) USED FOR DUNNETT'S
PROCEDURE
Effluent
Cone (%)
Control
6.25
12.5
25.0
50.0
i
1
2
3
4
5
1
1
0
0
0
Replicate
1
.017
.157
.998
.873
.715
Test
0
0
0
0
0
2
.745
.914
.793
.935
.907
Vessel
3
0.
0.
1.
0.
1.
i
Total
862
992
021
839
044
(
2
3
2
2
2
i
.624
.063
.812
.647
,666
Mean
c
0
i
0
0
0
f,-)
.875
.021
.937
.882
.889
419
-------
1.3 One way to obtain an estimate of the pooled variance is to construct an
ANOVA table including all sums of squares, using the following formulas:
Where: p = number of effluent concentrations including the control:
the total sample size;
n- = the number of replicates for concentration "i"
Total Sum of Squares
SSB=LTi2/n±-G2/N Between Sum of Squares
^L Y±J2-G2/N
SSW=SST-SSB
Within Sum of Squares
6 = the grand total of all sample observations; G=2^Ti
T- = the total of the replicate measurements for concentration
i
N
the total sample size;
n- = the number of replicates for concentration i
Yr = the jth observation for concentration i
1.4 For the data in this example:
n, - n2 = n3 = n4 = n5 = 3
N = 20
Ti - Y-, + Y12 + Y13 = 2.624
T2 - Y21 + Y22 + Y23 - 3.063
YSI
+ Y + Y = 2.812
Y -f
2.647
TS - + + Y53 - 2.666
G = T, + T2 + T3 + T4 + T5 = 13.812
420
-------
12.922 - (13.812)715
0.204
= 12.763 - (13.812)715
= 0.045
SSW=SST-SSB
= 0.204 - 0.045
= 0.159
1.5 Summarize these data in the ANOVA table (Table C.2). j
TABLE C.2. ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source
Between
Within
Total
df Sum of Mean Square (MS)
Squares (SS) (SS/df)
P - 1 SSB SB = SSB/(p-l)
N - p SSW Sj = SSW/(N-p)
N - 1 SST
421
-------
1.6 Summarize data for ANOVA (Table C.3).
TABLE C.3. COMPLETED ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source
Total
df
SS
14
0.204
Mean Square
Between
Within
5-1=4
15 - 5 = 10
0.045
0.159
0.011
0.016
1.7 To perform the individual comparisons, calculate the t statistic for each
concentration and control combination, as follows:
I- (1/fll)
Where: Yf = mean for each concentration i.
Y, - mean for the control
SH = square root of the within mean square
n1 = number of replicates in the control.
n- - number of replicates for concentration i
422
-------
1.8 Table C.4 includes the calculated t values for each concentration and
control combination.
TABLE C.4. CALCULATED T VALUES
Effluent
Concentration i t.
6.25
12.5
25.0
50.0
2
3
4
5
- 1.414
- 0.600
- 0.068
- 0.136 !
1.9 Since the purpose of the test is only to detect a decrease in growth from
the control, a one-sided test is appropriate. The critical value for the
one-sided comparison (2.47), with an overall alpha level of 0.05, 10 degrees
of freedom and four concentrations excluding the control is read from the
table of Dunnett's "T" values (Table C.5; this table assumes an equal number
of replicates in all treatment concentrations and the control). Comparing
each of the calculated t values in Table C.4 with the critical value, no
decreases in growth from the control were detected. Thus the NOEC is 50.0%.
1.10 To quantify the sensitivity of the test, the minimum significant
difference (MSD) may be calculated. The formula is as follows:
MSD =
Where: d = critical value for the Dunnett's Procedure
Sw = the square root of the within mean square
n = the number of replicates at each concentration, assuming an
equal number of replicates at all treatment concentrations
n1 = number of replicates in the control
For example:
AffiD=2. 47 (0.126) [V(1/3)+(1/3)] =2.47(0.126)
= 2.47 (0.126)(0.816)
= 0.254
423
-------
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424
-------
1.11 For this set of data, the minimum difference between the control mean
and a concentration mean that can be detected as statistically significant is
0.254 mg. This represents a decrease in growth of 29% from the control.
1.11.1 If the data have not been transformed, the MSD (and the percent
decrease from the control mean that it represents) can be reported as is.
1.11.2 In the case where the data have been transformed, the MSD would be in
transformed units. In this case carry out the following conversion to
determine the MSD in untransformed units.
1.11.2.1 Subtract the MSD from the transformed control moan. Call this
difference D. Next, obtain untransformed values for the control mean and the
difference, D. !
MSDU = controlu - Du I
I
Where: MSDU = the minimum significant difference for untransformed data
Controlu = the untransformed control mean
Du = the untransformed difference |
•
1.11.2.2 Calculate the percent reduction from the control that MSDU
represents as:
MSDU !
Percent Reduction = X 100
Controlu i
i
1.11.3 An example of a conversion of the MSD to untransformed units, when the
arc sine square root transformation was used on the data, follows.
Step 1. Subtract the MSD from the transformed control mean. As an
example, assume the data in Table C.I were transformed by the arc
sine square root transformation. Thus:
0.875 - 0.254 = 0.621 i
i
Step 2. Obtain untransformed values for the control mean (0.875) and the
difference (0.621) obtained in Step 1, above.
[ Sine (0.875)]2 = 0.589
[ Sine (0.621)]2 = 0.339 j
I
Step 3. The untransformed MSD (MSDU) is determined by subtracting the
untransformed values obtained in Step 2.
MSDU = 0.589 - 0.339 = 0.250
425
-------
In this case, the MSD would represent a 42% decrease in survival from the
control [(0.250/0.589)(100)].
2. COMPUTER CALCULATIONS
2.1 This computer program incorporates two analyses: an analysis of variance
(ANOVA), and a multiple comparison of treatment means with the control mean
(Dunnett's Procedure). The ANOVA is used to obtain the error value.
Dunnett's Procedure indicates which toxicant concentration means (if any) are
statistically different from the control mean at the 5% level of significance.
The program also provides the minimum difference between the control and
treatment means that could be detected as statistically significant, and tests
the validity of the homogeneity of variance assumption by Bartlett's Test.
The multiple comparison is performed based on procedures described by Dunnett
(1955).
2.2 The source code for the Dunnett's program is structured into a series of
subroutines, controlled by a driver routine. Each subroutine has a specific
function in the Dunnett's Procedure, such as data input, transforming the
data, testing for equality of variances, computing p values, and calculating
the one-way analysis of variance.
2.3 The program compares up to seven toxicant concentrations against the
control, and can accommodate up to 50 replicates per concentration.
2.4 If the number of replicates at each toxicant concentration and control
are not equal, a t test with the Bonferroni adjustment is performed instead of
Dunnett's Procedure (see Appendix D).
2.5 The program was written in IBM-PC FORTRAN by Computer Sciences
Corporation, 26 W. Martin Luther King Drive, Cincinnati, OH 4526.8. A compiled
version of the program can be obtained from EMSL-Cincinnati by sending a
diskette with a written request.
2.6 DATA INPUT AND OUTPUT
2.6.1 Data on the number of surviving mysids, Mysidopsis bahia, from a
survival, growth and fecundity test (Table C.6) are used to illustrate the
data input and output for this program.
2.6.2 Data Input
2.6.2.1 When the program is entered, the user is asked to select the type of
data to be analyzed:
1. Response proportions, like survival or fertilization proportions data.
2. Counts and measurements, like offspring counts, cystocarp and algal cell
counts, weights, chlorophyll measurements or turbidity measurements.
426
-------
2.6.2.2 After the type of analysis for the data is chosen, the user has the
following options:
i
1. Create a data file
2. Edit a data file
3. Perform analysis on existing data set
4. Stop ' . .
i
2.6.2.3 When Option 1 (Create a data file) is selected for response
proportions, the program prompts the user for the following information:
1. Number of concentrations, including control .;
2. For each concentration and replicate:
- number of organisms exposed per replicate
- number of organisms responding per replicate (organisms surviving,
eggs fertilized, etc.) ;
I
2.6.2.4 After the data have been entered, the user may save the file on a
disk, and the program returns to the main menu (see below).
2.6.2.5 Sample data input is shown in Figure C.I.
2.6.3. Program Output
2.6.3.1 When Option 3 (perform analysis on existing data set) is selected
from the menu, the user is asked to select the transformation desired, and
indicate whether they expect the means of the test groups to be less or
greater than the mean for the control group (see Figure C.2),
,
2.6.3.2 Summary statistics (Figure C.3) for the raw and transformed data, if
applicable, the ANOVA table, results of Bartlett's Test, the results of the
multiple comparison procedure, and the minimum detectable difference are
included in the program output.
427
-------
TABLE C.6. SAMPLE DATA FOR DUNNETT'S PROGRAM FOR SURVIVING MYSIDS,
MYSIDOPSIS BAHIA
Treatment
1 Control
2 50 ppb
3 100 ppg
A 210 ppb
5 450 ppb
Replicate
Chamber
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Total
Mysids
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
No.
Alive
4
4
5
5
5
5
5
4
4
5
4
4
5
5
4
5
3
5
5
5
5
3
4
4
5
4
1
4
3
4
4
4
0
1
0
1
0
0
0
2
428
-------
EMSL Cincinnati Dunnett Software
Version 1.5
1) Create a data file
2) Edit a data file
3) Analyze an existing data set
4) Stop
Your choice ? 3 j
j
Number of concentrations, including control ? 5
Number of replicates for cone. 1 (the control) ? 8
i
replicate number of organisms exposed number of organisms responding
(organisms surviving, eggs
fertilized, etc.)
1
2
3
4
5
6
7
8
5
5
5
5
5
5
5
5
Number of replicates for cone. 2 ? 8
Do you wish to save the data on disk ? y
Disk file for output ? mysidsur.dat
4
'4
Figure C.I. Sample Data Input for Dunnett's Program for Survival Data from
Table C.6.
429
-------
EMSL Cincinnati: Dunnett Software
Version 1.5
1) Create a data file
2) Edit a data file
3) Analyze an existing data set
4) Stop
Your choice ? 3
File name ? mysidsur.dat
Available Transformations
1) no transform
2) square root
3) loglO
4) arcsine square root
Your choice ? 4
Dunnett's test as implemented in this program is a one-sided test. You must
specify the direction the test is to be run; that is, do you expect the means for
the test concentrations to be less than or greater than the mean for the control
concentration.
Direction for Dunnetts test : L=less than, 6=greater than ? 1
Cone.
Summary Statistics for Raw Data
n Mean s.d.
cv%
1
» control
2
3
4
5
8
8
8
8
8
.9250
.9000
.8500
.7250
.1000
.1035
.1069
.1773
.2375
.1512
11.2
11.9
20.9
32.8
151.2
Hysid Survival Example with Data in Table C.6
Figure C.2. Example of Choosing Option 3 from the Main Menu of the Dunnett
Program.
430
-------
Mysid Survival Example with Data in Table C.6
Summary Statistics and ANOVA |
Transformation = Arcsine Square Root
I
Mean s.d. cv%
1 = control
2
3
4*
5*
8
8
8
8
8
1.2560
1.2262
1.1709
1.0288
.3424
.1232 9.8
.1273 10.4
.2042
17.4
.2593 25.2
.1752 51.2
*) the mean for this cone, is significantly less than
the control mean at alpha = 0.05 (1-sided) by Dunnett's test
i
i
Minimum detectable difference for Dunnett's test = -.208074
This corresponds to a difference of -.153507 in original units
This difference corresponds to -16.98 percent of control
i
*' • i
Between concentrations
sum of squares = 4.632112 with 4 degrees of freedom.
Error mean square = .034208 with 35 degrees of freedom.
Bartlett's test p-value for equality of variances = .257
Do you wish to restart the program ? i
Figure C.3. Example of Program Output for the Dunnett's Program Using the
Survival Data in Table C.6.
431
-------
APPENDIX D
T TEST WITH BONFERRONI'S ADJUSTMENT
1. The t test with Bonferroni's adjustment is used as an alternative to
Dunnett's Procedure when the number of replicates is not the same for all
concentrations. This test sets an upper bound of alpha on the overall error
rate, in contrast to Dunnett's Procedure, for which the overall error rate is
fixed at alpha. Thus, Dunnett's Procedure is a more powerful test.
2. The t test with Bonferroni's adjustment is based on the same assumptions
of normality of distribution and homogeneity of variance as Dunnett's
Procedure (See Appendix B for testing these assumptions), and, like Dunnett's
Procedure, uses a pooled estimate of the variance, which is equal to the error
value calculated in an analysis of variance.
3. An example of the use of the t test with Bonferroni's adjustment is
provided below. The data used in the example are the same as in Appendix C,
except that the third replicate from the 50% effluent treatment is presumed to
have been lost. Thus, Dunnett's Procedure cannot be used. The weight data
are presented in Table D.I.
TABLE D.I. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH
DATA (WEIGHT IN MG) USED FOR THE T TEST WITH BONFERRONI'S
ADJUSTMENT
Ef f 1 uent
Cone {%)
Control
6.25
12.5
25.0
50.0
i
1
2
3
4
5
Replicate Test Vessel
1
1.017
1.157
0.998
0.873
0.715
2
0.745
0.914
0.793
0.935
0.907
3
0.862
0.992
1.021
0.839
(Lost)
Total
(T,-)
2.624
3.063
2.812
2.647
1.622
Mean
-------
n,- = the number of replicates for concentration i
Total Sum of Squares
Between Sum of Squares
SSW=SST-SSB
Within Sum of Squares
Where:
The grand total of all sample observations;
T,- = The total of the replicate measurements for
concentration i
YfJ. = The jth observation for concentration i
3.2 For the data in this example:
ni =
= n3 = n4 = 3
N - 20
ill
+ Y12 + Y13 = 2.624
+ Y22 + Y23 = 3.063
+ Y32 + Y33 = 2.812
+ Y,, + Y7, = 2.647
= 1.622
! = T1 + T2 + T3 + T4 + T5 = 12.768
SSB=ETi2/ni-G2/N
±1
11.709 - (12.768)714
0.064
Y±J2-G2/N
11.832 - (12.768)714
0.188
433
-------
SSW=SST-SSB
= 0.188 - 0.064
= 0.124
3.3 Summarize these data in the ANOVA table (Table D.2):
TABLE D.2. ANOVA TABLE FOR BONFERRONI'S ADJUSTMENT
Source df
Between p - 1
Within N - p
Total N - 1
Sum of
Squares (SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
SB = SSB/(p-l) .
S* = SSW/(N-p)
3.4 Summarize these calculations in the ANOVA table (Table D.3):
TABLE D.3. COMPLETED ANOVA TABLE FOR THE T TEST WITH BONFERRONI'S
ADJUSTMENT
Source
Between
Within
df
5-1=4
14 - 5 = 9
SS
0.064
0.124
Mean Square
0.016
0.014
Total
13
0.188
434
-------
3.5 To perform the individual comparisons, calculate the 't statistic for each
concentration and control combination, as follows:
Where: Y, = mean for concentration i
Y, = mean for the control
Sw = square root of the within mean square
n1 = number of replicates in the control.
n,- = number of replicates for concentration i.
3.6 Table D.4 includes the calculated t values for each concentration and
control combination. !
TABLE D.4. CALCULATED T VALUES
Effluent
Concentration
6.25
12.5
25.0
50.0
2
3
4
5
- 1.511
- 0.642
- 0.072
- 0.592
3.7 Since the purpose of the test is only to detect a decrease in growth from
the control, a one-sided test is appropriate. The critical value for the
one-sided comparison (2.686), with an overall alpha level of 0.05, nine
degrees of freedom and four concentrations excluding the control, was obtained
from Table D.5. Comparing each of the calculated t values in Table D.4 with
the critical value, no decreases in growth from the control were detected.
Thus the NOEC is 50.0%.
435
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APPENDIX E
STEEL'S MANY-ONE RANK TEST
1. Steel's Many-one Rank Test is a nonparametric test for comparing
treatments with a control. This test is an alternative to Dunnett's
Procedure, and may be applied to data when the normality assumption has not
been met. Steel's Test requires equal variances across the treatments and the
control, but it is thought to be fairly insensitive to deviations from this
condition (Steel, 1959). The tables for Steel's Test require an equal number
of replicates at each concentration. If this is not the case, use Wilcoxon's
Rank Sum Test, with Bonferroni's adjustment (See Appendix F).
2. For an analysis using Steel's Test, for each control and concentration
combination, combine the data and arrange the observations in order of size
from smallest to largest. Assign the ranks to the ordered observations (1 to
the smallest, 2 to the next smallest, etc.). If ties occur in the ranking,
assign the average rank to the observation. (Extensive ties would invalidate
this procedure). The sum of the ranks within each concentration and within
the control is then calculated. To determine if the response in a
concentration is significantly different from the response in the control, the
minimum rank sum for each concentration and control combination is compared to
the significant values of rank sums given later in the section. In this
table, k equals the number of treatments excluding the control and n equals
the number of replicates for each concentration and the control.
3. An example of the use of this test is provided below. The test employs
survival data from a mysid 7-day, chronic test. The data are listed in Table
E.I. Throughout the test, the control data are taken from the site water
control. Since there is 0% survival for all eight replicates for the 50%
concentration, it is not included in this analysis and is considered a
qualitative mortality effect.
4. For each control and concentration combination, combine the data and
arrange the observations in order of size from smallest to largest. Assign
the ranks (1, 2, 3, ..., 16) to the ordered observations (1 to the smallest, 2
to the next smallest, etc.). If ties occur in the ranking, assign the average
rank to each tied observation.
5. An example of assigning ranks to the combined data for the control and
3.12% effluent concentration is given in Table E.2. This ranking procedure is
repeated for each control and concentration combination. The complete set of
rankings is listed in Table E.3. The ranks are then summed for each effluent
concentration, as shown in Table E.4.
6. For this set of data, determine if the survival in any of the effluent
concentrations is significantly lower than the survival of the control
organisms. If this occurs, the rank sum at that concentration would be
significantly lower than the rank sum of the control. Thus, compare the rank
sums for the survival at each of the various effluent concentrations with some
"minimum" or critical rank sum, at or below which the survival would be
considered to be significantly lower than the control. At a probability level
438
-------
TABLE E.I. EXAMPLE OF STEEL'S MANY-ONE RANK TEST:
MYSIDOPSIS BAHIA, 7-DAY CHRONIC TEST
DATA FOR MYSID,
Effluent
Concentration
Start of Test
Control
-------
TABLE E.2. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: ASSIGNING
RANKS TO THE CONTROL AND 3.12% EFFLUENT CONCENTRATIONS
Rank
1
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
14
14
14
14
14
Number of Live
My s ids, Mysidopsis bahia
3 .
4
4
4
4
4
4
4
4
• 4
4
5
5
5
5
5
Control or % Effluent
3.12
Control
Control
Control
Control
Control
3.12
3.12
3.12
3.12
3.12
Control
Control
Control
3.12
3.12
TABLE E.3. TABLE OF RANKS
Repl
icate
Chamber Control
1
2
3
4
5
6
7
8
4 (6.5,6,6.5,5)
4 (6.5,6,6.5,5)
5 (14,13.5,13.5,12.5)
4 (6.5,6,6.5,5)
5 (14,13.5,13.5,12.5)
4 (6.5,6,6.5,5)
4 (6.5,6,6.5,5)
5 (14,13.5,13.5,12.5)
Effluent Concentration (%)
3
4
4
4
5
4
4
5
3
.12
(6.5)
(6.5)
(6.5)
(14)
(6.5)
(6.5)
(14)
(1)
3
4
5
4
4
4
5
5
6.25
(1)
(6)
(13.5)
(6)
(6)
(6)
(13.5)
(13.5)
12.5
5
4
5
3
5
4
4
3
(13
(6.
(13
(1.
(13
(6.
(6.
(1.
.5)
5)
.5)
5)
.5)
5)
5)
5)
5
5
5
5
3
5
4
4
25.0
(12
(12
(12
(12
(1)
12.
(5)
(5)
.b)
.5)
.5)
.5)
5)
Control ranks are given in the order of the concentration with which
they were ranked.
440
-------
TABLE E.4. RANK SUMS
Effluent
Concentration
Rank Sum
3.12
6.25
12.50
25.00
61.5
65.5
63.0
73.5
of 0.05, the critical rank sum in a test with four concentrations and eight
replicates per concentration, is 47 (see Table F.4).
i
7. Of the rank sums in Table E.4, none are less than 47. Therefore, due to
the qualitative effect at the 50% effluent concentration, the NOEC is 25%
effluent and the LOEC is 50% effluent.
441
-------
TABLE E.5. SIGNIFICANT VALUES OF RANK SUMS: JOINT CONFIDENCE
COEFFICIENTS OF 0.95 (UPPER) and 0.99 (LOWER) FOR
ONE-SIDED ALTERNATIVES (Steel, 1959)
n
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
2
11
18
15
27
23
37
32
49
43
63
56
79
71
97
87
116
105
138
125
161
147
186
170
213
196
241
223
272
252
304
282
339
315
k =
3
10
17
--
26
22
36
31
48
42
62
55
77
69
95
85
114
103
135
123
158
144
182
167
209
192
237
219
267
248
299
278
333
310
number of
4
10
17
--
25
21
35
30
47
41
61
54
76
68
93
84
112
102
133
121
155
142
180
165
206
190
234
217
264
245
296
275
330
307
treatments
5
10
16
--
25
21
35
30
46
40
60
53
75
67
92
83
111
100
132
120
154
141
178
164
204
188
232
215
262
243
294
273
327
305
6
10
16
--
24
--
34
29
46
40
59
52
74
66
91
82
110
99
130
119
153
140
177
162
203
187
231
213
260
241
292
272
325
303
(excluding
7
--
16
--
24
--
34
29
45
40
59
52
74
66
90
81
109
99
129
118
152
139
176
161
201
186
229
212
259
240
290
270
323
301
control )
8
--
16
--
24
--
33
29
45
39
58
51
73
65
90
81
108
98
129
117
151
138
175
160
200
185
228
211
257
239
288
268
322
300
9
--
15
--
23
--
33
29
44
39
58
51
72
65
89
80
108
98
128
117
150
137
174
160
199
184
227
210
256
238
287
267
320
299
442
-------
APPENDIX F i
WILCOXON RANK SUM TEST
i
1. Wilcoxon's Rank Sum Test is a nonparametric test, to be used as an
alternative to Steel's Many-one Rank Test when the number of replicates are
not the same at each concentration. A Bonferroni's adjustment of the pairwise
error rate for comparison of each concentration versus the control is used to
set an upper bound of alpha on the overall error rate, in contrast to Steel's
Many-one Rank Test, for which the overall error rate is fixed at alpha. Thus,
Steel's Test is a more powerful test.
j
2. The use of this test may be illustrated with fecundity data from the mysid
test in Table F.I. The site water control and the 12.5% effluent •
concentration each have seven replicates for the proportion of females bearing
eggs, while there are eight replicates for each of the remaining three
concentrations.
3. For each concentration and control combination, combine the data and
arrange the values in order of size, from smallest to largest. Assign ranks
to the ordered observations (a rank of 1 to the smallest, 2 to the next
smallest, etc.). If ties in rank occur, assign the average rank to each tied
observation. ;
i
4. An example of assigning ranks to the combined data for the control and
effluent concentration 3.12% is given in Table F.2. This ranking procedure is
repeated for each of the three remaining control versus test concentration
combinations. The complete set of ranks is listed in Tab'le F.3. The ranks
are then summed for each effluent concentration, as shown in Table F.4.
5. For this set of data, determine if the fecundity in any of the test
concentrations is significantly lower than the fecundity in the control. If
this occurs, the rank sum at that concentration would be significantly lower
than the rank sum Thus, compare the rank sums for fecundity of each of the
various effluent concentrations with some "minimum" or critical rank sum, at
or below which the fecundity would be considered to be significantly lower
than the control. At a probability level of 0.05, the critical rank in a test
with four concentrations and seven replicates in the control is 44 for those
concentrations with eight replicates, and 34 for those concentrations with
seven replicates (see Table F.5, for K = 4). i
6. Comparing the rank sums in Table F.4 to the appropriate critical rank,
only the 25% effluent concentration does not exceed its critical value of 44.
Thus, the NOEC and LOEC for fecundity are 12.5% and 25%, respectively.
443
-------
TABLE F.I. EXAMPLE OF WILCOXON'S RANK SUM TEST: FECUNDITY DATA FOR
MYSID, MYSIDOPSIS BAHIA, 7-DAY CHRONIC TEST
Effluent
Concentration
Control
(Site Water)
Control
(Brine &
Dilution Water)
3.12%
6.25%
2.5X
25. OX
50.0X
Replicate
Chamber
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Number of
Mysids at
Start of Test
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Number of
Live Mysids
at End of Test
4
4
5
4
5
4
4
5
3
5
3
3
4
4
3
3
4
4
4
5
4
4
5
3
3
4
5
4
4
4
5
5
5
4
5
3
5
4
4
3
5
5
5
5
3
5
4
4
0
0
0
0
0
0
0
0
Proportion
of Females
with Eggs
0.50
0.75
0.67
0.67
0.50
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.50
0.50
0.50
1.00
0.50
0.67
1.00
0.50
1.00
1.00
0.00
0.50
0.00
0.75
1.00
1.00
1.00
0.67
0.67
0.33
0.50
1.00
1.00
0.00
0.33
0.50
0.00
0.50
0.13
0.00
0.50
0.00
0.50
0.50
444
-------
TABLE F.2. EXAMPLE OF WILCOXON'S RANK SUM TEST: ASSIGNING RANKS TO THE
CONTROL AND 3.12% EFFLUENT CONCENTRATIONS
Rank
Proportion of
Females W/Eggs
Site Water Control
or Effluent
1
3.5
3.5
3.5
3.5
7
7
7
9
12.5
12.5
12.5
12.5
12.5
12.5
0.00
0.50
0.50
0.50
0.50
0.67
0.67
0.67
0.75
1.00
1.00
1.00
1.00
1.00
1.00
3.12
Control
Control
3.12
3.12
Control
Control
3.12
Control
Control
Control
3.12
3.12
3.12
3.12
445
-------
TABLE F.3. TABLE OF RANKS
1
Rep
1
2
3
4
5
6
7
8
Proper- Site Water
tlon Control Rank '
0.50
0.75
0.67
0.67
0.50
1.00
1.00
(3.5,3,5.5,7.5)
— —
(9,9.5,10,13)
(7,6.5,8.5,11.5)
(7,6.5,8.5,11.5)
(3.5,3,5.5,7.5)
(12.5,13,12.5,14.5)
(12.5,13,12.5,12.5)
Effluent Concentration (%V
3.12
1.00 (12.5)
0.50 (3.5)
0.67 (7)
1.00 (12.5)
0.50 (3.5)
1.00 (12.5)
1.00 (12.5)
0.00 (1)
6.25
0.50 (3)
0.00 (1)
0.75 (9.5)
1.00 (13)
1.00 (13)
1.00 (13)
0.67 (6.5)
0.67 (6.5)
12.5
0.33 (2.5)
0.50 (5.5)
1.00 (12.5)
'
1.00 (12.5)
0.00 (1)
0.33 (2.5)
0.50 (5.5)
25.0
0.00 (2)
0.50 (7.5)
0.33 (4)
0.00 (2)
0.50 (7.5)
0.00 (2)
0.50 (7.5)
0.50 (7.5)
'Control ranks are given in the order of the concentration with which they, were ranked.
TABLE F.4. RANK SUMS
Effluent
Concentration
Rank Sum
No. of
Replicates
Critical
Rank Sum
3.12
6.25
12.50
25.00
65
65.5
42
40
8
8
7
8
44
44
34
44
446
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH
BONFERRONI'S ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K"
TREATMENTS VERSUS A CONTROL FIVE PERCENT CRITICAL LEVEL
(ONE-SIDED ALTERNATIVE: TREATMENT CONTROL)
K No. Replicates No. of Reolicates Per Effluent
in Control
1 3
4
5
6
7
8
9
10
2 3
4
5
6
7
8
9
10
3 3
4
5
6
7
8
9
10
4 3
4
5
6
7
8
9
10
3
6
6
7
8
8
9
10
10
...
--
6
7
7
8
8
9
.,
--
--
6
7
7
7
8
..
--
--
.6
6
7
7
7
4
10
11
12
13
14
15
16
17
10
11
12
13
14
14
15
„
10
11
11
12
13
13
14
--
10
11
12
12
13
14
5
16
17
19
20
21
23
24
26
15
16
17
18
20
21
22
23
16
17
18
19
20
21
22
15
16
17
18
19
20
21
6
23
24
26
28
29
31
33
35
22
23
24
26
27
29
31
32
21
22
24
25
26
28
29
31
21
22
23
24
26
27
28
30
7
30
32
34
36
39
41
43
45
29
31
33
34
36
38
40
42
29
. 30
32
33
35
37
39
41
28
30
31
33
34
36
38
40
8
39
41
44
46
49
51
54
56
38
40
42
44
46
49,
51
• 53
37
39
41
43
45
47
49
51
37
38
40
42
44
46
48
50
Concentration
9
49
51
54
57
60
63
66
69
47
49
52
55
57
60
62
65
46
48
51
53
56
58
61
63
46
48
50
52
55
57
60
62
10
59
62
66
69
72
72
79
82
58
60
63
66
69
72
75
78
57
59
62
65
68
70
73
76
56
59
•61
64
67
69
72
75
447
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH BONFERRONI'S
ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K" TREATMENTS
VERSUS A CONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED
ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)
K No. Replicates No. of Reolicates Per Effluent
in Control
5 3
4
5
6
7
8
9
10
6 3
4
5
6
7
8
9
10
7 3
4
5
6
7
8
9
10
8 3
4
5
6
7
8
9
10
3
__
__
__
6
6
7
7
_.
__
__
6
6
6
7
__
--
-_
_-
6
6
7
__
--
--
__
6
6
6
4
-_
10
11
11
12
13
13
_-
10
11
11
12
12
13
--
--
10
11
11
12
13
_-
--
10
11
11
12
12
5
15
16
17
18
19
20
21
15
16
16
17
18
19
20
--
15
16
17
18
19
20
--
15
16
17
18
19
19
6
22
23
24
25
27
28
29
21
22
24
25
26
27
.29
_.
21
22
23
25
26
27
28
21
22
23
24
25
27
28
7
28
29
31
32
34
35
37
39
28
29
30
32
33
35
37
38
..
29
30
32
33
35
36
38
..
29
30
31
33
34
36
37
8
36
38
40
42
43
45
47
49
36
38
39
41
43
45
47
49
36
37
39
41
43
44
46
48
36
37
39
40
42
44
46
48
Concentration
9
46
48
50
52
54
56
59
61
45
47
49
51
54
56
58
60
45
47
49
51
53
55
58
60
45
47
49
51
53
55
57
59
10
56
58
61
63
66
68
71
74
56
58
60
63
65
68
70
73
56
58
60
62
65
67
70
72
55
57
59
62
64
67
69
72
448
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH BONFERRONI'S
ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K" TREATMENTS
VERSUS A CONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED
ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)
K No. Replicat
in Control
9 3
4
5
6
7
8
9
10
10 3
4
5
6
7
8
9
10
es No. of Reolicates Per Effluent
3 4
..
__
__
10
10
11
6 11
6 12
..
__
__
10
10
11
6 11
6 12
5
--
15
16
17
18
18
19
--
15
16
16
17
18
19
6
21
22
23
24
25
26
28
21
22
23
24
25
26
27
7
28
30
31
33
34
35
37
28
29
31
32
34
35
37
' IB
I
37
39
40
42
44
46
47
1_
37
38
40
42
43
45
47
.
Concentration
9
45
46
48
50
52
55
57
59
45
46
48
50
52
54
56
58
10
55
57
59
62
64
66
69
71
55
57
59
61
64
66
68
71
449
-------
APPENDIX G
SINGLE CONCENTRATION TOXICITY TEST - COMPARISON OF CONTROL
WITH 100% EFFLUENT OR RECEIVING WATER
1. To statistically compare a control with one concentration, such as 100%
effluent or the instream waste concentration, a t test is the recommended
analysis. The t test is based on the assumptions that the observations are
independent and normally distributed and that the variances of the
observations are equal between the two groups.
2. Shapiro-Wilk's test may be used to test the normality assumption (See
Appendix B for details). If the data do not meet the normality assumption,
the nonparametric test, Wilcoxon's Rank Sum Test, may be used to analyze the
data. An example of this test is given in Appendix F. Since a control and
one concentration are being compared, the K = 1 section of Table F.5 contains
the needed critical values.
3. The F test for equality of variances is used to test the homogeneity of
variance assumption. When conducting the F test, the alternative hypothesis
of interest is that the variances are not equal.
4. To make the two-tailed F test at the 0.01 level of significance, put the
larger of the two variances in the numerator of F.
F =
si
where
5. Compare F with the 0.005 level of a tabled F value with n., - 1 and n2 - 1
degrees of freedom, where n, and n2 are the number of replicates for each of
the two groups.
6. A set of mysid growth data from an effluent (single concentration) test
will be used to illustrate the F test. The raw data, mean and variance for
the control and 100% effluent are given in Table 6.1.
7. Since the variability of the 100% effluent is greater than the variability
of the control, S for the 100% effluent concentration is placed in the
numerator of the F statistic and S for the control is placed in the
denominator.
F= °-00131 =1.52
0.000861
8. There are 8 replicates for the effluent concentration and 8 replicates for
the control. Thus, both numerator and denominator degrees of freedom are
equal to 7. For a two-tailed test at the 0.01 level of significance, the
450
-------
TABLE 6.1. MYSID, MYSIDOPSIS BAHIA, GROWTH DATA FROM AM EFFLUENT (SINGLE
CONCENTRATION) TEST
Replicate
1
8
Control 0.183 0.148 0.216 0.199 0.176 0.243 0.213 0.180 0.195 0.000861
100% --..,.. ,..:-•. ;
Effluent 0.153 0.117 0.085 0.153 0..086 0.193 0.137 0.129 0.132 0.00131
critical F value is obtained from a table of the F distribution (Snedecor and
Cochran, 1980). The criticalF value for this test is 8.89. Since 1.52 is
not greater thani 8.89, the conclusion is that the variances of the control and
100% eff1uent are homogeneous. . ; ,
9. Equal Variance T Test, ... ! .;.'.-
,,.,..• . i
9.1 To perform the t test, calculate the following test statistic:
Where:
: t =
n2
= mean for the control
= mean for the effluent concentration
SP =
ni+n2-2
S^ = estimate of the variance for the control
-- ' '. . , i
S2 = estimate of the variance for the effluent
concentration |
n1 = number of replicates for the control
n2 = number of replicates for the effluent
concentration
451
-------
9 2 Since we are usually concerned with a decreased response from the
control, such as a decrease in survival or a decrease in reproduction, a.
one-tailed test is appropriate. Thus, you would compare the calculated t with
a critical t, where the critical t is at the 5% level of significance with n,
+ n2 - 2 degrees of freedom. If the calculated t exceeds the critical t, the
mean responses are declared different.
9.3 Using the data from Table G.I to illustrate the t test, the calculation
of t is as follows:
t = 0.195-0.132 = 3>83
0.0329* -=•+-=•
_ ,/(8-l)0.000861+(8-l) 0.00131 ^n
Where:
9 4 For an 0.05 level of significance test with 14 degrees of freedom, the
critical t is 1.762 (Note: Table D.5 for K = 1 includes the critical t values
for comparing two groups). Since 3.83 is greater than 1.762, the cone usion
is that the growth for the 100% effluent concentration is significantly lower
than growth for the control.
10. UNEQUAL VARIANCE T TEST.
10 1 If the F test for equality of variance fails, the t test is still a
valid test. However, the denominator of the t statistic is adjusted as
follows:
Where:
Y, =
mean for the control
mean for the effluent concentration
estimate of the variance for the control
estimate of the variance for the effluent
concentration
number of replicates for the control
number of replicates for the effluent
concentration
452
-------
I
10.2 Additionally, the degrees of freedom for the test are adjusted using the
following formula: I
df'=-
-l) C2+(i-c)
Where:
C =
ni n2 ;
|
10.3 The modified degrees of freedom is usually not an integer. Common
practice is to round down to the nearest integer. i
10.4 The t test is then conducted as the equal variance t test. The
calculated t is compared to the critical t at the 0.05 significance level with
the modified degrees of freedom. If the calculated t exceeds the critical t,
the mean responses are found to be statistically different.
453
-------
APPENDIX H
PROBIT ANALYSIS
1. This program calculates the EC1 and EC50 (or LCI and LC50), and the
associated 95% confidence intervals.
2. The program is written in IBM PC Basic for the IBM compatible PC by
Computer Sciences Corporation, 26 W. Martin Luther King Drive, Cincinnati, OH
45268 A compiled, executable version of the program and supporting
documentation can be obtained from EMSL-Cincinnati by sending a written
request to EMSL at 3411 Church Street, Cincinnati, OH 45244.
2 1 Data input is illustrated by a set of mortality data (Figure H.I) from a
sheepshead minnow embryo-larval survival and teratogenicity test. The program
begins with a request for the following information:
1. Desired output of abbreviated (A) or full (F) output? (Note: only
abbreviated output is shown below.)
2. Output designation (P = printer, D = disk file).
3. Title for the output.
4. The number of exposure concentrations.
5. Toxicant concentration data.
2.2 The program output for the abbreviated output includes the following:
1. A table of the observed proportion responding and the proportion
responding adjusted for the controls (see Figure H.2)
2. The calculated chi-square statistic for heterogeneity and the
tabular value. This test is one indicator of how well the data fit
the model. The program will issue a warning when the test
indicates that the data do not fit the model.
3. The estimated LCI and LC50 values and associated 95% confidence
intervals (see Table H.2).
454
-------
Do you wish abbreviated (A) or full (F) input/output? A
Output to printer (P) or disk file (D)? P
Title ? Example of Probit Analysis
Number responding in the control group = ? 17
Mumbep of animals exposed in the concurrent control group = ? 100
Number of exposure concentrations, exclusive of controls ? 5
Input data starting with the lowest exposure concentration
Concentration = ? 6.25
Number responding = ? 14
Number exposed = ? 100
Concentration = ? 12.5
Number responding =' ? 16
Number exposed = ? 102
Concentration = ? 25.0
Number responding = ? 35
Number exposed = ? 100
Concentration = ? 50.0
Number responding = ? 72
Number exposed = ? 99
Concentration = ? 100
Number responding = ? 99
Number exposed = ? 99
Number
Cone.
6.2500
12.5000
25.0000
50.0000
100.0000
Number
Resp.
16
35
72
99
Number
Exposed
100
102
100
99
99
Do you wish to modify your data ? N
The number of control animals which responded
The number of control animals exposed = 100
Do you wish to modify these values ? N
= 17
Figure H.I. Sample Data Input for USEPA Probit Analysis Program,
Version 1.5.
455
-------
Example of Probit Analysis
Cone.
Control
6.2500
12.5000
25.0000
50.0000
100.0000
Number
Exposed
100
100
102
100
99
99
Number
Resp.
17
14
16
35
72
99
Observed
Proportion
Responding
0.1700
0.1400
0.1569
0.3500
0.7273
1.0000
Proportion
Responding
Adjusted for
Controls
0.0000
0.0201
0.0001
0.2290
0.6765
1.0000
Chi - Square for Heterogeneity (calculated)
Chi - Square for Heterogeneity
(tabular value at 0.05 level)
3.472
7.815
Example of Probit Analysis
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
12.917
37.667
Lower Upper
95% Confidence Limits
8.388
32.898
16.888
42.081
Figure H.2. USEPA Probit Analysis Program used for Calculating LC/EC
Values, Version 1.5.
456
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APPENDIX I
SPEARMAN-KARBER METHOD
i
1. The Spearman-Karber Method is a nonparametric statistical procedure for
estimating the LC50 and the associated 95% confidence interval (Finney, 1978).
The Spearman -Karber Method estimates the mean of the distribution of the Iog10
of the tolerance. If the log tolerance distribution is symmetric, this
estimate of the mean is equivalent to an estimate of the median of the log
tolerance distribution.
2. If the response proportions are not monotonically non-decreasing with
increasing concentration (constant or steadily increasing with concentration),
the data must be smoothed. Abbott's procedure is used to "adjust" the
concentration response proportions for mortality occurring in the control
replicates.
3. Use of the Spearman -Karber Method is recommended when partial mortalities
occur in the test solutions, but the data do not fit the Rrobit model.
4. To calculate the LC50 using the Spearman-Karber Method, the following must
be true: 1) the smoothed adjusted proportion mortality for the lowest
effluent concentration (not including the control) must be zero, and 2) the
smoothed adjusted proportion mortality for the highest effluent concentration
must be one.
5. To calculate the 95% confidence interval for the LC50 estimate, one or
more of the smoothed adjusted proportion mortalities must be between zero and
one.
6. The Spearman-Karber Method is illustrated below using a set of mortality
data from a Sheepshead Minnow Larval Survival and Growth test. These data are
listed in Table I.I.
•I
7. Let p0, p1} ..., pk denote the observed response proportion mortalities
for the control and k effluent concentrations. The first step is to smooth
the p. if they do not satisfy p0 < p1 < ... < pk. The smoothing process
replaces any adjacent pt's that do not conform to p0 < p, < ... < p. with their
average. For example, if p. is less than p,., then:
P/-I = Pi =
Where: p? = the smoothed observed proportion mortality for effluent
concentration i.
7.1 For the data in this example, because the observed mortality proportions
for the control and the 6.25% effluent concentration are greater than the
observed response proportions for the 12.5% and 25.0% effluent concentrations,
the responses for these four groups must be averaged:
457
-------
s s s
Po =Pi =Pa =
0.05 + 0.05+0.00+0.00 _ 0.10 _
-- --
TABLE I.I. EXAMPLE OF SPEARMAN-KARBER METHOD: MORTALITY DATA FROM
A SHEEPSHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST (40
ORGANISMS PER CONCENTRATION)
Ef f 1 uent
Concentration
Control
6.25
12.5
25.0
50.0
100.0
Number of
Mortalities
2
2
0
0
26
40
Mortality
Proportion
0.05
0.05
0.00
0.00
0.65
1.00
7.2 Since p. = 0.65 is larger than pj, set p^ = 0.65. Similarly, p5 = 1.00 is
larger than p£, so set p| = 1.00. Additional smoothing is not necessary. The
smoothed observed proportion mortalities are shown in Table 1.2.
TABLE 1.2. EXAMPLE OF SPEARMAN-KARBER METHOD: SMOOTHED, ADJUSTED
MORTALITY DATA FROM A SHEEPSHEAD MINNOW LARVAL SURVIVAL AND
GROWTH TEST
Effluent
Concentration
01
/o
Control
6.25
12.5
25.0
50.0
100.0
Mortality
Proportion
0.05
0.05
0.00
0.00
0.65
1.00
Smoothed
Mortality
Proportion
0.025
0.025
0.025
0.025
0.650
1.000
Smoothed,
Adjusted
Mortality
Proportion
0.000
0.000
0.000
0.000
0.641
1.000
8. Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney, 1971). The adjustment takes the form:
Where: pj - (p' - p*) / (1 - p?)
458
-------
PO = the smoothed observed proportion mortality for the control
i
pf = the smoothed observed proportion mortality for effluent
concentration i.
i
8.1 For the data in this example, the data for each effluent concentration
must be adjusted for control mortality using Abbott's formula, as follows:
= 0.650-0.025 = 0.0625
1-0.025
0.975
a _ P5S~PoS _ 1.000-0.025 0.975
-- —
1-P0S
1-0.025
0.975
nnn
= 1.000
The smoothed, adjusted response proportions for the effluent concentrations
are shown in Table 1.2. A plot of the smoothed, adjusted data is shown in
Figure I.I.
9. Calculate the Iog10 of the estimated LC50, m, as follows:
m =
Where: p° = the smoothed adjusted proportion mortality at concentration i
Xf = the Iog10 of concentration i
k = the number of effluent concentrations tested, not including the
control. i
9.1 For this example, the Iog10 of the estimated LC50, m, is calculated as
follows:
m = [(0.000 - 0.000) (0.7959 + 1.0969)]/2 + '
[(0.000 - 0.000) (1.0969 + 1.3979)]/2 +
[(0.641 - 0.000) (1.3979 + 1.6990)]/2 +
[(1.000 - 0.641) (1.6990 + 2.0000)]/2
= 1.656527
459
-------
o
-o
rO
QJ
.c
00
ca.
O)
OJ
s-
o
00
c
o
S-
o
o.
o
Q_
o
Q.
O)
S-
•o
-a s-
O) 3
J= 00
+->
O -
o co
E 3
oo •*->
ro
Q) -I—
> s~
s- a>
CD ^
00
4- O
o c
ID
S_
cn
460
-------
10. Calculate the estimated variance of m as follows:
' v(a) -
1-2
Where: X,- = the Iog10 of concentration i
n,- = the number of organisms tested at effluent concentration i
pf = the smoothed adjusted observed proportion mortality at effluent
concentration i
k = the number of effluent concentrations tested, not including the
control .
10.1 For this example, the estimated variance of m, V(m)., is calculated as
f ol 1 ows :
V(m) = (0.000)(1.000)(1.3979 - 0.7959)?/4(39)
(0.000)(1.000)(1.6990 - 1.0969)74(39)
(0.641)(0.359)(2.0000 - 1.3979)74(39)
rf^jy;
+
= 0.00053477
I
11. Calculate the 95% confidence interval for m: m ± 2.0
11.1 For this example, the 95% confidence interval for m is calculated as
follows:
• 1.656527 ±2^0.00053477 = (1.610277, 1.702777)
12. The estimated LC50 and a 95% confidence interval for the estimated LC50
can be found by taking base10 antilogs of the above values.
12.1 For this example, the estimated LC50 is calculated as follows:
LC50 = antilog(m) = antilog(l.656527) = 45.3%.
12.2 The limits of the 95% confidence interval for the estimated LC50 are
calculated by taking the antilogs of the upper and lower limits of the 95%
confidence interval for m as follows: I
j
lower limit: antilog(l.610277) = 40.8%
upper limit: antilog(l.702777) = 50.4%
461
-------
APPENDIX J
TRIMMED SPEARMAN-KARBER METHOD
1. The Trimmed Spearman-Karber Method is a modification of the Spearman-
Karber Method, a nonparametric statistical procedure for estimating the LC50
and the associated 95% confidence interval (Hamilton, et al, 1977). The
Trimmed Spearman-Karber Method estimates the trimmed mean of the distribution
of the log1Q of the tolerance. If the log tolerance distribution is
symmetric, this estimate of the trimmed mean is equivalent to an estimate of
the median of the log tolerance distribution.
E. If the response proportions are not monotonically non-decreasing with
increasing concentration (constant or steadily increasing with concentration),
the data must be smoothed. Abbott's procedure is used to "adjust" the
concentration response proportions for mortality occurring in the control
replicates.
3. Use of the Trimmed Spearman-Karber Method is recommended only when the
requirements for the Probit Analysis and the Spearman-Karber Method are not
met.
4. To calculate the LC50 using the Trimmed Spearman-Karber Method, the
smoothed, adjusted, observed proportion mortalities must bracket 0.5.
5. To calculate the 95% confidence interval for the LC50 estimate, one or
more of the smoothed, adjusted, observed proportion mortalities must be
between zero and one.
6 Let PO, p«, ..., pk denote the observed proportion mortalities for the
control and the k effluent concentrations. The first step is to smooth the p5
if they do not satisfy p0 < p, < ... < pk. The smoothing process replaces any
adjacent p^s that do not conform to p0 < p, < ... < pk, with their average.
For example, if p,- is less than pM then:
Where:
Pi-1
Pi
- Pi = (Pi + Pi-i)/2
= the smoothed observed proportion mortality for effluent
concentration i.
7. Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney, 1971). The adjustment takes the form:
Where:
Po
- (Pi - Po) / (1 - Po)
- the smoothed observed proportion mortality for the control
=" the smoothed observed proportion mortality for effluent
concentration i.
462
-------
1
8. Calculate the amount of trim to use in the estimation of the LC50 as
follows:
Where: Trim = max(p^, 1-pj!)
I
p^ = the smoothed, adjusted proportion mortality for the lowest
effluent concentration, exclusive of the control
pj^ = the smoothed, adjusted proportion mortality for the highest
effluent concentration
k = the number of effluent concentrations, exclusive of the
control.
The minimum trim should be calculated for calculated for each data set rather
than using a fixed amount of trim for each data set.
1
9. Due to the intensive nature of the calculation for the estimated LC50 and
the calculation of the associated 95% confidence interval using the Trimmed
Spearman-Karber Method, it is recommended that the data be analyzed by
computer.
10. A computer program which estimates the LC50 and associated 95% confidence
interval using the Trimmed Spearman-Karber Method, can be obtained through the
EMSL, 3411 Church Street, Cincinnati, OH 45244. The program can be obtained
from EMSL-Cincinnati by sending a written request to the above address.
11. The Trimmed Spearman-Karber program automatically performs the following
functions: i
,
a. Smoothing.
b. Adjustment for mortality in the control.
c. Calculation of the necessary trim. i
d. Calculation of the LC50.
e. Calculation of the associated 95% confidence interval.
12. To illustrate the Trimmed Spearman-Karber method using the Trimmed
Spearman-Karber computer program, a set of data from a Sheepshead Minnow
Larval Survival and Growth test will be used. The data are listed in
Table J.I. I
12.1 The program requests the following input (Figure J.I):
a. Output destination (D = disk file or P = printer).
b. Control data.
c. Data for each toxicant concentration. \
12.2 The program output includes the following (Figure J.2):
a. A table of the concentrations tested, number of organisms exposed,
and the mortalities. \
b. The amount of trim used in the calculation. i
c. The estimated LC50 and the associated 95% confidence interval.
463
-------
TABLE J.I. EXAMPLE OF TRIMMED SPEARMAN-KARBER METHOD: MORTALITY
DATA FROM A SHEEPSHEAD MINNOW LARVAL SURVIVAL AND
GROWTH TEST (40 ORGANISMS PER CONCENTRATION)
Eff1uent
Concentration
Number of
Mortalities
"I
fa
Mortality
Proportion
Control
6.25
12.5
25.0
50.0
100.0
2
0
2
0
0
32
0.05
0.00
0.05
0.00
0.00
0.80
464
-------
A:>TSK
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
ENTER DATE OF TEST:
1
ENTER TEST NUMBER:
2
WHAT IS TO BE ESTIMATED?.
(ENTER "L" FOR LC50 AND "E" FOR EC50)
L
ENTER TEST SPECIES NAME:
Sheepshead minnow
ENTER TOXICANT NAME:
eff1uent
ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT :
y
E°NTER THE NUMBER OF INDIVIDUALS IN THE CONTROL:
40
ENTER THE NUMBER OF MORTALITIES IN THE CONTROL:
2
ENTER THE NUMBER OF CONCENTRATIONS
(NOT INCLUDING THE CONTROL; MAX = 10):
5
ENTER THE 5 EXPOSURE CONCENTRATIONS (IN INCREASING ORDER):
6.25 12.5 25 50 100
ARE THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION EQUAL(Y/N)?
y
ENTER THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION:
40
ENTER UNITS FOR DURATION OF EXPERIMENT
(ENTER "H" FOR HOURS, "D" FOR DAYS, ETC.):
Days
ENTER DURATION OF TEST:
7
ENTER THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION:
0 2 0 0 32
WOULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)?
y
Figure J.I. Example input for Trimmed Spearman-Karber Method.
465 I
-------
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
DATE: 1
TOXICANT:
SPECIES:
RAW DATA:
TEST
eff 1 uent
sheepshead minnow
Concentration
[0/\
(/o)
.00
6.25
12.50
25.00
50.00
100.00
NUMBER: 2
Number
Exposed
40
40
40
40
40
40
D
Mortalities
2
0
2
0
0
32
DURATION:
7 Days
SPEARMAN-KARBER TRIM:
SPEARMAN-KARBER ESTIMATES:
20.41%
LC50: 77.28
95% CONFIDENCE LIMITS
ARE NOT RELIABLE.
NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING.
ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION.
Figure 0.2. Example output for Trimmed Spearman-Karber Method.
466
-------
APPENDIX K
GRAPHICAL METHOD
1. The Graphical Method is used to calculate the LC50. It is a mathematical
procedure which estimates the LC50 by linearly interpolating between points of
a plot of observed percent mortality versus the base 10 logarithm (Iog10) of
percent effluent concentration. This method does not provide a confidence
interval for the LC50 estimate and its use is only recommended when there are
no partial mortalities. The only requirement for the Graphical Method is that
the observed percent mortalities bracket 50%.
2. For an analysis using the Graphical Method the data must first be smoothed
and adjusted for mortality in the control replicates. The procedure for
smoothing and adjusting the data is detailed in the following steps.
i
3. The Graphical Method is illustrated below using a set of mortality data
from an Inland Silverside Larval Survival and Growth test. These data are
listed in Table K.I. !
TABLE K.I. EXAMPLE OF GRAPHICAL METHOD: MORTALITY DATA FROM AN
INLAND SILVERSIDE LARVAL SURVIVAL AND GROWTH TEST (40
ORGANISMS PER CONCENTRATION)
Ef f 1 uent
Concentration
Control
6.25
12.5
25.0
50.0
100.0
Number of Mortality
Mortalities
Proportion
2 0.05
0 0.00
0
0
0.00
0.00
40 1.00
40
1.00
4. Let p0, p., ..., p^ denote the observed proportion mortalities for the
control and tne k effluent concentrations. The first step is to smooth the p,-
if they do not satisfy p0 < p. < ... < pk. The smoothing process replaces any
adjacent p/s that do not conform to p0 < p1 < ... < pk with their average.
For example, if p? is less than p,-^ then:
Where:
P/-I = Pi =
-i) /2
the smoothed observed proportion mortality for effluent
concentration i.
467
-------
4.1 For the data in this example, because the observed mortality proportions
for the 6.25%, 12.5%, and 25.0% effluent concentrations are less than the
observed response proportion for the control, the values for these four groups
must be averaged:
PcT
^s ^s s 0.05+0.00+0.00+0.00
Pi - P2 - Ps = -. :
0.05
= 0.0125
4.2 Since p4 - p5 = 1.00 are larger then 0.0125, set p^ = p!j = 1.00.
Additional smoothing is not necessary. The smoothed observed proportion
mortalities are shown in Table K.2.
5. Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney, 1971). The adjustment takes the form:
Where:
Pi = (P/-Pos) /d-Pos)
PQ = the smoothed observed proportion mortality for the control
p? = the smoothed observed proportion mortality for effluent
concentration i.
5.1 Because the smoothed observed proportion mortality for the control group
is greater than zero, the responses must be adjusted using Abbott's formula,
as follows:
a _ s
^a_^a_^a „ a _ Pi Po
Po - Pi ~ Pz = P3 —
1-PO
0.0125-0.0125 = 0.0
1 - 0.0125 0.9875
= 0.0
1-PO
1.00 -0.0125
1 -0.0125
0.9875
0.9875
= 1.00
A table of the smoothed, adjusted response proportions for the effluent
concentrations are shown in Table K.2.
5.2 Plot the smoothed, adjusted data on 2-cycle semi-log graph paper with the
logarithmic axis (the y axis) used for percent effluent concentration and the
linear axis (the x axis) used for observed percent mortality. A plot of the
smoothed, adjusted data is shown in Figure K.I.
468
-------
TABLE K.2. EXAMPLE OF GRAPHICAL METHOD: SMOOTHED, ADJUSTED
MORTALITY DATA FROM AN INLAND SILVERSIDE LARVAL
SURVIVAL AND GROWTH TEST
Ef f 1 uent
Concentration
%
Control
6.25
12.5
25.0
50.0
100.0
Mortality
Proportion
0.05
0.00
0.00
0.00
1.00
1.00
Smoothed
Mortality
Proportion
0.0125
0.0125
0.0125
0.0125
1.0000
1.0000
Smoothed,
Adjusted
Mortality
Proportion
0.00
0.00
0.00
0.00
1.00
1.00
6. Locate the two points on the graph which bracket 50% mortality and connect
them with a straight line.
7. On the scale for percent effluent concentration, read the value for the
point where the plotted line and the 50% mortality line intersect. This value
is the estimated LC50 expressed as a percent effluent concentration.
7.1 For this example, the two points on the graph which bracket the 50%
mortality line (0% mortality at 25% effluent, and 100% mortality at 50%
effluent) are connected with a straight line. The point at which the plotted
line intersects the 50% mortality line is the estimated LC50. The estimated
LC50 = 35% effluent.
469
-------
100
50
LLJ
S
Z>
£
LLI
LL.
LL
LL1
z:
LLJ
O
DC
111
Q_
10
1
0 10 20 30 40 50 60 70 80 90 100
PERCENT MORTALITY
Figure K.I. Plot of the smoothed adjusted response proportions for
inland silverside, Menidia beryllina, survival data.
470
-------
APPENDIX I-
LINEAR INTERPOLATION METHOD
1. GENERAL PROCEDURE
1.1 The Linear Interpolation Method is used to calculate [a point estimate of
the effluent or other toxicant concentration that causes a given percent
reduction (e.g., 25%, 50%, etc.) in the reproduction or growth of the test
organisms (Inhibition Concentration, or 1C). The procedure v/as designed for
general applicability in the analysis of data from short-term chronic toxicity
tests, and the generation of an endpoint from a continuous; model that allows a
traditional quantitative assessment of the precision of the endpoint, such as
confidence limits for the endpoint of a single test, and a mean and
coefficient of variation for the endpoints of multiple tests-
1.2 The Linear Interpolation Method assumes that the responses (1)
monotonically non-increasing, where the mean response for each higher
concentration is less than or equal to the mean response for the previous
concentration, (2) follow a piecewise linear response function, and (3) are
from a random, independent, and representative sample of test data. If the
data are not monotonically non-increasing, they are adjusted by smoothing
(averaging). In cases where the responses at the low toxicant concentrations
are much higher than in the controls, the smoothing process may result in a
large upward adjustment in the control mean. Also, no assumption is made
about the distribution of the data except that the data within a group being
resampled are independent and identically distributed. i
2. DATA SUMMARY AND PLOTS
•
2.1 Calculate the mean responses for the control and each toxicant
concentration, construct a summary table, and plot the data.
3. MONOTONICITY
3.1 If the assumption of monotonicity of test results is met, the observed
response means (Yj) should stay the same or decrease as the toxicant
concentration increases. If the means do not decrease monotonically, the
responses are "smoothed" by averaging (pooling) adjacent means.
3.2 Observed means at each concentration are considered in order of
increasing concentration, starting with the control mean (Y.,). If the mean
observed response at the lowest toxicant concentration (Y;>) is equal to or
smaller than the control mean (Y,,), it is used as the response. If it is
larger than the control mean, it is averaged with the control, and this
average is used for both the control response (M,,) and the lowest toxicant
concentration response (M?) . This mean is then compared to the mean observed
response for the next higher toxicant concentration (Y3) . Again, if the mean
observed response for the next higher toxicant concentration is smaller than
the mean of the control and the lowest toxicant concentration, it is used as
the response. If it is higher than the mean of the first two, it is averaged
with the first two, and the mean is used as the response for the control and
471 I
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two lowest concentrations of toxicant. This process is continued for data
from the remaining toxicant concentrations. A numerical example of smoothing
the data is provided below. (Note: Unusual patterns in the deviations from
Where Y, decrease
monotonicity may require an additional step of smoothing).
monotonically, the Y,- become Mf without smoothing.
4. LINEAR INTERPOLATION METHOD
4.1 The method assumes a linear response from one concentration to the next.
Thus, the ICp is estimated by linear interpolation between two concentrations
whose responses bracket the response of interest, the (p) percent reduction
from the control.
4.2 To obtain the estimate, determine the concentrations Cj and CJ+1 which
bracket the response M, (1 - p/100), where M, is the smoothed control mean
response and p is the percent reduction in response relative to the control
response. These calculations can easily be done by hand or with a computer
program as described below. The linear interpolation estimate is calculated
as follows:
ICp = Cj. + [ Mx (l - p/ioo) -
"J +• 1
- CT)
(M.7
+ i
Where: Ca - tested concentration whose observed mean response
is greater than M.,(l - p/100).
Cj+1 = tested concentration whose observed mean response
is less than M,,(l - p/100).
M., = smoothed mean response for the control.
Mj = smoothed mean response for concentration J.
Mj + 1 = smoothed mean response for concentration J + 1.
p = percent reduction in response relative to the
control response.
ICp = estimated concentration at which there is a
percent reduction from the smoothed mean control
response. The ICp is reported for the test,
together with the 95% confidence interval
calculated by the ICPIN.EXE program described
below.
4.3 If the Cj is the highest concentration tested, the ICp would be specified
as greater than C,. If the response at the lowest concentration tested is
used to extrapolate the ICp value, the ICp should be expressed as a 7ess than
the lowest test concentration.
472
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5. CONFIDENCE INTERVALS
5.1 Due to the use of a linear interpolation technique to calculate an
estimate of the ICp, standard statistical methods for calculating confidence
intervals are not applicable for the ICp. This limitation is avoided by use a
technique known as the bootstrap method as proposed by Efron (1982) for
deriving point estimates and confidence intervals.
5.2 In the Linear Interpolation Method, the smoothed response means are used
to obtain the ICp estimate reported for the test. The bootstrap method is
used to obtain the 95% confidence interval for the true mean. In the
bootstrap method, the test data YM is randomly resampled with replacement to
produce a new set of data Y,,*, that is statistically equivalent to the
original data, but a new and slightly different estimate of the ICp (ICp*) is
obtained. This process is repeated at least 80 times (Marcus and Holtzman,
1988) resulting in multiple "data" sets, each with an associate ICp* estimate.
The distribution of the ICp* estimates derived from the sets of resampled data
approximates the sampling distribution of the ICp estimate. The standard
error of the ICp is estimated by the standard deviation of the individual ICp*
estimates. Empirical confidence intervals are derived from the quantiles of
the ICp* empirical distribution. For example, if the test data are resampled
a minimum of 80 times, the empirical 2.5% and the 97.5% confidence limits are
approximately the second smallest and second largest ICp* estimates (Marcus
and Holtzman, 1988).
5.3 The width of the confidence intervals calculated by the bootstrap method
is related to the variability of the data. When confidence intervals are
wide, the reliability of the 1C estimate is in question. However, narrow
intervals do not necessarily indicate that the estimate is highly reliable,
because of undetected violations of assumptions and the fact that the
confidence limits based on the empirical quantiles of a bootstrap distribution
of 80 samples may be unstable.
i
5.4 The bootstrapping method of calculating confidence intervals is
computationally intensive. For this reason, all of the calculations
associated with determining the confidence intervals for the ICp estimate have
been incorporated into a computer program. Computations are most easily done
with a computer program such as the revision of the BOOTSTRP program (USEPA,
1988; USEPA, 1989) which is now called "ICPIN" which is described below in
subsection 7.
6. MANUAL CALCULATIONS
6.1 DATA SUMMARY AND PLOTS
6.1.1 The data used in this example are the mysid growth data used in the
example in Section 14. The data is presented as the mean weight per original
number of organisms. Table L.I includes the raw data and the mean growth for
each concentration. A plot of the data is provided in Figure L.I.
473
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TABLE L.I. MYSID, MYSIDOPSIS BAHIA, GROWTH DATA
Replicate Control
Toxicant Concentration (ppb)
50
100
210
450
1
2
3
4
5
6
7
8
0.146
0.118
0.216
0.199
0.176
0.243
0.213
0.144
0.154
0.193-
0.190
0.190
0.256
0.191
0.122
0.177
0.114
0.172
0.160
0.199
0.165
0.145
0.207
0.186
0.153 0
0.094 0.012
0.017 0
0.122 0.002
0.052
0.154
0
0
0.110 0
0.103 0.081
Mean (Y;) 0.182 0.184 0.168 0.101
i 1 2 3 4
0.012
5
6.2 MONOTONICITY
I
6.2.1 As can be seen from the plot in Figure L.I, the observed means are not
monotonically non-increasing with respect to concentration. Therefore, the
means must be smoothed prior to calculating the 1C. i
6.2.2_ Starting with the control mean Y., = = 0.186 and Y~2 = 0.184, we see that
Y-, < Y2 . Calculate the smoothed means:
ML = M2 = (Ti + T2) /2 = 0.193 •
6.2.3 Since Y5 = 0.025 < Y, = 0.101 < Y3 = 0.168 < M2, set, M3 = 0.168 and M,
0.101, and M5 = 0.025. Table L.2 contains the smoothed means and Figure L.l
gives a plot of the smoothed response curve.
6.3 LINEAR INTERPOLATION \
i
6.3.1 Estimates of the IC25 and IC50 can be calculated using the Linear
Interpolation Method. A 25% reduction in mean weight, compared to the
controls, would result in a mean weight of 0.139, where M^l-p/100) =
0.185(1-25/100). A 50% reduction in mean weight, compared to the controls,
would result in a mean weight of 0.093 mg. Examining the smoothed means and
their associated concentrations (Table L.2), the two effluent concentrations.
bracketing the mean weight per original of 0.139 mg are C3 = 100 ppb and C4 =
475
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TABLE L.2. MYSID, MYSIDOPSIS BAHIA, MEAN GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(ppb)
Control
50
100
210
450
i
1
2
3
4
5
Smoothed
Mean
Mf (mg)
0.183
0.183
0.168
0.101
0.025
210 ppb. The two effluent concentrations bracketing a response of 0.093 mg
per total original number of organisms are C4 = 210 ppb and C5 = 450 ppb.
6.3.2 Using the equation from section 4.2, the estimate of the IC25 is
calculated as follows:
ICp = Cj. + [ Mx (i - p/100) - Mj ;
(Mj
- AfT)
IC25 - 100 + [0.193 (1 - 25/100) - 0.164]
= 151 ppb
/ft »\
(0 . 101 - 0 . 164)
6.3.3 Using Equation 1 from 4.2, the estimate of the IC50 is calculated as
fol1ows:
/1-< /-< \
ICp = Cj + [ ML (1 - p/100) - Mj ]
(MJ + 1 -
(450 - 210)
(0.028 - 0.101)
JC50 = 210 + [0.193 (1 - 50/100) - 0.101]
- 239 ppb
6.4 CONFIDENCE INTERVALS
6.4.1 Confidence intervals for the ICp are derived using the bootstrap
method. As described above, this method involves randomly resampling the
individual observations and recalculating the ICp at least 80 times, and
determining the mean ICp, standard deviation, and empirical 95% confidence
intervals. For this reason, the confidence intervals are calculated using a
computer program called ICPIN. This program is described below and is
available to carry out all the calculations of both the interpolation estimate
(ICp) and the confidence intervals.
476
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7. COMPUTER CALCULATIONS
7.1 The computer program, ICPIN, prepared for the Linear Interpolation
Methods was written in TURBO PASCAL for IBM compatible PCs. The program
(version 2.0) has been modified by Computer Science Corporation, Duluth, MN
with funding provided by the Environmental Research Laboratory, Duluth, MN
(Norberg-King, 1993). The program was originally developed by Battelle
Laboratories, Columbus, OH through a government contract supported by the
Environmental Research Laboratory, Duluth, MN (USEPA, 1988). A compiled,
executable version of the program and supporting documentation can be obtained
by sending a written request to EMSL-Cincinnati, 3411 Church Street,
Cincinnati, OH 45244.
7.2 The ICPIN.EXE program performs the following functions;: 1) it
calculates the observed response means (Y-) (response means); 2) it
calculates the standard deviations; 3) checks the responses for monotonicity;
4) calculates smoothed means (M,-) (pooled response means) if necessary; 5)
uses the means, Mp to calculate the initial ICp of choice by linear
interpolation; 6) performs a user-specified number of bootstrap resamples
between 80 and 1000 (as multiples of 40); 7) calculates the mean and standard
deviation of the bootstrapped ICp estimates; and 8) provides an original 95%
confidence intervals to be used with the initial ICp when the number of
replicates per concentration is over six and provides both original and
expanded confidence intervals when the number of replicates per concentration
are less than seven (Norberg-King, 1993).
7.3 For the ICp calculation, up to twelve treatments can be input (which
includes the control). There can be up to 40 replicates per concentration,
and the program does not require an equal number of replicates per
concentration. The value of p can range from 1% to 99%.
7.4 DATA INPUT
7.4.1 Data is entered directly into the program onscreen. ' A sample data
entry screen in shown in Figure L.2. The program documentation provides
guidance on the entering and analysis of data for the Linear Interpolation
Method. i
7.4.2 The user selects the ICp estimate desired (e.g., IC25 or IC50) and the
number of resamples to be taken for the bootstrap method of calculating the
confidence intervals. The program has the capability of performing any number
of resamples from 80 to 1000 as multiples of 40. However, Marcus and Holtzman
(1988) recommend a minimum of 80 resamples for the bootstrap method be used
and at least 250 resamples are better (Norberg-King, 1993).;
477
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Current File:
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
Response 11
Response 12
Response 13
Response 14
Response 15
Response 16
Response 17
Response 18
Response 19
Response 20
1
2
3
4
5
F10 for Command Menu
Use Arrow Keys to Switch Fields
Figure L.2. ICp data entry/edit screen. Twelve concentration
identifications can be used. Data for concentrations
are entered in columns 1 through 6. For concentrations
7 through 12 and responses 21-40 the data is entered in
additional fields of the same screen.
478
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7.5 DATA OUTPUT
7.5.1 The program output includes the following (Figures L.3 and L.4)
1. A table of the concentration identification, the concentration tested
and raw data response for each replicate and concentration.
2. A table of test concentrations, number of replicates, concentration
(units), response means (Y,.), standard deviations for each response
mean, and the pooled response means (smoothed means; M,-).
3. The linear interpolation estimate of the ICp using the means (M,-).
Use this value for the ICp estimate.
4. The mean ICp and standard deviation from the bootstrap resampling.
5. The confidence intervals calculated by the bootstrap method for the ICp.
Provides an original 95% confidence intervals to be used with the
initial ICp when the number of replicates per concentration is over six
and provides both original and expanded confidence intervals when the
number of replicates per concentration are less than seven.
7.6 ICPIN program output for the analysis of the mysid growth data in Table
L.I is provided in Figures L.3 and L.4.
7.6.1 When the ICPIN program was used to analyze this set of data, requesting
80 resamples, the estimate of the IC25 was 147.1702 (ppb). The empirical 95%
confidence intervals for the true mean was 97.0905 to 186.6383 (ppb).
7.6.2 When the ICPIN program was used to analyze this set of data, requesting
80 resamples, the estimate of the IC50 was 233.3311 (ppb). The empirical 95%
confidence intervals for the true mean were 184.8692 to 283.3965 (ppb).
479
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Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
, 1
0
.146
.118
.216
.199
.176
.243
.213
.144
2
50
.154
.193
.190
.190
.256
.191
.122
.177
3
100
.114
.172
.160
.199
.165
.145
.207
.186
4
210
.153
.094
.017
.122
.052
.154
.110
.103
5
450
0
.012
0
.002
0
0
0
.081
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date: Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.i25
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
ug/1
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Std.
Dev.
Means
0.043
0.038
0.030
0.047
0.028
Pooled
Response
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate: 133.5054 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 147.1702 Standard Deviation: 23.7984
Original Confidence Limits: Lower: 96.8623 Upper: 186.6383
Resampling time in Seconds: 0.16 Random Seed: -1623038650
Figure L.3. Example of ICPIN program output for the IC25.
480
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Cone. ID
Cone. Tested
50
100
210
5
450
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
.146
.118
.216
.199
.176
.243
.213
.144
.154
.193
.190
.190
.256
.191
.122
,177
.114
.172
.160
.199
.165
.145
.207
.186
.153
.094
.017
.122
.052
.154
.110
.103
0
.012
0
.002
0
0
0
.081
*** Inhibition Concentration Percentage Estimate ***
Toxi cant/Eff1uent:
Test Start Date: Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.iSO
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration Response Std. Pooled
ug/1 Means Dev. Response Means
0.000
50.000
100.000
210.000
450.000
0.182
0.184
0.168
0.101
0.012
0.043
0.038
0.030
0.047
0.028
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate: 234.6761 Entered P Value: 50
Number of Resamplings: .80 :
The Bootstrap Estimates Mean: 233.3311 Standard Deviation: 28.9594
Original Confidence Limits: Lower: 184.8692 Upper: 283.3965
Resampling time in Seconds: 0.11 Random Seed: 1103756486
Figure L.4. Example of ICPIN program output for the IC50.
481
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