&EPA
United States
Environmental Protection
Agency
Short-term Methods for Estimating
the Chronic Toxicity of Effluents and
Receiving Waters to Marine and
Estuarine Organisms
Third Edition
October 2002
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-02-014
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DISCLAIMER
The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and
approved this report for publication. Neither the United States Government nor any of its employees, contractors,
or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any
third party's use of or the results of such use of any information, apparatus, product, or process discussed in this
report, or represents that its use by such party would not infringe on privately owned rights.
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CONTENTS
Page
Figures viii
Tables xiii
Section Number Page
1. Introduction 1
2. Short-Term Methods for Estimating Chronic Toxicity 3
Introduction 3
Types of Tests 5
Static Tests 6
Advantages and Disadvantages of Toxicity Test Types 6
3. Health and Safety 7
General Precautions 7
Safety Equipment 7
General Laboratory and Field Operations 7
Disease Prevention 8
Safety Manuals 8
Waste Disposal 8
4. Quality Assurance 9
Introduction 9
Facilities, Equipment, and Test Chambers 9
Test Oranisms 9
Laboratory Water Used for Culturing and and Test Dilution Water 9
Effluent and Receiving Water Sampling and Handling 10
Test Conditions 10
Quality of Test Organisms 10
Food Quality 11
Acceptability of Chronic Toxicity Tests 11
Analytical Methods 12
Calibration and Standardization 12
Replication and Test Sensitivity 12
Variability in Toxicity Test Results 12
Test Precision 12
Demonstrating Acceptable Laboratory Performance 15
Documenting Ongoing Laboratory Performance 15
Reference Toxicants 16
Record Keeping 18
5. Facilities, Equipment, and Supplies 19
General Requirements 19
Test Chambers 20
Cleaning Test Chambers and Laboratory Apparatus 20
Apparatus and Equipment for Culturing and Toxicity Tests 20
Reagents and Consumable Materials 20
Test Organisms 21
Supplies 21
6. Test Organisms 22
Test Species 22
Sources of Test Organisms 22
in
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CONTENTS (CONTINUED)
Section Number Page
Life Stage 23
Laboratory Culturing 23
Holding and Handling Test Organisms 24
Transportation to the Test Site 24
Test Organism Disposal 25
7. Dilution Water 26
Types of Dilution Water 26
Standard, Synthetic Dilution Water 26
Use of Receiving Water as Dilution Water 28
Use of Tap Water as Dilution Water 29
Dilution Water Holding 30
8. Effluent and Receiving Water Sampling, Sample Handling,and Sample Preparation for
Toxicity Tests 31
Effluent Sampling 31
Effluent Sample Types 31
Effluent Sampling Recommendations 32
Receiving Water Sampling 32
Effluent and Receiving Water Sample Handling, Preservation, and Shipping 33
Sample Receiving 34
Persistence of Effluent Toxicity During Sample Shipment and Holding 34
Preparation of Effluent and Receiving Water Samples for Toxicity Tests 34
Preliminary Toxicity Range-finding Tests 38
Multiconcentration (Definitive) Effluent Toxicity Tests 38
Receiving Water Tests 38
9. Chronic Toxicity Test Endpoints and Data Analysis 40
Endpoints 40
Relationship between Endpoints Determined by Hypothesis Testing and Point
Estimation Techniques 40
Precision 42
Data Analysis 42
Choice of Analysis 44
Hypothesis Tests 46
Point Estimation Techniques 47
10. Report Preparation and Test Review 49
Report Preparation 49
Test Review 51
11. Test Method: Sheepshead Minnow, Cyprinodon variegatus, Larval
Survival and GrowthTest Method 1004.0 55
Scope and Application 55
Summary of Method 55
Interferences 55
Safety 57
Apparatus and Equipment 57
Reagents and Consumable Materials 59
Effluent and Receiving Water Collection, Preservation, and Storage 67
Calibration and Standardization 67
Quality Control 67
iv
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CONTENTS (CONTINUED)
Section Number Page
Test Procedures 67
Summary of Test Conditions and Test Acceptability Criteria 76
Acceptability of Test Results 76
Data Analysis 76
Precision and Accuracy 107
12. Test Method: Sheepshead Minnow, Cyprinodon variegatus, Embryo-larval Survival and
Teratogenicity Test Method 1005.0 117
Scope and Application 117
Summary of Method 117
Interferences 117
Safety 119
Apparatus and Equipment 119
Reagents and Consumable Materials 121
Effluent and Receiving Water Collection, Preservation, and Storage 130
Calibration and Standardization 130
Quality Control 130
Test Procedures 130
Acceptability of Test Results 136
Summary of Test Conditions and Test Acceptability Criteria 136
Data Analysis 136
Precision and Accuracy 152
13. Test Method: Inland Silverside, Menidia beryllina, Larval
Survival and Growth Method 1006.0 155
Scope and Application 155
Summary of Method 155
Interferences 155
Safety 157
Apparatus and Equipment 157
Reagents and Consumable Materials 159
Effluent and Receiving Water Collection, Preservation, and Storage 168
Calibration and Standardization 168
Quality Control 168
Test Procedures 168
Summary of Test Conditions and Test Acceptability Criteria 175
Acceptability of Test Results 175
Data Analysis 175
Precision and Accuracy 205
14. Test Method: Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity
Test Method 1007.0 214
Scope and Application 214
Summary of Method 214
Interferences 214
Safety 216
Apparatus and Equipment 216
Reagents and Consumable Materials 217
Effluent and Receiving Water Collection, Preservation, and Storage 226
Calibration and Standardization 226
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CONTENTS (CONTINUED)
Section Number Page
Quality Control 226
Test Procedures 227
Summary of Test Conditions and Test Acceptability Criteria 235
Acceptability of Test Results 235
Data Analysis 236
Precision and Accuracy 285
15. Test Method: SeaUrchin,Arbaciapunctulata, Fertilization Test Method 1008.0 293
Scope and Application 293
Summary of Method 293
Interferences 293
Safety 293
Apparatus and Equipment 293
Reagents and Consumable Materials 295
Effluent and Receiving Water Collection, Preservation, and Storage 303
Calibration and Standardization 303
Quality Control 303
Test Procedures 303
Summary of Test Conditions and Test Acceptability Criteria 307
Acceptability of Test Results 307
Data Analysis 309
Precision and Accuracy 325
16. Test Method: Red Macroalga, Champia parvula, Reproduction Test Method 1009.0 332
Scope and Application 332
Summary of Method 332
Interferences 332
Safety 333
Apparatus and Equipment 333
Reagents and Consumable Materials 334
Effluent and Receiving Water Collection, Preservation, and Storage 341
Calibration and Standardization 341
Quality Control 341
Test Procedures 341
Summary of Test Conditions and Test Acceptability Criteria 346
Acceptability of Test Results 346
Data Analysis 346
Precision and Accuracy 361
Cited References 367
Bibliography 376
Appendices 383
A. Independence, Randomization, and Outliers 384
B. Validating Normality and Homogeneity of Variance Assumptions 390
C. Dunnett's Procedure 401
D. T test with Bonferroni's Adjustment 414
E. Steel's Many-one Rank Test 420
F. Wilcoxon Rank Sum Test 425
G. Single Concentration Toxicity Test - Comparison of Control with 100% Effluent or
Receiving Water 432
vi
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CONTENTS (CONTINUED)
Section Number Page
H. Probit Analysis 436
I. Spearman-Karber Method 439
J. Trimmed Spearman-Karber Method 444
K. Graphical Method 448
L. Linear Interpolation Method 452
Cited References 463
vn
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FIGURES
SECTION 1-10
Number Page
1. Control charts 17
2. Flowchart for statistical analysis of test data 45
SECTION 11
Number Page
1. Embryonic development of sheepshead minnow, Cyprinodon variegatus 65
2. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Daily
record of larval survival and test conditions 72
3. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Dry
weights of larvae 75
4. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Summary
of test results 77
5. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval
survival data by hypothesis testing 81
6. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval
survival data by point estimation 82
7. Plot of mean survival proportion data in Table 5 85
8. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus,
larval growth data 92
9. Plot of weight data from sheepshead minnow, Cyprinodon variegatus, larval survival
and growth test 95
10. Plot of raw data, observed means, and smoothed means for the sheepshead minnow,
Cyprinodon variegatus, growth data from Tables 4 and 20 103
11. ICPIN program output for the IC25 105
12. ICPIN program output for the IC50 106
Vlll
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FIGURES (CONTINUED)
SECTION 12
Number Page
1. Data form for sheepshead minnow, Cyprinodon variegatus, embryo-larval survival/teratogenicity
test. Daily record of embryo-larval survival/terata and test conditions 122
2. Embryonic development of sheepshead minnow, Cyprinodon variegatus 134
3. Flowchart for statistical analysis of sheepshead minnow, Cyprinodon variegatus,
embryo-larval survival and teratogenicity test. Survival and terata data 140
4. Plot of sheepshead minnow, Cyprinodon variegatus, total mortality data from the
embryo-larval test 142
5. Output for USEPAProbit Analysis Program, Version 1.5 151
SECTION 13
Number Page
1. Glass chamber with sump area 158
2. Inland silverside, Menidia beryllina 167
3. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Daily
record of larval survival and test conditions 172
4. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Dry
weights of larvae 176
5. Data form for the inland silverside, Menidia beryllina, larval survival and growth test.
Summary of test results 177
6. Flowchart for statistical analysis of the inland silverside, Menida beryllina, survival data by hypothesis
testing 181
7. Flowchart for statistical analysis of the inland silverside, Menida beryllina, survival data by
point estimation 182
8. Plot of mean survival proportion of the inland silverside, Menidia beryllina, larvae 183
9. Output for USEPA Probit Analysis Program, Version 1.5 192
10. Flowchart for statistical analysis of the inland silverside, Menidia beryllina, growth data 193
11. Plot of mean weights of inland silverside, Menidia beryllina, larval survival and growth test 195
12. Plot of the raw data, observed means, and smoothed means from Tables 12 and 19 204
IX
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FIGURES (CONTINUED)
SECTION 13
Number Page
13. ICPIN program output for the IC25 207
14. ICPIN program output for the IC50 208
SECTION 14
Number Page
1. Apparatus (brood chamber) for collection of juvenile mysids, Mysidopsis bahia 225
2. Data form for the mysid, Mysidopsis bahia, water quality measurements 231
3. Mature female mysid, Mysidopsis bahia, with eggs in oviducts 232
4. Mature female mysid, Mysidopsis bahia, with eggs in oviducts and developing
embryos in the brood sac 233
5. Mature male mysid, Mysidopsis bahia 235
6. Immature mysid, Mysidopsis bahia, (A) lateral view, (B) dorsal view 236
7. Data form for the mysid, Mysidopsis bahia, survival and fecundity data 237
8. Data form for the mysid, Mysidopsis bahia, dry weight measurements 239
9. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by
hypothesis testing 245
10. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by
point estimation 246
11. Plot of survival proportions of mysids, Mysidopsis bahia, at each treatment level 247
12. Output for USEPA Probit Analyis Program, Version 1.5 254
13. Flowchart for statistical analysis of mysid, Mysidopsis bahia, growth data 256
14. Plot of mean growth data for mysid, Mysidopsis bahia, test 257
15. Plot of raw data, observed means, and smoothed means for the mysid, Mysidopsis bahia,
growth data from Tables 13 and 20 266
16. ICPIN program output for the IC25 268
17. ICPIN program output for the IC50 269
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FIGURES (CONTINUED)
SECTION 14
Number Page
18. Flowchart for statistical analysis of mysid, Mysidopsis bahia, fecundity data 270
19. Proportion of female mysids, Mysidopsis bahia, with eggs 272
20. Plot of the mean proportion of females mysids, Mysidopsis bahia, with eggs 282
21. ICPIN program output for the IC25 283
22. ICPIN program output for the IC50 284
SECTION 15
Number Page
1. Dataform (1) for fertilization test using sea urchin, Arbacia punctulata 297
2. Dataform (2) for fertilization test using sea urchin, Arbacia punctulata 298
3. Dataform (3) for fertilization test using sea urchin, Arbacia punctulata 299
4. Flowchart for statistical analysis of sea urchin, Arbacia punctulata, by point estimation 312
5. Plot of mean percent of fertilized sea urchin, Arbacia punctulata, eggs 313
6. ICPIN program output for the IC25 323
7. ICPIN program output for the IC50 324
SECTION 16
Number Page
1. Life history of the red macroalga, Champia parvula 338
2. Apex of branch of female plant, showing sterile hairs and reproductive
hairs (trichogynes) 339
3. A portion of the male thallus showing spermatial sori 340
4. A magnified portion of a spermatial sorus 340
5. Apex of a branch on a mature female plant that was exposed to spermatia from a male plant 340
6. Data form for the red macroalga, Champia parvula, sexual reproduction test. Receiving
water summary sheet 342
XI
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FIGURES (CONTINUED)
SECTION 16
Number Page
7. A mature cystocarp 345
8. Comparison of a very young branch and an immature cystocarp 345
9. An aborted cystocarp 345
10. Data form for the red macroalga, Champia parvula, sexual reproduction test. Cystocarp
data sheet 347
11. Flowchart for statistical analysis of the red macroalga, Champia parvula, data 351
12. Plot of the number of cystocarps perplant 353
13. ICPIN program output for the IC25 362
14. ICPIN program output for the IC50 363
xn
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TABLES
SECTION 1- 10
Number Page
1. National interlaboratory study of chronic toxicity test precision, 1991: Summary of responses using two
reference toxicants 14
2. National interlaboratory study of chronic toxicity test precision, 2000: Precision of responses using
effluent, receiving water, and reference toxicant sample types 14
3. Preparation of GP2 artificial seawater using reagent grade chemicals 28
4. Oxygen solubility (mg/L) in water at equilibrium with air at 760 mm Hg 36
5. Percent unionized NH3 in aqueous ammonia solutions: temperature 15-26°C and pH 6.0-8.9 37
6. Variability criteria (upper and lower PMSD bounds) for sublethal hypothesis testing endpoints submitted
under NPDES permits 54
SECTION 11
Number Page
1. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the sheepshead minnow,
Cyprinodon variegatus, toxicity test 61
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 69
3. Summary of test conditions and test acceptability criteria for sheepshead minnow, Cyprinodon variegatus,
larval survival and growth test with effluents and receiving waters (Test Method 1004.0) 78
4. Summary of survival and growth data for sheepshead minnow, Cyprinodon variegatus, larvae exposed to
an effluent for seven days 80
5. Sheepshead minnow, Cyprinodon variegatus, survival data 84
6. Centered observations for Shapiro-Wilk's example 84
7. Ordered centered observations for the Shapiro-Wilk's example 86
8. Coefficients and differences for Shapiro-Wilk's example 87
9. Assigning ranks to the control and 6.25% effluent concentration for Steel's Many-one Rank Test 88
10. Table of ranks 88
11. Rank sums 88
Xlll
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TABLES (CONTINUED)
SECTION 11
Number Page
12. Data for example of Spearman-Karber Analysis 90
13. Sheepshead minnow, Cyprinodon variegatus, growth data 93
14. Centered observations for Shapiro-Wilk's example 93
15. Ordered centered observations for Shapiro-Wilk's example 94
16. Coefficients and differences for Shapiro-Wilk's example 96
17. ANOVA table 98
18. ANOVA table for Dunnett's Procedure example 100
19. Calculated lvalues 100
20. Sheepshead minnow, Cyprinodon variegatus, mean growth response after smoothing 102
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 109
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 110
23. Single-laboratory precision of the Sheepshead minnow, Cyprinodon 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 Ill
24. Single-laboratory precision of the Sheepshead minnow, Cyprinodon 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 112
25. 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, and hexavalent chromium as the reference toxicant 113
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 seawater 114
xiv
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TABLES (CONTINUED)
SECTION 11
Number Page
27. Data from interlaboratory study of the sheepshead minnow, Cyprinodon variegatus, larval
survival and growth test using an industrial effluent as a reference toxicant 115
28. Precision of point estimates for various sample types 116
29. Frequency distribution of hypothesis testing results for various sample types 116
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 126
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 (Test Method 1005.0) 137
3. Sheepshead minnow, Cyprinodon variegatus, embryo-larval total mortality data 141
4 Centered observation for Shapiro-Wilk's example 141
5. Ordered centered observations for Shapiro-Wilk's example 143
6. Coefficients and differences for Shapiro-Wilk's example 144
7. ANOVA table 146
8. ANOVA table for Dunnett's Procedure example 147
9. Calculated t values 148
10. Data for Probit Analysis 150
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
toxicant 153
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
reference toxicant 154
xv
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TABLES (CONTINUED)
SECTION 13
Number 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 162
2. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the inland silverside,
Menidia beryllina, toxicity test 163
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 (Test Method 1006.0) 178
4. Inland silverside, Menidia beryllina, larval survival data 184
5. Centered observations for Shapiro-Wilk's example 184
6. Ordered centered observations for Shapiro-Wilk's example 185
7. Coefficients and differences for Shapiro-Wilk's example 186
8. ANOVAtable 187
9. ANOVA table for Dunnett's Procedure example 189
10. Calculated lvalues 190
11. Data for Probit Analysis 191
12. Inland silverside, Menidia beryllina, growth data 194
13. Centered observations for Shapiro-Wilk's example 194
14. Ordered centered observations for Shapiro-Wilk's example 196
15. Coefficients and differences for Shapiro-Wilk's example 197
16. ANOVAtable 199
17. ANOVA table for Dunnett's Procedure example 200
18. Calculated lvalues 201
19. Inland silverside mean growth response after smoolhing 202
20. Single-laboratory precision of Ihe inland silverside, Menidia beryllina, survival and growlh lesl performed
in nalural seawaler, using larvae from fish mainlained and spawned in nalural seawaler, and copper (Cu) as
a reference loxicanl 209
xvi
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TABLES (CONTINUED)
SECTION 13
Number Page
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 toxicant 210
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 211
23. Precision of point estimates for various sample types 212
24. Frequency distribution of hypothesis testing results for various sample types 213
SECTION 14
Number Page
1. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the mysid,
Mysidopsis bahia, toxicity test 220
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 221
3. Summary of test conditions and test acceptability criteria for the my sid, Mysidopsis bahia, seven day
survival, growth, and fecundity test with effluents and receiving waters (Test Method 1007.0) 241
4. Data tor:Mysidopsis bahia 7-day survival, growth, and fecundity test 243
5. Mysid, Mysidopsis bahia, survival data 248
6. Centered observations for Shapiro-Wilk's example 249
7. Ordered centered observations for Shapiro-Wilk's example 250
8. Coefficients and differences for Shapiro-Wilk's example 251
9. Assigning ranks to the control and 50 ppb concentration level for
Steel's Many-one Rank Test 252
10. Table of ranks 252
11. Rank sums 253
12. Data for Probit Analysis 255
XVII
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TABLES (CONTINUED)
SECTION 14
Number Page
13. Mysid, Mysidopsis bahia, growth data 255
14. Centered observations for Shapiro-Wilk's example 258
15. Ordered centered observations for Shapiro-Wilk's example 259
16. Coefficients and differences for Shapiro-Wilk's example 260
17. ANOVA table 262
18. ANOVA table for Dunnett's Procedure example 263
19. Calculated t values 264
20. Mysid, Mysidopsis bahia, mean growth response after smoothing 265
21. Mysid, Mysidopsis bahia, fecundity data: Percent females with eggs 273
22. Centered observations for Shapiro-Wilk's example 273
23. Ordered centered observations for Shapiro-Wilk's example 274
24. Coefficients and differences for Shapiro-Wilk's example 275
25. ANOVA table 276
26. ANOVA table for the t test with Bonferroni's Adjustment example 278
27. Calculated t values 279
28. Mysid, Mysidopsis bahia, mean proportion of females with eggs 280
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 286
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 287
31. 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
sodium dodecyl sulfate (SDS) as a reference toxicant 288
xvm
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TABLES (CONTINUED)
SECTION 14
Number Page
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 289
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 290
34. Precision of point estimates for various sample types 291
35. Frequency distribution of hypothesis testing results for various sample types 292
SECTION 15
Number Page
1. Preparation of test solutions at a salinity of 30%o using natural seawater, hypersaline brine, or artificial sea
salts 301
2. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the sea urchin, Arbacia
punctulata toxicity test 302
3. Summary of test conditions and test acceptability criteria for sea urchin, Arbacia punctulata, fertilization
test with effluent and receiving waters (Test Method 1008.0) 308
4. Data from sea urchin, Arbacia punctulata, fertilization test 310
5. Sea urchin, Arbacia punctulata, fertilization data 314
6. Centered observations for Shapiro-Wilk's example 314
7. Ordered centered observations for Shapiro-Wilk's example 315
8. Coefficients and differences for Shapiro-Wilk's example 316
9. ANOVAtable 317
10. ANOVA table for Dunnett's Procedure example 319
11. Calculated t values 320
12. Sea urchin, Arbacia punctulata, mean proportion of fertilized eggs 321
xix
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TABLES (CONTINUED)
SECTION 15
Number Page
13. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in FORTY
FATHOMS® artificial seawater, using gametes from adults maintained in FORTYFATHOMS® artificial
seawater, or obtained directly from natural sources, and copper (Cu) sulfate as a reference toxicant . . 326
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 327
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 328
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 329
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 toxicants 330
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 and sodium
dodecyl sulfate (SDS) as reference toxicants 331
SECTION 16
Number Page
1. Nutrients to be added to natural seawater and to artificial seawater (GP2) described in
Table 2 336
2. Reagent grade chemicals used in the preparation of GP2 artificial seawater for use in conjunction with
natural seawater for the red macroalga, Champiaparvula, culturing and toxicity testing 337
3. Summary of test conditions and test acceptability criteria for the red macroalga, Champia parvula, sexual
reproduction test with effluents and receiving waters 348
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 350
5. Red macroalga, Champia parvula, sexual reproduction data 352
6. Centered observations for Shapiro-Wilk's example 353
xx
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TABLES (CONTINUED)
SECTION 16
Number Page
7. Ordered centered observations for Shapiro-Wilk's example 354
8. Coefficients and differences for Shapiro-Wilk's example 354
9. ANOVAtable 356
10. ANOVA table for Dunnett's Procedure example 358
11. Calculated t values 359
12. Red macroalga, Champiaparvula, mean number of cystocarps 360
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 364
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) 365
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 366
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) .... 366
xxi
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SECTION 1
INTRODUCTION
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. With the exception of the Red Macroalga, Champia parvula, Reproduction Test Method 1009.0, 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. The Red Macroalga, Champia parvula, Reproduction Test Method 1009.0 is not listed at
40 CFR Part 136 for nationwide use.
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 serf-monitoring permit requirements,
compliance biomonitoring inspections, toxics sampling inspections, and special investigations. Data from chronic
toxicity tests performed 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, 2002a), the short-term chronic toxicity test methods for freshwater organisms (USEPA,
2002b), and the manual for evaluation of laboratories performing aquatic toxicity tests (USEPA, 1991c). In 2002,
EPA revised previous editions of each of the three methods manuals (USEPA, 1993a; USEPA, 1994a; USEPA,
1994b).
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.
1.7 The five species for which toxicity test methods are provided are: the sheepshead minnow, Cyprinodon
variegatus; the inland silverside, Menidia beryllina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia
punctulata', and the red macroalga, Champia parvula.
1.7.1 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
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demonstrated infield studies (USEPA, 1986d).
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.
1.11 The manual was prepared in the established EMSL-Cincinnati format (USEPA, 1983).
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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 offish 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) offish to establish
water quality criteria.
2.1.6 Macek and Sleight (1977) found that exposure of critical life stages offish 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
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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).
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.
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., 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 DURSB AN®, 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 %o (USEPA, 1987a).
2.1.15 Lussieretal. (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.
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2.1.16 Nacci and Jackim( 1985) andUSEPA (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 (r2 = 0.85). However, the
results of the fertilization test with the five metals did not correlate well with the results from the acute tests.
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 parvula, 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 intheNPDES 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)72, (3) RWC, (4) RWC/2, and (5)
RWC/4. More specifically, if the RWC = 50%, appropriate effluent concentrations may 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 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.
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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.
2. Reduced possibility of loss of toxicants through volatilization and/or adsorption to the exposure vessels.
3. Test organisms that rapidly deplete energy reserves 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 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.
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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
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).
4.3 TEST ORGANISMS
4.3.1 The test organisms used in the procedures described in this manual are the sheepshead minnow, Cyprinodon
variegatus', the inland silverside, Menidia beryllina', the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata,
and the red macroalga, Champiaparvula. 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
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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
MARTNEMTX®, 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 ug/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
4.6.1 Water temperature and salinity should be maintained within the limits specified for each test. The temperature
of test 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 should be maintained within the limits 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 The health of test organisms is primarily assessed by the performance (survival, growth, and/or reproduction) of
organisms in control treatments of individual tests. The health and sensitivity of test organisms is also assessed by
reference toxicant testing. In addition to documenting the sensitivity and health of test organisms, reference toxicant
testing is used to initially demonstrate acceptable laboratory performance (Subsection 4.15) and to document ongoing
laboratory performance (Subsection 4.16).
4.7.2 Regardless of the source of test organisms (in-house cultures or purchased from external suppliers), the testing
laboratory must perform at least one acceptable reference toxicant test per month for each toxicity test method
conducted in that month (Subsection 4.16). If a test method is conducted only monthly, or less frequently, a reference
toxicant test must be performed concurrently with each effluent toxicity test.
4.7.3 When 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 toxicant and test conditions (see Section 6, Test Organisms).
4.7.4 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.
4.7.5 If a routine reference toxicant test fails to meet test acceptability criteria, then the reference toxicant test must be
immediately repeated.
10
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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 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.
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 ug/g wet weight, or the concentration of total
organochlorine pesticides plus PCBs exceeds 0.30 ug/g wet weight, or toxic metals (Al, As, Cr, Cd, Cu, Pb, Ni, Zn,
expressed as total metal) exceed 20 ug/g wet weight, the food should not be used (for analytical methods, see AOAC,
1990; and USD A, 1989).
4.84 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
4.9.1 The results of the sheepshead minnow, Cyprinodon variegatus, inland silverside, Menidia beryllina, 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 70%. 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, Cyprindon variegatus, 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 must 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, Menidia
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 must 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 must 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 specifications, depending on the 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.
11
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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 offish) and the 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 data from a 1991 study of chronic toxicity tests using two reference toxicants with the
mysid, Mysidopsis bahia, and the inland silverside, Menidia beryllina, is listed in Table 1. Table 2 shows
interlaboratory precision data from a study of three chronic toxicity test methods using effluent, receiving water, and
reference toxicant sample types (USEPA, 200 la; USEPA, 200 Ib). For the Mysidopsis bahia and the Cyprinodon
variegatus test methods, the effluent sample was a municipal wastewater spiked with KC1, the receiving water sample
was a river water spiked with KC1, and the reference toxicant sample was bioassay-grade FORTY FATHOMS®
12
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synthetic seawater spiked with KC1. For the Menidia beryllina test method, the effluent sample was an industrial
wastewater spiked with CuSO4, the receiving water sample was a natural seawater spiked with CuSO4, and the
reference toxicant sample was bioassay-grade FORTY FATHOMS® synthetic seawater spiked with CuSO4. Additional
precision data for each of the tests described in this manual are presented in the sections describing the individual test
methods.
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, 199la).
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 No-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).
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.
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TABLE 1. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST PRECISION,
1991: SUMMARY OF RESPONSES USING TWO REFERENCE TOXICANTS1'2
Organism
Endpoint
No. Labs
KCl(mg/L)4
SD
CV(%)3
Mysidopsis
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
Organism
Endpoint
No. Labs
Cu(mg/L)4
SD
CV(%)3
Menidia
beryllina
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
1 From a national study of interlaboratory 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.
2 Static renewal test, using 25 %o modified GP2 artificial seawater.
3 Percent coefficient of variation = (standard deviation X 100)/mean.
4 Expressed as mean.
TABLE 2. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST PRECISION,
2000: PRECISION OF RESPONSES USING EFFLUENT, RECEIVING WATER, AND
REFERENCE TOXICANT SAMPLE TYPES1
Organism
Endpoint
Number of Tests2
CV (%)3
Cyprinodon variegatus
Growth, IC25
21
10.5
Menidia beryllina
Growth, IC25
30
Mysidopsis bahia
Growth, IC25
36
41.3
1 From EPA's WET Interlaboratory Variability Study (USEPA, 200la; USEPA, 200Ib).
2 Represents the number of valid tests (i.e., those that met test acceptability criteria) that were used in the analysis
of precision. Invalid tests were not used.
3 CVs based on total interlaboratory variability (including both within-laboratory and between-laboratory
components of variability) and averaged across sample types. IC25s or IC50s were pooled for all laboratories
to calculate the CV for each sample type. The resulting CVs were then averaged across sample types.
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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 toxicity test method conducted in the laboratory during that month. 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. Each laboratory's reference toxicity data will reflect conditions unique to that facility, including dilution
water, culturing, and other variables; however, each laboratory's reference toxicity results should reflect good
repeatability.
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, LC50s, etc.) should be plotted and examined to determine if the results (X^ are
within prescribed limits (Figure 1). The chart should plot logarithm of concentration on the vertical axis against the
date of the test or test number on the horizontal axis. The types of control charts illustrated (see USEPA, 1979a) are
used to evaluate the cumulative trend of results from a series of samples, thus reference toxicant test results should not
be used as a de facto criterion for rejection of individual effluent or receiving water tests. For endpoints that are point
estimates (LC50s and IC25s), the cumulative mean (X) and upper and lower control limits (± 2S) are re-calculated 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 chart
should be maintained using only the 20 most recent data points.
4.16.3 Laboratories should compare the calculated CV (i.e., standard deviation / mean) of the IC25 for the 20 most
recent data points to the distribution of laboratory CVs reported nationally for reference toxicant testing (Table 3-2 in
USEPA, 2000b). If the calculated CV exceeds the 75th percentile of CVs reported nationally, the laboratory should use
the 75th and 90th percentiles to calculate warning and control limits, respectively, and the laboratory should investigate
options for reducing variability. Note: Because NOECs can only be a fixed number of discrete values, the mean,
standard deviation, and CV cannot be interpreted and applied in the same way that these descriptive statistics are
interpreted and applied for continuous variables such as the IC25 or LC50.
4.16.4 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 (LC50s and IC25s), at the
P0 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 laboratory should investigate sources of
variability, take corrective actions to reduce identified sources of variability, and perform an additional reference
toxicant test during the same month. Control limits for the NOECs will also be exceeded occasionally, regardless of
how well a laboratory performs. In those instances when the laboratory can document the cause for the outlier (e.g.,
operator error, culture health or test system failure), the outlier should be excluded from the future calculations of the
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control limits. If two or more consecutive tests do not fall within the control limits, the results must be explained and
the reference toxicant test must be immediately repeated. Actions taken to correct the problem must be reported.
4.16.5 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 laboratory should investigate sources of
variability, take corrective actions to reduce identified sources of variability, and perform an additional reference
toxicant test during the same month. 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 in determining whether or not a reference toxicant
test result falls "well" outside the expected range. The width of the control limits may be evaluated by comparing the
calculated CV (i.e., standard deviation / mean) of the IC25 for the 20 most recent data points to the distribution of
laboratory CVs reported nationally for reference toxicant testing (Table 3-2 in USEPA, 2000b). In determining
whether or not a reference toxicant test result falls "well" outside the expected range, the result also may be compared
with upper and lower bounds for ± 3 S, as any result outside these control limits would be expected to occur by chance
only 1 out of 100 tests (Environment Canada, 1990). When a result from a reference toxicant test is outside the 99%
confidence intervals, the laboratory must conduct an immediate investigation to assess the possible causes for the
outlier.
4.16.6 Reference toxicant test results should not be used as a de facto criterion for rejection of individual effluent or
receiving water tests. Reference toxicant testing is used for evaluating the health and sensitivity of organisms
over time and for documenting initial and ongoing laboratory performance. While reference toxicant test results should
not be used as a de facto criterion for test rejection, effluent and receiving water test results should be reviewed and
interpreted in the light of reference toxicant test results. The reviewer should consider the degree to which the
reference toxicant test result fell outside of control chart limits, the width of the limits, the direction of the deviation
(toward increased test organism sensitivity or toward decreased test organism sensitivity), the test conditions of both
the effluent test and the reference toxicant test, and the objective of the test.
4.17 REFERENCE TOXICANTS
4.17.1 Reference toxicants such as sodium chloride (NaCl), potassium chloride (KC1), cadmium chloride (CdCl2),
copper sulfate (CuSO4), sodium dodecyl sulfate (SDS), and potassium dichromate (K2Cr2O7), 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. Standard reference materials 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.
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o
III
o
0
O
0
r i
LU
O
UPPER CONTROL LIMIT
CENTRAL TENDENCY
LOWER CONTROL LIMIT
i i i i 1 i i i i 1 i i i i 1 i i i i 1
5 10 15 20
UPPER CONTROL LIMIT (X + 2 S)
CENTRAL TENDENCY
LOWER CONTROL LIMIT (X - 2 S)
I I I I I I I I I I I I I I I I I I I I .
A
^\
B
O 5 1O 15 2O
TOXICITY TESTS WITH REFERENCE TOXICANTS
S =
n-\
Where: Xf = Successive toxicity values from toxicity tests.
n = Number of tests.
X = Mean toxicity value.
S = Standard deviation.
Figure 1.
Control charts. (A) hypothesis testing results; (B) point estimates (LC, EC, or 1C).
17
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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.
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)].
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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 BALSTON8 Grade BX or equivalent filters, and
oil and other organic vapors can be removed using activated carbon filters (BALSTON*, 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.
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.
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
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.
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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.
5.4 APPARATUS AND EQUIPMENT FOR CULTURING AND TOXICITY TESTS
5.4.1 Apparatus and equipment requirements for culturing and toxicity tests are specified in each toxicity test method.
Also, see USEPA, 2002a.
5.4.2 WATER PURIFICATION SYSTEM
5.4.2.1 A good quality, laboratory grade deionized water, providing a resistance of 18 megaohm-cm, must be
available in the laboratory and in sufficient quantity for laboratory needs. Deionized water may be obtained from
MILLIPORE®' MILLI-Q®, MILLIPORE® QPAK™2 or equivalent system. If large quantities of high quality deionized
water are needed, it may be advisable to supply the laboratory grade water deionizer with preconditioned water from a
Culligan®, Continental®, or equivalent mixed-bed water treatment system.
5.5 REAGENTS AND CONSUMABLE MATERIALS
5.5.1 SOURCES OF FOOD FOR CULTURE AND TOXICITY TESTS
1. Brine Shrimp, Artemia sp. cysts ~ Many commercial sources of brine shrimp cysts are available.
2. Frozen Adult Brine Shrimp, Artemia — Available from most pet supply shops or other commercial
sources.
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.
5.5.1.1 All food should be tested for nutritional suitability and chemically analyzed for organochlorine pesticides,
PCBs, and toxic metals (see Section 4, Quality Assurance).
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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
5.7.1 See toxicity test methods (see Sections 11-16) for specific supplies.
21
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SECTION 6
TEST ORGANISMS
6.1 TEST SPECIES
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, 2002a).
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 beryllina, the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata\
and the red macroalga, Champia parvula.
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 Sub section 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.
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 (2002a).
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,
1993b).
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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
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 (1993b).
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. 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, 2002a).
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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.
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 BIOPJL® 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 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.
6.6.3 A group of organisms must not be used for a test if they appear to be unhealthy, discolored, or otherwise
stressed, or if mortality appears to 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
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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 obj ectives of the study.
7.1.1.1 If the objective of the test is to estimate the absolute chronic toxicity of the effluent, 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.
7.1.2 An acceptable dilution water is one which is appropriate for the objectives of the test; supports adequate
performance of the test organisms with respect to survival, growth, reproduction, or other responses that may be
measured in the test (i.e., consistently meets test acceptability criteria for control responses); is consistent in quality;
and does not contain contaminants that could produce toxicity. Receiving waters, synthetic waters, or synthetic waters
adjusted to approximate receiving water characteristics may be used for dilution provided that the water meets the
above listed qualifications for an acceptable dilution water. USEPA (2000a) provides additional guidance on selecting
appropriate dilution waters.
7.1.3 When dual controls (one control using culture water and one control using dilution water) are used (see
Subsections 7.1.1.1 -7.1.1.3 above), the dilution water control should be used to determine test acceptability. It is also
the dilution water control that should be compared to effluent treatments in the calculation and reporting of test results.
The culture water control should be used to evaluate the appropriateness of the dilution water source. Significant
differences between organism responses in culture water and dilution water controls could indicate toxicity in the
dilution water and may suggest an alternative dilution water source. USEPA (2000a) provides additional guidance on
dual controls.
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 MAPJNEMIX®) (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).
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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),
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 3 l%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 deionized 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 (3 l%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 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® 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
Compound
NaCl
Na2SO4
KC1
KBr
Na2B4O7-10H2O
MgCl2 -6 H2O
CaCl2 -2 H2O
SrCl2 • 6 H2O
NaHCO3
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
1 Modified GP2 from Spotte et al. (1984).
2 The constituent salts and concentrations were taken from USEPA (2002a). The salinity is 30.89 g/L.
3 GP2 can be diluted with deionized (DI) water to the desired test salinity.
7.3 USE OF RECEIVING WATER AS DILUTION 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.
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 0-6°C during or immediately following collection, and maintained at that temperature
prior to use in the test.
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
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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 um mesh openings prior to use.
7.3.5 HYPERSALINE BRINE
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 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.
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 mm 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 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 HSB should be filtered a second time through a 1-um filter and
poured directly into portable containers (20-L CUBIT AINERS® 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
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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 WATER SAMPLING, SAMPLE HANDLING,
AND SAMPLE PREPARATION FOR TOXICITY TESTS
8.1 EFFLUENT SAMPLING
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, 2002a).
8.1.3 Aeration during collection and transfer of effluents should be minimized to reduce the loss of volatile
chemicals.
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.
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.
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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)
CUBIT AINER® will provide sufficient sample volume for most tests.
8.3.4 THE FOLLOWING EFFLUENT SAMPLING METHODS ARE RECOMMENDED:
8.3.4.1 Continuous Discharges
8.3.4.1.1 If the facility discharge is continuous, a single 24-h composite sample is to be taken.
8.3.4.2 Intermittent Discharges
8.3.4.2.1 If the facility discharge is intermittent, a composite sample is to be collected for the duration of the
discharge but not more than 24 hours.
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.
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 (or hand delivered to the testing
laboratory for use on the day of collection), it is recommended that they be held at 0-6°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 each grab or composite sample 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), should 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, each grab or composite
sample may also be used to prepare test solutions for renewal at 24 h and/or 48 h after first use, if stored at 0-6°C,
with minimum head space, as described in Subsection 8.5. If shipping problems (e.g., unsuccessful Saturday
delivery) are encountered with renewal samples after a test has been initiated, the permitting authority may allow the
continued use of the most recently used sample for test renewal. 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 0-6°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 should not be placed between the ice and the sample in the shipping container unless required to
prevent breakage of glass sample containers.
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 Mail is delivered seven days a week. Saturday and Sunday shipping and receiving schedules of private
carriers vary with the carrier.
33
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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 0-6°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 sample 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 reclosing, 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 should be filtered through a sieve with 60-um mesh
openings. Since filtering may increase the dissolved oxygen (DO) in an effluent, the DO should be checked both
before and after filtering. Low dissolved oxygen concentrations will indicate a potential problem in performing the
test. Caution: filtration may remove some toxicity.
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, samples may be warmed slowly in
open test containers. If DO is still above 100% saturation, based on temperature and salinity (Table 4), after
warming to test temperature, samples should be aerated moderately (approximately 500 mL/min) for a few minutes
using an airstone. If DO is below 4.0 mg/L, the solutions must be aerated moderately (approximately 500 mL/min)
for a few minutes, using an airstone, 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 should 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
34
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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
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.
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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
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
SALINITY (%«)
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
0.2
0.0
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
36
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TABLE 5. PERCENT UNIONIZED NH3 IN AQUEOUS AMMONIA SOLUTIONS: TEMPERATURE
15-26°C AND pH 6.0-8.91
pH
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
TEMPERATURE (°C)
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
o o o
J.JJ
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 18
0.0318 0.0343
0.0400 0.0431
0.0504 0.0543
0.0634 0.0683
0.0799 0.0860
0.1005 0.1083
0.127 0.136
0.159 0.171
0.200 0.216
0.252 0.271
0.317 0.342
0.399 0.430
0.502 0.540
0.631 0.679
0.793 0.854
0.996 1.07
1.25 1.35
1.57 1.69
1.97 2.12
2.46 2.65
3.08 3.31
3.85 4.14
4.80 5.15
5.97 6.40
7.40 7.93
9.14 9.78
11.2 12.0
13.8 14.7
16.7 17.8
20.2 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
0.0530
0.0667
0.0901
0.1134
0.133
0.167
0.210
0.265
0.333
0.419
0.527
0.663
0.833
1.05
1.31
1.65
2.07
2.59
3.24
4.04
5.03
6.25
7.75
9.56
11.7
14.4
17.4
21.0
25.1
29.6
25
0.0568
0.0716
0.0901
0.1134
0.143
0.180
0.226
0.284
0.358
0.450
0.566
0.711
0.893
1.12
1.41
1.77
2.21
2.77
3.46
4.32
5.38
6.68
8.27
10.2
12.5
15.2
18.5
22.2
26.4
31.1
26
0.0610
0.0768
0.0966
0.1216
0.153
0.19
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 see Emerson et al. (1975), Thurston et al.
(1974), and USEPA (1985a).
37
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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. Caution: 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.
8.10.2 The tests consist of a control and a minimum of five effluent concentrations. USEPA recommends the use
of a >0.5 dilution factor for selecting effluent test concentrations. Effluent test concentrations of 6.25%, 12.5%,
25%, 50%, and 100% are commonly used, however, test concentrations should be selected independently for each
test based on the objective of the study, the expected range of toxicity, the receiving water concentration, and any
available historical testing information on the effluent. USEPA (2000a) provides additional guidance on choosing
appropriate test concentrations.
8.10.3 When these tests are used in determining compliance with permit limits, effluent test concentrations should
be selected to bracket the receiving water concentration. This may be achieved by selecting effluent test
concentrations in the following manner: (1) 100% effluent, (2) [RWC + 100]/2, (3) RWC, (4) RWC/2, and (5)
RWC/4. For example, where the RWC = 50%, appropriate effluent concentrations may 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).
38
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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.
39
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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 quantal, "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 the 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 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
40
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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 offish 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.
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 "no-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 (2) 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.
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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 variegatus, 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, Ceriodaphnia 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 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.
9.3.2.2 It should be noted that software used to calculate point estimates occasionally may not provide associated
95% confidence intervals. This situation may arise when test data do not meet specific assumptions required by the
statistical methods, when point estimates are outside of the test concentration range, and when specific limitations
imposed by the software are encountered. USEPA (2000a) provides guidance on confidence intervals under these
circumstances.
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
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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 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 lexicologists 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
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, and should
be based on the objectives for obtaining the toxicity data.
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.
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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.
9.5.3 ANALYSIS OF GROWTH AND REPRODUCTION DATA
9.5.3.1 Growth data from the sheepshead minnow, Cyprinodon variegatus, and inland silverside, Menidia
beryllina, 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, Champiaparvula, 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.
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DATA (SURVIVAL, GROWTH, REPRODUCTION, ETC.)
POINT
ESTIMATION
HYPOTHESIS TESTING
TRANSFORMATION?
ENDPOINT ESTIMATE
LC, EC, 1C
SHAPIRO-WILK'S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
NO STATISTICAL ANALYSIS
RECOMMENDED
NO
^
4 OR
REPLIC
YES
MORE
;ATES?
NO
r
EQUAL NUMBER OF
REPLICATES?
}
YES
r
EQUAL NUMBER OF
REPLICATES?
YES
NO
FWITH
.RRONI
TMENT
DUNNETT'S
TEST
STEEL'S MANY-ONE
RANK TEST
WILCOXON
TEST
BONFERRONI
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 2. Flowchart for statistical analysis of test data
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9.5.4 ANALYSIS OF THE SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA
9.5.4.1 Data from the sea urchin, Arbaciapunctulata, 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 H-
K) (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 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.
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.
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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).
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.
9.7.1.3 A discussion of Probit Analysis, and examples of computer program input and output, are found in
Appendix H.
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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.
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.
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SECTION 10
REPORT PREPARATION AND TEST REVIEW
10.1 REPORT PREPARATION
The toxicity data are reported, together with other appropriate data. The following general format and content are
recommended for the report:
10.1.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body
5. Contract Laboratory (if the test was performed under contract)
a. Name of firm
b. Phone number
c. Address
6. Objective of test
10.1.2 PLANT OPERATIONS
1. Product(s)
2. Raw materials
3. Operating schedule
4. Description of waste treatment
5. Schematic of waste treatment
6. Retention time (if applicable)
7. Volume of waste flow (MOD, CFS, GPM)
8. Design flow of treatment facility at time of sampling
10.1.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Effluent Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Mean daily discharge on sample collection date
f. Lapsed time from sample collection to delivery
g. Sample temperature when received at the laboratory
2. Receiving Water Samples
a. Sampling point (including latitude and longitude)
b. Collection dates and times
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
10.1.4 TEST METHODS
1. Toxicity test method used (title, number, source)
2. Endpoint(s) of test
3. Deviations) 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)
15. Specify if (and how) pH control measures were implemented
10.1.5 TEST ORGANISMS
1. Scientific name and how determined
2. Age
3. Life stage
4. Mean length and weight (where applicable)
5. Source
6. Diseases and treatment (where applicable)
7. Taxonomic key used for species identification
10.1.6 QUALITY ASSURANCE
1. Reference toxicant used routinely; source
2. Date and time of most recent reference toxicant test; test results and current control (cusum) chart
3. Dilution water used in reference toxicant test
4. Results (NOEC or, where applicable, LOEC, LC50, EC50, IC25 and/or IC50); report percent
minimum significant difference (PMSD) calculated for sublethal endpoints determined by hypothesis
testing in reference toxicant test
5. Physical and chemical methods used
10.1.7 RESULTS
1. Provide raw toxicity data in tabular form, including daily records of affected organisms in each
concentration (including controls) and replicate, and in graphical form (plots of toxicity data)
2. Provide table of LC50s, NOECs, IC25, IC50, etc. (as required in the applicable NPDES permit)
3. Indicate statistical methods to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
6. Provide percent minimum significant difference (PMSD) calculated for sublethal endpoints
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10.1.8 CONCLUSIONS AND RECOMMENDATIONS
1. Relationship between test endpoints and permit limits.
2. Action to be taken.
10.2 TEST REVIEW
10.2.1 Test review is an important part of an overall quality assurance program (Section 4) and is necessary for
ensuring that all test results are reported accurately. Test review should be conducted on each test by both the
testing laboratory and the regulatory authority.
10.2.2 SAMPLING AND HANDLING
10.2.2.1 The collection and handling of samples are reviewed to verify that the sampling and handling procedures
given in Section 8 were followed. Chain-of-custody forms are reviewed to verify that samples were tested within
allowable sample holding times (Subsection 8.5.4). Any deviations from the procedures given in Section 8 should
be documented and described in the data report (Subsection 10.1).
10.2.3 TEST ACCEPTABILITY CRITERIA
10.2.3.1 Test data are reviewed to verify that test acceptability criteria (TAG) requirements for a valid test have
been met. Any test not meeting the minimum test acceptability criteria is considered invalid. All invalid tests must
be repeated with a newly collected sample.
10.2.4 TEST CONDITIONS
10.2.4.1 Test conditions are reviewed and compared to the specifications listed in the summary of test condition
tables provided for each method. Physical and chemical measurements taken during the test (e.g., temperature, pH,
and DO) also are reviewed and compared to specified ranges. Any deviations from specifications should be
documented and described in the data report (Subsection 10.1).
10.2.4.2 The summary of test condition tables presented for each method identify test conditions as required or
recommended. For WET test data submitted under NPDES permits, all required test conditions must be met or the
test is considered invalid and must be repeated with a newly collected sample. Deviations from recommended test
conditions must be evaluated on a case-by-case basis to determine the validity of test results. Deviations from
recommended test conditions may or may not invalidate a test result depending on the degree of the departure and
the objective of the test. The reviewer should consider the degree of the deviation and the potential or observed
impact of the deviation on the test result before rejecting or accepting a test result as valid. For example, if
dissolved oxygen is measured below 4.0 mg/L in one test chamber, the reviewer should consider whether any
observed mortality in that test chamber corresponded with the drop in dissolved oxygen.
10.2.4.3 Whereas slight deviations in test conditions may not invalidate an individual test result, test condition
deviations that continue to occur frequently in a given laboratory may indicate the need for improved quality control
in that laboratory.
10.2.5 STATISTICAL METHODS
10.2.5.1 The statistical methods used for analyzing test data are reviewed to verify that the recommended
flowcharts for statistical analysis were followed. Any deviation from the recommended flowcharts for selection of
statistical methods should be noted in the data report. Statistical methods other than those recommended in the
statistical flowcharts may be appropriate (see Subsection 9.4.1.2), however, the laboratory must document the use of
and provide the rationale for the use of any alternate statistical method. In all cases (flowchart recommended
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methods or alternate methods), reviewers should verify that the necessary assumptions are met for the statistical
method used.
10.2.6 CONCENTRATION-RESPONSE RELATIONSHIPS
10.2.6.1 The concept of a concentration-response, or more classically, a dose-response relationship is "the most
fundamental and pervasive one in toxicology" (Casarett and Doull, 1975). This concept assumes that there is a
causal relationship between the dose of a toxicant (or concentration for toxicants in solution) and a measured
response. A response may be any measurable biochemical or biological parameter that is correlated with exposure
to the toxicant. The classical concentration-response relationship is depicted as a sigmoidal shaped curve, however,
the particular shape of the concentration-response curve may differ for each coupled toxicant and response pair. In
general, more severe responses (such as acute effects) occur at higher concentrations of the toxicant, and less severe
responses (such as chronic effects) occur at lower concentrations. A single toxicant also may produce multiple
responses, each characterized by a concentration-response relationship. A corollary of the concentration-response
concept is that every toxicant should exhibit a concentration-response relationship, given that the appropriate
response is measured and given that the concentration range evaluated is appropriate. Use of this concept can be
helpful in determining whether an effluent possesses toxicity and in identifying anomalous test results.
10.2.6.2 The concentration-response relationship generated for each multi-concentration test must be reviewed to
ensure that calculated test results are interpreted appropriately. USEPA (2000a) provides guidance on evaluating
concentration-response relationships to assist in determining the validity of WET test results. All WET test results
(from multi-concentration tests) reported under the NPDES program should be reviewed and reported according to
USEPA guidance on the evaluation of concentration-response relationships (USEPA, 2000a). This guidance
provides review steps for 10 different concentration-response patterns that may be encountered in WET test data.
Based on the review, the guidance provides one of three determinations: that calculated effect concentrations are
reliable and should be reported, that calculated effect concentrations are anomalous and should be explained, or that
the test was inconclusive and the test should be repeated with a newly collected sample. It should be noted that the
determination of a valid concentration-response relationship is not always clear cut. Data from some tests may
suggest consultation with professional lexicologists and/or regulatory officials. Tests that exhibit unexpected
concentration-response relationships also may indicate a need for further investigation and possible retesting.
10.2.7 REFERENCE TOXICANT TESTING
10.2.7.1 Test review of a given effluent or receiving water test should include review of the associated reference
toxicant test and current control chart. Reference toxicant testing and control charting is required for documenting
the quality of test organisms (Subsection 4.7) and ongoing laboratory performance (Subsection 4.16). The reviewer
should verify that a quality control reference toxicant test was conducted according to the specified frequency
required by the permitting authority or recommended by the method (e.g., monthly). The test acceptability criteria,
test conditions, concentration-response relationship, and test sensitivity of the reference toxicant test are reviewed to
verify that the reference toxicant test conducted was a valid test. The results of the reference toxicant test are then
plotted on a control chart (see Subsection 4.16) and compared to the current control chart limits (± 2 standard
deviations).
10.2.7.2 Reference toxicant tests that fall outside of recommended control chart limits are evaluated to determine
the validity of associated effluent and receiving water tests (see Subsection 4.16). An out of control reference
toxicant test result does not necessarily invalidate associated test results. The reviewer should consider the degree to
which the reference toxicant test result fell outside of control chart limits, the width of the limits, the direction of the
deviation (toward increasing test organism sensitivity or toward decreasing test organism sensitivity), the test
conditions of both the effluent test and the reference toxicant test, and the objective of the test. More frequent
and/or concurrent reference toxicant testing may be advantageous if recent problems (e.g., invalid tests, reference
toxicant test results outside of control chart limits, reduced health of organism cultures, or increased within-test
variability) have been identified in testing.
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10.2.8 TEST VARIABILITY
10.2.8.1 The within-test variability of individual tests should be reviewed. Excessive within-test variability may
invalidate a test result and warrant retesting. For evaluating within-test variability, reviewers should consult EPA
guidance on upper and lower percent minimum significant difference (PMSD) bounds (USEPA, 2000b).
10.2.8.2 When NPDES permits require sublethal hypothesis testing endpoints from Methods 1006.0 or 1007.0
(e.g., growth NOECs and LOECs), within-test variability must be reviewed and variability criteria must be applied
as described in this section (10.2.8.2). When the methods are used for non-regulatory purposes, the variability
criteria herein are recommended but are not required, and their use (or the use of alternative variability criteria) may
depend upon the intended uses of the test results and the requirements of any applicable data quality objectives and
quality assurance plan.
10.2.8.2.1 To measure test variability, calculate the percent minimum significant difference (PMSD) achieved in
the test. The PMSD is the smallest percentage decrease in growth or reproduction from the control that could be
determined as statistically significant in the test. The PMSD is calculated as 100 times the minimum significant
difference (MSD) divided by the control mean. The equation and examples of MSD calculations are shown in
Appendix C. PMSD may be calculated legitimately as a descriptive statistic for within-test variability, even when
the hypothesis test is conducted using a non-parametric method. The PMSD bounds were based on a representative
set of tests, including tests for which a non-parametric method was required for determining the NOEC or LOEC.
The conduct of hypothesis testing to determine test results should follow the statistical flow charts provided for each
method. That is, when test data fail to meet assumptions of normality or heterogeneity of variance, a non-parametric
method (determined following the statistical flowchart for the method) should be used to calculate test results, but
the PMSD may be calculated as described above (using parametric methods) to provide a measure of test variability.
10.2.8.2.2 Compare the PMSD measured in the test with the upper PMSD bound variability criterion listed in
Table 6. When the test PMSD exceeds the upper bound, the variability among replicates is unusually large for the
test method. Such a test should be considered insufficiently sensitive to detect toxic effects on growth or
reproduction of substantial magnitude. A finding of toxicity at a particular concentration may be regarded as
trustworthy, but a finding of "no toxicity" or "no statistically significant toxicity" at a particular concentration should
not be regarded as a reliable indication that there is no substantial toxic effect on growth or reproduction at that
concentration.
10.2.8.2.3 If the PMSD measured for the test is less than or equal to the upper PMSD bound variability criterion in
Table 6, then the test's variability measure lies within normal bounds and the effect concentration estimate (e.g.,
NOEC or LOEC) would normally be accepted unless other test review steps raise serious doubts about its validity.
10.2.8.2.4 If the PMSD measured for the test exceeds the upper PMSD bound variability criterion in Table 6, then
one of the following two cases applies (10.2.8.2.4.1, 10.2.8.2.4.2).
10.2.8.2.4.1 If toxicity is found at the permitted receiving water concentration (RWC) based upon the value of the
effect concentration estimate (NOEC or LOEC), then the test shall be accepted and the effect concentration estimate
may be reported, unless other test review steps raise serious doubts about its validity.
10.2.8.2.4.2 If toxicity is not found at the permitted RWC based upon the value of the effect concentration estimate
(NOEC or LOEC) and the PMSD measured for the test exceeds the upper PMSD bound, then the test shall not be
accepted, and a new test must be conducted promptly on a newly collected sample.
10.2.8.2.5 To avoid penalizing laboratories that achieve unusually high precision, lower PMSD bounds shall also
be applied when a hypothesis test result (e.g., NOEC or LOEC) is reported. Lower PMSD bounds, which are based
on the 10th percentiles of national PMSD data, are presented in Table 6. The 10th percentile PMSD represents a
practical limit to the sensitivity of the test method because few laboratories are able to achieve such precision on a
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regular basis and most do not achieve it even occasionally. In determining hypothesis test results (e.g., NOEC or
LOEC), a test concentration shall not be considered toxic (i.e., significantly different from the control) if the relative
difference from the control is less than the lower PMSD bounds in Table 6. See USEPA, 2000b for specific
examples of implementing lower PMSD bounds.
10.2.8.3 To assist in reviewing within-test variability, EPA recommends maintaining control charts of PMSDs
calculated for successive effluent tests (USEPA, 2000b). A control chart of PMSD values characterizes the range of
variability observed within a given laboratory, and allows comparison of individual test PMSDs with the
laboratory's typical range of variability. Control charts of other variability and test performance measures, such as
the MSD, standard deviation or CV of control responses, or average control response, also may be useful for
reviewing tests and minimizing variability. The log of PMSD will provide an approximately normal variate useful
for control charting.
TABLE 6. VARIABILITY CRITERIA (UPPER AND LOWER PMSD BOUNDS) FOR SUBLETHAL
HYPOTHESIS TESTING ENDPOINTS SUBMITTED UNDER NPDES PERMITS.1
Test Method
Method 1006.0, Inland Silverside
Larval Survival and Growth Test
Method 1007.0, Mysidopsis bahia
Survival, Growth, and Fecundity Test
Endpoint
growth
growth
Lower PMSD
Bound
11
11
Upper PMSD
Bound
28
37
Lower and upper PMSD bounds were determined from the 10th and 90th percentile, respectively, of PMSD
data from EPA's WET Interlaboratory Variability Study (USEPA, 200 la; USEPA, 2000b).
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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, Cyprinodon variegatus, 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.
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-hLC50s).
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.
11.1.5 This method 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.
11.2 SUMMARY OF METHOD
11.2.1 Sheepshead minnow, Cyprinodon variegatus, 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.
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.
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.
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11.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent toxicants
(such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection 8.8.6), so
upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with increasing pH.
Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel and zinc were
more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed to be
artifactual and due to pH drift (as determined by parallel testing as described in Subsection 11.3.6.1), the regulatory
authority may allow for control of sample pH during testing using procedures outlined in Subsection 11.3.6.2. It
should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1 pH
unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for toxicity.
11.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with
controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the
test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 11.3.6.2.
The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If the
objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be
maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective of
the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the
sample after adjusting the sample salinity for use in marine testing.
11.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning (i.e., initial pH) and
end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial
pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day
for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was
effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and
the mean final pH should be within ±0.3 pH units of the target pH. Test procedures for conducting toxicity
identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests
(USEPA, 1996).
11.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total
ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test
may be due to ammonia (USEPA, 1992).
11.3.6.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to
the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the
permitted instream waste concentration, then the results from this test should be used for determining compliance. If
the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pH-
controlled test should be used for determining compliance, provided that this test meets test acceptability criteria and
pH was properly controlled (see Subsection 11.3.6.1.1).
11.3.6.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results
of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled
treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of
artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 11.3.6.2) is applied routinely to
subsequent testing of the effluent.
11.3.6.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled
atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding IN NaOH or IN HC1
(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional ions
(Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This may be
accomplished by placing test solutions and test organisms in closed headspace test chambers, and then injecting a
predetermined volume of CO2 into the headspace of each test chamber (USEPA, 199 Ib; USEPA, 1992); or by
placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996). Prior
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experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to inject.
This volume will depend upon the sample pH, sample volume, container volume, and sample constituents. If more
than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the headspace with no more than 5%
CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the receiving
water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving water
(measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the absolute
toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the
sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount (1992)
provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-controlled
testing, control treatments must be subjected to all manipulations that sample treatments are subjected to. These
manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled testing,
the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to confirm
that pH was effectively controlled at the target pH level.
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 240 per
test, if 10 larvae are used in each of four 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.
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).
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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.
11.5.21 Test chambers ~ four 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.
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 culturingArtemia nauplii.
11.5.27 Pipets, volumetric - Class A, 1 -100 mL.
11.5.28 Pipets, automatic - adjustable, 1-100 mL.
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 um, 500 um, 3 to 5 mm) ~ for collecting Artemia nauplii
and fish embryos, and for spawning baskets, respectively.
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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-- 24 per test, containing 4% formalin or 70% ethanol, to preserve larvae (optional).
11.6.4 Weighing pans, aluminum ~ 24 per test.
11.6.5 Tape, colored ~ for labeling 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.
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.
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.
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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.
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 um before placing
into the brine generator. Water should be collected on an incoming tide to minimize the possibility of
contamination.
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 um 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 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® 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 al., 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 al., 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.
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
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between 21 -26°C. The artificial seawater must be conditioned (aerated) for 24 h before use as the testing medium.
If the solution is to be autoclaved, sodium bicarbonate is added after the solution has cooled. A stock solution of
sodium bicarbonate is made up by dissolving 33.6 g NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this
stock solution for each liter of the GP2 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
NaCl
Na2SO4
KC1
KBr
Na2B4O7-10H2O
MgCl2-6 H2O
CaCl2-2H2O
SrCl2-6 H2O
NaHCO3
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
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.
3 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, 2002a) 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.
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. 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 ug/g wet weight or the total
concentration of organochlorine pesticides plus PCBs exceeds 0.30 ug/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
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with incubation temperature and the geographic strain of Artemia used (USEPA, 1985a; USEPA,
2002a; ASTM, 1993).
3. After 24 h, cut off the air supply in the separately 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 um 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.
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 "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.
11.6.15 TEST ORGANISMS, SHEEPSHEAD MINNOWS, CYPRINODON VARIEGATUS
11.6.15.1 Brood Stock
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.
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 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®
or MARDEL AQUARIAN® Tropical Fish Flakes 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 equivalent.
11.6.15.1.5 The system is equipped with an undergravel or outside biological filter of shells (Spotte, 1973; Bower,
1983) for conditioning the biological filter), or a cartridge filter, such as a MAGNUM® Filter, or an EHEIM® Filter,
62
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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)
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
human chorionic gonadotrophin (HCG) hormone. 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.
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 um 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.
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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 periodically 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 can then be gently rolled on a NITEX® screen and culled
(see Section 6, Test Organisms).
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 um 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 um 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 contamination of the newly spawned embryos after they have been manipulated, they
should be placed in a 250 um sieve and briskly sprayed with seawater from a squeeze bottle.
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-G, 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.
64
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Figure 1. Embryonic development of sheepshead minnow, Cyprinodon 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; 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).
65
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N
o
Figure 1. Embryonic development of sheepshead minnow, Cyprinodon 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; O. Young fish 12 mm in length (CONTINUED).
From Kuntz (1916).
66
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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. Atestuarine 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 um 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.
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 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
67
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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.
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.
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.
11.10.1.3 Dilution Water
11.10.1.3.1 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 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
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 for the first time 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).
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TABLE 2. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20%«, USING 20%« 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
Volume of
Effluent Solution
6800 mL
3400 mL Solution 1
3400 mL Solution 2
3400 mL Solution 3
3400 mL Solution 4
To Be Combined
Volume of Diluent
Seawater (20%o)
+ 3400 mL
+ 3400 mL
+ 3400 mL
+ 3400 mL
3400 mL
17000 mL
1 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%oby 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.
2 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.
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%o 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
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controls) must have a minimum of four 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).
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 a minimum of 10 larvae, for a total of a minimum of 40 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.
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
uE/W/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, 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 uneatenArtemia nauplii as possible from each chamber daily to ensure that
the larvae principally eat newly hatched nauplii.
11.10.5.3 On days 0-2, weigh 4 g wet weight or pipette 4 mL of concentrated, rinsedArtemia 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 enoughArtemia in the pipette or syringe for one test
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chamber or settling ofArtemia may occur, resulting in unequal amounts ofArtemia being distributed to the replicate
test chambers.
11.10.5.4 On days 3-6, weigh 6 g wet weight or pipette 6 mLArtemia suspension for a test with five treatments and a
control. Resuspend the 6 gArtemia 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.
11.10.5.5 If the survival rate in any test replicate on any day falls below 50%, reduce the volume ofArtemia 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
11.10.6.1 Before the daily renewal of test solutions, uneaten and deadArtemia, 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
1.10.7.1.1 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.
11.10.7.1.4 Record all the measurements on the data sheet (Figure 2).
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Test Dates:
Type Effluent:_
Effluent Tested:
_ Species:_
Field
Lab
Test
CONCENTRATION:
REPLICATE:
DAYS
#LIVE
LARVAE
TEMP
(°C)
SALINITY
(%o)
DO
(mg/L)
# LARVAE/
DRYWT
0
1
2
3
4
5
MEAN
WEIGHT/
LARVAE (mg)
±SD
6
7
REPLICATE:
0
1
# LARVAE/
DRYWT
2
3
4
5
6
MEAN
WEIGHT/
LARVAE (mg)
±SD
7
REPLICATE:
0
1
# LARVAE/
DRYWT
2
3
4
5
6
MEAN
WEIGHT/
LARVAE (mg)
±SD
7
REPLICATE:
0
1
# LARVAE/
DRYWT
2
3
4
5
6
MEAN
WEIGHT/
LARVAE (mg)
±SD
7
CONCENTRATION:
#LIVE
LARVAE
TEMP
(°C)
SALINITY
(%o)
DO
(mg/L)
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
CONCENTRATION:
#LIVE
LARVAE
TEMP
(°C)
SALINITY
(%o)
DO
(mg/L)
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
#
LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
TIME
FED
Figure 2.
COMMENTS:
Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Daily record of larval survival and test conditions. (From USEPA, 1987b).
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Test Dates:
Type Effluent:_
_ Species:_
Field
Lab
Test
Effluent Tested:
CONCENTRATION:
REPLICATE:
DAYS
#LIVE
LARVAE
TEMP
(°C)
SALINITY
(%o)
DO
(mg/L)
# LARVAE/
DRYWT
0
1
2
3
4
5
MEAN
WEIGHT/
LARVAE (mg)
±SD
6
7
REPLICATE:
0
1
# LARVAE/
DRYWT
2
3
4
5
6
MEAN
WEIGHT/
LARVAE (mg)
±SD
7
REPLICATE:
0
1
# LARVAE/
DRYWT
2
3
4
5
6
MEAN
WEIGHT/
LARVAE (mg)
±SD
7
REPLICATE:
0
1
# LARVAE/
DRYWT
2
3
4
5
6
MEAN
WEIGHT/
LARVAE (mg)
±SD
7
CONCENTRATION:
#LIVE
LARVAE
TEMP
(°C)
SALINITY
(%o)
DO
(mg/L)
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
CONCENTRATION:
#LIVE
LARVAE
TEMP
(°C)
SALINITY
(%o)
DO
(mg/L)
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
#
LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
# LARVAE/
DRYWT
MEAN
WEIGHT/
LARVAE (mg)
±SD
COMMENTS:
Figure 2.
TIME
FED
Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Daily record of larval survival and test conditions. (CONTINUED)
(From USEPA, 1987b).
73
-------
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 2), 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
0-6°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 um 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 mg, and record
the weights (Figure 3).
74
-------
Test Dates:
Species:
Pan
No.
Cone.
&
Rep.
Initial
Wt.
(mg)
Final
Wt.
(mg)
Diff.
(mg)
No.
Larvae
Av. Wt./
Larvae
(mg)
Figure 3. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Dry
weights of larvae (fromUSEPA 1987b).
75
-------
11.10.9.4 Immediately prior to drying, rinse the preserved larvae in distilled (or deionized) water. The rinsed larvae
from each test chamber are 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 Subsection 11.12). 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 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.
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.
76
-------
Test Dates:
Species:
Effluent Tested:
NO. LIVE
LARVAE
SURVIVAL
(%)
MEAN DRY WT/
LARVAE (MG)
±SD
SIGNIF. DIFF.
FROM CONTROL
(o)
MEAN
TEMPERATURE
(°Q
±SD
MEAN SALINITY
%0
±SD
AVE DISSOLVED
OXYGEN
(MG/L) ± SD
COMMENTS:
Figure 4. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test.
Summary of test results (from USEPA, 1987b).
77
-------
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 (TEST METHOD 1004.0)1
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 (required)
20%o to 32%o (± 2%o of the selected test salinity)
(recommended)
25 ± 1°C (recommended)
Test temperatures must not deviate (i.e., maximum minus
minimum temperature) by more than 3°C during the test
(required)
Ambient laboratory illumination (recommended)
10-20 uE/m2/s (50-100 ft-c) (ambient laboratory levels)
(recommended)
16 h light, 8 h darkness (recommended)
600 mL -1 L beakers or equivalent (recommended)
500-750 mL/replicate (loading and DO restrictions must be met)
(recommended)
Daily (required)
Newly hatched larvae (less than 24 h old; less than or equal to
24-h range in age) (required)
10 (required minimum)
4 (required minimum)
40 (required minimum)
Newly hatched Artemia nauplii, (less than 24-h old) (required)
Feed once a day 0.10 g wet weight^rfe/w/'a nauplii per replicate
on Days 0-2; Feed 0.15 g wet weight Artemia nauplii per
replicate on Days 3-6 (recommended)
Siphon daily, immediately before test solution renewal and
feeding (required)
None, unless DO falls below 4.0 mg/L, then aerate all
chambers. Rate should be less than 100 bubbles/minimum
(recommended)
1 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is
identified as required or recommended (see Subsection 10.2 for more information on test review). Additional
requirements may be provided in individual permits, such as specifying a given test condition where several options
are given in the method.
78
-------
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 (TEST METHOD 1004.0)
(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
MARTNEMIX® FORTY FATHOMS®, GP2 or equivalent)
(available options)
Effluent: 5 and a control (required)
Receiving Waters: 100% receiving water (or minimum of 5)
and a control (recommended)
Effluents: > 0.5 (recommended)
Receiving waters: None, or > 0.5 (recommended)
7 days (required)
Survival and growth (weight) (required)
80% or greater survival in controls; average dry weight per
surviving organism in control chambers must 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 (required)
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 (e.g., 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) (required)
6 L per day (recommended)
79
-------
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) in
Replicate Chambers
A
1.29
1.27
1.32
1.29
1.62
—
B
1.32
1.00
1.37
1.33
0.56
—
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.
11.13.2 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 Forthe 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.
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).
80
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STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL HYPOTHESIS TESTING
SURVIVAL DATA
PROPORTION SURVIVING
T
ARC SINE
TRANSFORMATION
1
r
SHAPIRO-WIUCSTEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
NO
EQUAL NUMBER OF
REPLICATES?
i
YES
'
EQUAL NUMBER OF
REPLICATES?
YES
NO
T-TESTWITH
BONFERRONI
ADJUSTMENT
DUNNETTS
TEST
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.
81
-------
STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW
LARVAL SURVIVAL AND GROWTH TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
T
TWO OR MORE
PARTIAL MORTALITIES?
I YES
NO
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2 TEST)
NO
ONE OR MORE
PARTIAL MORTALITIES?
YES
IYES
GRAPHICAL METHOD
LC50
PROBIT METHOD
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
YES
NO
SPEARMAN-KARBER
METHOD
TRIMMED SPEARMAN-
KARBER METHOD
LC50AND95%
CONFIDENCE
INTERVAL
Figure 6. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval
survival data by point estimation.
82
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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 and 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 =
Where: Xj = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
83
-------
TABLE 5. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, SURVIVAL DATA
Effluent Concentration (%)
Replicate Control
6.25 12.5 25.0 50.0
RAW
ARC SINE
TRANSFORMED
Mean(Y)
s,2
i
A
B
C
D
A
B
C
D
1.0
1.0
1.0
1.0
1.412
1.412
1.412
1.412
1.412
0.0
1
1.0
1.0
0.9
1.0
1.412
1.412
1.249
1.412
1.371
0.007
2
1.0
1.0
1.0
1.0
1.412
1.412
1.412
1.412
1.412
0.0
3
1.0
1.0
1.0
0.8
1.412
1.412
1.412
1.107
1.336
0.023
4
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 12.5 25.0 50.0
A
B
C
D
0.0
0.0
0.0
0.0
0.041
0.041
-0.122
0.041
0.0
0.0
0.0
0.0
0.076
0.076
0.076
-0.229
0.084
0.084
-0.032
-0.137
11.13.2.6.3 For this set of data,
n = 20
X = — (-0.001) = 0.000
20
D = 0.1236
84
-------
INDIVIDUAL REPLICATE SURVIVAL PROPORTIONS
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
0.8
0.7-
CfL
O
Q_
O
> 0.4^
co 0.3-
0.2-
0.1-
0.0
O.OO
6.25 12.50 25.00
EFFLUENT CONCENTRATION (%)
Figure 7. Plot of mean survival proportion data in Table 5.
-X-
85
-------
11.13.2.6.4 Order the centered observations from smallest to largest
X(l) < X(2) < < X(n)
where X(l) denotes the ith ordered observation. The ordered observations for this example are listed in Table 7.
11.13.2.6.5 From Table 4, AppendixB, forthe number of observations, n, obtain the coefficients ab 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 ^ values are listed
in Table 8.
11.13.2.6.6 Compute the test statistic, W, as follows:
The differences X(M+1) - X(l) are listed in Table 8. For the data in this example,
W = -— (0.3178)2 = 0.8171
0.1236
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR THE SHAPIRO-WILK'S EXAMPLE
X® i X(l)
1
2
o
3
4
5
6
7
8
9
10
-0.229
-0.137
-0.122
-0.032
0.0
0.0
0.0
0.0
0.0
0.0
11
12
13
14
15
16
17
18
19
20
0.0
0.0
0.041
0.041
0.041
0.076
0.076
0.076
0.084
0.084
11.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 11.13.2.6.6 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 = 0.817 is less than the critical value, conclude that the data are not normally
distributed.
86
-------
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.
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1 0.4734
2 0.3211
3 0.2565
4 0.2085
5 0.1686
6 0.1334
7 0.1013
8 0.0711
9 0.0422
10 0.0140
0.313
0.221
0.198
0.108
0.076
0.041
0.041
0.041
0.0
0.0
X(20)
X(19)
X(18)
X(")
X(16)
x(15)
X(14)
Xd3)
X(12)
X(1D
-x(1)
-x(2)
-x(3)
_x<4>
-x(5)
-X(6)
-x(7)
-X®
-x(9)
.x(10)
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.
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 is 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.
87
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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
1.249
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
TABLE 10. TABLE OF RANKS
Replicate Control
Effluent Concentration (%)
6.25
12.5
25.0
TABLE 11. RANK SUMS
50.0
A
B
C
D
1.412 (5,4.5,5,6.5)
1.412 (5,4.5,5,6.5)
1.412 (5,4.5,5,6.5)
1.412 (5,4.5,5,6.5)
1.412(5)
1.412(5)
1.249(1)
1.412(5)
1.412 (4.5)
1.412 (4.5)
1.412 (4.5)
1.412 (4.5)
1.412(5)
1.412(5)
1.412(5)
1.107(1)
1.107(3.5)
1.107(3.5)
0.991 (2)
0.886(1)
Effluent Concentration (%)
6.25
12.5
25.0
50.0
Rank Sum
16
18
16
10
-------
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:
s s s 0.00+0.025+0.00 0.025 „ „„„,,
Po = Pi = Pi = ; = —— = °-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 be adjusted using Abbott's formula (Finney, 1971). The adjustment takes the
form:
Where: ps0 = the smoothed observed proportion mortality for the control
psj = the smoothed observed proportion mortality for effluent concentration i
11.13.2.8.4.1 Forthe data in this example, the data for each effluent concentration must be adjusted for control
mortality using Abbott's formula, as follows:
a Pi'Po 0.0083-0.0083 0.00 „ „
Pn = Pi = P? = - = - = - = 0.0
° ' 2 _s 1-0.0083 0.9917
a P3-Po 0.05-0.0083 0.0417 „„.,
p, = - = - = - = 0.042
!_„* 1-0.0083 0.9917
1 Po
0.275-0.0083 0.2667
1-0.0083 0.9917
s-Po 1.000-0.0083 0.9917
= 1.000
l_ps 1-0.0083 0.9917
The smoothed, adjusted response proportions for the effluent concentrations are shown in Table 12.
89
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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
11.13.2.8.5 Calculate the Iog10 of the estimated LC50, m, as follows:
Where: p\ = the smoothed adjusted proportion mortality at concentration i
Xj = 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
= 1.755873
11.13.2.8.6 Calculate the estimated variance of mas follows:
V(m) =
Where: Xj = the Iog10 of concentration i
HJ = the number of organisms tested at effluent concentration i
paj = the smoothed adjusted observed proportion mortality at effluent concentration i
k = the number of effluent concentrations tested, not including the control
11.13.2.8.6.1 Forthis example, the estimated variance of m, V(m), is calculated as follows:
V(m) = (0.000)(1.000)(1.3979 - 0.7959)2/4(39) +
(0.042)(0.958)(1.6990 - 1.0969)2/4(39)+
(0.269)(0.731)(2.0000 - 1.3979)2/4(39)
= 0.0005505
90
-------
11.13.2.8.7 Calculate the 95% confidence interval for m: m ± 2.0 /~"V (m)
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(l.756974) = 57.1%
11.13.3 EXAMPLE OF ANALYSIS OF SHEEPSHEAD MINNOW, CYPRINODON VARIEGA TUS, 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. Because this measurement is based on the number
of original organisms exposed (rather than the number surviving), the measured response is a combined survival and
growth endpoint that can be termed biomass. 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 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.
91
-------
STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
GROWTH
POINT ESTIMATION
ENDPCUNT ESTIMATE
IC25, IC50
GFO/VTHDATA
MEAN DRY WEIGHT
HYPOT1-ESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FCR SURVIVAL)
SHAPIRO-WILKS TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
1
EQU
R
NO
r
T-TESTW1TH
BONFERRONI
ADJUSTMENT
IAL NUMBER OF
EPLICATES?
1 YES
DUNNETTS
TEST
1
B
YES
r
STEEL'S MANY-ONE
RANK TEST
aUAL NUMBER OF
REPLICATES?
1 NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
t
ENDFOINT ESTIMATES
NOEC, LOEC
Figure 8. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval growth data.
92
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TABLE 13. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, GROWTH DATA
Effluent Concentration (%)
Replicate
A
B
C
D
Mean (Y )
s2,
i
Control
1.29
1.32
1.59
1.27
1.368
0.0224
1
6.25
1.27
1.00
0.97
0.97
1.053
0.0212
2
12.5
1.32
1.37
1.35
1.34
1.345
0.0004
3
25.0 50.0
1.29
1.33
1.20
0.94
1.190
0.0307
4 5
100.0
-
-
-
_
-
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 within a concentration from each observation in that concentration. The centered observations are
summarized in Table 14.
TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Effluent Concentration (%)
Replicate
A
B
C
D
Control
-0.078
-0.048
0.222
-0.098
6.25
0.217
-0.053
-0.083
0.083
12.5
-0.025
0.025
0.005
-0.005
25.0
0.100
0.140
0.010
-0.250
11.13.3.5.2 Calculate the denominator, D, of the test statistic:
D =
Where: Xj = 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
93
-------
X = — (-0.004) = 0.00024 = 0.00
16
D = 0.2245
11.13.3.5.3 Order the centered observations from smallest to largest:
X(l) < X(2) < < X(n)
Where X(l) is the ith ordered observation. These ordered observations are listed in Table 15.
TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
X®
-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
X®
-0.005
0.005
0.010
0.025
0.100
0.140
0.217
0.222
94
-------
X
O
LU
LU
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0
CONNECTS MEAN VALUE FOR EACH CONCENTRATION
REPRESENTS THE CRITICAL VALUE FOR DUNNETT'S TEST
(ANY MEAN WEIGHT BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
6.25 12.50
EFFLUENT CONCENTRATION (%)
25.00
Figure 9. Plot of weight data from sheepshead minnow, Cyprinodon variegatus, larval survival and growth test.
95
-------
11.13.3.5.4 From Table 4, Appendix B, for the number of observations, n, obtain the coefficients ab 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 = 16 and k = 8. The ^ values
are listed in Table 16.
TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1 0.5056
2 0.3290
3 0.2521
4 0.1939
5 0.1447
6 0.1005
7 0.0593
8 0.0196
0.472
0.315
0.223
0.183
0.103
0.063
0.053
0.020
x(16) - x(1)
X(15).X(2)
X(14).X(3)
X(13).X(4)
X(12).X(5)
X(11).X(6)
X(10).X(7)
X(9) _X(8)
11.13.3.5.5 Compute the test statistic, W, as follows:
w =
The differences X(M+1) - X(l) are listed in Table 16.
For this set of data:
W = — (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 0.844. Since W = 0.938 is
greater than the critical value, the conclusion of the test is that the data are normally distributed.
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 Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
follows:
96
-------
B = -J=L
p p
[(EF,) In S2 - EF; In S2]
Where: V; = degrees of freedom for each effluent concentration and control, V; = (n; - (1))
HJ = 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
C =
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.
1 1. 13.3.6.3 Bartlett's statistic is therefore:
.
B = [(12)ln(0.0187) - 3Eln(,Sf)]/1.139
Z = l
= [12(-3.979) - 3(-18.876)]/1.139
= 8.882/1.139
= 7.798
11.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees of freedom, when the variances are
in fact the same. Therefore, the appropriate critical value for this test, at a significance level of 0.0 1 with three
degrees of freedom, is 11.345. Since B = 7.798 is less than the critical value of 11.345, conclude that the
variances are not different.
97
-------
11.13.3.7 Dunnett's Procedure
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
Mean Square (MS)
(SS/df)
Si = SSBI(p-\)
S'w = SSW/(N-p)
Where: p = number of concentration levels including the control
N = total number of observations n{ + n2... + n p
HJ = number of observations in concentration i
SSB =
Between Sum of Squares
p "i
SST = EE7,.?-G2/A^
Total Sum of Squares
SSW = SST-SSB
Within Sum of Squares
G = the grand total of all sample observations, G = E71.
z = l
Tj = the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the mean dry weight of the my sids for
concentration i in test chamber j)
11.13.3.7.2 For the data in this example:
H! = n2 = n3 = n4 = 4
N =16
T=Y +Y +Y +Y =5 47
J-i * n ^ * 12 ^ * is ^ * 14 3-4/
98
-------
T2 = Y21+Y22 + Y23 + Y24 = 4.21
T3 = Y31+Y32 + Y33 + Y34 = 5.38
T=Y +Y +Y +Y = 476
14 141 T i 42 -t- i43 -t- i44 t. /o
G = 1\ + T2 + T3 + T4 = 19.82
SSB =
= 1 (99.247) - (19.82)2 =0.260
4 16
•SST =
= 25.036 - (19.82)2 =0.484
16
= SST-SSB
= 0.484 - 0.260 = 0.224
SB2 = SSB/(p-l) = 0.2607(4-1) = 0.087
Sw2 = SSW/(N-p) = 0.2247(16-4) = 0.019
11.13.3.7.3 Summarize these calculations in the ANOVA table (Table 18).
99
-------
TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
3
12
15
Sum of Squares
(SS)
0.260
0.224
0.484
Mean Square(MS)
(SS/df)
0.087
0.019
11.13.3.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control
combination as follows:
ti —
'f11> (1/fl,-)
Where: Y; = mean dry weight for effluent concentration i
Y! = mean dry weight for the control
Sw = square root of the within mean square
H! = number of replicates for the control
HJ = number of replicates for concentration i.
11.13.3.7.5 Table 19 includes the calculated lvalues for each concentration and control combination. Inthis
example, comparing the 6.25% concentration with the control, the calculation is as follows:
TABLE 19. CALCULATED T VALUES
Effluent Concentration (%)
6.25
12.5
25.0
i
2
3
4
ti
3.228
0.236
1.824
100
-------
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
tj is greater than the critical value. Since 12 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 = dSllnJ +(!/«)
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)
H! = the number of replicates in the control.
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, forthis 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
101
-------
means by
11.13.3.8.2 parting with the control mean, Y ! = 1.368 and Y2= 1.053, we see that Yj >Y2.
Comparing Y2 to Y 3, Y 2
-------
1.6
1.5
1.4
1.3
1.2
0) 1.1
< °-9
| °-8
CQ 0.7
LU
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.00
INDIVIDUAL REPLICATE MEAN BIOMASS
CONNECTS THE OBSERVED MEAN VALUES
CONNECTS THE SMOOTHED MEAN VALUES
6.25 12.50 25.00
EFFLUENT CONCENTRATION (%)
50.00
100.00
Figure 10. Plot of raw data, observed means, and smoothed means for the sheepshead minnow, Cyprinodon varieagatus, growth data from Tables 4 and
20.
103
-------
11.13.3.8.6 AnIC25 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/100) = 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 C 5 = 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:
(C -C)
ICp =
J
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 from Section 4.2 of Appendix L, the estimate of the IC50 is calculated as follows:
IC50 = 50.0 + [1.368(1-50/100) - 0525] (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 3 1. 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 forthe true mean was 39.1011% and
49.0679%. The computer program output is shown in Figure 12.
104
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
0
1.29
1.32
1.59
1.27
6.25
1.27
1
.972
.97
12.5
1.32
1.37
1.35
1.34
25
1.29
1.33
1.2
.936
50
.62
.560
.46
.46
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. Number Concentration
ID
1
2
3
4
5
6
The
Replicates
4
4
4
4
4
4
Linear Interpolation Estimate:
%
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
31.1512
Std.
Dev.
0.150
0.145
0.021
0.177
0.079
0.000
Entered? Value:
Pooled
Response Means
1.368
1.199
1.199
1.189
0.525
0.000
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.
105
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 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.iSO
Con
ID
1
2
3
4
5
6
The
c. Number
Replicates
4
4
4
4
4
4
Linear Interpolation Estimate:
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
44.0230
Std.
Dev.
0.150
0.145
0.021
0.177
0.079
0.000
Entered? Value: 50
Pooled
Response Means
1.368
1.199
1.199
1.189
0.525
0.000
Number of Resamplings: 80
The Bootstrap Estimates Mean: 44.3444 Standard Deviation: 1.7372
Original Confidence Limits: Lower: 40.9468 Upper: 47.1760
Expanded Confidence Limits: Lower: 39.1011 Upper: 49.0679
Resampling time in Seconds: 1.70 Random Seed: -156164614
Figure 12. ICPIN program output for the IC50.
106
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11.14 PRECISION AND ACCURACY
11.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below (Subsections
11.14.1.1 and 11.14.1.2). Single-laboratory precision is a measure of the reproducibility of test results when tests are
conducted using a specific method under reasonably constant conditions in the same laboratory. Single-laboratory
precision is synonymous with the terms within-laboratory precision and intralaboratory precision. Multilaboratory
precision is a measure of the reproducibility of test results from different laboratories using the same test method and
analyzing the same test material. Multilaboratory precision is synonymous with the term interlaboratory precision.
Interlaboratory precision, as used in this document, includes both within-laboratory and between-laboratory
components of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined (termed total
interlaboratory variability). The total interlaboratory variability that is reported from these studies is synonymous
with interlaboratory variability reported from other studies where individual variability components are not
separated.
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 GP2 with copper sulfate, sodium dodecyl
sulfate, and hexavalent chromium, as reference toxicants, are given in Tables 21-26. 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.
11.14.1.1.2 EPA evaluated within-laboratory precision of the Sheepshead Minnow, Cyprinodon variegatus, Larval
Survival and Growth Test using a database of routine reference toxicant test results from five laboratories (USEPA,
2000b). The database consisted of 57 reference toxicant tests conducted in 5 laboratories using reference toxicants
including: cadmium and potassium chloride. Among the 5 laboratories, the median within-laboratory CV calculated
for routine reference toxicant tests was 13% for the IC25 growth endpoint. In 25% of laboratories, the within-
laboratory CV was less than 9%; and in 75% of laboratories, the within-laboratory CV was less than 14%.
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 interlaboratory
precision. The coefficient of variation (IC25) was 44.2% and (IC50) was 56.9%, indicating acceptable precision.
11.14.1.2.2 In 2000, EPA conducted an interlaboratory variability study of the Sheepshead Minnow, Cyprinodon
variegatus, Larval Survival and Growth Test (USEPA, 200la; USEPA, 200Ib). In this study, each of 7 participant
laboratories tested 4 blind test samples that included blank, effluent, reference toxicant, and receiving water sample
types. The blank sample consisted of bioassay-grade FORTY FATHOMS* synthetic seawater, the effluent sample
was a municipal wastewater spiked with KC1, the receiving water sample was a natural seawater spiked with KC1,
and the reference toxicant sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater spiked with
KC1. Of the 28 Sheepshead Minnow Larval Survival and Growth Tests conducted in this study, 100% were
successfully completed and met the required test acceptability criteria. Of 7 tests that were conducted on blank
samples, none showed false positive results for the survival endpoint or the growth endpoint. Results from the
reference toxicant, effluent, and receiving water sample types were used to calculate the precision of the method.
Table 28 shows the precision of the IC25 for each of these sample types. Averaged across sample types, the total
interlaboratory variability (expressed as a CV%) was 10.5% for IC25 results. Table 29 shows the frequency
distribution of survival and growth NOEC endpoints for each sample type. For the survival endpoint, NOEC values
spanned two concentrations for the reference toxicant sample type and one concentration for the effluent and
107
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receiving water sample types. The percentage of values within one concentration of the median was 100% for each
of the sample types. For the growth endpoint, NOEC values spanned one concentration for the reference toxicant
sample type and two concentrations for the effluent and receiving water sample types. The percentage of values
within one concentration of the median was 100% for each of the sample types.
11.14.2 ACCURACY
11.14.2.1 The accuracy of toxicity tests cannot be determined.
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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, USING COPPER (CU)
SULFATE AS A REFERENCE TOXICANT1'2'3'4'5
Test
Number
1
2
o
5
4
5
6
7
8
n:
Mean:
CV(%):
1 Data from USEPA
2 Tests performed by
3 All tpsts WPHP nprfn
NOEC
(^g/L)
50
<507
<507
50
<507
50
50
50
5
NA
NA
(1988a) and USEPA (1991a).
IC25
(^g/L)
113.3
54.3
41.8
63.2
57.7
48.3
79.6
123.5
8
72.7
41.82
Donald J. Klemm, Bioassessment and Ecotoxicology
,1-mprl iidntr FORTY FA TROMS® «v
nthptir spawatpr
IC50
(|Ag/L)
152.3
97.5
71.4
90.8
99.8
132.5
159.7
236.4
8
130.0
40.87
Branch, EMSL, Cincinnati,
Thnpp npnliratp p^nnsiirp rV
Most
Sensitive
Endpoint6
S
G
G
S
S
G
G
G
OH.
lamhprs parh
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 mg/L. Copper concentrations in Tests 7-8 were: 25, 50,100, 200 and 400 mg/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.
Lowest concentration tested was 50 ug/L (NOEC Range: < 50* - 50 ug/L).
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TABLE 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 TOXICANT1'2'3'4'5'6
Test
Number
1
2
3
4
5
6
n:
Mean:
CV(%):
NOEC
(mg/L)
1.0
1.0
1.0
0.5
1.0
0.5
6
NA
NA
IC25
(mg/L)
1.2799
1.4087
2.3051
1.9855
1.1901
1.1041
6
1.5456
31.44
IC50
(mg/L)
1.5598
1.8835
2.8367
2.6237
1.4267
1.4264
6
1.9595
31.82
Most
Sensitive
Endpoint7
S
S
S
G
S
G
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 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
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TABLE 23. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON
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 TOXICANT1'2'3'4'5'6
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(Hg/L)
125
31
125
125
125
5
NA
NA
IC25
(Hg/L)
320.3
182.3
333.4
228.4
437.5
5
300.4
33.0
IC50
(Hg/L)
437.5
323.0
484.4
343.8
NC8
4
396.9
19.2
Most
Sensitive
Endpoint7
S
G
S
S
S
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by George Morrison and Elise Torello, ERL-N, USEPA, 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 ug/L.
NOEC Range: 31 - 125 ug/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.
Ill
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TABLE 24. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON
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
TOXICANT1'2'3'4'5'6
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(mg/L)
2.5
1.3
1.3
1.3
1.3
5
NA
NA
IC25
(mg/L)
2.9
NCI8
1.9
2.4
1.5
4
2.2
27.6
IC50
(mg/L)
3.6
NC29
2.4
NC2
1.8
o
3
2.6
35.3
Most
Sensitive
Endpoint7
S
G
S
G
S
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by George Morrison and Elise Torello, 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.
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.
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TABLE 25. 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, AND HEXAVALENT
CHROMIUM AS THE REFERENCE TOXICANT1'2'3'4'5
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(mg/L)
2.0
1.0
4.0
2.0
1.0
5
NA
NA
IC25
(mg/L)
5.8
2.9
6.9
2.4
3.1
5
4.2
47.6
IC50
(mg/L)
11.4
9.9
11.5
9.2
10.8
5
10.6
9.7
Most
Sensitive
Endpoint6
G
G
G
G
G
1 Tests performed by Donald Klemm, Bioassessment and Ecotoxicology Branch, EMSL, Cincinnati, OH.
2 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.
3 NOEC Range: 1.0 - 4.0 mg/L (this represents a difference of four exposure concentrations)
4 Adults collected in the field.
5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
6 Endpoints: G=growth; S=survival.
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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 SEAWATER1'2'3'4
Survival
Growth
SDS (mg/L)
GP2
NSW
GP2
NSW
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
Copper(ug/L)
GP2
NSW
GP2
NSW
Mean
CV(%)
455
467
390
437
9.4
412
485
528
475
12.3
341
496
467
435
18.9
333
529
776
546
40.7
1 Tests performed by George Morrison and Glen Modica, ERL-N, USEPA, Narragansett, RI.
2 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.
3 Adults collected in the field.
4 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
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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
Test
Number
1
2
1
2
NOEC
(%)
3.2 (S,G)
3.2 (S,G)
3.2 (S,G)
3.2 (S,G)
Most Sensitive Endpoint4
IC25
(%)
7.4 (S)
7.6 (S)
5.7 (G)
5.7 (G)
IC50
(%)
7.4 (G)
14.3 (G)
9.7 (G)
8.8 (G)
Laboratory C
Laboratory D
n:
Mean:
CV(%):
1 1.0 (S)
1 3.2 (S,G)
2 1.0 (G)
7
NA
NA
4.7 (S)
7.4 (G)
5.2 (S)
7
5.5
44.2
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.
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TABLE 28. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES1
Test Endpoint
Sample Type
CV (%)2
IC25
Reference toxicant
Effluent
Receiving water
18.4
6.12
7.15
Average
10.5
FromEPA's WET Intel-laboratory Variability Study (USEPA, 2001a; USEPA, 200Ib).
CVs were calculated based on the total interlaboratory variability (including both within-laboratory and between-
laboratory components of variability). Individual within-laboratory and between-laboratory components of variability
could not be calculated since the study design did not provide within-laboratory replication for this sample type.
TABLE 29. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR VARIOUS SAMPLE
TYPES1
Test Endpoint
Survival NOEC
Growth
NOEC
Sample Type
Reference toxicant
Effluent
Receiving water
Reference toxicant
Effluent
Receiving water
Median
NOEC
Value
25%
25%
25%
25%
12.5%
12.5%
% of Results at
the Median
57.1
100
100
100
57.1
71.4
% of Results
±12
42.9
0.00
0.00
0.00
42.9
28.6
% of Results
>23
0.00
0.00
0.00
0.00
0.00
0.00
1 FromEPA's WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 200Ib).
2 Percent of values at one concentration interval above or below the median. Adding this percentage to the percent of
values at the median yields the percent of values within one concentration interval of the median.
3 Percent of values two or more concentration intervals above or below the median.
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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 (1987b), estimates the chronic toxicity of
effluents and receiving waters to the sheepshead minnow, Cyprinodon variegatus, 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-hLC50s).
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 variegatus, 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).
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.
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12.3.5 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent
toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection
8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with
increasing pH. Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel
and zinc were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed
to be artifactual and due to pH drift (as determined by parallel testing as described in Subsection 12.3.5.1), the
regulatory authority may allow for control of sample pH during testing using procedures outlined in Subsection
12.3.5.2. It should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large
(more than 1 pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold
for toxicity.
12.3.5.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with
controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the
test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 12.3.5.2.
The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If
the objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be
maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective
of the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the
sample after adjusting the sample salinity for use in marine testing.
12.3.5.1.1 During parallel testing, the pH must be measured in each treatment at the beginning (i.e., initial pH) and
end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial
pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day
for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was
effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and
the mean final pH should be within ± 0.3 pH units of the target pH. Test procedures for conducting toxicity
identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests
(USEPA, 1996).
12.3.5.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total
ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test
may be due to ammonia (USEPA, 1992).
12.3.5.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to
the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the
permitted instream waste concentration, then the results from this test should be used for determining compliance.
If the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pH-
controlled test should be used for determining compliance, provided that this test meets test acceptability criteria
and pH was properly controlled (see Subsection 12.3.5.1.1).
12.3.5.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results
of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled
treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of
artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 12.3.5.2) is applied routinely to
subsequent testing of the effluent.
12.3.5.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled
atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding IN NaOH or IN HC1
(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional
ions (Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This
may be accomplished by placing test solutions and test organisms in closed headspace test chambers, and then
injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992);
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or by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996).
Prior experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to
inject. This volume will depend upon the sample pH, sample volume, container volume, and sample constituents.
If more than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the headspace with no more
than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the
receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving
water (measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the
absolute toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount
(1992) provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-
controlled testing, control treatments must be subjected to all manipulations that sample treatments are subjected to.
These manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled
testing, the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to
confirm that pH was effectively controlled at the target pH level.
12.4 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.
12.5.2 Sheepshead minnow culture unit ~ see Subsection 12.6.12 below. To perform toxicity tests on-site or in the
laboratory, sufficient numbers of newly fertilized eggs must be available, preferably from an in-house sheepshead
minnow culture unit. If necessary, embryos can be obtained from outside sources if shipped in well oxygenated
water in insulated containers.
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 of the test.
12.5.3 Brine Shrimp, Artemia, Culture Unit - for feeding sheepshead minnow larvae in the continuous culture unit
(see Subsection 12.6.12 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.
12.5.9 Air Pump ~ for oil free air supply.
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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 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). 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 Pipets, 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.
12.5.28 Siphon with bulb and clamp ~ for cleaning test chambers.
12.5.29 NITEX® or stainless steel mesh sieves, (< 150 um, 500 um, and 3-5 mm) ~ for collecting Artemia nauplii
and fish embryos, and for spawning baskets, respectively.
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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).
12.6.2 Data sheets (One set per test) ~ for data recording (see Figure 1).
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 Winkler 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%o 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. 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.
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Test Dates:
Type Effluent:
Effluent Tested:
Original pH:
Species:
Field:
Lab:
Test:
Salinity:
DO:
CONCENTRATION:
Replicate I:
DAY
#Live/Dead
Embryo-Larvae
Terata
Temp. (°C)
Salinity (ppt)
DO (mg/L)
PH
0
1
2
3
4
5
6
7
8
9
CONCENTRATION:
Replicate II:
DAY
#Live/Dead
Embryo-Larvae
Terata
Temp. (°C)
Salinity (ppt)
DO (mg/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 12.10.9 and 12.13).
Figure 1. Data form for sheepshead minnow, Cyprinodon variegatus, embryo-larval survival/teratogenicity test.
Daily record of embryo-larval survival/terata and test conditions.
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CONCENTRATION:
Replicate III:
DAY
#Live/Dead
Embryo-Larvae
Terata
Temp. (°C)
Salinity (ppt)
DO (mg/L)
PH
0
1
2
3
4
5
6
7
8
9
CONCENTRATION:
Replicate IV:
DAY
#Live/Dead
Embryo-Larvae
Terata
Temp. (°C)
Salinity (ppt)
DO (mg/L)
PH
0
1
2
o
J
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 12.10.9 and 12.13).
Figure 1. Data form for sheepshead minnow, Cyprinodon variegatus, embryo-larval survival/teratogeniciry test.
Daily record of embryo-larval survival/terata and test conditions (CONTINUED).
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12.6.10.3 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% HSB is used as a diluent, the maximum concentration of effluent that can be tested
using HSB is limited to 80% at 20%o salinity, and 70% at 30%o salinity.
12.6.10.3.1 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 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.
12.6.10.3.2 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.
12.6.10.3.3 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.
12.6.10.3.4 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.
12.6.10.3.5 After the required salinity is attained, the HSB should be filtered a second time through a 1 um 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 at room temperature until used.
12.6.10.3.6 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.
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 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 sea water. The difference, 800 mL, is the quantity of deionized water
required.
12.6.10.3.8 Table 1 illustrates the composition of test solutions at20%oif 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 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 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).
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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, 2002a). 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.
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. A sample of
newly batched 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 ug/g wet weight or the total
concentration of organochlorine pesticides plus PCBs exceeds 0.30 ug/g wet weight. (For analytical methods see
USEPA, 1982).
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TABLE 1. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20%«, USING 20%«
NATURAL OR ARTIFICAL SEA WATER, HYPERSALINE BRINE, OR ARTIFICAL SEA
SALTS
Effluent
Effluent Cone.
Solution (%)
1 1001'2
2 50
3 25
4 12.5
5 6.25
Control 0.0
Solutions
Volume of
Effluent
Solution
4000 mL
2000 mL Solution 1
2000 mL Solution 2
2000 mL Solution 3
2000 mL Solution 4
To Be Combined
Volume of Diluent
Seawater (20%o)
—
+ 2000 mL
+ 2000 mL
+ 2000 mL
+ 2000 mL
2000 mL
Total 10000 mL
1 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%o
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 20%o salinity, and 70% at 30%o salinity.
2 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.
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,
2002a; 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 five to 10 minutes. To prevent mortality, do
not leave the concentrated nauplii at the bottom of the funnel more than 10 minutes without aeration.
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4. Drain the nauplii into a beaker or funnel fitted with a < 150 um NITEX® or stainless steel screen, and
rinse with seawater or equivalent before use.
12.6.11.4 Testing Artemia nauplii as food for toxicity test 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.
12.6.11 A.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.
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 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.
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.
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 hatchedArtemia 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®
or MARDEL AQUARIAN® Tropical Fish Flakes, 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 equivalent.
12.6.12.1.5 The system is equipped with an undergravel or outside biological filter of shells (see Spotte, 1973;
Bower, 1983) for conditioning the biological filter, or a cartridge filter, such as a MAGNUM® Filter, or an EHEIM®
Filter, or equivalent, at a salinity of 20-30%o and a photoperiod of 16 h light/8 h dark.
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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
human chorionic gonadotrophin (HCG) hormone. 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
four 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 10 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 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.
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
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 um 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 minutes, 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 minutes. After incubation, wash the eggs on a NITEX®
screen and resuspend them in clean seawater.
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12.6.12.2A Natural Spawning
12.6.12.2 A. I 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 10 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 um 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 250-500 um 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 forthe test, the adult fish are returned to the (18-20°C)
culture tanks.
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 (285 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 um 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 predationby 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
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
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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
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 um NITEX® 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 ^ 0.5 dilution
factor. If 100%o salinity 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.
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 1-2
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.
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12.10.1.2.4 Maintain the effluent at 0-6 ° 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.
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 in Section 7,
Dilution Water). Other artificial 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
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 for the first time 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.
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.
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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 uE/m2/s, or
50-100 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 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, 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 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, daily (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),
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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
12.10.7.1 Since feeding is not required, test chambers are not cleaned daily unless accumulation of paniculate
matter at the bottom 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 1,3, and 5. Maintain the
samples at 0-6 °C until used.
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Figure 2 Embryonic development of sheepshead minnow, Cyprinodon 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; 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).
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J
M
N
0
Figure 2. Embryonic development of sheepshead minnow, Cyprinodon 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 five days after hatching,
actual length 5 mm; N. Young fish 9 mm in length; O. Young fish 12 mm in length
(CONTINUED). From Kuntz (1916).
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12.10.8.2 The test solutions are adjusted to the correct salinity and renewed daily using freshly collected samples.
During the daily renewal process, 7-10 mm of water is left in the chamber to ensure that the embryos and larvae remain
submerged during the renewal process. New test solution (400 mL) should be added slowly by pouring 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 concentrations 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, select a salinity which approximately matches the salinity of the receiving waters. Table 1
illustrates the 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.
12.10.9 TERMINATION OF THE TEST
12.10.9.1 The test 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 each. The deformed
larvae are treated as dead. Keep a separate record of the total number of deformed larvae for use in reporting the
teratogenicity of the test solution.
12.11 ACCEPTABILITY OF TEST RESULTS
12.11.1 For the test results to be acceptable, survival in the controls must be at least 80% or better.
12.12 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
12.12.1 A summary of test conditions and test acceptability criteria is listed in Table 2.
12.13 DATA ANALYSIS
12.13.1 General
12.13.1.1 Tabulate and summarize the data.
12.13.1.2 The endpointsof 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
NOEC values, for total mortality, are obtained using a hypothesis test approach such as Dunnett's Procedure (Dunnett,
1955) or Steel's Many-one Rank Test (Steel, 1959; Miller, 1981). See the Appendices for examples of the manual
computations, program listings, and examples of data input and program output.
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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 (TEST
METHOD 1005.0)1
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. of embryos per chamber:
12. No. replicate test chambers
per concentration:
13. No. embryos per concentration:
14. Feeding regime:
15. Aeration:
16. Dilution water:
Static renewal (required)
5%o to 32%o (±2%o of the selected test salinity)
(recommended)
25 ± 1 °C (recommended)
Test temperatures must not deviate (i.e., maximum minus
minimum temperature) by more than 3 ° C during the test
(required)
Ambient laboratory light (recommended)
10-20 uE/nf/s, or 50-100 ft-c (ambient laboratory levels)
(recommended)
16 h light, 8 h darkness (recommended)
400-500 mL (recommended)
250-400 mL per replicate (loading and DO restrictions must
be met) (recommended)
Daily (required)
Less than 24 h old (required)
15 (recommended)
10 (required minimum)
4 (recommended)
3 (required minimum)
60 (recommended)
30 (required minimum)
Feeding not required
None unless DO falls below 4.0 mg/L (recommended)
Uncontaminated source of natural seawater; deionized water
mixed with hypersaline brine or artificial sea salts (HW
MARTNEMIX®, FORTY FATHOMS®, GP2, or equivalent)
(available options)
1 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is
identified as required or recommended (see Subsection 10.2 for more information on test review). Additional
requirements may be provided in individual permits, such as specifying a given test condition where several options
are given in the method.
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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 (TEST
METHOD 1005.0)1
17. Test concentrations: Effluents: 5 and a control (required minimum)
Receiving waters: 100% receiving water (or minimum of 5)
and a control (recommended)
18. Dilution factor: Effluent: >0.5 (recommended)
Receiving waters: None, or >0.5 (recommended)
19. Test duration: 9 days (required)
20. Endpoints: 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
(required)
21. Test acceptability criteria: 80% or greater survival in controls (required)
22. Sampling requirements: For on-site tests, samples collected daily and used within
24 h of the time they are removed from the sampling device.
For off-site tests, a minimum of three samples (e.g.,
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)
(required)
23. Sample volume required:
5 L per day (recommended)
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 inFigure 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 is 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 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
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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 fit the Probit Analysis, the Spearman-Karber Method, the Trimmed
Spearman-Karber Method or the Graphical Method may be used (see Appendices H-K).
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% 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.
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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
ENDPOINT ESTIMATE
ECs
ARC SINE
TRANSFORMATION
1
r
SHAPIRO-WILK'S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
YES
EQUAL NUMBER OF
REPLICATES?
YES
NO
T-TEST WITH
BONFERRONI
ADJUSTMENT
DUNNETTS
TEST
STEEL'S MANY-ONE
RANK TEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 3. Flowchart for statistical analysis of sheepshead minnow, Cyprinodon variegatus, embryo-larval
survival and teratogenicity test. Survival and terata data.
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TABLE 3. SHEEPSHEAD MINNOW, CYPRINODON VARIEGA TUS, EMBRYO-LARVAL TOTAL
MORTALITY DATA
SDS Concentration (mg/L)
Replicate Control
0.5
1.0
2.0
4.0
RAW
ARC SINE
TRANS-
FORMED
Mean (Y,)
s2,
i
A
B
C
D
A
B
C
D
0.1
0.0
0.1
0.0
0.322
0.159
0.322
0.159
0.241
0.009
1
0.0
0.2
0.2
0.1
0.159
0.464
0.464
0.322
0.352
0.021
2
0.0
0.1
0.1
0.2
0.159
0.322
0.322
0.464
0.317
0.016
3
0.3
0.1
0.2
0.4
0.580
0.322
0.464
0.685
0.513
0.024
4
0.9
0.7
0.8
0.8
1.249
0.991
1.107
1.107
1.114
0.011
5
1.0
1.0
1.0
1.0
-
—
—
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
SDS Concentration (mg/L)
Replicate
Control
0.5
1.0
2.0
4.0
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
-0.049
0.172
0.135
-0.123
-0.007
-0.007
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INDIVIDUAL REPLICATE PROPORTION MORTALITY
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
1.0
0.9
0.8
0.7
0.6
0.5
u_
O
| 0.4-
O 0.3
Q_
O
§: 0.2
0.1
0.0
0.00
3.12 6.25 12.50
EFFLUENT CONCENTRATION (%)
25.00
50.00
Figure 4.
Plot of Sheepshead minnow, Cyprinodon variegatus, total mortality data from the embryo-larval test
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12.13.2.6.2 Calculate the denominator, D, of the statistic:
D = £(xrx)2
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
X = — (-0.005) = 0.000
20
D= 0.2428
12. 13.2.6.4 Order the centered observations from smallest to largest
Where: X(l) = the ith ordered observation
The ordered observations for this example are listed in Table 5.
TABLE 5. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO- WILK'S EXAMPLE
i X®
1 -0.193
2 -0.191
3 -0.158
4 -0.123
5 -0.082
6 -0.082
7 -0.049
8 -0.030
9 -0.007
10 -0.007
i
11
12
13
14
15
16
17
18
19
20
X®
0.005
0.005
0.067
0.081
0.081
0.112
0.112
0.135
0.147
0.172
12.13.2.6.5 From Table 4, Appendix B, for the number of observations, n, obtain the coefficients ab 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 6.
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TABLE 6. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
a, x(n-1+1)-X(l)
1 0.4734
2 0.3211
3 0.2565
4 0.2085
5 0.1686
6 0.1334
7 0.1013
8 0.0711
9 0.0422
10 0.0140
0.365
0.338
0.293
0.295
0.194
0.163
0.130
0.097
0.012
0.012
X(20).X(1)
X(19).X(2)
X(18).X(3)
X(17).X(4)
X(16).X(5)
X(15).X(6)
X(14).X(7)
X(13).X(8)
X(12).X(9)
X(ii) _x(10)
12.13.2.6.6 Compute the test statistic, W, as follows:
i k
w = — £>, (x("-I+1) - x®)]
D ;=i
The differences x(n"1+1) - X(l) are listed in Table 6. For the data in this example,
W = -— (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 12. 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 control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as follows:
B
[(X) V;) In S2 - X) v> ln
— _ -
C
Where: V; = degrees of freedom for each copper concentration and control, V; = (n -1)
p = number of concentration levels including the control
In = loge
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i = 1, 2,..., p where p is the number of concentrations including the control
HJ = the number of replicates for concentration i
(£vlSl)2
1 V
•<
C = 1 + [3 (p-1)]-1 [£i/Vl - (£v;)-l]
1=1 1=1
12.13.2.7.2 SinceB 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.
12.13.2.7.3 For the data in this example, V; = 3, p=5, S2 = 0.0162, and C= 1.133. The calculated B value is:
(15) [In (0.01262)]-3]
B =
1.33
= 15 (-4.1227) - 3 (-20.9485)
1.33
= 0.886
12.13.2.7.4 SinceB 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. SinceB = 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.
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TABLE?. ANOVATABLE
Source
Between
Within
Total
df
p-1
N-p
N-l
Sum of Squares (SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
S^ = SSB/(p-l)
Sw= SSW/(N-p)
Where: p = number of concentration levels including the control
N = total number of observations n{ + n2... + r^
HJ = number of observations in concentration i
SSB =
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;
1=1
Tj = the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the mean dry weight of the mysids for
concentration i in test chamber j)
12.13.2.8.2 For the data in this example:
H! = n2 = n3 = n4 = n5 = 4
N = 20
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T1=Y11+Y12 + Y13+Y14 = 0.962
T2 = Y21 + Y22 + Y23 + Y24 = 1.409
T3 = Y31+Y32 + Y33+Y34= 1.267
T4 = Y41+Y42 + Y43+Y44 = 2.051
T5 = Y51+Y52 + Y53+Y54 = 4.454
SSB =
1=1
(28.561) - - = 1.996
4 20
= 7.383 - (10-143) = 1.996
20
SSW = SST-SSB = 2.239 - 1.996 = 0.243
S^ = SSB/(p-l) = 1.9967(5-1) = 0.499
S^ = SSW/(N-p) = 0.2437(20-5) = 0.016
12. 13.2.8.3 Summarize these calculations in the ANOVA table (Table 8).
TABLE 8. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
4
15
19
Sum of Squares
(SS)
1.996
0.243
2.239
Mean Square (MS)
(SS/df)
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:
(1/11;)
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Where: Y; = mean proportion surviving for concentration i
Yj = mean proportion surviving for the control
Sw = square root of the within mean square
H! = number of replicates for the control
H = 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 _12/|1
[0.1265^(1/4) + (1/4)]
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 total mortality for concentration "i" is considered significantly less than the mean proportion of
total mortality for the control if tj 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.
TABLE 9. CALCULATED T VALUES
SDS Concentration (mg/L) i t,
0.5
1.0
2.0
4.0
2
3
4
5
1.241
0.850
3.041
9.760
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 SwV/(l/nj) + (1/n)
Where: d = the critical value for Dunnett's procedure
Sw = the square root of the within mean square
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H! = 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) ^(1/4) + (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.
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 (MSDJ 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.
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TABLE 10. DATA FOR PROBIT ANALYSIS
SDS Concentration (mg/L)
Number Dead
Number Exposed
Control
2
40
0.5
5
40
1.0
4
40
2.0
10
40
4.0
32
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.
150
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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.
2
5
4
10
32
40
Observed
Proportion
Responding
0.5000
0.1250
0.1000
0.2500
0.8000
1.0000
Proportion
Responding
Adjusted for
Controls
0.0000
0.0369
0.0094
0.1745
0.7799
1.0000
Chi - Square for Heterogeneity (calculated) = 0.782
Chi - Square for Heterogeneity (tabular value) = 7.815
Probit Analysis of Sheephead Minnow Embryo-Larval Survival and Teratogenicity Data
Estimated LC/EC Values and Confidence Limits
Point
Exposure
Cone.
Lower 95%
Confidence
Upper
Limits
LC/EC
LC/EC
1.00
50.00
1.187
2.912
0.643
2.432
1.601
3.361
Figure 5. Output for USEPA Probit Analysis Program, Version 1.5
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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 ug Cu/L and 270 ug 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 ug) concentration intervals, to more precisely identify the
threshold concentration. The NOEC and LOEC for these tests are 200 ug and 220 ug 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 Data on the multilaboratory precision of this test are not yet available.
12.14.2 Accuracy
12.14.2.1 The accuracy of toxicity tests cannot be determined.
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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
1
2
3
4
5
6
7
8
n:
Mean:
CV (%):
EC1
173
*
*
182
171
*
*
195
4
180
6.1
EC5
189
*
*
197
187
*
*
203
4
194
3.8
EC10
198
*
*
206
197
*
*
208
4
202
2.8
EC50
234
*
*
240
234
*
*
226
4
233
2.5
NOEC
240
240
240
240
240
< 200
220
220
7
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 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 ug/L. Copper concentrations for Test 6 were: 220, 240, 260, 280, and 300 ug/L. Copper
concentrations for Tests 7-8 were: 200, 220, 240, 260, and 280 ug/L. Tests were conducted over a two-week
period.
Adults collected in the field.
NOEC Range: 200-240 ug/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.
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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 (SDS) AS REFERENCE TOXICANT1'2'3'4'5'6'7
Test
Number
1
2
o
6
4
5
n:
Mean:
CV (%):
EC1
(mg/L)
1.7
*
0.4
1.9
1.3
4
1.3
51.2
ECS
(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) andUSEPA (1991a).
Tests performed by Terry Hollister, Aquatic Biologist, Houston Facility, Environmental Services Division,
Region 6, USEPA, Houston, Texas.
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.
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.
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SECTION 13
TEST METHOD
INLAND SILVERSIDE, M£7V/M4 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,
Menidia beryllina, using seven to 11-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-hLC50s).
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.
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, Menidia beryllina, seven to 11-day 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.
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.
13.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent
toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection
8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with
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increasing pH. Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel
and zinc were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed
to be artifactual and due to pH drift (as determined by parallel testing as described in Subsection 13.3.6.1), the
regulatory authority may allow for control of sample pH during testing using procedures outlined in Subsection
13.3.6.2. It should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large
(more than 1 pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold
for toxicity.
13.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with
controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the
test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 13.3.6.2.
The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If
the objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be
maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective
of the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the
sample after adjusting the sample salinity for use in marine testing.
13.3.6.1.1 During parallel testing, thepH must be measured in each treatment at the beginning (i.e., initial pH) and
end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial
pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day
for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was
effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and
the mean final pH should be within ± 0.3 pH units of the target pH. Test procedures for conducting toxicity
identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests
(USEPA, 1996).
13.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total
ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test
may be due to ammonia (USEPA, 1992).
13.3.6.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to
the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the
permitted instream waste concentration, then the results from this test should be used for determining compliance.
If the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pH-
controlled test should be used for determining compliance, provided that this test meets test acceptability criteria
and pH was properly controlled (see Subsection 13.3.6.1.1).
13.3.6.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results
of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled
treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of
artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 13.3.6.2) is applied routinely to
subsequent testing of the effluent.
13.3.6.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled
atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding IN NaOH or IN HC1
(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional
ions (Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This
may be accomplished by placing test solutions and test organisms in closed headspace test chambers, and then
injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992);
or by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996).
Prior experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to
inject. This volume will depend upon the sample pH, sample volume, container volume, and sample constituents.
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If more than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the headspace with no more
than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the
receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving
water (measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the
absolute toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount
(1992) provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-
controlled testing, control treatments must be subjected to all manipulations that sample treatments are subjected to.
These manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled
testing, the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to
confirm that pH was effectively controlled at the target pH level.
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 (2002a) for detailed culture methods. This test requires from 180-360 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 ~ Millipore 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
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.
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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 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).
0
0)
GLASS
REINFORCEMENTS
SUMP
Figure 1. Glass chamber with sump area. Modified from Norberg and Mount (1985). From USEPA (1987c).
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 um 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
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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).
13.5.21 Beakers - 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.
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 cultwmgArtemia.
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.
13.5.34 NITEX® Mesh Sieves (< 150 um, 500 um, 3-5 mm) ~ for collecting Artemia nauplii and fish larvae.
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).
13.6.6 Weighing pans, aluminum ~ 26/test (two extra).
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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 DO 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.
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.
13.6.13.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine
systems contain little or no measurable salts. Exposure ofMenidia beryllina 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 (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% 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 um 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
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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 um 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 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 and 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 NaHCO3 in 500 mL deionized water. Add 2.5 mL of this stock
solution for each liter of the GP2 artificial seawater.
13.6.14 ROTIFER CULTURE -for feeding cultures and test organisms
13.6.14.1 At hatching Menidia beryllina larvae are too small to ingest Artemia nauplii and must be fed rotifers,
Brachionus plicatilis. 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
Volume of
Effluent
Effluent (0%o)
Concentration (mL)
80 2400
40 1200
20 600
10 300
5 150
Control 0
Total 4,650
Volume of
Deionized
Water
(mL)
0
1200
1800
2100
2250
2400
9,750
Volume of
Hypersaline
Brine
(mL)
600
600
600
600
600
600
3,600
Total
Volume
(mL)
3000
3000
3000
3000
3000
3000
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
TEST1'2'3
Compound
NaCl
Na2SO4
KC1
KBr
Na2B4O7-10H2O
MgCl2-6 H2O
CaCl2-2 H2O
SrCl2-6 H2O
NaHCO3
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
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.
3 GP2 can be diluted with deionized (DI) water to the desired test salinity.
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 um 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 seven to 10 days and is sustainable for two to three 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 minutes). After cooling to room temperature, the carboys are placed in a temperature chamber
controlled at 18-20°C. One liter of T. suecica or Chlorella sp. starter culture and 100 mL of nutrients are added to
each carboy.
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13.6.15.2 Formula for algal culture nutrients.
13.6.15.2.1 Addl80gNaNO3, 12 gNaH2PO4, and 6.16 gEDTAto 12L 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 H2O 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 to 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 to 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 intoxicity tests. Although
there are many commercial sources of brine shrimp cysts, the Brazilian or Colombian strains are being used because
the supplies examined have had low concentrations of chemical residues and produce nauplii of suitably small size.
13.6.16.2 Each new batch ofArtemia 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. 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 ug/g wet weight or that the
total concentration of organochlorine pesticides plus PCBs does not exceed 0.30 ug/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 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,
2002a; 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 um 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.
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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 seven to nine 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 VeraCruz, Mexico (Johnson, 1975). It can tolerate a wide range of temperature, 2.9-32.5 °C (Tagatzand
Dudley, 1961; Smith, 1971) and salinity, of 0-58%o (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 six to seven days when incubated at 25 °C and maintained in seawater ranging from 5-30%o (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, 2002a for detailed culture
methods) may be cultured in the laboratory or obtained from the Gulf of Mexico or Atlantic coast estuaries
throughout the 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 one to two months and will generally spawn for 4-6 months.
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.
13.6.17.4 Samples may contain a mixture of inland silverside, Menidia beryllina, and Atlantic silverside, Menidia
menidia, on the Atlantic coast or inland silverside and tidewater silverside, Menidia 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,!/.
beryllina juveniles and adults are usually considerably smaller thanM. menidia juveniles and adults (Bengtson,
1984), and can be separated easily in the field on that basis.
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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 20-32%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 andArtemia 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 six to eight days. Larvae are 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 rotifers, to
provide a transition period. After Day 7, only nauplii are fed, and the age range for the nauplii can be increased
from 12 h old to 24 h old.
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A. Adult, ca. 64 mm SL
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, two and one half days
after fertilization. From Martin and Drewry (1978).
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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 well onArtemia 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 seven 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.
13.10 TEST PROCEDURES
13.10.1 TEST SOLUTIONS
13.10.1.1 Receiving Waters
13.10.1.1.1 The sampling point is determined by the objectives of the test. Atestuarine 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 um 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. 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. If 100% salinity 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.
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 1-2
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
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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 within24 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 labeled 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 3 in Section 7,
Dilution Water). Other artificial sea salts may be used for culturing inland silverside 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 to 32%o. If the test salinity ranges from 16 to 32%o, the salinity for spawning,
incubation, and culture of the embryos and larvae should be maintained within this salinity range. If the test salinity
is in the range of 5 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 Priorto 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, 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
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should contain a minimum of 10 larvae and it is required that there be four replicates minimum 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 (two to three 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 (two to three 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.
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
uE/m2/s, or 50-100 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-
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 pipet with a 1-2 mm orifice such as a 1 mL KIMAX® serological pipet, 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 hold) 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.
170
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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 seawaterina 100 mL
beaker. Bring to a volume of 80 mL and dispense as described above.
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.
13.10.6 DAILY CLEANING OF TEST CHAMBERS
13.10.6.1 Before the daily renewal of test solutions, uneaten and deadArtemia, 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 pipet
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)
171
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MEAN WEIGHT/
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MEAN WEIGHT/
LARVAE (mg) ±
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#
LARVAE/
DRYWT
MEAN WEIGHT/
LARVAE (mg) ±
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LARVAE (mg) ±
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LARVAE/
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LARVAE (mg) ±
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MEAN WEIGHT/
LARVAE (mg) ±
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172
-------
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13.10.7.2 Routine Biological Observation
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 7-10 mm, leaving 10-15% 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 0-6°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 seven 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 seven days. 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 um 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. 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.
13.10.9.4 Immediately prior to drying, rinse the preserved larvae in distilled (or deionized) water. The rinsed
larvae from each test chamber are transferred, using forceps, 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 to cool and to prevent the adsorption of moisture from the air until weighed. Weigh all weighing pans
containing the dried larvae to 0.01 mg, subtract the tare weight to determine dry weight of larvae in each replicate.
Record (Figure 4) the weights. Divide the dry weight by the number of original larvae per replicate to determine the
average dry weight, and record (Figures 4 and 5) on the data sheets. For the controls, also calculate the mean
weight per surviving fish in the test chamber to evaluate if weights met test acceptability criteria (see
Subsection 13.11). Complete the summary data sheet (Figure 5) after calculating the average measurements and
174
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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 seven-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 seven 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.
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.
175
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Test Dates:
Species:,
Pan
No.
Cone.
&
Rep.
Initial
Wt.
(mg)
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 (fromUSEPA, 1987b).
176
-------
Test Dates:
Species:.
Effluent Tested:
TREATMENT
NO. LIVE
LARVAE
SURVIVAL
(%)
MEAN DRY WT/
LARVAE (MG)
±SD
SIGNIF. DIFF.
FROM CONTROL
(o)
MEAN
TEMPERATURE
(°C)
±SD
MEAN SALINITY
%0
±SD
AVE. DISSOLVED
OXYGEN
(MG/L) ± SD
COMMENTS:
Figure 5. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Summary of
test results (fromUSEPA, 1987c).
177
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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 (TEST METHOD 1006.0)1
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 (required)
5%o to 32%o (± 2%o of the selected test salinity)
(recommended)
25 ± 1°C (recommended)
Test temperatures must not deviate (i.e., maximum minus
minimum temperature) by more than 3°C during the test
(required)
Ambient laboratory illumination (recommended)
10-20 uE/m2/s (50-100 ft-c) (Ambient laboratory levels)
(recommended)
16 h light, 8 h darkness (recommended)
600 mL-1 L containers (recommended)
500-750 mL/replicate (loading and DO restrictions must be
met) (recommended)
Daily (required)
7-11 days post hatch; less than or equal to 24-h range in age
(required)
10 (required minimum)
4 (required minimum)
40 (required minimum)
Newly hatched Artemia nauplii (survival of 7-9 days old
Menidia beryllina larvae improved by feeding 24 h old
Artemia) (required)
Feed 0.10 g wet weight^rte/w/'a nauplii per replicate on days
0-2; Feed 0.15 g wet weight^rte/w/'a nauplii per replicate on
days 3-6 (recommended)
Siphon daily, immediately before test solution renewal and
feeding (required)
1 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is
identified as required or recommended (see Subsection 10.2 for more information on test review). Additional
requirements may be provided in individual permits, such as specifying a given test condition where several options
are given in the method.
178
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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 (TEST METHOD 1006.0) (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/minimum (recommended)
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) (available options)
Effluent: 5 and a control (required)
Receiving Waters: 100% receiving water (or minimum of 5)
and a control (recommended)
Effluents: > 0.5 (recommended)
Receiving waters: None, or > 0.5 (recommended)
7 days (required)
Survival and growth (weight) (required)
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 (required)
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 (e.g., 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
forToxicity Tests, Subsection 8.5.4) (required)
6 L per day (recommended)
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 Forthe 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
179
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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.
180
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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-WILK'S TEST
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
EQUAL NUMBER OF
REPLICATES?
NO
T-TEST WITH
BONFERRONI
ADJUSTMENT
YES
NON-NORMAL DISTRIBUTION
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
YES
DUNNETT'S
TEST
STEEL'S MANY-ONE
RANK TEST
y
NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 6. Flowchart for statistical analysis of the inland silverside, Menidia beryllina, survival data by
hypothesis testing.
181
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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
ONE OR MORE
PARTIAL MORTALITIES?
NO
^
YES
IYES
GRAPHICAL METHOD
LC50
PROBIT METHOD
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONG.?
YES
NO
SPEARMAN-KARBER
METHOD
TRIMMED SPEARMAN-
KARBER METHOD
LC50 AND 95%
CONFIDENCE
INTERVAL
Figure 7. Flowchart for statistical analysis of the inland silverside, Menidia beryllina, survival data by point
estimation.
182
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D_
O
1.0
0.9
0.8
05
U'°
< 0.4
| 0.3
00 0.2
0.1
0.0
0.00
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
REPRESENTS THE CRITICAL VALUE FOR DUNNETT'S TEST
(ANY PROPORTION BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
6.25 12.50
EFFLUENT CONCENTRATION (%
--*
25.00
Figure 8.
Plot of mean survival proportion of the inland silverside, Menidia beryllina, larvae.
183
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TABLE 4. INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL DATA
Concentration
Replicate
Control
6.25
12.5
25.0
50.0
100.0
RAW
ARC SINE
TRANS-
FORMED
Mean(Y;)
S2
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
6.25
Effluent Concentration (%)
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
184
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13.13.2.6.2 Calculate the denominator. D, of the statistic:
n
D = Y
Where: Xj = 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 = J_(0.002) = 0.0
12
D = 0.3214
13.13.2.6.4 Order the centered observations from smallest to largest:
x(1)< x(2)< ...< x(n)
where X(l) denotes the ith ordered observation. The ordered observations for this example are listed in Table 6.
TABLE 6. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i X®
1 -0.321
2 -0.256
3 -0.097
4 -0.087
5 -0.004
6 -0.002
i
7
8
9
10
11
12
x©
0.018
0.091
0.096
0.099
0.226
0.239
13.13.2.6.5 From Table 4, AppendixB, forthe number of observations, n, obtain the coefficients ab 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 ^ values are listed in
Table 7.
13.13.2.6.6 Compute the test statistic, W, as follows:
185
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The differences X(M+1) - X(l) are listed in Table 7. For the data in this example,
W = J_(0.5513)2 = 0.945
0.3214
TABLE 7. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1 0.5475 0.560 X(12)-X(1)
2 0.3325 0.482 X(11)-X(2)
3 0.2347 0.196 X(10)-X(3)
4 0.1586 0.183 X(9) -X(4)
5 0.0922 0.095 X(8) -X(5)
6 0.0303 0.020 X(7) -X(6)
13.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 13.13.2.6.6 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:
V)
B = -^
Where: V; = degrees of freedom for each effluent concentration and control, V; = (n; -1)
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
HJ = the number of replicates for concentration i.
p
(E vtsf)
s2 = ^i
186
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c =
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, V; = 2 for all i.
13.13.2.7.3 Bartlett's statistic is therefore:
B = [(8)In(0.0402) -2^ In(S~)]/1.2083
Z=l
= [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 withp -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.
TABLES. ANOVA TABLE
Source
Between
Within
Total
df
p-1
N-p
N-l
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
SB = SSB/(p-l)
Sl= SSW/(N-p)
Where: p = number of SDS concentration levels including the control
N = total number of observations n{ + n 2... + np
HJ = number of observations in concentration i
187
-------
SSB = Y, T?/ni-G2/N Between Sum of Squares
SST = Y, Y.YJ-G2/N Total Sum of Squares
=l _/ = !
TF = SST - SSB Within Sum of Squares
G = the grand total of all sample observations, G = ^ Ti
z=l
Tj = the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the proportion surviving for toxicant
concentration i in test chamber j)
13.13.2.8.2 Forthe data in this example:
H! = n2 = n3 = n4 = 3
N = 12
T =Y +Y +Y =3
-1! * 11 ^ * 12 ^ * 13 J-
T2 = Y21+Y22 + Y23 = 3.333
T3 = Y31+Y32 + Y33 = 2.605
T4 = Y41+Y42 + Y43= 1.768
4= 11.318
SSB =
z=l
= J. (34.067) - £1L318}2 =0.681
3 12
= 11.677-(11.318')2 = 1.002
12
SSW = SST-SSB= 1.002 - 0.681 = 0.321
Sjj, = SSB/(p-l) = 0.6817(4-1) = 0.227
Sw = SSW/(N-p) = 0.3217(12-4) = 0.040
13.13.2.8.3 Summarize these calculations in the ANOVA table (Table 9).
188
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TABLE 9. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
df
o
3
8
Sum of Squares
rssi
0.681
0.321
Mean Square(MS)
rSS/df)
0.227
0.040
Total 11 1.002
13.13.2.8.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control
combination as follows:
t
Where: Y; = mean proportion surviving for effluent concentration i
Yl = mean proportion surviving for the control
Sw= square root of the within mean square
H! = number of replicates for the control
HJ = 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:
t - (L2°4 - U11> = 0.570
[0.020 ^(1/3) + (1/3)]
189
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TABLE 10. CALCULATED T VALUES
Effluent Concentration (%)
6.25 2 0.570
12.5 3 2.058
25.0 4 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 tj 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%.
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 Sl/nj)+(!/«)
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)
H! = 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
190
-------
2. Obtain the untransformed values for the control mean and the difference calculated in step 1.
[Sine (1.204) ]2 = 0.871
[ Sine (0.809) ]2 = 0.524
3. The untransformed MSD (MSDJ is determined by subtracting the untransformed values
from step 2.
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.
13.13.2.8.11 This represents a 40% decrease in survival from the control.
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
Effluent Concentration (%)
Control 6.25 12.5 25.0 50.0 100.0
Number Dead
Number Exposed
6
45
9
45
19
45
45
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, MENIDIA 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. Because this measurement is based on
the number of original organisms exposed (rather than the number surviving), the measured response is a combined
survival and growth endpoint that can be termed biomass. 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
191
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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.
Probit Analysis of Inland Silverside Larval Survival Data
Cone.
Number
Exposed
Number
Resp.
Observed
Proportion
Responding
Proportion
Responding
Adjusted for
Controls
Control
6.2500
12.5000
25.0000
50.0000
100.0000
45
45
45
45
45
45
6
9
19
30
45
45
0.1333
0.2000
0.4222
0.6667
1.0000
1.0000
0.0000
0.0488
0.3130
0.6037
1.0000
1.0000
Chi - Square for Heterogeneity (calculated)
Chi - Square for Heterogeneity (tabular value)
= 4.149
= 7.815
Probit Analysis of Inland Silverside Larval Survival Data
Estimated LC/EC Values and Confidence Limits
Point
Exposure
Cone.
Lower Upper
95% Confidence Limits
LC/EC 1.00
LC/EC 50.00
4.980
18.302
2.023
13.886
7.789
22.175
Figure 9. Output for USEPA Probit Analysis Program, Version 1.5.
192
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STATISTICAL ANALYSIS OF INLAND SILVERSIDE 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-WILJCS TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
I
\
EQUAL NUMBER OF
REPLICATES?
NO 1 YES
T-TESTWITH niiMMcrno
BONFERRONI DUNNfTrS
ADJUSTMENT TEST
\
EQ
YES
r
STEEL'S MANY-ONE
RANK TEST
i
ENDPOINT ESTIMATES
NOEC, LOEC
UAL NUMBER OF
REPLICATES?
1 NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
Figure 10.
Flowchart for statistical analysis of the inland silverside, Menida beryllina, growth data.
193
-------
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.
TABLE 12. INLAND SILVERSIDE, MENIDIA BERYLLINA, GROWTH DATA
Effluent Concentration %
Replicate Control 6.25 12.5 25.0 50.0 100.0
A
B
C
Mean(Y)
Si2
i
0.751
0.849
0.907
0.836
0.0062
1
0.737
0.922
0.927
0.862
0.0117
2
0.722
0.285
0.718
0.575
0.0631
3
0.196
0.312
0.079
0.196
0.0136
4 5
-
-
_
-
6
13.13.3.5 Test for Normality
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-WILK'S EXAMPLE
Effluent Concentration (%)
Replicate Control 6.25 12.5
A -0.085 -0.125 0.147
B 0.013 0.060 -0.290
C 0.071 0.065 0.143
194
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CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
CD
LU
<
LU
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.00
REPRESENTS THE CRITICAL VALUE FOR DUNNETTS TEST
(ANY WEIGHT BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
6.25
EFFLUENT CONCENTRATION (%)
12.50
Figure 11. Plot of mean weights of inland silverside, Menidia beryllina, larval survival and growth test.
195
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13.13.3.5.2 Calculate the denominator, D, of the test statistic:
D = E(^-i)2
;=1
Where: Xj = 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 = 9
X = J_(-0.002) = 0.000
9
D = 0.162
13.13.3.5.3 Order the centered observations from smallest to largest:
x(1)< x(2) <...
-------
The differences X(M+1) - X(l) are listed in Table 15. For this set of data:
W = 1_ (0.3800)2 = 0.89
0.162
TABLE 15. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1
2
3
4
0.5888
0.3244
0.1976
0.0947
0.437
0.268
0.156
0.052
x(9) - x(1)
X(8).X(2)
X(7).X(3)
X(6) _ 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:
C
Where: V; = degrees of freedom for each effluent concentration and control, V; = (n - 1)
p = number of levels of effluent concentration including the control
i = 1,2,..., p where p is the number of concentrations including the control
In = loge
HJ = number of replicates for concentration i
197
-------
s2 =
£v#
"Ix"
C =
13.13.3.6.2 For the data in this example, (See Table 13) all effluent concentrations including the control have the
same number of replicates (r^ = 3 for all i). Thus, V; = 2 for all i.
13.13.3.6.3 Bartlett's statistic is therefore:
B = [(6)In(0.027) -2^J In(Sf)/ 1.222
= [6(-3.612)-2(-12.290)]/1.222
= 2.909/1.222
= 2.38
13.13.3.6.4 B is approximately distributed as chi-square withp -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 = 2.38 is less than the critical value of 9.210, conclude that the variances are not
different.
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.
198
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TABLE 16. ANOVA TABLE
Source
Between
Within
Total
df
p-1
N-p
N-l
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
= SSB/(p-l)
= SSW/(N-p)
Where: p = number of effluent concentrations including the control
N = total number of observations nl+n2...+i\p
H = number of observations in concentration i
SSB = TlnrG2IN
SST = _
;=i j=i
SSW = SST-SSB
Between Sum of Squares
Total Sum of Squares
Within Sum of Squares
G = the grand total of all sample observations,
j = the total of the replicate measurements for concentration i
JJ = the jth observation for concentration i (represents the mean dry weight of the fish for toxicant
concentration i in test chamber j)
13.13.3.7.2 For the data in this example:
= n2 = n3 = 3
N =9
T! = Yn + Y12 + Y13 = 0.751 + 0.849 + 0.907 = 2.507
T2 = Y21 + Y22 + Y23 = 0.727 + 0.922 + 0.927 = 2.576
T3 = Y31 + Y32 + Y33 = 0.722 + 0.285 + 0.718 = 1.725
G = T! + T2 + T3 = 6.808
199
-------
= 1 (15.8961 - (6.80812 =0.1488
3 9
= 5.463 - (6.808)2 =0.3131
9
= 0.3131-0.1488 = 0.1643
= SSB/(p-l) = 0.14887(3-1) = 0.0744
= SSW/(N-p) = 0.16437(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 2 0.1488 0.0744
Within 6 0.1643 0.0274
Total 8 0.3131
13.13.3.7.4 To perform the individual comparisons, calculate the t statistic for each concentration and control
combination as follows:
^lln, + I/H.)
t, =
Where: Y; = mean dry weight for effluent concentration i
Y[ = mean dry weight for the control
Sw = square root of the within mean square
H! = number of replicates for the control
HJ = number of replicates for concentration i.
200
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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)]
TABLE 18. CALCULATED T VALUES
Effluent Concentration (ppb)
6.25 2 -0.120
12.5 3 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 tj 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.
MSD =
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)
H! = the number of replicates in the control.
13.13.3.7.8 In this example:
MSD = 2.34(0.1655)^(1/3)+(1/3)
= 2.34(0.1655)(0.8165)
= 0.316
201
-------
13.13.3.7.9 Therefore, forthis set of data, the minimum difference that can be detected as statistically significant is
0.316mg.
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 19andFigure 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 Mr
13.13.3.8.2 Starting with the control mean, Y\ = 0.836 and Y2= 0.859, we see that Y\ < Y2. Set M, = Yr
13.13.3.8.3 Calculate the smoothed means:
M! = M2 = (Yj + Y2)/2 = 0.847
13.13.3.8.4 Since Y5 = 0 < Y4= 0.196 < Y3= 0.575
-------
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 = c +KO
IC25 = 6.25 + [0.847(1 - 25/100) - 0.847] (12.50-6.251
(0.575 - 0.847)
= 11.1%.
203
-------
O
CQ
1
1.D
D.D
0.1 ^
D.D-
i
D.DD
-X-
INDMDUAL REPLICATE MEAN
DDNNECT& THE OBSERVED MEAN VALUES
CDNNEDT& THE SOUTHED MEAN VALUES
6.25 12.50
EFFLUENT CONCENTRATION (%)
25.00
Figure 12. Plot of the raw data, observed means, and smoothed means from Tables 12 and 19.
50.00
204
-------
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 = C +[iw1(l-jp/100)-M]-§l1~C/)
IC50 = 6.25 + [0.847(1 - 50/100) - 0.847] (12.50-6.25)
(0.575 - 0.847)
= 17.5%.
13.13.3.8.9 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of
theIC25 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.
1314 PRECISION AND ACCURACY
13.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below (Subsections
13.14.1.1 and 13.14.1.2). Single-laboratory precision is a measure of the reproducibility of test results when tests are
conducted using a specific method under reasonably constant conditions in the same laboratory. Single-laboratory
precision is synonymous with the terms within-laboratory precision and intralaboratory precision. Multilaboratory
precision is a measure of the reproducibility of test results from different laboratories using the same test method and
analyzing the same test material. Multilaboratory precision is synonymous with the term interlaboratory precision.
Interlaboratory precision, as used in this document, includes both within-laboratory and between-laboratory
components of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined (termed total
interlaboratory variability). The total interlaboratory variability that is reported from these studies is synonymous
with interlaboratory variability reported from other studies where individual variability components are not
separated.
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.
13.14.1.1.2 EPA evaluated within-laboratory precision of the Inland Silverside, Menidia beryllina, Larval Survival
and Growth Test using a database of routine reference toxicant test results from 16 laboratories (USEPA, 2000b).
The database consisted of 193 reference toxicant tests conducted in 16 laboratories using a variety of reference
toxicants including: chromium, copper, potassium chloride, and sodium dodecyl sulfate. Among the 16 laboratories,
the median within-laboratory CV calculated for routine reference toxicant tests was 27% for the IC25 growth
endpoint. In 25% of laboratories, the within-laboratory CV was less than 18%; and in 75% of laboratories, the
within-laboratory CV was less than 43%.
205
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13.14.1.2 Multilaboratory Precision
13.14.1.2.1 In 2000, EPA conducted an interlaboratory variability study of the Inland Silverside, Menidia beryllina,
Larval Survival and Growth Test (USEPA, 200 la; USEPA, 200 Ib). In this study, each of 10 participant laboratories
tested 4 blind test samples that included some combination of blank, effluent, reference toxicant, and receiving water
sample types. The blank sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater, the effluent
sample was an industrial wastewater spiked with CuSO4, the receiving water sample was a natural seawater spiked
with CuSO4, and the reference toxicant sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater
spiked with CuSO4. Of the 40 Menidia beryllina Larval Survival and Growth tests conducted in this study, 100%
were successfully completed and met the required test acceptability criteria. Of seven tests that were conducted on
blank samples, none showed false positive results for survival endpoints or for the growth endpoint. Results from the
reference toxicant, effluent, and receiving water sample types were used to calculate the precision of the method.
Table 23 shows the precision of the IC25 for each of these sample types. Averaged across sample types, the total
interlaboratory variability (expressed as a CV%) was 43.8% for IC25 results. Table 24 shows the frequency
distribution of survival and growth NOEC endpoints for each sample type. For the survival endpoint, NOEC values
spanned five concentrations for the effluent, four concentrations for the reference toxicant sample type, and three
concentrations for the receiving water sample type. The percentage of values within one concentration of the median
was 90.9%, 84.6%, and 85.7% for the reference toxicant, effluent, and receiving water sample types, respectively.
For the growth endpoint, NOEC values spanned four concentrations for the reference toxicant and effluent sample
types and three concentrations for the receiving water sample type. The percentage of values within one
concentration of the median was 90.9%, 91.7%, and 85.7% for the reference toxicant, effluent, and receiving water
sample types, respectively.
13.14.2 ACCURACY
13.14.2.1 The accuracy of toxicity tests cannot be determined.
206
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 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.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
Means
0.836
0.859
0.575
0.196
0.000
0.000
Std.
Dev.
0.079
0.114
0.251
0.117
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
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds: 1.43
Standard Deviation:
Lower:
Lower:
Random Seed:
2.1155
8.5413 Upper:
5.7119 Upper:
-1912403737
14.9696
19.2112
Figure 13. ICPIN program output for the IC25.
207
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Cone. ID
Cone. Tested
Response 1
Response 2
Response 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.iSO
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.
Dev.
0.079
0.114
0.251
0.117
0.000
0.000
Pooled
Response Means
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
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds: 1.43
Standard Deviation: 2.49.73
Lower: 12.2513 Upper:
Lower: 6.4891 Upper:
Random Seed: -1440337465
19.8638
22.4754
Figure 14. ICPIN program output for the IC50.
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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 SEAWATER, AND COPPER (CU)
AS A REFERENCE TOXICANT1'2'3'4'5'6'7
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(fig/L)
63
125
63
125
31
5
NA
NA
IC25
(H8/L)
96.2
207.2
218.9
177.5
350.1
5
209.9
43.7
IC50
(H8/L)
148.6
NC8
493.4
241.4
479.8
4
340.8
50.7
Most
Sensitive
Endpoint6
S
S
G
S
G
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 copper concentration.
Copper concentrations were: 31, 63, 125, 250, and 500 ug/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 ug/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.
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.
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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 NOEC IC25
Number (mg/L) (mg/L)
1
2
3
4
5
n:
.3 0.3
.3 1
6
.3 1.5
.3 1
.3 1
5 5
Mean: NA 1
CV(%): NA 43
5
6
o
5
2
IC50
(mg/L)
1.7
1.9
1.9
1.9
2.2
5
1.9
9.4
Most
Sensitive
Endpoint
S
S
S
S
S
1 Data from USEPA (1988a) and USEPA (1991a)
2 Tests performed by George Morrison and Elise Torello, ERL-N, USEPA, Narragansett, RI.
3 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.
4 Adults collected in the field.
5 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.
7 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
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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
Survival
SDS (mg/L)
GP2
NSW
Copper (ug/L) GP2
NSW
Growth
GP2
NSW
Mean
CV (%)
3.59
4.87
5.95
4.81
24.6
3.69
4.29
8.05
5.34
44.2
3.60
5.54
6.70
5.28
29.6
3.55
5.27
8.53
5.79
43.8
GP2
NSW
Mean
CV (%)
247
215
268
243
10.9
256
211
240
236
9.8
260
236
NC5
248
6.9
277
223
238
246
11.2
1 Tests performed by George Morrison and Glen Modica, ERL-N, USEPA, Narragansett, RI.
2 Three replicate exposure chambers with 10-15 larvae per treatment.
3 Adults collected in the field.
4 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
5 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.
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TABLE 23. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES1
Test Endpoint Sample Type
Within-lab3 Between-lab4 Total5
IC25 Reference toxicant 22.0 29.1 36.4
Effluent 7.24 55.5 56.0
Receiving water - - 39.1
Average 14.6 42.3 43.8
1 From EPA's WET Interlaboratory Variability Study (USEPA, 200la; USEPA, 200Ib).
2 CVs were calculated based on the within-laboratory component of variability, the between-laboratory
component of variability, and the total interlaboratory variability (including both within-laboratory and between-
laboratory components). For the receiving water sample type, within-laboratory and between-laboratory
components of variability could not be calculated since the study design did not provide within-laboratory
replication for this sample type.
3 The within-laboratory (intralaboratory) component of variability for duplicate samples tested at the same time in
the same laboratory.
4 The between-laboratory component of variability for duplicate samples tested at different laboratories.
5 The total interlaboratory variability, including within-laboratory and between-laboratory components of
variability. The total interlaboratory variability is synonymous with interlaboratory variability reported from
other studies where individual variability components are not separated.
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TABLE 24. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR VARIOUS
SAMPLE TYPES1
Test Endpoint
Survival NOEC
Growth
NOEC
Sample Type
Reference toxicant
Effluent
Receiving water
Reference toxicant
Effluent
Receiving water
Median
NOEC
Value
12.5%
25%
25%
12.5%
25%
25%
% of Results at
the Median
72.7
38.5
57.1
72.7
41.7
57.1
% of Results
±12
18.2
46.1
28.6
18.2
50.0
28.6
% of Results
>23
9.09
15.4
14.3
9.09
8.33
14.3
1 From EPA's WET Interlaboratory Variability Study (USEPA, 200la; USEPA, 200Ib).
2 Percent of values at one concentration interval above or below the median. Adding this percentage to the
percent of values at the median yields the percent of values within one concentration interval of the median.
3 Percent of values two or more concentration intervals above or below the median.
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SECTION 14
TEST METHOD
, MYSIDOPSIS 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-hLC50s).
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).
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.
14.3.4 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent
toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection
8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with
increasing pH. Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel
and zinc were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed
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to be artifactual and due to pH drift (as determined by parallel testing as described in Subsection 14.3.4.1), the
regulatory authority may allow for control of sample pH during testing using procedures outlined in Subsection
14.3.4.2. It should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large
(more than 1 pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold
for toxicity.
14.3.4.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with
controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the
test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 14.3.4.2.
The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If
the objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be
maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective
of the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the
sample after adjusting the sample salinity for use in marine testing.
14.3.4.1.1 During parallel testing, thepH must be measured in each treatment at the beginning (i.e., initial pH) and
end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial
pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day
for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was
effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and
the mean final pH should be within ± 0.3 pH units of the target pH. Test procedures for conducting toxicity
identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests
(USEPA, 1996).
14.3.4.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total
ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test
may be due to ammonia (USEPA, 1992).
14.3.4.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to
the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the
permitted instream waste concentration, then the results from this test should be used for determining compliance.
If the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pH-
controlled test should be used for determining compliance, provided that this test meets test acceptability criteria
and pH was properly controlled (see Subsection 14.3.4.1.1).
14.3.4.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results
of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled
treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of
artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information
or additional parallel testing before pH control (as described in Subsection 14.3.4.2) is applied routinely to
subsequent testing of the effluent.
14.3.4.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled
atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding IN NaOH or IN HC1
(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional
ions (Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This
may be accomplished by placing test solutions and test organisms in closed headspace test chambers, and then
injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992);
or by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996).
Prior experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to
inject. This volume will depend upon the sample pH, sample volume, container volume, and sample constituents.
If more than 5% CO2 is needed, adjust the solutions with acids (IN HC1) and then flush the headspace with no more
than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the
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receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving
water (measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the
absolute toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH
of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount
(1992) provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-
controlled testing, control treatments must be subjected to all manipulations that sample treatments are subjected to.
These manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled
testing, the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to
confirm that pH was effectively controlled at the target pH level.
14.4 SAFETY
14.4.1 See Section 3, Health and Safety.
14.5 APPARATUS AND EQUIPMENT
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 14.13 below. This test requires a minimum of 240
7-day old (juvenile) mysids. It is preferable to obtain the test organisms from an in-house culture unit. If it is not
feasible to culture mysids in-house, juveniles can be obtained from other sources, if shipped in well oxygenated
saline water in insulated containers.
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).
14.5.6 Water purification system ~ Millipore Milli-Q®, deionized water or equivalent.
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.
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.
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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 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-L — Two-four for cultunngArtemia.
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.
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 um, 500-1000 um, 3-5 mm) ~ for concentrating organisms.
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 2, 7, and 8).
14.6.3 Tape, colored ~ for labeling test chambers.
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14.6.4 Markers, waterproof ~ for marking containers, etc.
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).
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 um or smaller openings).
14.6.11.1 Saline test and dilution water ~ The salinity of the test water must be in the range of 20%o to 30%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.
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%o 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.
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14.6.11.3.3 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.
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.
14.6.11.3.5 After the required salinity is attained, the HSB should be filtered a second time through a 1 mm filter
and poured directly into portable containers (20-L cubitainers or polycarbonate water cooler jugs are suitable). The
containers should be capped and labeled 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.
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.
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), deionized 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 Artificial sea salts: FORTY FATHOMS® brand sea salts 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® 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).
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 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 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 before use as the testing medium.
If the solution is to be autoclaved, sodium bicarbonate is added after the solution has cooled. A stock solution of
sodium bicarbonate is made up by dissolving 33.6 g NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this
stock solution for each liter of the GP2 artificial seawater.
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TABLE 1. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2 ARTIFICIAL
SEAWATER FOR THE MYSID, MYSIDOPSISBAHIA, TOXICITY TEST1'2'3
Compound
NaCl
Na2SO4
Kcl
KBr
Na2B4O7-10H2O
MgCl2-6 H2O
CaCl2-2 H2O
SrCl2-6 H2O
NaHCO3
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
20L
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.
3 GP2 can be diluted with deionized (DI) water to the desired test salinity.
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 produce nauplii of suitably small size.
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TABLE 2. QUANTITIES OF EFFLUENT, DEIONIZED WATER, AND HYPERSALINE BRINE (100%«)
NEEDED TO PREPARE 1800 ML VOLUMES OF TEST SOLUTION WITH A SALINITY OF
20%«
Effluent
Concentration
(%)
80
40
20
10
5
Control
Volume of
Effluent
(0%o)
(mL)
1440
720
360
180
90
0
Volume of
Deionized
Water
(mL)
0
720
1080
1260
1350
1440
Volume of
Hypersaline
Brine
(mL)
360
360
360
360
360
360
Total Volume
(mL)
1800
1800
1800
1800
1800
1800
Total 2790 5850 2160 10800
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. 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 exceeds 0.15 ug/g wet weight or the total
concentration of organochlorine pesticides plus PCBs exceeds 0.30 ug/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, 2002a; 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 um 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.
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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, 2002a for information on
mysid ecology). The genus name of this organism was formally changed to Americamysis (Price et al., 1994);
however, the method manual will continue to refer to Mysidopsis bahia to maintain consistency with previous
versions of the method.
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.
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.
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5. After the nitrite level falls below 0.05 mg/L, add another 30 mL ofArtemia nauplii concentrate
and check the nitrite concentration every day.
6. Continue this cycle until the addition ofArtemia 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.
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 seawateris recommended as the culture medium, but HSB maybe used to makeup 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/night 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 um 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.
14.6.13.1.16 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
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.
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14.6.13.1.17 Culture Maintenance (Also See USEPA, 2002a)
14.6.13.1.17.1 Cultures inclosed, 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 Inclosed, 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 be 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, gravid females must be obtained from the stock culture eight days in advance of
the test. Whenever possible, brood stock should be obtained from cultures having similar salinity, temperature, light
regime, etc., as are to be used in the toxicity test.
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 um) flow-through containers (Figure 1) held within 4-L glass, wide-mouth 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.
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INFLOW
OUTFLOW
NETTED
CHAIVBER
SEPARATORS
FUNNEL
Figure 1.
NETTED
CHAIVBER
CULTURE DISH
Apparatus (brood chamber) for collection of juvenile mysids,
Mysidopsis bahia. From USEPA (1987d).
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.
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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.
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.
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.
14.6.13.2.11 The pre-test holding conditions of test organisms (as well as the test conditions) have been shown to
significantly influence the success of achieving the test acceptability criteria for the fecundity endpoint (egg
production by 50% or more of control females). Temperature, feeding, and organism density are important factors
in the rate of mysid development. Laboratories should optimize these factors (within the limits of the test
procedure) during both the pre-test holding period and the testing period to encourage achieving the test
acceptability criteria for the fecundity endpoint. If test organisms are purchased, the testing laboratory should also
confer with the supplier to ensure that pre-test holding conditions are optimized to successfully achieve the
fecundity endpoint. Lussier et al. (1999) found that by increasing holding temperature and test temperature from
26°C ± 1°C to 26°C - 27°C and maintaining holding densities to < 10 organisms / L, the percentage of tests meeting
the test acceptability criteria for fecundity increased from 60% to 97%. While the fecundity endpoint is an optional
endpoint, it is often the most sensitive measure of toxicity, and the 7-d mysid test estimates the chronic toxicity of
effluents most effectively when all three endpoints (survival, growth, and fecundity) are measured (Lussier et al.
1999).
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
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.
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14.10 TEST PROCEDURES
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.
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 um 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 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.
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.
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 labeled with the test concentration and replicate
number. Dispense 150 mL of the appropriate effluent dilution to each test chamber.
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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 artificial seawater prepared from FORTY FATHOMS® or GP2 sea salts (see Table 1 and Section 7,
Dilution Water). Other artificial 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).
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 uE/m2/s, 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.
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.
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, 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
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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 AeadArtemia, 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 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 7). 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 disturbance 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 labeled 100 mL plastic screw capped
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jar, and 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 in the oviduct. NOTE: Adult females without eggs in the oviduct or brood sac look like
immature mysids (see Figure 6).
230
-------
TEST:
START DATE:.
SALINITY:
DAY1
DAY 2
DAYS
DAY 4
DAYS
DAY 6
DAY?
TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
TEMP
SALINITY
D.O.
pH
TRTMT
TEMP
SALINITY
D.O.
pH
DAY1
DAY 2
DAYS
DAY 4
DAYS
DAY 6
DAY?
TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
RFP
TEMP
SALINITY
D.O.
pH
TRTMT
TEMP
SALINITY
D.O
pH
Figure 2.
Data form for the mysid, Mysidopsis bahia, water quality measurements. From USEPA (1987d).
231
-------
MATURE FEMALE, EGGS IN OVIDUCTS
antennule
eyestalk
carapace
statocyst
telson
developing brood sac
oviducts with developing ova
Figure 3.
Mature female mysid, Mysidopsis bahia, with eggs in oviducts. From USEPA (1987d).
232
-------
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
Figure 4. Mature female mysid, Mysidopsis bahia, with eggs in oviducts and developing embryos in the
brood sac. Above: lateral view. Below: dorsal view. FromUSEPA (1987d).
233
-------
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.
14.10.10.3.4 Immediately upon removal from the drying oven, the weighing pans 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 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 weight by the number of original mysids per replicate to determine the
average individual dry weight and record data. For the controls also calculate the mean weight per surviving mysid
in the test chamber to evaluate if weights met test acceptability criteria (see Subsection 14.12).
14.10.9.3.5 Pieces of aluminum foil (1-cm square) or small aluminum weighing pans can be used for dry weight
analyses. The weighing pans should not exceed 10 mg in weight.
14.10.9.3.6 Number each pan with a waterproof pen with the treatment concentration and replicate number.
Forty-eight (48) weigh pans are required per test if all the organisms survive.
234
-------
MATURE MALE
eyestalk
carapace
antennule
antenna
statocyst
telson
uropod
gonad
^w oil globules
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/surviving mysid in the controls. If fecundity in the controls is adequate (egg production by 50% of females),
fecundity should be used as a criterion of effect in addition to survival and growth.
235
-------
IMMATURE
. eyestalk
carapace
antennule
statocyst
telson
Figure 6.
Immature mysid, Mysidopsis bahia, (A) lateral view, (B) dorsal view. From USEPA (1987d).
14.13 DATA ANALYSIS
14.13.1 GENERAL
14.13.1.1 Tabulate and summarize the data. Table 4 presents a sample set of survival, growth, and fecundity data.
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.
236
-------
TEST:
START DATE:
SALINITY:
TREATMENT/
REPLICATE
C
1
2
DAY1
# ALIVE
DAY 2
# ALIVE
DAYS
# ALIVE
DAY 4
# ALIVE
DAYS
# 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).
237
-------
TEST:
START DATE:.
SALINITY:
TREATMENT/
REPLICATE
1
2
3
3 4
5
6
7
8
1
2
3
4 4
5
6
7
8
1
2
3
5 4
5
6
7
8
DAY1
# ALIVE
DAY 2
# ALIVE
DAY 3
# ALIVE
DAY 4
# ALIVE
DAYS
# 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 (CONTINUED). From
USEPA (1987d).
238
-------
TEST:
START DATE:.
SALINITY:
TREATMENT/REPLICATE
1
2
3
C 4
5
6
7
8
1
2
3
1 4
5
6
7
8
1
2
3
2 4
5
6
7
8
PAN#
TARE WT.
TOTAL WT.
ANIMAL WT.
# OF ANIMALS
WT./ ANIMAL
Figure 8.
Data form for the mysid, Mysidopsis bahia, dry weight measurements. From USEPA (1987d).
239
-------
TEST:
START DATE:
SALINITY:
TREATMENT/REPLICATE
1
2
3
3 4
5
6
7
8
1
2
3
4 4
5
6
7
8
1
2
3
5 4
5
6
7
8
PAN#
TARE WT.
TOTAL WT.
ANIMAL WT.
# OF ANIMALS
WT./ ANIMAL
Figure 8. Data form for the mysid, Mysidopsis bahia, dry weight measurements (CONTINUED). From
USEPA (1987d).
240
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
MY$ID,MYSIDOPSISBAHIA, SEVEN DAY SURVIVAL, GROWTH, AND FECUNDITY
TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1007.0)1
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. organisms per test chamber:
12. No. replicate chambers per concentration:
13. No. larvae per concentration:
14. Source of food:
15. Feeding regime:
16. Cleaning:
Static renewal (required)
20%o to 30%o (± 2%o of the selected test salinity)
(recommended)
26 ± 1°C (recommended)
Test temperatures must not deviate (i.e., maximum minus
minimum temperature) by more than 3°C during the test
(required)
Ambient laboratory illumination (recommended)
10-20 uE/m2/s (50-100 ft-c.)
(ambient laboratory levels) (recommended)
16 h light, 8 h darkness, with phase in/out period
(recommended)
8 oz plastic disposable cups, or 400 mL glass beakers
(recommended)
150 mL per replicate (recommended minimum)
Daily (required)
7 days (required)
5 (required minimum)
8 (required minimum)
40 (required minimum)
Newly hatched Artemia nauplii (less than 24 h old)(required)
Feed 150 24 h old nauplii per mysid daily, half after test
solution renewal and half after 8-12 h (recommended)
Pipette excess food from cups daily immediately before test
solution renewal and feeding (recommended)
1 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above
is identified as required or recommended (see Subsection 10.2 for more information on test review). Additional
requirements may be provided in individual permits, such as specifying a given test condition where several
options are given in the method.
241
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
MY$ID,MYSIDOPSISBAHIA, SEVEN DAY SURVIVAL, GROWTH, AND FECUNDITY
TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1007.0)
(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
(recommended)
Uncontaminated source of natural seawater, deionized water mixed
with hypersaline brine or artificial sea salts (HW MARINEMIX®,
FORTY FATHOMS®, GP2 or equivalent) (available options)
Effluents: 5 and a control (required)
Receiving waters: 100% receiving water (or minimum of 5) and a
control (recommended)
Effluents: > 0.5 series (required)
Receiving waters: None, or > 0.5 (recommended)
7 days (required)
Survival and growth (required); and egg development
(recommended)
80% or greater survival, average dry weight 0.20 mg or greater in
controls (required); fecundity may be used if 50% or more of females
in controls produce eggs (required if fecundity endpoint used)
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 (e.g., 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) (required)
3 L per day (recommended)
242
-------
TABLE 4. DATA FOR MYSIDOPSISBAHIA 7-DAY SURVIVAL, GROWTH, AND FECUNDITY TEST1
Treatment
Control
50ppb
100 ppb
210ppb
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
5
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
Total
Females
1
2
3
1
2
5
2
3
2
3
3
0
5
2
4
3
3
2
1
2
3
1
4
0
1
2
1
3
1
2
1
3
0
0
0
0
0
0
0
1
Females
w/Eggs
1
2
2
1
2
4
2
3
1
1
2
0
2
1
1
1
1
1
0
1
2
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Mean
Weight
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
0.094
0.017
0.122
0.052
0.154
0.110
0.103
0.012
0.002
0.081
1 Data provided by Lussier, Kuhn and Sewall, Environmental Research Laboratory, U.S. Environmental
Protection Agency, Narragansett, RI.
243
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14.13.2 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSISBAHIA, 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 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 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-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.
14.13.2.3 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 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 I-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.
244
-------
STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST
SURVIVAL HYPOTHESIS TESTING
SURVIVAL DATA
PROPORTION SURVIVING
ARC SINE
TRANSFORMATION
1
SHAPIRO-WILK'S TEST
NORMAL DISTRIBUTION
1
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
v
HETEROGENEOUS
VARIANCE
v
1
EQUAL NUME
REPLICAT
rNO
JEROF
ES?
YES
T-TEST WITH n, |MMPTT,o
BONFERRONI DUNNETTS
ADJUSTMENT ' t& '
EQ
1 YES
STEEL'S MANY-ONE
RANK TEST
t
ENDPOINT ESTIMATES
NOEC, LOEC
UAL NUMBER OF
REPLICATES?
1 NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
Figure 9. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by hypothesis testing.
245
-------
STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
SURVIVAL, GROWTH, AND FECUNDITY TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
T
TWO OR MORE
PARTIAL MORTALITIES?
YES
NO
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2 TEST)
YES
NO
ONE OR MORE
PARTIAL MORTALITIES?
I YES
NO
GRAPHICAL METHOD
LC50
PROBIT METHOD
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONC.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
YES
NO
SPEARMAN-KARBER
METHOD
T
TRIMMED SPEARMAN-
KARBER METHOD
LC50 AND 95%
CONFIDENCE
INTERVAL
Figure 10.
Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by point estimation.
246
-------
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
1.0-
0.9
0.8
0.7
g 0.6
Q.
O
Qi Q5
0_ u-°
> 0.4
3 0.3
0.2
0.1
0.0
50 100
CONCENTRATION (PPB)
210
450
Figure 11.
Plot of survival proportions of mysids, Mysidopsis bahia, at each treatment level.
247
-------
TABLE 5. MYSID, MYSIDOPSISBAHIA, SURVIVAL DATA
Replicate
Control
Concentration (ppb)
50.0
100.0 210.0
450.0
1
2
3
RAW 4
5
6
7
8
1
ARC SINE 2
TRANS- 3
FORMED 4
5
6
7
8
0.80 0.80
0.80 1.00
1.00 0.80
1.00 0.80
1.00 1.00
1.00 1.00
1.00 0.80
0.80 1.00
.107 1.107
.107 .345
.345 .107
.345 .107
.345 .345
.345 .345
.345 .107
.107 .345
MeanCY,) 1.256 1.226
S;2 0.015 0.016
i
[ 2
0.60
1.00
1
00
1.00
1
00
0.60
0.80
0
0
80
886
.345
345
345
.345
0
886
.107
.107
1.171
0.042
o
6
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
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 -
Where: Xj = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations.
248
-------
TABLE 6. CENTERED OBSERVATIONS FOR SHARIRO-WILK'S EXAMPLE
Concentration (ppb)
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
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 = J_(-0.006) = 0.0
40
0=1.197
14.13.2.6.4 Order the centered observations from smallest to largest:
X^ < X^ < < X^
Where X(l) is the ith ordered observation. These ordered observations are listed in Table 7.
14.13.2.6.5 From Table 4, Appendix B, for the number of observations, n, obtain the coefficients ab 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 = 40 and k = 20. The ^ values are listed in Table 8.
14.13.2.6.6 Compute the test statistic, W, as follows:
W = —
D i=i
The differences x(n"1+1) - X(l) are listed in Table 8. For this data in this example:
W = 1 (1.0475)2 = 0.9167
1.197
249
-------
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
X® i X®
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
14.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.2.6.6 with the critical value
found in Table 6, Appendix B. If the computed W is less than the critical value, conclude that the data are not normally
distributed. For this set of data, the critical value at a significance level of 0.01 andn = 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 observation, etc. If ties occur when ranking, assign the average
rank to each tied observation.
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.
250
-------
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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
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
X(40) x(l)
X(39).X(2)
X(38) _ x(3)
X(37).X(4)
X(36).X(5)
V(35) v(6)
-A. ~ -A.
X(34).X(7)
X(33).X(8)
X(32).X(9)
X(31) _ x(10)
X(30) x(l 1)
X(29) _ X(12)
X(28).X(13)
X(27).X(14)
v(26) VO^)
X(25).X(16)
X(24).X(17)
X(23).X(18)
X(22).X(19)
X(21) _ X(20)
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).
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 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.
251
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TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 50 PPB CONCENTRATION LEVEL FOR
STEEL'S MANY-ONE RANK TEST
Rank
Transformed Proportion
of Total Mortality
Concentration
4
4
4
4
4
4
4
12
12
12
12
12
12
12
12
12
.107
.107
.107
.107
.107
.107
.107
.571
.571
.571
.571
.571
.571
.571
.571
.571
Control
Control
Control
50 ppb
50ppb
50 ppb
50 ppb
Control
Control
Control
Control
Control
50 ppb
50 ppb
50 ppb
50 ppb
14.13.3 EXAMPLE OF ANALYSIS OF MYSID,A4YSIDOPSISBAHIA, 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. Because this measurement is based
on the number of original organisms exposed (rather than the number surviving), the measured response is a combined
survival and growth endpoint that can be termed biomass. 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.
TABLE 10. TABLE OF RANKS1
Replicate
Control
Concentration (ppb)
50
100
210
'Control ranks are given in the order of the concentration with which they were ranked.
252
450
1
2
3
4
5
6
7
8
1.107(4,5,6.5,10)
1.107(4,5,6.5,10)
1.345(12,12,13.5,14)
1.345(12,12,13.5,14)
1.345(12,12,13.5,14)
1.345(12,12,13.5,14)
1.345(12,12,13.5,14)
1.107(4,5,6.5,10)
.107(4)
.345(12)
.107(4)
.107(4)
.345(12)
.345(12)
.107(4)
.345(12)
0.886(1.5)
1.345(12)
1.345(12)
1.345(12)
1.345(12)
0.886(1.5)
1.107(5)
1.107(5)
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)
0.225(3)
0.464(6.5)
0.225(3)
0.464(6.5)
0.225(3)
0.225(3)
0.225(3)
0.685(8)
-------
TABLE 11. RANK SUMS
Concentration Rank Sum
50 64
100 61
210 49
450 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.
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 nonparametric alternative. For detailed
information on the Bonferroni adjustment, see Appendix D.
253
-------
Probit Analysis ofMysidopsis bahia 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
0.0750
0.1000
0.1500
0.2750
0.9000
Proportion
Responding
Adjusted for
Controls
0.0000
-0.0080
0.0480
0.1880
0.8880
Chi - Square for Heterogeneity (calculated)
Chi - Square for Heterogeneity (tabular value)
0.725
5.991
Probit Analysis ofMysidopsis bahia Survival Data
Estimated LC/EC Values and Confidence Limits
Point
Exposure Lower Upper
Cone. 95% Confidence Limits
LC/EC 1.00
LC/EC 50.00
123.112
288.873
65.283
239.559
165.552
335.983
Figure 12. Output for USEPA Probit Analysis Program, Version 1.5.
254
-------
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
36
40
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.
TABLE 13. MYS1D,A4YSIDOPSISBAHIA, GROWTH DATA
Replicate
Control
Concentration (ppb)
50.0
100.0
210.0
450.0
1
2
o
6
4
5
6
7
8
Mean (Y;)
s?
i
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
0.153
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
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 concentration from each observation in that concentration. The centered observations are listed
in Table 14.
255
-------
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-WILK'S TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTS TEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
1
YES
EQUAL NUMBER OF
REPLICATES?
YES
NO
T-TEST WITH
BONFERRONI
ADJUSTMENT
DUNNETT'S
TEST
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.
256
-------
O)
0.30
0.28
0.26
0.24
0.22
0.20
0.18
I
CD
LU 0.16
E
Q
<
LU
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
REPRESENTS THE CRITICAL VALUE FOR DUNNETT'S TEST
(ANY MEAN WEIGHT BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
50 100
CONCENTRATION (PPB)
-X-
210
Figure 14.
Plot of mean growth data for mysid, Mysidopsis bahia, test.
257
-------
TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
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
Concentration (ppb)
100.0
-0.054
0.004
-0.008
0.031
-0.003
-0.023
0.039
0.018
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:
n _
r> - V (Y y\2
U — / j \A ~A)
Where: Xj = 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 =J. (0.007) = 0.000
32
0 = 0.0451
14.13.3.5.4 Order the centered observations from smallest to largest
X(D < X(2) < < X(n)
Where X(l) denotes the ith ordered observation. The ordered observations for this example are listed in Table 15.
258
-------
TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHARIRO-WILK'S EXAMPLE
X© j X(i)
1 -0.084
2 -0.064
3 -0.062
4 -0.054
5 -0.049
6 -0.038
7 -0.036
8 -0.030
9 -0.023
10 -0.008
11 -0.007
12 -0.007
13 -0.006
14 -0.003
15 0.002
16 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 ab 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:
1 k
W = — [£ ar.(x<"-''+1>-x«]2
The differences X(IM+1) - X(l) are listed in Table 16. For this set of data:
W = 1 (0.2097)2 = 0.9752
0.045
14.13.3.5.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.3.5.6 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. Forthis set of data, the critical value at a significance level of 0.01 and n = 32 observations is 0.904. Since
W = 0.9752 is greater than the critical value, conclude that the data are normally distributed.
259
-------
TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
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
XPO-XO)
X(31).X(2)
-V<30) x(3)
X(29).X(4)
X(28) _ X(5)
X(27) . X(6)
X^ - X(7)
X(25) _ X(8)
x<24>-x(9)
X(23).X(10)
X(22).X(11)
X(21).X(12)
X(20) _ x(13)
X(19) _ X(14)
X(18) _ x(15)
X(17) _ 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
follows:
D
D =
c
Where: V; = degrees of freedom for each copper concentration and control, V; = (^ -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
HJ = the number of replicates for concentration i.
260
-------
p
(E
s2 = ^
C =
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 (r^ = 8 for all i). Thus, V; = 7 for all i.
14.13.3.6.3 Bartlett's statistic is therefore:
B = [(28)ln(0.00162)-7^1n(5'12)]/l-06
z = l
= [28(-6.427) - 7(-25.9329)]/1.06
= [-179.973-(-181.530)]/1.06
= 1.469
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 11.34. SinceB= 1.469 is less than the critical value of 11.34, 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.
261
-------
TABLE 17. 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)
Si = SSB/(p-l)
Si = SSW/(N-p)
Where: p = number of concentration levels including the control
N = total number of observations n{ + n2... + rip
HJ = number of observations in concentration i
SSB = Y Tj/nt-G2/N
z=l
SST =
p n
SSW = SST-SSB
Between Sum of Squares
Total Sum of Squares
Within Sum of Squares
G = the grand total of all sample observations, G = 2^ Ti
z = l
Tj = the total of the replicate measurements for concentration i
YJJ = 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:
H! = n2 = n3 = n4 = 8
N=32
T1=Y11+Y12 + ...+Y18= 1.455
T2 = Y21+Y22 + ...+Y28= 1.473
T3 = Y31+Y32 + ...+Y38= 1.348
T4 = Y41+Y42 + ...+Y48 = 0.805
262
-------
G =
= 5.081
SSB = £ T/ni-G2/N
= 1 (6.752) - (5.081)2 =0.0372
8 32
SST =
Yl-G2IN
= 0.889-(5.081)2 =0.0822
32
SSW = SST-SSB = 0.0822-0.0372 = 0.0450
SB = SSB/(p-l) = 0.03727(4-1) = 0.0124
S%, = SSW 7 (N-p) = 0.0450 7 (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
df
Sum of Squares
(SS)
Mean Square(MS)
(SS/df)
Between
Within
3
28
0.0372
0.0450
0.0127
0.0016
Total
31
0.0822
14.13.3.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control
combination as follows:
t =
Wj)+ (!/«,.)
Where: 7. = mean dry weight for concentration i
7j = mean dry weight for the control
Sw = square root of the within mean square
263
-------
H! = number of replicates for the control
HJ = number of replicates for concentration 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:
(0.182-0.184)
—
[0.040^(1/8) + (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 weight for the control if t; is
greater 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.
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.
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)
H! = the number of replicates in the control.
264
-------
14.13.3.7.8 In this example:
MSD = 2.15(0.04V(l/8) + (1/8)
= 2.15(0.04)(0.5)
= 0.043
14.13.3.7.9 Therefore, for this set of data, the minimum difference 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 ICp. In the following discussion, the observed means are represented by 7. and the smoothed means by
H.
14.13.3.8.2 Starting with the control mean, 7j =0.182and 72 =0.184, we see that Yl <72. Calculate the smoothed
means:
M1=M2 = (71 + 72)/2 = 0.183
14.13.3.8.3 Since 75 = 0.025 < 74 =0.101< 73 =0.168
-------
0
INDIVIDUAL REPLICATE MEAN BIOMASS
CONNECTS THE OBSERVED MEAN VALUES
CONNECTS THE SMOOTHED MEAN VALUES
50 100 210
TOXICANT CONCENTRATION (PPB)
Figure 15. Plot of raw data, observed means, and smoothed means for the mysid, Mysidopsis bahia, growth data from Tables 13 and 20.
266
-------
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 = C. + [Mj(l
IC25 = 100+ [0.183(1-25/100)-0.168] (210 - 100)
(0.101-0.168)
= 151 ppb.
14.13.3.8.6 Using Equation 1 from Appendix L, the estimate of the IC50 is calculated as follows:
/Cp = C. + tMj (1-/7/100)-
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 150.6446 ppb. The empirical 95.0% confidence interval forthe 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 234.6761 ppb. The empirical 95.0% confidence interval for the true mean was (183.8187 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.
267
-------
Cone. ID
4.
Cone. Tested 0
50
100
210
450
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
.146 .154
.118 .19
.216 .193
.199 .190
.176 .190
.243 .191
.213 .122
.144 .177
.114
.172
.160
.199
.165
.145
.207
.186
.153 0
.094 .012
.017 0
.122 .002
.052 0
.154 0
.110 0
.103 .081
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date : Test Ending Date :
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration: growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysid.i25
Cone.
ID
1
2
o
6
4
5
Number Concentration
Replicates /ug/l
8 0.000
8 50.000
8 100.000
8 210.000
8 450.000
Response
Means
0.182
0.184
0.168
0.101
0.102
Standard. Pooled
Dev. Response Means
0.043 0.183
0.038 0.183
0.030 0.168
0.047 0.101
0.028 0.012
The Linear Interpolation Estimate:
150.6446
Entered? Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 147.1702 Standard Deviation:
Original Confidence Limits: Lower: 97.0905 Upper:
Resampling time in Seconds: 0.11 Random Seed:
23.7984
186.6383
-1623038650
Figure 16. ICPIN program output for the IC25.
268
-------
Cone. ID
Cone. Tested
50
100
210
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 ***
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.iSO
Cone
ID
1
2
3
4
5
8
8
8
8
8
Number
Replicates
0.000
50.000
100.000
210.000
450.000
Concentration
Mg/L
0.182
0.184
0.168
0.101
0.012
Response
Means
0.043
0.038
0.030
0.047
0.028
Standard. Pooled
Dev. Response Means
0.183
0.183
0.168
0.101
0.01
The Linear Interpolation Estimate: 234.6761 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 230.7551 Standard Deviation: 30.6781
Original Confidence Limits: Lower: 183.8197 Upper: 277.9211
Resampling time in Seconds: 0.16 Random Seed: -628896314
Figure 17. ICPIN program output for the IC50.
269
-------
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)
ENDPOINT ESTIMATE
IC25, IC50
ARC SINE TRANSFORMATION
SHAPIRO-WILK'S TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
EQ
r
1 NO
T-TEST WITH
BONFERRONI
ADJUSTMENT
UAL NUMBER OF
REPLICATES?
1 YES
DUNNETT'S
TEST
}
EQ
YES
'
STEEL'S MANY-ONE
RANK TEST
UAL NUMBER OF
REPLICATES?
1 NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
*
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 18. Flowchart for statistical analysis of mysid, Mysidopsis bahia, fecundity data.
270
-------
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. 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.
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 Bonferroni 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
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 =
Where: X1 = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
271
-------
1.0
0.9
0.8
CD
LJJ
LU
<
LJJ
0.6
0.5
I 0.3
Q_
o
£ 0.2
0.1
0.0
0
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
REPRESENTS THE CRITICAL VALUE FOR DUNNETT'S TEST
(ANY PROPORTION BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
50
CONCENTRATION (PPB)
100
Figure 19. Proportion of female mysids, Mysidopsis bahia, with eggs.
272
-------
TABLE 21. MYSID, MYSIDOPSISBAHIA, FECUNDITY DATA: PERCENT FEMALES WITH EGGS
Replicate
Test Concentration (ppb)
Control
50.0
100.0
210.0
RAW
ARC SINE
TRANS-
FORMED1
MeanOQ
s2,
i
1
2
3 (
4
5
6 (
7
8
1
2
3 (
4
5
6
7
8
(
LOO 0.50 0
LOO 0.33 0
).67 0.67 0
LOO - 0
LOO 0.40 0
).80 0.50 0
LOO 0.25 0
LOO 0.33
.57 0.78 0
.57 0.61 0
).96 0.96 0
.57 - 0
.57 0.68 0
.12 0.78 0
.57 0.52 0
.57 0.61
L44 0.71 0
).064 0.021 0
L 2 3
o o
JJ
50
00
50
67
00
25
61
78
00
78
96
00
52
52
147
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
-
-
-
-
-
-
-
-
4
1 Since the denominator of the proportion of females with eggs varies with the number of females occurring in that
replicate, the adjustment of the arc sine square root transformation for 0% and 100% is not used for this data.
TABLE 22. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Test Concentration (ppb)
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
-
273
-------
14.13.4.5.3 For this set of data, n=22
X = J_ (0.000) = 0.000
22
0=1.4412
14.13.4.5.4 Order the centered observations from smallest to largest:
X(D < X(2) < < X(n)
Where X(l) 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 ab a2,... ak where k
is n/2 if n is even and (n-1)/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:
The differences x(n"1+1) - X(I) are listed in Table 24. For the data in this example:
W 1 (1.1389)2 = 0.900
1.4412
TABLE 23. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
X®
-0.52
-0.52
-0.48
-0.32
-0.19
-0.10
-0.10
0.03
0.00
0.07
0.07
i
12
13
14
15
16
17
18
19
20
21
22
X®
0.09
0.13
0.13
0.13
0.13
0.13
0.13
0.25
0.26
0.26
0.44
274
-------
TABLE 24. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
i a, X&M+D.X©
1 0.4590
2 0.3156
3 0.2571
4 0.2131
5 0.1764
6 0.1443
7 0.1150
8 0.0878
9 0.0618
10 0.0368
11 0.0122
0.96
0.78
0.74
0.57
0.32
0.23
0.23
0.16
0.13
0.06
0.02
X(22).X(1)
X(2D.X(2)
X(20)_X(3)
X(19) _ X(4)
X(18).X(5)
X(17).X(6)
X(16).X(7)
X(15).X(8)
X(14) _ X(9)
X(13) _ x(10)
X(12).X(11)
14.13.4.5.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.4.5.6 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. Forthis set of data, the critical value at a significance level of 0.01 and n = 22 observations is 0.878. Since
W = 0.900 is greater than the critical value, conclude that the data are normally distributed.
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:
- £ Vt In Sf]
c
Where: V; = degrees of freedom for each copper concentration and control, V; = (n -1)
p = number of concentration levels including the control
p
(E vt sf)
02 i,\
p
£*",
z=l
In = loge
i = 1, 2,..., p where p is the number of concentrations including the control
HJ = the number of replicates for concentration i.
275
-------
c =
14.13.4.6.2 For the data in this example (see Table 21), ^ = 8, n2 = 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)]/1.07
= 4.868
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.
14.13.4.7 T test with the Bonferroni Adjustment
14.13.4.7.1 At 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
Between
Within
Total
df
p-1
N-p
N-l
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
Si = SSB/(p-l)
S2W = SSW/(N-p)
Where: p = number of concentration levels including the control
N = total number of observations n{ + n2... + r^
HJ = number of observations in concentration i
276
-------
SSB = T/n -G2/N Between Sum of Squares
i
z=l
p "j
SST = Y" Y" Y2-G2/N Total Sum of Squares
Z = l j-l
SSW =SST-SSB ,,,.t, . 0 ._
Within Sum of Squares
p
G = the grand total of all sample observations, G = ^ Ti
z = l
Tj = the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the mean dry weight of the mysids 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
T=Y +Y + +Y =115
M J- 11 ^ J- 12 ^ ••• ^ J- is J-1-3
T2 = Y21+Y22 + ...+Y27= 4.94
T3 = Y31+Y32 + ...+Y37= 3.65
G = T! + T2 + T3 = 20.09
SSB = £ TlnrG2IN
i=\
= 132.25 + 24.40 + 13.32 - 403.61 = 3.57
8 7 7 22
P "J
= 23.396-403.61 =5.05
22
SSW = SST - SSB = 5.05 - 3.57 = 1.48
SB = SSB/(p-l) = 3.577(3-1) = 1.785
Sw = SSW/(N-p) = 1.487(22-3) = 0.078
277
-------
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
Between 2
Within 19
Total 21
Sum of Squares
(SS)
3.57
1.48
5.05
Mean Square (MS)
(SS/df)
1.785
0.078
14.13.4.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control
combination as follows:
Where: 7. = mean proportion of females with eggs for concentration i
7j = mean proportion of females with eggs for the control
Sw = square root of the within mean square
H! = number of replicates for the control
HJ = 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^(1/8)-(1/7)]
= 5.05
278
-------
TABLE 27. CALCULATED T VALUES
Test Concentration (ppb)
50.0 2 5.05
100.0 3 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 S
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)
H! = 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.
279
-------
1. Subtract the MSD from the transformed control mean.
1.44-0.30= 1.14
2. Obtain the untransformed values for the control mean and the difference calculated in 4.10.1.
[Sine(1.44)]2 =0.983
[ Sine (1.14) ]2 = 0.823
3. The untransformed MSD (MSDJ is determined by subtracting the untransformed values from
14.13.4.7.10.2.
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, MYSIDOPSISBAHIA, MEAN MEAN PROPORTION OF FEMALES WITH EGGS
Toxicant Response Smoothed
Cone. Means Mean
(ppb) i Y1 (mg) 1^ (mg)
Control 1 0.934 0.934
50.0 2 0.426 0.426
100.0 3 0.317 0.317
210.0 4 0.000 0.000
450.0 5 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 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 could 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 Q = 0 ppb and C2 = 50 ppb. The response, 0.467, is bracketed by Q = 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 = C. + [Mj(l
280
-------
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:
J+ l J (M^-M}
IC50 = 0 + [0.934(1 - 50/100) - 0.934] (50 - 0)
(0.426 - 0.934)
= 46 ppb.
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 29.9745 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.
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 was 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.
281
-------
CO
o
O
LU
CO
LU
LU
LL
LL
O
Z
O
1.2
1.0
0.9
0.8
0.7
0.6
0.4
o:
Q- 0.2
z
S5 0.1
^
0.0
INDIVIDUAL PROPORTION OF FEMALES WITH EGGS
CONNECTS THE OBSERVED MEAN VALUE
50 100 210
TOXICANT CONCENTRATION (PPB)
450
Figure 20. Plot of the mean proportion of female mysids, Mysidopsis bahia, with eggs
282
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
1
0
1
1
.67
1
1
.8
1
1
2
50
.5
.33
.67
.4
.5
.25
.33
o
5
100
o
.J
.5
0
.5
.67
0
.25
4
210
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: 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 8
27
3 7
48
Number
Replicates
0.000
50.000
100.000
210.000
Concentration
Mg/1
0.934
0.426
0.317
0.000
Response
Means
0.127
0.142
0.257
0.000
Standard.
Dev.
0.934
0.426
0.317
0.000
Pooled
Response Means
The Linear Interpolation Estimate:
29.9745
Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 23.8871 Standard Deviation:
Original Confidence Limits: Lower: 20.0499 Upper:
3.0663
30.5765
Resampling time in Seconds: 1.37
Random Seed:
1918482350
Figure 21. ICPIN program output for the IC25.
283
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
1
0
1
1
.67
1
1
.8
1
1
2
50
.5
.33
.67
.4
.5
.25
.33
3
100
o
.J
.5
0
.5
.67
0
.25
21
0
0
0
0
0
0
0
0
4
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date : Test Ending Date :
Test Species: MYSID SHRIMP
Test Duration: fecundity
DATA FILE: mysidfe.icp
OUTPUT FILE: mysidfe.iSO
-Cone.
ID
1 8
2 7
3 7
4 8
Number
Replicates
0.000
50.000
100.000
210.000
Concentration
0.934
0.426
0.317
0.000
Response
Means
0.127
0.142
0.257
0.000
Std.
Dev.
0.934
0.426
0.317
0.000
Pooled
Response Means
The Linear Interpolation Estimate:
45.9490
Entered?Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 47.8720 Standard Deviation: 8.2908
Original Confidence Limits: Lower: 40.1467 Upper:
Resampling time in Seconds: 1.32 Random Seed:
63.0931
-391064242
Figure 22. ICPIN program output for the IC50.
284
-------
14.14 PRECISION AND ACCURACY
14.14.1 PRECISION - Data on single-laboratory and multilaboratory precision are described below (Subsections
14.14.1.1 and 14.14.1.2). Single-laboratory precision is a measure of the reproducibility of test results when tests are
conducted using a specific method under reasonably constant conditions in the same laboratory. Single-laboratory
precision is synonymous with the terms within-laboratory precision and intralaboratory precision. Multilaboratory
precision is a measure of the reproducibility of test results from different laboratories using the same test method and
analyzing the same test material. Multilaboratory precision is synonymous with the term interlaboratory precision.
Interlaboratory precision, as used in this document, includes both within-laboratory and between-laboratory
components of variability. In recent multilaboratory studies, these two components of interlaboratory precision have
been displayed separately (termed within-laboratory and between-laboratory variability) and combined (termed total
interlaboratory variability). The total interlaboratory variability that is reported from these studies is synonymous with
interlaboratory variability reported from other studies where individual variability components are not separated.
14.14.1.1 Single-Laboratory Precision
14.14.1.1.1 Data on the single-laboratory precision of the my sid 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. 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.
14.14.1.1.2 EPA evaluated within-laboratory precision of the Mysid, Mysidopsis bahia, Survival, Growth, and
Fecundity Test using a database of routine reference toxicant test results from 10 laboratories (USEPA, 2000b). The
database consisted of 130 reference toxicant tests conducted in 10 laboratories using a variety of reference toxicants
including: chromium, copper, and potassium chloride. Among the 10 laboratories, the median within-laboratory CV
calculated for routine reference toxicant tests was 28% for the IC25 growth endpoint. In 25% of laboratories, the
within-laboratory CV was less than 24%; and in 75% of laboratories, the within-laboratory CV was less than 32%.
14.14.1.2 Multilaboratory Precision
14.14.1.2.1 In 2000, EPA conducted an interlaboratory variability study of the Mysid, Mysidopsis bahia, Survival,
Growth, andFecundity Test (USEPA, 2001a; USEPA, 200 Ib). In this study, each of 11 participant laboratories tested
4 blind test samples that included some combination of blank, effluent, reference toxicant, and receiving water sample
types. The blank sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater, the effluent sample was
a municipal wastewater spiked with KC1, the receiving water sample was a natural seawater spiked with KC1, and the
reference toxicant sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater spiked with KC1. Of
the 44 Mysidopsis bahia Survival, Growth, and Fecundity tests conducted in this study, 97.7% were successfully
completed and met the required test acceptability criteria. Of seven tests that were conducted on blank samples, none
showed false positive results for survival, growth, or fecundity endpoints. Results from the reference toxicant, effluent,
and receiving water sample types were used to calculate the precision of the method. Table 34 shows the precision of
the IC25 for each of these sample types. Averaged across sample types, the total interlaboratory variability (expressed
as a CV%) was 41.3% for growth IC25 results. Table 35 shows the frequency distribution of survival and growth
NOEC endpoints for each sample type. For the survival endpoint, NOEC values spanned three concentrations for the
reference toxicant, effluent, and receiving water sample types. The percentage of values within one concentration of
the median was 100% for each of the sample types. For the growth endpoint, NOEC values spanned four
concentrations for the reference toxicant sample type and three concentrations for the effluent and receiving water
sample types. The percentage of values within one concentration of the median was 92.3%, 100%, and 100%forthe
reference toxicant, effluent, and receiving water sample types, respectively. For the fecundity endpoint, NOEC values
spanned three concentrations for the reference toxicant, the effluent, and the receiving water sample types. The
percentage of values within one concentration of the median was 75.0%, 87.5%, and 66.7% for the reference toxicant,
effluent, and receiving water sample types, respectively.
285
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14.14.2 ACCURACY
14.14.2.1 The accuracy of toxicity tests cannot be determined.
TABLE 29. SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSISBAHIA, 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 TOXICANT1'2'3'4'5'6
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
1 Data from USEPA
2 Tests performed by
3 Fipht remliratp: fwnr
NOEC
(H8/L)
63
125
125
125
125
5
NA
NA
(1988a) and USEPA
IC25
(H8/L)
96.1
138.3
156.3
143.0
157.7
5
138.3
18.0
(1991a).
Randy Cameleo, ERL-N, USEPA, Narraj
isiirp: rhambpirs parh
with five; iiivpni1p<; \
IC50
(H8/L)
NC8
175.5
187.5
179.9
200.3
4
185.8
5.8
pnsett, RI.
vere, ii<;pfl fnr thp mntrnl
Most
Sensitive
Endpoint7
S
S
S
S
S
anH parh tnxirant
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 mg/L. Copper concentrations in Tests 3-6 were,
16, 31, 63, 125, and 250 ug/L.
NOEC Range: 63 -125 ug/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.
286
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TABLE 30. SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSISBAHIA, 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 NOEC
Number (mg/L)
1 2.5
2 <0.3
3 <0.6
4 5.0
5 2.5
6 5.0
n: 4
Mean: NA
CV(%): NA
IC25
(mg/L)
4.5
NC8
NC8
7.8
3.6
7.0
4
5.7
35.0
IC50
(mg/L)
NC9
NC9
NC9
NC9
4.6
9.3
2
6.9
47.8
Most
Sensitive
Endpoint7
S
S
S
S
S
S
1 Data from USEPA (1988a) and USEPA (1991a).
2 Tests performed by Randy Cameleo, ERL-N, USEPA, Narragansett, RI.
3 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.
4 SDS concentrations in Tests 1-2 were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L. SDS concentrations in Tests 3-4 were: 0.6,
1.3, 2.5, 5.0 and 10.0 mg/L. SDS concentrations in Tests 5-6 were: 1.3, 2.5, 5.0, 10.0, and 20.0 mg/L.
5 NOEC Range: < 0.3 - 5.0 mg/L (this represents a difference of four exposure concentrations).
6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
7 Endpoints: G=Growth; S=Survival.
8 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.
9 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.
287
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TABLE 31.
COMPARISON OF SURVIVAL (LC50)1, GROWTH AND FECUNDITY (IC50)1 RESULTS
FROM 7-DAY TESTS WITH THE MYSID, MYSIDOPSISBAHIA, USING NATURAL
SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS DILUTION WATER AND
SODIUM DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT
Test
1
2
3
Survival L
NSW
16.2
20.5
— 2
,C50
GP2
16.3
19.2
21.9
Growth
NSW
16.8
24.2
~2
IC50
GP2
16.3
23.3
24.4
Fecundity
NSW
12.0
20.1
2
IC50
GP2
10.9
18.5
21.7
1 All LC50/IC50 values in mg/L.
2 No test performed.
288
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TABLE 32. COMPARISON OF SURVIVAL (LC50)1, GROWTH AND FECUNDITY (IC50)1 RESULTS
FROM 7-DAY TESTS WITH THE MYSID, MYSIDOPSISBAHIA, USING NATURAL
SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS DILUTION WATER AND
COPPER (Cu) SULFATE AS A REFERENCE TOXICANT
Test
1
2
3
Survival L
NSW
177
~2
190
,C50
GP2
182
173
174
Growth IC50
NSW GP2
208 186
-2 210
195 179
Fecundity IC50
NSW GP2
177 125
-2 142
168 186
1 All LC50/IC50 values in ug/L.
2 No test performed.
289
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TABLE 3 3. CONTROL RESULTS FROM 7-DAY SURVIVAL, GROWTH, AND FECUNDITY TESTS
WITH THE MYSID, MYSIDOPSISBAHIA, USING NATURAL SEAWATER AND ARTIFICIAL
SEAWATER (GP2) AS A DILUTION WATER
Control '
Survival (%)
Test NSW GP2
1 98 93
2 80 90
3 -2 95
4 94 84
5 -2 94
6 80 75
Growth (mg)
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
-2 83
79 93
1 Survival as percent of mysids alive after 7 days; growth as mean individual dry weight; fecundity as percent
females with eggs.
2
No test performed.
290
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TABLE 34. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES1
Test Endpoint Sample Type
CV (%)
Within-lab3 Between-lab4
Total5
IC25 for
Growth
Reference toxicant
Effluent
Receiving water
8.69
5.26
40.0
36.6
40.9
37.0
45.9
Average
6.98
38.3
41.3
FromEPA's WET Intel-laboratory Variability Study (USEPA, 200la; USEPA, 200Ib).
CVs were calculated based on the within-laboratory component of variability, the between-laboratory component of
variability, and the total interlaboratory variability (including both within-laboratory and between-laboratory
components). For the receiving water sample type, within-laboratory and between-laboratory components of
variability could not be calculated since the study design did not provide within-laboratory replication for this
sample type.
The within-laboratory (intralaboratory) component of variability for duplicate samples tested at the same time in the
same laboratory.
The between-laboratory component of variability for duplicate samples tested at different laboratories.
The total interlaboratory variability, including within-laboratory and between-laboratory components of variability.
The total interlaboratory variability is synonymous with interlaboratory variability reported from other studies
where individual variability components are not separated.
291
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TABLE 35. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR VARIOUS
SAMPLE TYPES1
Test Endpoint
Survival
NOEC
Growth
NOEC
Fecundity
NOEC
Sample Type
Reference toxicant
Effluent
Receiving water
Reference toxicant
Effluent
Receiving water
Reference toxicant
Effluent
Receiving water
Median
NOEC
Value
25%
12.5%
12.5%
25%
12.5%
12.5%
18.8%
25%
9.38%
% of Results
at the Median
53.8
46.7
37.5
53.8
46.7
50.0
_4
62.5
_4
% of Results
±12
46.2
53.3
62.5
38.5
53.3
50.0
75.0
25.0
66.7
% of Results
>23
0.00
0.00
0.00
7.69
0.00
0.00
25.0
12.5
33.3
FromEPA's WET Interlaboratory Variability Study (USEPA, 200la; USEPA, 200Ib).
Percent of values at one concentration interval above or below the median. Adding this percentage to the percent of
values at the median yields the percent of values within one concentration interval of the median.
Percent of values two or more concentration intervals above or below the median.
The median NOEC fell between test concentrations, so no test results fell precisely on the median.
292
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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).
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 15.6.19, culturing methods
below and Section 4, Quality Assurance. To test effluent or receiving water toxicity, sufficient eggs and sperm
must be available.
293
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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.
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 washing 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.
15.5.18 Sedgwick-Rafter counting chamber - for counting egg stock and examining fertilized eggs.
15.5.19 Hemacy tometer, 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.
294
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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.
15.5.3 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, Arbaciapunctulata minimum 12 of each sex.
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.
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 labeling 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.
15.6.12 Formalin, 1%, in 2 mL of seawater ~ for preserving eggs (see Subsection 15.10.9 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 USEPA Method 150.1, USEPA, 1979b).
15.6.14 Membranes and filling solutions for dissolved oxygen probe (see USEPA Method 360.1, USEPA, 1979b),
or reagents ~ for modified Winkler analysis.
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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.
296
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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 (1) for fertilization test using sea urchin, Arbacia punctulata.
297
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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 = SPM
SOLUTION E x 20 = SOLUTION B = SPM
SOLUTION E x 5 = SOLUTION D = SPM
SOLUTION SELECTED FOR TEST ( = 5 x 107 SPM):
DILUTION: SPM/(5 x 107) = DF
[(DF) x 10) -10 = + SW, mL
FINAL SPERM COUNTS =
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
TEST TIMES:
SPERM COLLECTED:
EGGS COLLECTED:
SPERM ADDED:
EGGS ADDED:
FIXATIVE ADDED:
SAMPLES READ:
Figure 2. Data form (2) for fertilization test using sea urchin, Arbacia punctulata.
298
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DATE TESTED:
SAMPLE:
TOTAL AND UNFERTILIZED EGG COUNT AT END OF TEST:
EFFLUENT
CONC (%)
REPLICATE VIAL
1
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.
299
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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 desirable to match the test salinity with that of the receiving water. Two methods are available to
adjust salinities - hypersaline brine (HSB) derived from natural seawater or artificial sea salts.
15.6.18.3 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.
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 would 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.
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 umbefore 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 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 HSB should be filtered a second time through a 1 mm filter and
poured directly into portable containers, (20 L cubitainers or polycarbonate water cooler jugs are suitable). The
containers should be capped and labeled 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.
15.6.18.3.8 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.
300
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15.6.18.4 Artificial sea salts: FORTY FATHOMS® brand sea salts 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.
15.6.18.4.1 Synthetic sea salts are packaged in plastic bags and mixed with deionized water or equivalent. The
instructions on the package of sea salts should be followed carefully, and the salts should be mixed in a separate
container - not in the culture tank. The deionized water used in hydration should be in the temperature range of 21-
26°C. Seawater made from artificial sea salts is conditioned (Spotte, 1973; Spotte, et al., 1984; Bower, 1983).
15.6.18.4.2 The GP2 reagent grade chemicals (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 g NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this stock solution for each liter of the
GP2 artificial seawater.
TABLE 1. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 30%« USING NATURAL
SEAWATER, HYPERSALINE BRINE, OR ARTIFICIAL SEA SALTS '
Solutions To Be Combined
Effluent
Solution
1
2
3
4
5
Control
Effluent
Concentration
(%)
1001
50
25
12.5
6.25
0.0
Volume of
Effluent
Solution
(mL)
840
420
420
420
420
Volume of Diluent
Seawater (30%o)
(mL)
—
Solution 1 + 420
Solution 2 + 420
Solution 3 + 420
Solution 4 + 420
420
Total
2080
1 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
NaCl
Na2SO4
KC1
KBr
Na2B4O7-10H2O
MgCl2-6 H2O
CaCl2-2 H2O
SrCl2-6 H2O
NaHCO,
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
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.
3 GP2 can be diluted with deionized (DI) water to the desired test salinity.
15.6.19 TEST ORGANISMS, SEA URCHINS, ARBACIA PUNCTULATA
15.6.19.1 Adult sea urchins, Arbacia punctulata, can be obtained from commercial suppliers. After acquisition, the
animals are sexed by briefly stimulating them with current from a 12 V transformer. 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.
15.6.19.2 The sexes are separated and maintained in 20-L, aerated fiberglass tanks, each holding about 20 adults. The
tanks are supplied continuously (approximately 5 L/min) with filtered natural seawater, or salt water prepared from
commercial sea salts is recirculated. The animals are checked daily and any obviously unhealthy animals are discarded.
15.6.19.3 The culture unit should be maintained at 15 ± 3°C, with a water temperature control device.
15.6.19.4 The food consists of kelp, Laminaria sp., gathered from known uncontaminated zones or obtained from
commercial supply houses whose kelp comes from known uncontaminated areas, or romaine lettuce. Fresh food is
introduced into the tanks at approximately one week intervals. Decaying food is removed as necessary. Ample
supplies of food should always be available to the sea urchins.
15.6.19.5 Natural or artificial seawater with a salinity of 30%o is used to maintain the adult animals, for all washing
and dilution steps, and as the control water in the tests (see Subsection 15.6.18).
15.6.19.6 Adult male and female animals used in field studies are transported in separate or partitioned insulated
boxes or coolers packed with wet kelp or paper toweling. Upon arrival at the field site, aquaria (or a single partitioned
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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.
15.6.19.7 To successfully maintain about 25 adult animals for 7 days at a field site, a screen-partitioned, 40-L glass
aquarium using aerated, recirculating, clean saline water (30%o) and a gravel bed filtration system, is housed within a
water bath, such as FORTY FATHOMS® or equivalent (15°C). The inner aquarium is used to avoid contact of animals
and water bath with cooling coils.
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.
15.8 CALIBRATION AND STANDARDIZATION
15.8.1 See Section 4, Quality Assurance.
159 QUALITY CONTROL
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 um NITEX® filter and compared without dilution against a control. Using four replicate
chambers per test, each 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 for the first time 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).
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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 labeled 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 artificial 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 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, barely 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
15.10.3.1 Using control water, dilute the pooled sperm sample to a concentration of about 5 X107 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:
a. Add 400 uL 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 (100X). Average the counts from the two sides.
e. SPM in Vial E = 104 x average count.
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3. Calculate the SPM in all other suspensions using the SPM in Vial E above:
SPMinVialA = 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 ly sed 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 pipet 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
dissecting microscope. Confirm that the final egg count = 2000/mL (± 200).
15.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
15.10.5.1 The light quality and intensity should be at ambient laboratory levels 10-20 uE/m2/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.
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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 KDVIAX® serological pipet,
or equivalent.
15.10.7 OBSERVATIONS DURING THE TEST
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.
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.
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.
15.10.8 START OF THE TEST
15.10.8.1 Effluent/receiving water samples are adjusted to salinity of 30%o. Four replicates 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 uL 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 pipet 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.
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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 sperm:egg ratio routinely employed must result in fertilization of 70%-90%of the eggs in the control
chambers.
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR SEA
URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT AND
RECEIVING WATERS (TEST METHOD 1008.0)1
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 sperm cells per chamber:
10. No. replicate chambers per concentration:
11. Dilution water:
12. Test concentrations:
Static (required)
30%o (± 2%o of the selected test salinity) (recommended)
20 ± 1°C (recommended)
Test temperatures must not deviate (i.e., maximum minus
minimum temperature) by more than 3°C during the test
(required)
Ambient laboratory light during test preparation
(recommended)
10-20 uE/nf/s, or 50-100 ft-c (Ambient laboratory levels)
(recommended)
Disposable (glass) liquid scintillation vials (20 mL capacity),
presoaked in control water (recommended)
5 mL (recommended)
Pooled sperm from four males and pooled eggs from four
females are used per test (recommended)
About 2,000 eggs and 5,000,000 sperm cells per vial
(recommended)
4 (required minimum)
Uncontaminated source of natural seawater; deionized water
mixed with hypersaline brine or artificial sea salts (HW
MARTNEMIX®, FORTY FATHOMS®, GP2, or equivalent)
(available options)
Effluents: 5 and a control (required minimum)
Receiving waters: 100% receiving water (or minimum of 5)
and a control (recommended)
1 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is
identified as required or recommended (see Subsection 10.2 for more information on test review). Additional
requirements may be provided in individual permits, such as specifying a given test condition where several options
are given in the method.
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR SEA
URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT AND
RECEIVING WATERS (TEST METHOD 1008.0) (CONTINUED)
13. Dilution factor: Effluents: >0.5 (recommended)
Receiving waters: None or >0.5 (recommended)
14. Test duration: 1 h and 20 min (required)
15. Endpoint: Fertilization of sea urchin eggs (required)
16. Test acceptability criteria: 70% - 90% egg fertilization in controls (required)
17. Sampling requirements: For on-site tests, one sample collected at test initiation, and
used within 24 h of the time it is removed from the sampling
device. For off-site tests, holding time must not exceed 36 h
before first use (see Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample Preparation for
Toxicity Tests, Subsection 8.5.4) (required)
18. Sample volume required: 1 L per test (recommended)
15.13 DATA ANALYSIS
15.13.1 GENERAL
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.
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TABLE 4. DATA FROM SEA URCHIN, AREA CM PUNCTULATA, FERTILIZATION TEST
Copper
Concentration
(Hg/L)
Control
2.5
5.0
10.0
20.0
40.0
Replicate
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
No. of Eggs
Counted
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
No. of Eggs
Fertilized
85
78
87
81
65
71
63
74
78
63
66
51
41
41
37
12
30
26
Proportion
Fertilized
0.85
0.78
0.87
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
1 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,
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
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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, Arbaciapunctulata, 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.
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STATISTICAL ANALYSIS OF SEA URCHIN FERTILIZATION TEST
FERTILIZATION DATA
PROPORTION OF FERTILIZED EGGS
POINT ESTIMATION
T
ENDPOINT ESTIMATE
IC25, IC50
ARC SINE
TRANSFORMATION
1
r
SHAPIRO-WILK'S TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
NO
r
EQUAL NUMBER OF
REPLICATES?
}
YES
r
EQUAL NUMBER OF
REPLICATES?
YES
NO
T-TEST WITH
BONFERRONI
ADJUSTMENT
DUNNETT'S
TEST
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.
312
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1.0
0.9
§ 0.8
CD
LU
Q 0.7
LU
N
p 0.6
o:
LU
"- 0.5
0.4
O
Q.
O
0.2
0.1
0.0
0.0
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
REPRESENTS THE CRITICAL VALUE FOR DUNNETTS TEST
(ANY PROPORTION BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
2.5 5.0 10.0
COPPER CONCENTRATION (|jg/L)
20.0
40.0
Figure 5. Plot of mean percent of fertilized sea urchin, Arbacia punctulata, eggs.
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TABLE 5. SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA
Copper Concentration (us/L)
Replicate
A
RAW B
C
ARC SINE A
TRANSFORMED B
C
Mean (Y; )
S;2
i
Control
0.85
0.78
0.87
1.173
1.083
1.202
1.153
0.004
1
2.5
0.81
0.65
0.71
1.120
0.938
1.002
1.020
0.009
2
5.0
0.63
0.74
0.78
0.917
1.036
1.083
1.012
0.007
o
5
10.0
0.63
0.66
0.51
0.917
0.948
0.795
0.887
0.007
4
20.0
0.41
0.41
0.37
0.695
0.695
0.654
0.681
0.001
5
40.0
0.12
0.30
0.26
0.354
0.580
0.535
0.490
0.014
6
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Copper Concentration (us/L)
Replicate
A
B
C
Control
0.020
-0.070
0.049
2.5
0.100
-0.082
-0.018
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:
Where:
Xj = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
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15.13.2.5.3 For this set of data, n = 18
X = J_ (0) = 0
18
D = 0.0822
15.13.2.5.4 Order the centered observations from smallest to largest
X^ < X^ < < X®^
where X(l) denotes the ith ordered observation. The ordered observations for this example are listed in Table 7.
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i X® i X®
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 ab 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 = -*-££ ai(x("-'+r>-x®)]2
D 2=1
The differences, x(n"1+1) - X(l), are listed in Table 8. For the data in this example:
W = l-— (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 Subsection 15.13.2.5.6 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. Forthe data in this example, the critical value at a significance level of 0.01 andn= 18 observations is
0.858. Since W = 0.942 is greater than the critical value, conclude that the data are normally distributed.
315
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TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
x-.x-
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
0.236
0.185
0.163
0.143
0.119
0.072
0.048
0.010
0.006
X(18) x(l)
X(17).X(2)
x(16) _ x(3)
X(15).X(4)
X(14).X(5)
V(13) v(6)
-A. ~ -A.
X(12).X(7)
X(ll) _X(8)
X(10) 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:
B =
Where:
V; =
p =
nj =
In =
degrees of freedom for each copper concentration and control, V; = (n - 1)
number of levels of copper concentration including the control
the number of replicates for concentration i.
loge
1 ,2, . . . , p where p is the number of concentrations including the control
P
£*",
c =
316
-------
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 (r^ = 3 for all i). Thus, V; = 2 for all i.
15.13.2.6.3 Bartlett's statistic is, therefore:
B = [(12)ln(0.0007)-2£ ln(,Sf)]/1.194
= [12(-4.962) -2(-31.332)]/1.194
= 3.122/1.194
= 2.615
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 df
Between p-1
Within N - p
Sum of Squares
(SS)
SSB
SSW
Mean Square (MS)
(SS/df)
SB = SSB/(p-l)
S^=SSW/(N-p)
Total
N-l
SST
Where: p = number of concentration levels including the control
N = total number of observations n{ + n2... + rip
HJ = number of observations in concentration i
SSB = T/nj-G2/N
Between Sum of Squares
Total Sum of Squares
317
-------
SSW = SST - SSB Within Sum of Squares
p
G = the grand total of all sample observations, G = £ Tt
! = 1
Tj = the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the proportion of fertilized eggs for upper
concentration i in test chamber j)
15.13.2.7.2 For the data in this example:
H! = n2 = n3 = n4 = n5 = rig = 3
N = 18
T1=Y11+Y12 + Y13 = 3.458
T2 = Y21+Y22 + Y23 = 3.060
T3 = Y31+Y32 + Y33 = 3.036
T4 = Y41+Y42 + Y43 = 2.660
T5 = Y51+Y52 + Y53 = 2.044
T6 = Y61+Y62 + Y63 = 1.469
G = T! + T2 + T3 + T4 + T5 + T6 = 15.727
SSB = T T?/n.-G2/N
= (43.950)73 - (15.727)2/18 =0.909
SST = Y Y
= 14.732 - (15.727)2/18 = 0.991
SSW = SST-SSB
= 0.991-0.909 = 0.082
B = SSB/(p-l) = 0.9097(6-1) = 0.182
S^, = SSW/(N-p) = 0.0827(18-6) = 0.007
15.13.2.7.3 Summarize these calculations in the ANOVA table (Table 10).
318
-------
TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
df
5
12
Sum of Squares
(SS)
0.909
0.082
Mean Square(MS)
(SS/df)
0.182
0.007
Total 17 0.991
15.13.2.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
,
+ (llnj
Where: Y;= mean proportion fertilized eggs for copper concentration i
Yj = mean proportion fertilized eggs for the control
Sw = square root of the within mean square
H! = number of replicates for the control
HJ = 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.
15.13.2.7.5 Table 11 includes the calculated t values for each concentration and control combination. In this example,
comparing the 2.5 ug/L concentration with the control the calculation is as follows:
(1.153-1.020)
319
-------
TABLE 11. CALCULATED T VALUES
Copper Concentration (ug/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 tj is greater than the critical value. Therefore, the 10.0 ug/L, 20.0 ug/L
and 40.0 ug/L concentrations have a significantly lower mean proportion of fertilized eggs than the control. Hence the
NOEC is 5.0 ug/L and the LOEC is 10.0 ug/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)
U[ = the number of replicates in the control.
15.13.2.7.8 In this example,
MSD = 2.50 (0.084) V(l/3)+(1/3)
= 2.50(0.084)(0.816)
= 0.171
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 15.13.2.7.9.1
[ Sine (1.153) ]2 = 0.835
320
-------
[ Sine (0.982) ]2 = 0.692
3. The untransformed MSD (MSDU) is determined by subtracting the untransformed values from
step 2 in 15.13.2.7.9.
MSDU = 0.835 - 0.692 = 0.143
15.13.2.7.10 Therefore, forthis 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 AnIC25 andIC50 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 ug/L copper and C4 = 10.0 ug/L copper. The response, 0.417, is bracketed
by C4 = 10.0 ug/L copper and C5 = 20.0 ug/L copper.
TABLE 12. SEA URCHIN, ARBACIA PUNCTULATA, MEAN PROPORTION OF FERTILIZED EGGS
Copper
Cone.
(Hg/L)
Control
2.5
5.0
10.0
20.0
40.0
i
1
2
o
6
4
5
6
Response
Means Y;
(proportion)
0.833
0.723
0.717
0.600
0.397
0.227
Smoothed
MeanM;
(proportion)
0.833
0.723
0.717
0.600
0.397
0.227
15.13.2.8.3 Using the equation from Section 4.2 in Appendix L, the estimate of the IC25 is calculated as follows:
ICp =
(C -C)
— ^ - J—
J L iv * ' J' (U -i
vvl(j+l) lv±j>
IC25 = 5.0 + [0.833(1 - 25/100) - 0.717] (10.0-5.01
(0.600-0.717)
= 8.9 ug/L.
321
-------
15.13.2.8.4 Using the equation from Section 4.2 in Appendix L, the estimate of the IC50 is calculated as follows:
(C -C}
ICp = C. + |M(-- — ^ - J—
:
IC50 = 10.0 + [0.833(1 - 50/100) - 0.600] (20.0 - 10.0)
(0.397 - 0.600)
= 19.0 ug/L.
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 ug/L. The empirical 95.0% confidence interval for the true mean was 3.3036 ug/L to 14.6025
ug/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 19.0164 ug/L. The empirical 95.0% confidence interval for the true mean was 16. 1083 ug/L to
23.6429 ug/L. The computer program output for the IC50 for this data set is shown in Figure 7.
322
-------
Cone. ID
Cone. Tested
2
~Y.5~
3
To"
4
"Io7o"
5
~20~0~"
6
~40~0~"
Response 1
Response 2
Resrjpnse 3
.85
.78
.87
.81
.65
.71
.63
.74
.71
.63
.66
.51
.41
.41
.37
.12
.3
.2
*** Inhibition Concentration Percentage Estimate
***
Toxicant/Effluent:
Test Start Date:
Test Species:
Test Duration:
Copper
Test Ending Date:
sea urchin, Arbacia punctulata
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/L
0.000
2.500
5.000
10.000
20.000
40.000
Response
Means
0.833
.723
0.717
0.600
0.397
0.227
Standard.
Dev.
0.047
0.081
0.078
0.079
0.023
0.095
Pooled
Response Means
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
Original Confidence Limits: Lower: 6.2500
Expanded Confidence Limits Lower: 3.3036
Resampling time in Seconds: 1.59
Standard Deviation:
Upper:
Upper:
Random Seed:
0.8973
11.6304
14.6025
1834854321
Figure 6. ICPIN program output for the IC25.
323
-------
Cone. ID 1
Cone. Tested 0
2
~2.5~
3
To"
4
1576"
20.0
6
"4576"
Response 1
Response 2
Response 3
.85
.78
.87
.81
.65
.71
.63
.74
.78
.63
.66
.51
.41
.41
.37
.12
.3
.26
*** Inhibition Concentration Percentage Estimate
***
Toxicant/Effluent:
Test Start Date:
Test Species:
Test Duration:
DATA FILE:
OUTPUT FILE:
Copper
Test Ending Date:
MYSID SHRIMP
fecundity
mysidfe.icp
mysidfe.iSO
Cone.
ID
1
2
3
4
Number
Replicates
8
7
7
8
Concentration
ngfl
0.000
50.000
100.000
210.000
Response
Means
0.934
0.426
0.317
0.000
Standard.
Dev.
0.127
0.142
0.257
0.000
Pooled
Response Means
0.934
0.426
0.317
0.000
The Linear Interpolation Estimate: 19.0164
Entered? Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds: 1.65
19.0013 Standard Deviation: 0.8973
Lower: 17.6316 Upper: 21.2195
Lower: 16.1083 Upper: 23.6492
Random Seed: -823775279
Figure 7. ICPIN program output for the IC50.
324
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15 14 PRECISION AND ACCURACY
15.14.1 PRECISION
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.
325
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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) AS A REFERENCE TOXICANT1'2'3'4'5
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
LOEC
(ug/L)
5.0
12.5
<6.2
6.2
12.5
4
NA
NA
IC25
(ug/L)
8.92
26.35
11.30
34.28
36.67
5
23.51
54.60
IC50
(ug/L)
29.07
38.96
23.93
61.75
75.14
5
45.77
47.87
Data from USEPA (1991a)
Tests performed by Dennis McMullen, Technology Applications, Inc., EMSL, Cincinnati, OH.
All tests were performed using FORTY FATHOMS® synthetic seawater.
Copper test solutions were prepared with copper sulfate. Copper concentrations in Test 1 were: 2.5, 5.0,
10.0, 20.0, and 40.0 ug/L. Copper concentrations in Tests 2-5 were: 6.25, 12.5, 25.0, 50.0, and 100.0 ug/L.
NOEC Range: < 5.0 -12.5 ug/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.
326
-------
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
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(mg/L)
<0.9
0.9
1.8
0.9
1.8
4
NA
NA
IC25
(mg/L)
1.11
1.27
2.26
1.90
2.11
5
1.73
29.7
IC50
(mg/L)
1.76
1.79
2.87
2.69
2.78
5
2.38
23.3
1 Data from USEPA (1991a)
2 Tests performed by Dennis M. McMullen, Technology Applications, Inc., EMSL, Cincinnati, OH.
All tests were performed using FORTY FATHOMS synthetic seawater.
NOEC Range: <0.9 - 1.8 mg/L (this represents a difference of two exposure concentration).
SDS concentrations for all tests were: 0.9, 1.8, 3.6, 7.2, and 14.4 mg/L.
For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
327
-------
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 ''2'3'4'5'6
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(Hg/L)
12.2
12.2
24.4
<6.1
6.1
4
NA
NA
IC25
(Hg/L)
14.2
32.4
30.3
26.2
11.2
5
22.8
41.9
IC50
(Hg/L)
18.4
50.8
46.3
34.1
17.2
5
29.9
48.2
Data from USEPA (199 la)
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 ug/L.
NOEC Range: < 6.1 - 24.4 ug/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.
328
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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 1'2'3'4'5'6
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
NOEC
(mg/L)
1.8
1.8
1.8
0.9
1.8
5
NA
NA
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
Data from USEPA (199 la).
Tests performed by Ray Walsh and Wendy Greene, ERL-N, USEPA, Narragansett, RI.
SDS concentrations were: 0.9,1.8, 3.6, 7.3, and 14.5 mg/L.
NOEC Range: 0.9 -1.8 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.
329
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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
Cu (U.S/D
CI
27.3-31.1
44.6-50.8
29.8-35.8
73.3-83.9
41.0-50.7
SDS (ms/L)
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
1.8
2.2
2.3
1.8
2.0
0.2
10.0
CI
2.0-2.1
1.8-1.9
2.1-2.2
2.2-2.4
1.7-2.8
NOEC LOEC
.3 2.5
.3 2.5
.3 2.5
.3 2.5
.3 2.5
1 Tests performed by Pamela Comeleo, Science Application International Corp., ERL-N, USEPA, Narragansett, RI.
2 All tests were performed using GP2 artificial seawater.
Copper concentrations were: 6.25,12,5, 25.0, 50.0 and 100 ug/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.
330
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TABLE 18. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBAC1A PUNCTULATA,
FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER, USING GAMETES FROM
ADULTS MAINTAINED IN NATURAL SEAWATER AND COPPER (CU) SULFATE AND
SODIUM DODECYL SULFATE (SDS) AS REFERENCE TOXICANTS1'2'3'4
Test
1
2
o
5
4
5
Mean
SD
CV
LC50
28.6
13.0
67.8
36.7
356
36.3
20.0
55.1
Cu (us/L)
CI
26.7-30.6
11.9-14.2
63.2-72.6
33.9-398
33.6-37.7
SDS (mg/L)
NOEC
6.3
6.3
6.3
<6.3
<6.3
LOEC
12.5
12.5
12.5
6.3
6.3
LC50
12.5
12.5
12.5
6.3
6.3
2.5
0.58
23.2
CI
2.1-2.2
1.9-2.0
2.1-2.3
3.3-3.4
2.8-3.1
NOEC
1.3
1.3
1.3
<0.6
<0.6
LOEC
2.5
2.5
2.5
0.6
0.6
1 Tests performed by Anne Kuhn-Hines, Catherine Sheehan, GlenModica, and Pamela Comeleo, Science Application
International Corp., ERL-N, USEPA, Narragansett, RI.
2
Copper concentrations were prepared with copper sulfate. Concentrations were 6.25,12.5, 25.0, 50.0, and 100 ug/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.
331
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SECTION 16
TEST METHOD
RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST
METHOD 1009.0
16.1 SCOPE AND APPLICATION
16.1.1 CAUTION: The Red Macroalga, Champiaparvula, Reproduction Test Method 1009.0 is not listed at 40
CFR Part 136 for nationwide use.
16.1.2 This method, adapted in part from USEPA (19871) 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.
16.1.3 Detection limits of the toxicity of an effluent or chemical substance are organism dependent.
16.1.4 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, highly volatile and highly degradable toxicants present in
the source may not be detected in the test.
16.1.5 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.
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 days 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).
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.
16.3.4 Pathogenic and/or predatory organisms in the dilution water and effluent may affect test organism survival,
and confound test results.
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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, Champiaparvula, 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 ± 1°C).
16.5.5 Water purification system ~ Millipore 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.
16.5.1 Thermometers, glass or electronic, laboratory grade ~ for measuring water temperatures.
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.
16.5.22 Erlenmeyer flasks, 250 mL, or 200 mL disposable polystyrene cups, with covers ~ for use as exposure
chambers.
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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 uL - 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.
16.5.30 Pipet bulbs and fillers - PROPIPET®, or equivalent.
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 parvula, 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.
16.6.6 Tape, colored ~ for labeling 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.
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16.6.13.1 Saline test and dilution water - the use of natural seawateris 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%o,
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
parvula, 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 (HSB) 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, etal, 1984) has been used
successfully to perform the red macroalga sexual reproduction test. The preparation of artificial seawater (GP2) is
described in Table 2.
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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 MINIMUM (VITAMINS ARE AUTOCLAVED
SEPARATELY FOR 2 MINIMUM AND ADDED AFTER THE NUTRIENT STOCK
SOLUTION IS AUTOCLAVED). THE pH OF THE SOLUTION IS ADJUSTED TO
APPROXIMATELY pH 2 BEFORE AUTOCLAVING 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
NaNO3
NaH2P04 • H2O
Na2EDTA • 2 H2O
Na3C6H5O7 • 2 H2O
Iron2
Vitamins3
6.35 g
0.64 g
133 mg
51 mg
9.75 mL
10 mL
1.58 g
0.16 g
-
12.8 mg
2.4 mL
2.5 mL
1 Add 10 mL of appropriate nutrient stock solution per liter of culture or test medium.
2 A stock solution of iron is made that contains 1 mg iron/mL. Ferrous or ferric chloride can be used.
3 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 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.
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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 TESTING1'2'3'4'5'6'7
Compound
NaCl
Na2SO4
KC1
KBr
Na2B4O7-10H2O
MgCl2-6 H2O
CaCl2-.2 H2O
SrCl2-6 H2O
NaHCO3
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
1 Modified GP2 from Spotte et al. (1984).
Tlii^ /"•/"inotitm^rvt coltc on/1 /"•/~\n/"ii^rYtr'oti/~\no •f-i;f*rt* tol^i^n ~fVv~\m T TCT7"D A /1QQOT"\"\
The original formulation calls for autoclaving anhydrous and hydrated 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 minimum for 1-L volumes, and 20 minimum for 10-to-20-L volumes.
4 Prepare in 10-L to 20-L batches.
5 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.
6 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.
7 Nutrients listed in Table 1 should be added to the artificial seawater in the same concentration described for natural
seawater.
16.6.14 TEST ORGANISMS RED MACROALGA, CHAMPIA PARVULA
16.6.14.1 Cultures
16.6.14.1.1 Mature plants are illustrated in Figure 1. The adult plant body (thallus) is hollow, septate, and highly
branched. New cultures can be propagated asexually from excised branches, making it possible to maintain clonal
material indefinitely.
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tetrasporangia—
TETRASPOROPHYTE
spermatia
fertilization
cyst oca rp
5 mm
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.
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 uE/m2/s (500 ft-c) of
cool-white fluorescent light on a 16:8 h lightdark cycle. Depending on the type of culture chamber or room used, i.e.,
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the degree of reflected light, the light levels may have to be adjusted downward. The temperature is 22 to 24°C and the
salinity 28-3 0%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. 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 um 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.
sterile hairs
trichogynes
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 thallus, but are seen easiest at the "edges."
Receptive trichogynes occur only near the branch tips. From USEPA (1987f).
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1 cm
spermatial sorus
Figure:
A portion of the male thallus showing spermatial sori. The sorus areas are generally slightly
thicker and somewhat lighter in color. From USEPA (1987f).
K\xcuticle
^spermatia
lOOum
\
~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).
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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.
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 um 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 testusingaO.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.
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.
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SITE:
COLLECTION DATE:
TEST DATE:
LOCATION
INITIAL
SALINITY
FINAL
SALINITY
SOURCE OF SALTS FOR1
SALINITY ADJUSTMENT
'Natural 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. FromUSEPA (19871).
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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 labeled 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
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 for the first time
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).
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.
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.
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.
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 uE/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
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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. The aeration rate should not exceed 100
bubbles/minute, using a pipet with a 1-2 mm orifice, such as a ImL KIMAX® serological pipet, 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.
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.
16.10.7 TRANSFER OF PLANTS TO CONTROL WATER AFTER 48 H
16.10.7.1 Label the recovery vessels. These vessels can be 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.
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1 mm
Figure 7. A mature cystocarp. In the controls and lower effluent concentrations, cystocarps often occur in
clusters of 10 or 12. FromUSEPA (1987f).
1 mm
young branch
S
immature
cystocarp
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).
1 mm
Figure 9.
An aborted cystocarp. A new branch will eventually develop at the apex. From USEPA (1987f).
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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 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.
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. A sample set of reproduction data is listed in Table 4.
16.13.1.2 The endpoints of the red macroalga, Champiaparvula, 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.
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COLLECTION DATE
EXPOSURE BEGAN (date).
. RECOVERY BEGAN (date).
. COUNTED (date)
EFFLUENT OR TOXICANT
TREATMENT (% EFFLUENT, mG/L, or RECEIVING WATER SITES)
1 REPLICATES
CONTROL
1
A 1
2
o
6
4
MEAN
B 1
2
3
4
MEAN
C 1
2
3
4
MEAN
1 OVERALL
MEAN
1
Temperature
Salinity
Light
Source of Dilution Water
Figure 10. Data form for the red macroalga, Champia parvula, sexual reproduction test. Cystocarp data sheet.
From USEPA (19871).
347
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST WITH
EFFLUENTS AND RECEIVING WATERS
CAUTION: This method is not listed at 40 CFR Part 136 for nationwide use.
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. No. organisms
per test chamber:
10. No. replicate
per concentration:
11. No. organisms per
concentrations:
12. Dilution water:
13. Test concentrations:
14. Dilution factor:
Static, non-renewal
30%o (± 2%o of the selected test salinity)
23 ± 1°C
Cool-white fluorescent lights
75 uE/m2/s (500 ft-c)
16 h light, 8 h darkness
200 mL polystyrene cups, or 250 mL Erlenmeyer flasks
100 mL (minimum)
5 female branch tips and 1 male plant
4 (minimum of 3)
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
348
-------
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: 2 day exposure to effluent, followed by 5 to 7-day recovery period in
control medium for cystocarp development
16. Endpoints: Reduction in cystocarp production compared to controls
17. Test acceptability criteria 80% or greater survival, and an average of 10 cystocarps per plant in
controls
18. Sampling requirements: For on-site tests, one sample collected at test initiation, and used within
24 h of the time it is removed from the sampling device. For off-site
tests, holding time must not exceed 36 h before first use (see Section 8,
Effluent and Receiving Water Sampling, Sampling Handling, and
Sample Preparation for Toxicity Tests, Subsection 8.5.4)
19. Sample volume required: 2 L per test
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 Forthe 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.
349
-------
TABLE 4. DATA FROM THE RED MACRO ALGA, CHAMPIA 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
o
J
0
1
3
0
0
0
4
7
11
20
12
9
13
5
8
6
4
4
0
5
4
1
0
0
1
0
0
0
5
18
23
16
11
10
8
4
4
4
0
6
0
0
2
o
J
0
0
3
_
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.
-------
STATISTICAL ANALYSIS OF CHAMPIA PARVULA
SEXUAL REPRODUCTION TEST
REPRODUCTION DATA
MEAN CYSTOCARP COUNT
POINT ESTIMATION
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILK'S TEST
TRIBUTION ^
r
NON-N
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
NO
r
EQUAL NUMBER OF
REPLICATES?
1
YES
r
EQUAL NUMBER OF
REPLICATES?
YES
NO
T-TEST WITH
BONFERRONI
ADJUSTMENT
DUNNETT'S
TEST
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
351
-------
6.13.2.3 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.
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 observations 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.
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
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
MeanCY,)
s?
i
18.00
1.12
1
10.53
1.77
2
4.53
0.37
3
3.60
8.92
4
2.60
1.12
5
1.07
1.05
6
0.27
0.09
7
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.
352
-------
20
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION
REPRESENTS THE CRITICAL VALUE FOR DUNNETTS TEST
(ANY MEAN REPRODUCTION BELOW THIS VALUE WOULD BE
SIGNIFICANTLY DIFFERENT FROM THE CONTROL)
N
0.
0
F
C
Y
S
T
0
C
A
R
P
S
0.0
0.8
1.3 22
EFFLUENT CONCENTRATION (%)
~r
10
Figure 12. Plot of the number of cystocarps per plant.
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Effluent Concentration (%)
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
2.2
-1.20
3.40
-2.20
3.6
-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)
Where: Xj = the ith centered observation
x = the overall mean of the centered observations
n = the total number of centered observations.
353
-------
16.13.2.5.3 For this set of data, n= 18
X = 1(0.01) = 0.00
8
D = 28.7201
16. 13 .2.5.4 Order the centered observations from smallest to largest
XCD < X<2) < ___ < X(n)
Where X(l) is the ith ordered observation. These ordered observations are listed in Table 7.
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
X®
X®
1
2
o
6
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 ab 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.
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
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(18) x(l)
X(17).X(2)
X(17) _ X(3)
X(15).X(4)
X(14).X(5)
x(13) _ x(6)
X(12).X(7)
X(11)_X(8)
X(10) x(9)
354
-------
16.13.2.5.6 Compute the test statistic, W, as follows:
i k
W = —[Y.a^-'^-X®]2
D 2=1
The differences X(IH+1) - X(l) are listed in Table 8. For the data,
W = 1 (5.1425)2 = 0.921
28.7201
16.13.2.5.7 The decision rule for this test is to compare W as calculated in Subsection 16.3.2.5.6 with the critical value
found in Table 6, Appendix B. If the computed W is less than the critical value, conclude that the data are not normally
distributed. For this example, the critical value at a significance level of 0.01 and 18 observations (n) is 0.858. Since W
= 0.921 is greater than the critical value, conclude of the test is that the data are normally distributed.
16.13.2.6 Test for Homogeneity of Variance
16.13.2.6.1 The test used to examine whether the variation in mean cystocarp production is the same across all effluent
concentrations including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as follows:
V)
B = —
c
Where: V; = degrees of freedom for each effluent concentration and control, V; = fa-1)
p = number of levels of effluent concentration including the control
HJ = the number of replicates for concentration i
In = loge
i = 1, 2,..., p where p is the number of concentrations
p
(E ytsf)
16.13.2.6.2 For the data in this example (See Table 5) all effluent concentrations including the control have the same
number of replicates (A = 3 for all i). Thus, V; = 2 for all i.
c = i
355
-------
16.13.2.6.3 Bartlett's statistic is therefore:
B = [(12)ln(2.3917)-2
= [12(0.8720) -2(ln(1.12)+ln(1.77)+...+ln(1.05))]/l.1944
= (10.4640-4.0809)71.1944
= 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
Between
Within
Total
df
p-1
N-p
N-l
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square(MS)
(SS/df)
SB = SSB/(p - 1)
S£ = SSW/(N - p)
Where: p = number effluent concentrations including the control
N = total number of observations n{ + n2... + rip
HJ = number of observations in concentration i
SSB =
SST =
Between Sum of Squares
Total Sum of Squares
SSW = SST -SSB
Within Sum of Squares
356
-------
G = the grand total of all sample observations,
Tj = the total of the replicate measurements for concentration i
Yj j = the jth observation for concentration i (represents the mean (across plants) number of cy stocarps for
effluent concentration i in test chamber j)
16.13.2.7.2 For the data in this example:
H! = n2 = n3 = n4 = n5 = ng =3
N =18
T! = Yu + Y12 + Y13 = 17.6 + 17.2 + 19.2 = 54
T2 = Y21 + Y22 + Y23 = 12.0 + 9.4 + 10.2 = 31.6
T3 = Y31+Y32 + Y33= 4.4+ 5.2+ 4.0 = 13.6
T4 = Y41+Y42 + Y43= 2.4+ 7.0+ 1.4 = 10.8
T5 = Y51+Y52 + Y53= 1.8+ 3.8+ 2.2= 7.8
T6 = ¥«+¥« + ¥« = 0.2+ 0.8+ 2.2= 3.2
G = T, + T, + T, + T4 + T, + Tfi = 121.0
SSB = X Tj/nt-G2/N
! = 1
= J_ (4287.24) - (121.0)2 = 615.69
3 18
P "L^ .
•SST = E E ??--G2IN
Z-^ Z-^ ij
i=l J-=l
= 1457.8-(121.0)2 =644.41
18
SSW = SST-SSB
= 644.41-615.69 = 28.72
S% = SSB/(p-l) = 615.697(6-1) =123.14
SK = SSW/(N-p) = 28.727(18-6) = 2.39
16.13.2.7.3 Summarize these calculations in the ANOVA table (Table 10).
357
-------
TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between 5 615.69 123.14
Within 12 28.72 2.39
Total 17 644.41
16. 13.2.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control
combination as follows:
,
Where: 7. = mean number of cystocarps for effluent concentration i
7j = mean number of cystocarps for the control
Sw = square root of the within mean square
H! = number of replicates for the control
n, = number of replicates for concentration i
16. 13.2.7.5 Table 1 1 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) =
[1.55^(1/3) +(1/3)]
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 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%.
358
-------
TABLE 11. CALCULATED T VALUES
Effluent Concentration(%)
0.8
1.3
2.2
3.6
6.0
i
2
o
5
4
5
6
t,
5.90
10.64
11.38
12.17
13.38
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 = dS^l/nJ + ^/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)
H! = the number of replicates in the control.
16.13.2.7.8 In this example,
MSD = 2.50(1.55) ^(1/3)+(1/3)
= 2.50(1.55)(.8165)
= 3.16
16.13.2.7.9 Therefore, forthis 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.
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 12 for a plot of the response curve.
359
-------
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
o
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
MI (mg)
18.00
10.53
4.53
3.60
2.60
1.07
0.27
16.13.2.8.2 AnIC25 andIC50 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 Q = 0.0% effluent and C2 = 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 = C, + [M (1-/7/100) -Ml fi+ir^}
IC25 = 0.0 + [18.00(l-25/100)-18.00] (°'8 °-0)
(10.53-18.00)
= 0.5%.
16.13.2.8.4 Using the equation from Section 4.2 from Appendix L, the estimate of the IC50 is calculated as
follows:
(C -C)
ICp = C. + [M, (1 -/V100) -Ml —^ J—
3 •' -
/C50=0.8 + [18.00(l-50/100)-10.53] (L3 °'
(4.53-10.53)
= 0.9%
360
-------
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
16.14.1.1 Single-Laboratory Precision
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%.
16.14.1.1.2 EPA evaluated single-laboratory (within-laboratory) precision of the Red Macroalga, Champia parvula,
Reproduction Test using a database of routine reference toxicant test results from two laboratories (USEPA, 2000b).
The database consisted of 23 reference toxicant tests conducted in 2 laboratories using reference toxicants
including: copper and sodium dodecyl sulfate. The within-laboratory CVs calculated for routine reference toxicant
tests at these 2 laboratories were 58% and 59% for the IC25 reproduction endpoint.
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.
361
-------
Conc._ID
Cone. Tested
3
"IT
5
~3~6~
o
Response 1 19 10
Response 2 20 16
Response 3 24 11
Response 4 7 12
Response 5 18 11
Response 6 19 12
Response 7 12 10
Response 8 21 6
Response 9 11 9
Response 10 23 10
Response 11 17 12
Response 12 25 9
Response 13 18 9
Response 14 20 13
Response 15 16 8
10 1 2
0 2 1
3 5 1
545
400
673
494
496
844
462
430
424
223
6 0 1
403
1
0
0
0
0
1
2
1
0
0
0
4
o
3
i
o
J
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 MACRO ALGA, Champia parvula
Test Duration:
DATA FILE: champia.icp
OUTPUT FILE: champia.i25
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
Standard.
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
The Linear Interpolation Estimate: 0.4821 Entered P Value: 25
Number of Resamplings: 80
Original Confidence Limits:
Resampling time in Seconds:
3.68
The Bootstrap Estimates Mean: 0.4947 Standard Deviation:
Lower: 0.4013 Upper: 0.6075
Random Seed: 703617166
0.0616
Figure 13. ICPIN program output for the IC25.
362
-------
Cqnc._ID 1
Cone. Tested 0
1.3
4 5___
2.2 3.6
6_
6
10
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
19
20
24
7
18
19
12
21
11
23
17
25
18
20
16
10
16
11
12
11
12
10
6
9
10
12
9
9
13
8
10
0
3
5
4
6
4
4
8
4
4
4
2
6
4
1
2
5
4
0
7
9
9
4
6
3
2
2
0
0
2
1
1
5
0
3
4
6
4
2
0
4
3
1
3
1
0
0
0
0
1
2
1
0
0
0
4
o
J
1
3
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 MACRO ALGA, 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
Standard.
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
The Linear Interpolation Estimate: 0.9278 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 0.9263
Original Confidence Limits:
Resampling time in Seconds: 3.63
Standard Deviation: 0.0745
Lower: 0.7893 Upper: 1.0576
Random Seed: -1255453122
Figure 14. ICPIN program output for the IC50.
363
-------
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(%):
NOEC
(|Ag/L)
1.0
1.0
1.0
1.0
0.5
0.5
6
NA
NA
IC25
(Hg/L)
1.67
1.50
0.69
0.98
0.38
0.38
6
0.93
59.6
IC50
(Hg/L)
2.37
1.99
1.53
1.78
0.76
0.75
6
1.5
43.7
1 Data from USEPA (1991a).
2 Tests performed by Glen Thursby and Mark Tagliabue, ERL-N, USEPA, Narragansett, RI. Tests were
conducted at 22°C, in 50/50 GP2 and natural seawater at a salinity of 30%o.
3 Copper concentrations were: 0.5, 1.0, 2.5, 5.0, 7.5, and 1.0 ug/L.
4 NOEC Range: 0.5 -1.0 ug/L (this represents a difference of one exposure concentration).
5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
364
-------
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)1'2'3'4'5
Test
Number
1
2
3
4
5
6
7
8
9
n:
Mean:
CV(%):
NOEC
(mg/L)
<0.80
0.48
<0.48
<0.48
0.26
0.09
0.16
0.09
<0.29
5
NA
NA
IC25
(mg/L)
0.6
0.7
0.4
0.2
0.2
0.1
0.2
0.1
0.3
9
0.31
69.0
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
Data from USEPA (1991a).
Tests performed by Glen Thursby and Mark Tagliabue, 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.
365
-------
TABLE 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) SULFATE1'2'3
Cu (ue/L)
Test NOEC
1 1.00
2 0.50
3 0.50
4 0.50
n: 4
Mean: NA
CV(%): NA
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
1 Data from USEPA (1991a).
2 Copper concentrations were 0.5, 1.0, 2.5, 5.0, 7.5, and 10 ug/L. Concentrations of Cu were made from a 100
ug/mL CuSO4 standard obtained from Inorganic Ventures, Inc., Brick, NJ.
3 Prepared by Steven Ward and Glen Thursby, Environmental Research Laboratory, USEPA, Narragansett, RI.
TABLE 16. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA PARVULA,
REPRODUCTION TEST IN NATURAL SEAWATER (30%« SALINITY). THE REFERENCE
TOXICANT USED WAS SODIUM DODECYL SULFATE (SDS)1'2'3
Test
1
2
3
4
n:
Mean:
CV(%):
NOEC
0.60
0.60
0.30
0.15
4
NA
NA
SDS (ms/L)
IC25
0.05
0.48
0.69
0.60
4
0.46
62.29
IC50
0.50
0.81
0.89
0.81
4
0.75
22.92
1 Data from USEPA (1991a).
2 SDS concentrations were 0.0375, 0.075, 0.15, 0.03, 0.60, and 1.20 mg/L. Concentrations of SDS were made
from a 44.64 ± 3.33 mg/mL standard obtained from the EMSL-USEPA, Cincinnati, OH.
3 Prepared by Steven Ward and Glen Thursby, Environmental Research Laboratory, USEPA, Narragansett, RI.
366
-------
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382
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APPENDICES
Page
A. Independence, Randomization, and Outliers 384
1. Statistical Independence 384
2. Randomization 384
3. Outliers 389
B. Validating Normality and Homogeneity of Variance Assumptions 390
1. Introduction 390
2. Tests for Normal Distribution of Data 390
3. Test for Homogeneity of Variance 397
4. Transformations of the Data 399
C. Dunnett's Procedure 401
1. Manual Calculations 401
2. Computer Calculations 408
D. T test with Bonferroni's Adjustment 414
E. Steel's Many-one Rank Test 420
F. Wilcoxon Rank Sum Test 425
G. Single Concentration Toxicity Test - Comparison of Control with
100% Effluent or Receiving Water 432
H. Probit Analysis 436
I. Spearman-Karber Method 439
J. Trimmed Spearman-Karber Method 444
K. Graphical Method 448
L. Linear Interpolation Method 452
1. General Procedure 452
2. Data Summary and Plots 452
3. Monotonicity 452
4. Linear Interpolation Method 452
5. Confidence Intervals 453
6. Manual Calculations 454
7. Computer Calculations 458
Cited References 463
383
-------
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.
2.3 RANDOMIZATION OF FISH TO REPLICATE CHAMBERS EXAMPLE
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. 1.
Note that the double digits 00 and 97 through 99 were not used.
384
-------
TABLE A. 1. RANDOM ASSIGNMENT OF FISH TO REPLICATE CHAMBERS
EXAMPLE ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER
Assigned Numbers
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,
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
Replicate Chamber
Control,
Control,
Control,
Control,
6.25% effluent,
6.25% effluent,
6.25% effluent,
6.25% effluent,
12.5% effluent,
12.5% effluent,
12.5% effluent,
12.5% effluent,
25.0% effluent,
25.0% effluent,
25.0% effluent,
25.0% effluent,
50.0% effluent,
50.0% effluent,
50.0% effluent,
50.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
replicate chamber 1
replicate chamber 2
replicate chamber 3
replicate chamber 4
replicate chamber 1
replicate chamber 2
replicate chamber 3
replicate chamber 4
replicate chamber 1
replicate chamber 2
replicate chamber 3
replicate chamber 4
replicate chamber 1
replicate chamber 2
replicate chamber 3
replicate chamber 4
replicate chamber 1
replicate chamber 2
replicate chamber 3
replicate chamber 4
replicate chamber 1
replicate chamber 2
replicate chamber 3
replicate chamber 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. 1, 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.
385
-------
TABLE A.2. TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983)
1009732533
37 54 20 48 05
08 42 26 89 53
9901902529
12 80 79 99 70
6606574717
3106010805
85 26 97 76 02
6357332135
73 79 64 57 53
9852017767
1180505431
83 45 29 96 34
88 68 54 02 00
9959467348
6548117674
8012435635
74 35 09 98 17
69 91 62 68 03
09 89 32 05 05
9149914523
8033694598
4410481949
1255073742
63 60 64 93 29
61 19690446
1547445266
94 55 72 85 73
4248116213
2352378317
04 49 35 24 94
00 54 99 76 54
3596315307
59 80 80 83 91
46 05 88 52 36
32 17 90 05 97
6923461406
1956541430
4515514938
9486431994
98 08 62 48 26
3318516232
80 95 10 04 06
79 75 24 91 40
1863332537
74 02 94 39 02
5417845611
1166449883
48 32 47 79 28
6907494138
76 52 01 35 86
64 89 47 42 96
1964509303
0937670715
8015736147
3407276850
4557182406
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
8751764969
1746850950
1772708015
77 40 27 72 14
6625229148
14225685 14
68 47 92 76 86
26 94 03 68 58
8515747954
11 10002040
16 50 53 44 84
26 45 74 77 74
9527079953
67 89 75 43 87
9734408721
73 20 88 98 37
75 24 63 38 24
6405 1881 59
26 89 80 93 45
45 42 72 68 42
0139092286
8737925241
2011745204
01 75 87 53 79
1947607246
3616810851
45 24 02 84 04
4194150949
96 38 27 07 74
71 96 12 82 96
9814506571
77 55 73 22 70
80 99 33 71 43
52 07 98 48 27
3124964710
8763791976
34 67 35 43 76
24 80 52 40 37
23 20 90 25 60
3831 13 1165
64 03 23 66 53
36 69 73 61 70
35 30 34 26 14
6866574818
90 55 35 75 48
35 80 83 42 82
22 10 94 05 58
50 72 56 82 48
1374670078
36 76 66 79 51
91 82 60 89 28
58 04 77 69 74
45 31 82 23 74
43 23 60 02 10
36 93 68 72 03
46 42 75 67 88
46 16 28 35 54
70 29 73 41 35
32 97 92 65 75
12 86 07 46 97
40 21 95 25 63
51 92433729
59 36 78 38 48
54 62 24 44 31
16 86 84 87 67
68 93 59 14 16
45 86 25 10 25
9611 963896
33 35 13 54 62
83 60 94 97 00
77 28 14 40 77
05 56 70 70 07
1595660000
40 41 92 15 85
43 66 79 45 43
34 88 88 15 53
44 99 90 88 96
89 43 54 85 81
20 15 12 33 87
69 86 10 25 91
3101024674
97 79 01 71 19
05 33 51 29 69
5938171539
02 29 53 68 70
35 58 40 44 01
80959091 17
20 63 61 04 02
1595334764
8867674397
9895116877
6581339885
86 79 90 74 39
73 05 38 52 47
28 46 82 87 09
60 93 52 03 44
60 97 09 34 33
2940524201
1847540610
90 36 47 64 93
9378561368
73 03 95 71 86
21 11578253
4552164237
7662113990
96 29 77 88 22
94 75 08 99 23
5314033340
5760040881
96 64 48 94 39
4365177082
6539459593
82396101 18
91 19042592
0307112059
26 25 22 96 63
6196279335
5469282391
77 97 45 00 24
1302124892
9391083647
8674317157
1874392423
6667436806
59 04 79 00 33
01 54 03 54 56
39 09 47 34 07
8869541994
25 01 62 52 98
74 85 22 05 39
0545561427
5252758021
5612719255
09 97 33 34 40
32 30 75 75 46
1051821615
3929274945
0082291665
35 08 03 36 06
04 43 62 76 59
1227176833
11 19929170
23 40 30 97 32
1862388579
8349125624
35 27 38 84 35
50 50 07 39 98
5277567851
6871 177817
296091 1062
23478341 13
4021816544
1438553763
96 28 60 26 55
9440056418
5438214598
37 08 92 00 48
4205082341
2222206413
2870725815
0720731790
42 58 26 05 27
3321 159466
92 92 74 59 73
25 70 14 66 70
05 52 28 25 62
65 33 71 24 72
23 28 72 95 29
9010339333
78 56 52 01 06
7061742941
853941 1838
9711896338
84 96 28 52 07
20 82 66 95 41
0501451176
3544131880
37 54 87 30 43
9462461171
00 38 75 95 79
7793891936
8081451748
36 04 09 03 24
8846123356
1502009994
0184876938
386
-------
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. 1, 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.
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 taken from tank
fish taken from tank
fish taken from tank
fish taken from tank
fish taken from tank
fish taken from tank
fish taken from tank
fish taken from tank
fish taken from tank
fish taken from 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,
replicate chamber 1
replicate chamber 2
replicate chamber 4
replicate chamber 4
replicate chamber 1
replicate chamber 4
replicate chamber 1
replicate chamber 3
replicate chamber 2
replicate chamber 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. 1. 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.
387
-------
TABLE A.4. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE
LABELING THE POSITIONS WITHIN THE WATER BATH
1
7
13
19
2
8
14
20
3
9
15
21
4
10
16
22
5
11
17
23
6
12
18
24
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.
TABLE A.5. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE
ASSIGNED NUMBERS FOR EACH POSITION
Assigned Numbers Position
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,
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
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
388
-------
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 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.
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. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
EXAMPLE 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).
389
-------
APPENDIX B
VALIDATING NORMALITY AND HOMOGENEITY OF VARIANCE ASSUMPTIONS
1 INTRODUCTION
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
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 Kolmogorov "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.
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 1.017 1.157 0.998 0.837 0.715
2 0.745 0.914 0.793 0.935 0.907
3 0.862 0.992 1.021 0.839 1.044
MeanCY,)
s?
i
0.875
0.019
1
1.021
0.015
2
0.937
0.016
3
0.882
0.0031
4
0.889
0.027
5
390
-------
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.
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 Calculate the denominator, D, of the test statistic:
D =
Where: Xj = 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, andD = 0.1589.
2.4.1 For this set of data,
n= 15
X = 1/50 (0) = 0.0
D = 0.1589
2.5 Order the centered observations from smallest to largest,
X(D < X(2) < _ _ < X(n)
where X(l) denote the ith order statistic. The ordered observations are listed in Table B.3.
391
-------
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 ab a2,..., ak, where k is n/2 if nis
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.
The differences, X(IH+1) - X(l), are listed in Table B.5.
2.7 Compute the test statistic, W, as follows:
1 k
D i=\ !
392
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (Conover, 1980)
\ Number of Observations
iV
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
-
-
6
0.6431
0.2806
0.0875
-
-
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
\ Number of Observations
iV
1
2
3
4
5
6
7
8
9
10
11
0.5601
0.3315
0.2260
0.1429
0.0695
0.0000
-
-
-
-
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
-
-
-
15
0.5150
0.3306
0.2495
0.1878
0.1353
0.0880
0.0433
0.0000
-
-
16
0.5056
0.3209
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
\ Number of Observations
iV
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
21
0.4643
0.3185
0.2578
0.2119
0.1736
0.1399
0.1092
0.0804
0.0530
0.0263
0.0000
-
-
-
-
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
-
-
-
25
0.4450
0.3069
0.2543
0.2148
0.1822
0.1539
0.1283
0.1046
0.0923
0.0610
0.0403
0.0200
0.0000
-
-
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
3 O'}
393
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED)
\ Number of Observations
i V
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
-
-
-
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
-
-
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
0.0733
0.0622
0.0515
0.0409
0.0305
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
\ Number of Observations
i V
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
-
-
-
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
-
-
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
394
-------
TABLE B.5. EXAMPLE OF THE SHAPIRO-WILK'S TEST: TABLE OF COEFFICIENTS AND
DIFFERENCES
i a^ Xni -X1
1 0.4734
2 0.3211
3 0.2565
4 0.2085
5 0.1686
6 0.1334
7 0.1013
8 0.0711
9 0.0422
10 0.0140
0.181
0.128
0.105
0.097
0.076
0.048
0.034
0.025
0.008
0.005
X(20) .
X(19) .
X(18) .
x(17) -
X(16) _
x(15) -
X(14) .
x(13) -
x(12) -
X(11) -
X(l)
x®
X(3)
X(4)
X(5)
X®
X(7)
x®
x(9)
XC1Q>
395
-------
TABLE B.6. QUANTILES OF THE SHAPIRO WILK'S TEST STATISTIC (Conover, 1980)
n
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
0.01
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
0.02
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
0.05
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
0.10
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
0.50
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
0.90
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
0.95
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
0.98
1.000
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
0.99
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
396
-------
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.
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 Wilk'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 follows:
p P
KEF;.) in
B = -^
C
Where: V; = degrees of freedom for each effluent concentration and control, (V; = n -1)
p = number of levels of toxicant concentration including the control
In = loge
i = 1, 2,..., p where p is the number of concentrations including the control
HJ = the number of replicates for concentration i.
p P
z=l ' z=l
397
-------
TABLE B.7. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH DATA
(WEIGHT IN MG) USED FOR BARTLETT'S TEST FOR HOMOGENEITY OF
VARIANCE
Effluent Concentration (%)
Replicate Control 6.25 12.5 25.0 50.0
1
2
3
Mean
s?
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
0.873
0.935
0.839
0.882
0.0024
4
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, S"2 = 0.0158, and C = 1.2. The calculated B value is:
2[5(ln 0.0158)-£ln(,S;2)]
B = -
1.2
= 2[5(- 4.1477) - (-22.1247)]
1.2
= 2.3103
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.
398
-------
4 TRANSFORMATIONS OF THE DATA
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-Wilk'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.
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 P; (1 - Pj), where P; 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 Pj for different treatments, i. Also, when the observed proportions are based on small samples, or when Pj is
close to zero or one, the normality assumption may be invalid. The arc sine square root (arc sine /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.
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
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) = arc sine
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.
399
-------
Angle (in radians) = arc sine \J\I4N
Where: N = Number of animals/treatment replicate
Example: If 20 animals are used:
Angle = arc sine yTTSO
= arc sine 0.1118
= 0.1120 radians
4.2.4.3 Modification of the arc sine square root when RP = 1
Angle = 1.5708 radians - (radians forRP = 0)
Example: Using above value:
Angle =1.5708-0.1120
= 1.4588 radians
400
-------
APPENDIX C
DUNNETT'S PROCEDURE
1 MANUAL CALCULATIONS
1.1 Dunnett'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).
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. 1.
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
Replicate Test Vessel
i
1
2
o
5
4
5
1
1.017
1.157
0.998
0.873
0.715
2
0.745
0.914
0.793
0.935
0.907
o
J
0.862
0.992
1.021
0.839
1.044
Total
(Ti)
2.624
3.063
2.812
2.647
2.666
Mean
(YD
0.875
1.021
0.937
0.882
0.889
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:
AT" V^
N = the total sample size; ~ n>
HJ = the number of replicates for concentration "i"
SST=E Y2-G 2/N Total Sum of Squares
v
401
-------
SSB-ET?/n-G2/N Between Sum of Squares
SSW=SST-SSB Within Sum of Squares
p
G = the grand total of all sample observations; G = E Ti
Tj = the total of the replicate measurements for concentration i
N=£«
N = the total sample size; . >
HJ = the number of replicates for concentration i
YJJ = the jth observation for concentration i
1.4 For the data in this example:
N = 20
T=Y +Y +Y = 9 694
11 * 11 * 12 * 13 ^.ozt
T2 = Y21+Y22 + Y23 = 3.063
T3 = Y31+Y32 + Y33 = 2.812
T=Y +Y +Y =9 647
14 * 41 * 42 J- 43 ^.Ot /
T=Y +Y +Y =9 666
1 5 * 51 ^ * 52 ^ J- 53 ^.DOO
G = T1+T2 + T3 + T4 + T5= 13.812
SST=EYi:i-G2/N
= 12.922-(13.812)2/15
= 0.204
= 12.763-(13.812)2/15
= 0.045
SSW=SST-SSB
= 0.204 - 0.045
402
-------
= 0.159
1.5 Summarize these data in the ANOVA table (Table C.2).
TABLE C.2. ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source df
Between p - 1
Within N - p
Sum of
Squares (SS)
SSB
SSW
Mean Square (MS)
(SS/df)
S| = SSB/(p-l)
S^ = SSW/(N-p)
Total N -1 SST
1.6 Summarize data for ANOVA (Table C.3).
TABLE C.3. COMPLETED ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source
Between
Within
Total
df
5 - 1 = 4
15 - 5 = 10
14
SS
0.045
0.159
0.204
Mean Square
0.011
0.016
1.7 To perform the individual comparisons, calculate the t statistic for each concentration and control combination,
as follows:
Where: Y; = mean for each concentration i.
403
-------
Y! = mean for the control
Sw = square root of the within mean square
H! = number of replicates in the control.
HJ = number of replicates for concentration i.
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
6.25 2 - 1.414
12.5 3 -0.600
25.0 4 -0.068
50.0 5 -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=dSw.
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
H! = number of replicates in the control
404
-------
For example:
MSZ)=2.47(0.126)h/(l/3)+( 1/3)] =2.47(0.126X^273)
= 2.47(0.126)(0.816)
= 0.254
405
-------
oo
Os
W
PH
W
Q
in
w
1-1
406
-------
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 mean. Call this difference D. Next, obtain untransformed
values for the control mean and the difference, D.
MSDU= controlu-Du
Where: MSDU = the minimum significant difference for untransformed data
Controlu = the untransformed control mean
Du = the untransformed difference
1.II.2.2 Calculate the percent reduction from the control that MSDU represents as:
MSDu
Percent Reduction = x 100
Control,,
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. 1
were transformed by the arc sine square root transformation. Thus:
0.875-0.254 = 0.621
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
Step 3. The untransformed MSD (MSDU) is determined by subtracting the untransformed values obtained in Step
2.
MSDU = 0.589-0.339 = 0.250
In this case, the MSD would represent a 42% decrease in survival from the control [(0.250/0.589)(100)].
407
-------
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 45268. 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.
2.6.2.2 After the type of analysis for the data is chosen, the user has the following options:
1. Create a data file
2. Edit a data file
3. Perform analysis on existing data set
4. Stop
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.)
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).
408
-------
2.6.2.5 Sample data input is shown in Figure C. 1.
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) forthe 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.
409
-------
TABLE C.6. SAMPLE DATA FOR DUNNETT'S PROGRAM FOR SURVIVING MYSIDS,
MYSIDOPSIS BAHIA
Treatment
1 Control
2 50ppb
3 lOOppg
4 210 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
410
-------
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
Number of concentrations, including control ? 5
Number of replicates for cone. 1 (the control) ? 8
replicate number of organisms exposed number of organisms responding
(organisms surviving, eggs fertilized, etc.)
1 5 4
25 4
35 5
45 5
55 5
65 5
75 5
85 4
Number of replicates for cone. 2 ? 8
Do you wish to save the data on disk ? y
Disk file for output ? mysidsur.dat
Figure C.I. Sample Data Input for Dunnett's Program for Survival Data from Table C.6.
411
-------
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, G=greater than ? 1
Summary Statistics for Raw Data
Cone. 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
Mysid Survival Example with Data in Table C.6
Figure C.2. Example of Choosing Option 3 from the Main Menu of the Dunnett Program.
412
-------
Mysid Survival Example with Data in Table C.6
Summary Statistics and ANOVA
Transformation = Arcsine Square Root
Cone. n 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
.1273
.2042
.2593
.1752
9.8
10.4
17.4
25.2
51.2
*) the mean for this cone, is significantly less than
the control mean at alpha = 0.05 (1-sided) by Dunnett's test
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
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 ?
Figure C.3. Example of Program Output for the Dunnett's Program Using the Survival Data in Table C.6.
413
-------
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. 1.
TABLE D.I. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH DATA
(WEIGHT IN MG) USED FOR THE T TEST WITH BONFERRONI'S ADJUSTMENT
Effluent
Cone (%)
Control
6.25
12.5
25.0
50.0
Replicate Test Vessel
i
1
2
3
4
5
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
00
0.875
1.021
0.937
0.882
0.811
3.1 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
N=Yn
N = the total sample size; . '
HJ = the number of replicates for concentration i
SST=E Y^-G 2/N Total Sum of Squares
y
414
-------
SSB-ET2/n-G2/N Between Sum of Squares
SSW=SST-SSB Within Sum of Squares
p
Where: G = The grand total of all sample observations; G = E Ti
;=1
Tj = The total of the replicate measurements for concentration i
YJJ = The jth observation for concentration i
3.2 For the data in this example:
H! = n2 = n3 = n 4 = 3
N = 20
T! = Yn+Y12 + Y13 = 2.624
T2 = Y21+Y22 + Y23 = 3.063
T3 = Y31+Y32 + Y33 = 2.812
T4 = Y41+Y42 + Y43 = 2.647
T5 = Y51+Y52 + Y53= 1.622
G = T!+T2 + T3 + T4 + T5= 12.768
SSB=ET2/ni-G2/N
i
= 11.709 - (12.768)2/14
= 0.064
SST=EY2-G2/N
= 11.832 - (12.768)2/14
= 0.188
SSW=SST-SSB
= 0.188-0.064
= 0.124
415
-------
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)
S^ = SSB/(p-l)
s^ = SSW/CN-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
Total
df SS
5-1=4 0.064
14-5 = 9 0.124
13 0.188
Mean Square
0.016
0.014
3.5 To perform the individual comparisons, calculate the t statistic for each concentration and control combination, as
follows:
Where:
',• = -
Yj = mean for concentration i
Y! = mean for the control
Sw = square root of the within mean square
H! = number of replicates in the control.
416
-------
rij = 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%.
417
-------
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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. 1. 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 of 0.05, the critical rank sum in a test with four concentrations and eight replicates per
concentration, is 47 (see Table E.5).
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.
420
-------
TABLE E.I. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: DATA FOR MYSID, MYSIDOPSIS
BAHIA, 7-DAY CHRONIC TEST
Effluent
Concentration
Control
(Site Water)
Control
(Brine &
Dilution Water)
3.12%
6.25%
12.5%
25.0%
50.0%
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 Number of
Mysids at Live Mysids
Start of Test at End of Test
5 4
5 4
5 5
5 4
5 5
5 4
5 4
5 5
5 3
5 5
5 3
5 3
5 4
5 4
5 3
5 3
5 4
5 4
5 4
5 5
5 4
5 4
5 5
5 3
5 3
5 4
5 5
5 4
5 4
5 4
5 5
5 5
5 5
5 4
5 5
5 3
5 5
5 4
5 4
5 3
5 5
5 5
5 5
5 5
5 3
5 5
5 4
5 4
5 0
5 0
5 0
5 0
5 0
5 0
5 0
5 0
421
-------
TABLE E.2. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: ASSIGNING
RANKS TO THE CONTROL AND 3.12% EFFLUENT CONCENTRATIONS
Rank Number of Live Control or % Effluent
Mysids, Mysidopsis bahia
1 3 3.12
6.5 4 Control
6.5 4 Control
6.5 4 Control
6.5 4 Control
6.5 4 Control
6.5 4 3.12
6.5 4 3.12
6.5 4 3.12
6.5 4 3.12
6.5 4 3.12
14 5 Control
14 5 Control
14 5 Control
14 5 3.12
14 5 3.12
422
-------
TABLE E.3. TABLE OF RANKS
Replicate
Chamber
1
2
3
4
5
6
7
8
Effluent Concentration (%)
Control1
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)
3.12
4 (6.5)
4 (6.5)
4 (6.5)
5(14)
4 (6.5)
4 (6.5)
5(14)
3(1)
6.25
3(1)
4(6)
5(13.5)
4(6)
4(6)
4(6)
5(13.5)
5(13.5)
12.5
5(13.5)
4 (6.5)
5(13.5)
3 (1.5)
5(13.5)
4 (6.5)
4 (6.5)
3 (1.5)
25.0
5(12.5)
5 (12.5)
5(12.5)
5 (12.5)
3(1)
5 12.5)
4(5)
4(5)
Control ranks are given in the order of the concentration with which they were ranked.
TABLE E.4. RANK SUMS
Effluent Rank Sum
Concentration
3.12 61.5
6.25 65.5
12.50 63.0
25.00 73.5
423
-------
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
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
of treatments (excluding control)
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
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
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
424
-------
APPENDIX F
WILCOXON RANK SUM TEST
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.
2. The use of this test may be illustrated with fecundity data from the mysid test in Table F. 1. 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.
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 Table 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).
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.
425
-------
TABLE F. 1. EXAMPLE OF WILCOXON'S RANK SUM TEST: FECUNDITY DATA FOR MYSID,
MYSIDOPSISBAHIA, 7-DAY CHRONIC TEST
Effluent
Concentration
Control
(Site Water)
Control
(Brine &
Dilution Water)
3.12%
6.25%
12.5%
25.0%
50.0%
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
—
—
—
—
—
—
—
—
426
-------
TABLE F.2. EXAMPLE OF WILCOXON'S RANK SUM TEST: ASSIGNING RANKS TO THE CONTROL
AND 3.12% EFFLUENT CONCENTRATIONS
Rank Proportion of Site Water Control
Females W/Eggs or Effluent %
1 0.00 3.12
3.5 0.50 Control
3.5 0.50 Control
3.5 0.50 3.12
3.5 0.50 3.12
7 0.67 Control
7 0.67 Control
7 0.67 3.12
9 0.75 Control
12.5 1.00 Control
12.5 1.00 Control
12.5 1.00 3.12
12.5 1.00 3.12
12.5 1.00 3.12
12.5 1.00 3.12
427
-------
TABLE F.3. TABLE OF RANKS1
Rep
Proportion
Site Water
Control Rank 3.12
Effluent Concentration (%)
6.25 12.5
25.0
1
2
3
4
5
6
7
8
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)
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)
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)
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)
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 Rank Sum No. of Critical
Concentration Replicates Rank Sum
3.12 65 8 44
6.25 65.5 8 44
12.50 42 7 34
25.00 40 8 44
428
-------
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
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
No. of Replicates Per Effluent Concentration
3
6
6
7
8
8
9
10
10
—
6
7
7
8
8
9
~
—
6
7
7
7
8
4
10
11
12
13
14
15
16
17
10
11
12
13
14
14
15
10
11
11
12
13
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
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
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
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
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
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
429
-------
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
in Control
4 3
4
5
6
7
8
9
10
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
No. of Reulicates Per Effluent Concentration
3
—
~
6
6
7
7
7
~
—
~
6
6
7
7
~
~
—
6
6
6
7
—
~
~
—
6
6
7
4
—
10
11
12
12
13
14
~
10
11
11
12
13
13
~
10
11
11
12
12
13
—
~
10
11
11
12
13
5
15
16
17
18
19
20
21
15
16
17
18
19
20
21
15
16
16
17
18
19
20
—
15
16
17
18
19
20
6
21
22
23
24
26
27
28
30
22
23
24
25
27
28
29
21
22
24
25
26
27
29
21
22
23
25
26
27
28
7
28
30
31
33
34
36
38
40
28
29
31
32
34
35
37
39
28
29
30
32
33
35
37
38
29
30
32
33
35
36
38
8
37
38
40
42
44
46
48
50
36
38
40
42
43
45
47
49
36
38
39
41
43
45
47
49
36
37
39
41
43
44
46
48
9
46
48
50
52
55
57
60
62
46
48
50
52
54
56
59
61
45
47
49
51
54
56
58
60
45
47
49
51
53
55
58
60
10
56
59
61
64
67
69
72
75
56
58
61
63
66
68
71
74
56
58
60
63
65
68
70
73
56
58
60
62
65
67
70
72
430
-------
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
in Control
8 3
4
5
6
7
8
9
10
9 3
4
5
6
7
8
9
10
10 3
4
5
6
7
8
9
10
No. of Replicate Per Effluent
3
~
—
~
—
6
6
6
~
~
—
~
—
6
6
—
~
~
—
~
6
6
4
~
—
10
11
11
12
12
~
~
10
10
11
11
12
—
~
10
10
11
11
12
5
~
15
16
17
18
19
19
~
15
16
17
18
18
19
—
15
16
16
17
18
19
6
21
22
23
24
25
27
28
21
22
23
24
25
26
28
21
22
23
24
25
26
27
7
29
30
31
33
34
36
37
28
30
31
33
34
35
37
28
29
31
32
34
35
37
Concentration
8
36
37
39
40
42
44
46
48
37
39
40
42
44
46
47
37
38
40
42
43
45
47
9
45
47
49
51
53
55
57
59
45
46
48
50
52
55
57
59
45
46
48
50
52
54
56
58
10
55
57
59
62
64
67
69
72
55
57
59
62
64
66
69
71
55
57
59
61
64
66
68
71
431
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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
mimpratnr r*f P1
numerator of F.
S2
F = — where S2>S;
S2
5. Compare F with the 0.005 level of a tabled F value with ^ - 1 and n2 - 1 degrees of freedom, where ^ 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 G. 1.
7. Since the variability of the 100% effluent is greater than the variability of the control, S2 for the 100% effluent
concentration is placed in the numerator of the F statistic and S2 for the control is placed in the denominator.
F °-00131 =152
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 critical F
value is obtained from a table of the F distribution (Snedecor and Cochran, 1980). The critical F value for this test
is 8.89. Since 1.52 is not greater than 8.89, the conclusion is that the variances of the control and 100% effluent are
homogeneous.
432
-------
TABLE G. 1 . MYSID, MYSIDOPSIS BAHIA, GROWTH DATA FROM AN EFFLUENT (SINGLE
CONCENTRATION) TEST
Replicate
Control
100%
Effluent
1
0.183
0.153
2
0.148
0.117
3
0.216
0.085
4
0.199
0.153
5
0.176
0.086
6
0.243
0.193
7
0.213
0.137
8 X
0.180 0.195
0.129 0.132
S2
0.000861
0.00131
9. Equal Variance T Test.
9.1 To perform the t test, calculate the following test statistic:
t =
1 1
Where: Y = mean for the control
0 = mean for the effluent concentration
S =
p
Sl = estimate of the variance for the control
S2 = estimate of the variance for the effluent
concentration
H! = number of replicates for the control
n2 = number of replicates for the effluent
concentration
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 rij + 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. 1 to illustrate the t test, the calculation of t is as follows:
0.1950.132 „
( -
0.0329
1 + 1
433
-------
Where:
_ y/(8 - 1)0.000861 + (8 - 1)0.00131
p 8+8-2
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 conclusion 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:
\ Wj W2
Where: Yl = mean for the control
Y2 = mean for the effluent concentration
Sj = estimate of the variance for the control
S2 = estimate of the variance for the effluent concentration
H! = number of replicates for the control
n2 = number of replicates for the effluent concentration
10.2 Additionally, the degrees of freedom for the test are adjusted using the following formula:
(n, - 1) («9 - 1)
df = —————
J . - . ~o . - „ -> ,
Where:
434
-------
10.3 The modified degrees of freedom is usually not an integer. Common practice is to round down to the nearest
integer.
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.
435
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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 Figure H.2).
436
-------
EPA PROBIT ANALYSIS PROGRAM
USED FOR CALCULATING LC/EC VALUES
Version 1.5
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
Number 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 Number
Number Cone. Resp. Exposed
1 6.2500 14 100
2 12.5000 16 102
3 25.0000 35 100
4 50.0000 72 99
5 100.0000 99 99
Do you wish to modify your data ? N
The number of control animals which responded =17
The number of control animals exposed = 100
Do you wish to modify these values ? N
Figure H.I. Sample Data Input for USEPA Probit Analysis Program, Version 1.5.
437
-------
Example of Probit Analysis
Number
Cone. Exposed
Control
6.2500
12.5000
25.0000
50.0000
100.0000
100
100
102
100
99
99
Observed
Number Proportion
Resp. Responding
17
14
16
35
72
99
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) = 3.472
Chi - Square for Heterogeneity
(tabular value at 0.05 level) = 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.
438
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APPENDIX I
SPEARMAN-KARBER METHOD
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 Probit 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 1.1.
7. Let p0, pb ..., pk denote the observed response proportion mortalities for the control and k effluent
concentrations. The first step is to smooth the ft if they do not satisfy p0
-------
TABLE I.I. EXAMPLE OF SPEARMAN-KARBER METHOD: MORTALITY DATA FROM A
SHEEPSHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST (40 ORGANISMS
PER CONCENTRATION)
Effluent
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 p4 = 0.65 is larger than p3\ set />/ = 0.65. Similarly, p5 = 1.00 is larger thanp5 so set p4 = 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
%
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:
a s S 2
Pi = (P, -Po
Where : ps0 = the smoothed observed proportion mortality for the control
pts = the smoothed observed proportion mortality for effluent concentration i.
440
-------
. 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:
a P\-P0 0.025-0.025 0.0
Pn =Pl =P? = Pl = = =
° l 2 3 i__* 1-0.025 0.975
ta_P4~Po _ 0.650-0.025 0.0625 _Q6/|1
'4 i_n* 1-0.025 0.975
, a _ Ps-Po _ 1.000-0.025 0.975 1 QQQ
'5 i_n* 1-0.025 0.975
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 1.1.
9. Calculate the Iog10 of the estimated LC50, m, as follows:
k ( a \ (Y V \
m =£-1 " i+ M
! = 1 2
Where: pta= the smoothed adjusted proportion mortality at concentration i
Xj = the Iog10 of concentration i
k = the number of effluent concentrations tested, not including the control.
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
441
-------
p
R
O
P
O
R
T
I
O
N
M
O
R
T
A
L
I
T
Y
1.0 -
0.9 -
0.8 -^
0.7 -
0.6 -^
0.5 -^
0.4 -
0.3 -
0.2 -
0.1 -
0.0 -
0
.00
OBSERVED PROPORTION
y MORTALITY
SMOOTHED PROPORTION MORTALITY
SMOOTHED ADJUSTED PROPORTION MORTALITY
6.25 12.50 25.00
EFFLUENT CONCENTRATION (%)
50.00
100.00
Figure 1.1. Plot of observed, smoothed, and adjusted response proportions for sheepshead minnow, Cyprinodon variegatus, survival data.
442
-------
10. Calculate the estimated variance of m as follows:
^_i
Where: Xl = the Iog10 of concentration i
HJ = 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, not including the control.
10.1 For this example, the estimated variance of m, V(m), is calculated as follows:
V(m) = (0.000)(1.000)(1.3979 - 0.7959)2/4(39) +
(0.000)(1.000)(1.6990 - 1.0969)2/4(39) +
(0.641)(0.359)(2.0000 - 1.3979)2/4(39)
= 0.00053477
11. Calculate the 95% confidence interval for m: m ± 2.0i/V(m)
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(1.656527) = 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:
lower limit: antilog( 1.610277) = 40.8%
upper limit: antilog( 1.702777) = 50.4%
443
-------
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 Iog10 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.
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 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 p0, pb ..., pk denote the observed proportion mortalities for the control and the k effluent concentrations.
The first step is to smooth the ft if they do not satisfy p 0
-------
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.
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:
a. Smoothing.
b. Adjustment for mortality in the control.
c. Calculation of the necessary trim.
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.
12.1 The program requests the following input (Figure J. 1):
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.
c. The estimated LC50 and the associated 95% confidence interval.
TABLE J.I. EXAMPLE OF TRIMMED SPEARMAN-KARBER METHOD: MORTALITY DATA FROM A
SHEEPSHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST (40 ORGANISMS PER
CONCENTRATION)
Effluent
Concentration
Control
6.25
12.5
25.0
50.0
100.0
Number of
Mortalities
2
0
2
0
0
32
Mortality
Proportion
0.05
0.00
0.05
0.00
0.00
0.80
445
-------
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:
effluent
ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT :
%
ENTER 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; MAXIMUM = 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(YTN)?
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:
020032
WOULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(YTN)?
y
Figure J. 1. Example input for Trimmed Spearman-Karber Method.
446
-------
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
DATE: 1
effluent
SPECIES: sheepshead minnow
TEST NUMBER: 2
DURATION: 7 Days TOXICANT:
RAW DATA:
Concentration
.00
6.25
12.50
25.00
50.00
100.00
SPEARMAN-KARBER TRIM:
SPEARMAN-KARBER ESTIMATES:
imber
.)
40
40
40
40
40
40
20.41%
LC50:
Mortalities
Exposed
2
0
2
0
0
32
77.28
95% CONFIDENCE LIMITS
ARE NOT RELIABLE.
NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING.
ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION.
Figure J.2. Example output for Trimmed Spearman-Karber Method.
447
-------
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.
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. 1.
TABLE K.I. EXAMPLE OF GRAPHICAL METHOD: MORTALITY DATA FROM AN INLAND
SILVERSIDE LARVAL SURVIVAL AND GROWTH TEST (40 ORGANISMS PER
CONCENTRATION)
Effluent Number of Mortality
Concentration Mortalities Proportion
Control 2 0.05
6.25 0 0.00
12.5 0 0.00
25.0 0 0.00
50.0 40 1.00
100.0 40 1.00
4. Let PO, Pi,..., pk denote the observed proportion mortalities for the control and the k effluent concentrations.
The first step is to smooth the ft if they do not satisfy p0
-------
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:
s s s s 0.05+0.00+0.00+0.00 0.05 „ m~,
Po = P, = P2 = P3 = : = —— = °-0125
4.2 Since p4 = p5 = 1.00 are larger then 0.0125, set /?/ = p5s = 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:
Pg = 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 P\-po 0.0125-0.125 0.0 „„
Pn = Pi = P? = PT, = = = =0'°
° l 2 3 i_ * 1-0.0125 0.9875
a P4-P0 1.00-0.0125 0.9875 , „„
PA = P^ = = = = 1-00
4 5 i _„* 1-0.0125 0.9875
1 fo
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. 1.
449
-------
TABLE K.2. EXAMPLE OF GRAPHICAL METHOD: SMOOTHED, ADJUSTED MORTALITY DATA
FROM AN INLAND SILVERSIDE LARVAL SURVIVAL AND GROWTH TEST
Effluent
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.
450
-------
I
I
0 10 20 30 40 50 60 70 80 90 100
PERCENT MORTALITY
Figure K. 1. Plot of the smoothed adjusted response proportions for inland silverside, Menidia beryllina,
survival data.
451
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APPENDIX L
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 was 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) are 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.
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 (Y;) 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 jneans at each concentration are considered in order of increasing concentration, starting with the
control mean (Yj). If the mean observed response at the lowest toxicant concentration (Y2) is equal to or smaller
than the control mean (Yj), 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
(M2). 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 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. jTSfote: Unusual patterns in the deviations from monotonicity may require an
additional step of smoothing). Where Y; decrease monotonically, the Y; become M; 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.
452
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4.2 To obtain the estimate, determine the concentrations C, and CJ+1 which bracket the response Ml (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:
, (C, , -Cr)
ICp = C, + [M,(l-lp/lOO)-M,] — ^ - —
Where: Cj = tested concentration whose observed mean response is greater than M^l - p/100).
CJ+ j = 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+ j = 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 less than the lowest
test concentration.
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 Y^ 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
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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.
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. 1 includes the raw data and the mean
growth for each concentration. A plot of the data is provided in Figure L.I.
454
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o
CO
I
D.2B-:
D.26r
D.24H
D.22-1
D.2DH
D.1B-!
D.16-:
D.14-!
D.12H
D.1DH
D.DBr
D.D6-!
D.D4-!
D.D2-;
D.DDJ
i
D
INDIVIDUAL REPLIDATEMEAN
DDNNEDTt THE DEEERVED MEAN VALUES
DOMMEDTt THE SMOOTHED MEAN VALUES
50 1DD
TOXICANT CONCENTRATION (PPB)
21D
45D
Figure L.I.
Plot of raw data, observed means, and smoothed means for the mysid, Mysidopsis bahia, growth data.
455
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TABLE L. 1. MYSID, MYSIDOPSISBAHIA, GROWTH DATA
Toxicant Concentration (ppb)
Replicate Control 50 100 210 450
1
2
o
3
4
5
6
7
8
Mean (Y;)
i
0.146
0.118
0.216
0.199
0.176
0.243
0.213
0.144
0.182
1
0.154
0.193
0.190
0.190
0.256
0.191
0.122
0.177
0.184
2
0.114
0.172
0.160
0.199
0.165
0.145
0.207
0.186
0.168
3
0.153
0.094
0.017
0.122
0.052
0.154
0.110
0.103
0.101
4
0
0.012
0
0.002
0
0
0
0.081
0.012
5
6.2 MONOTONICITY
6.2.1 As can be seen from the plot in Figure L. 1, the observed means are not monotonically non-increasing with
respect to concentration. Therefore, the means must be smoothed prior to calculating the 1C.
6.2.2 Starting with the control mean Yl = 0.186 and 72= 0.184, we see that Yl < Y2. Calculate the smoothed means:
Ml = M2 = (Fj+72)/2 = 0.193
6.2.3 Since Y5 = 0.025<74 = 0.101<73 =0.168
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TABLE L.2. MYSID, MYSIDOPSISBAHIA, MEAN GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(ppb)
Control
50
100
210
450
i
1
2
3
4
5
Smoothed
Mean
Mj(mg)
0.183
0.183
0.168
0.101
0.025
6.3.2 Using the equation from section 4.2, the estimate of the IC25 is calculated as follows:
ICp = C. + [M,(l-1/7/100)-M'] —^-—]—
3 (M+1 -M)
IC25 = 100 + [0.93 (1-25/100) -0.164] (21° 100)
(0.101-0.164)
= 151 ppb
6.3.3 Using Equation 1 from 4.2, the estimate of the IC50 is calculated as follows:
ICp = C. + [M,(l-1/VlOO)-M'] —^—J—
J J (M^-M)
(450-210)
IC50 =210 + [210 + [0.193(1-50/100) -0.101]
(0.028-0.101)
= 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.
457
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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 Q
(response means); 2) it calculates the standard deviations; 3) checks the responses for monotonicity; 4) calculates
smoothed means (Mj) (pooled response means) if necessary; 5) uses the means, Mj, 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.
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).
458
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ICp Data Entry/Edit Screen
Cone. ID 1
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
Current File:
4
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.
459
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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 OO,
standard deviations for each response mean, and the pooled response means (smoothed means;
Mi).
3. The linear interpolation estimate of the ICp using the means (Mi). 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. 1 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 133.5054 (ppb). The empirical 95% confidence intervals for the true mean was 96.8623 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 234.6761 (ppb). The empirical 95% confidence intervals for the true mean were 184.8692 to 283.3965
(ppb).
460
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Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 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: mysid.i25
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
ngfl
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Standard.
Dev.
0.043
0.038
0.030
0.047
0.028
Pooled
Response Means
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate: 13 3.5054
Entered? 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.
461
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Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 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.iSO
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
ug/L
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Standard.
Dev.
0.043
0.038
0.030
0.047
0.028
Pooled
Response Means
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 ICPIN program output for the IC50.
462
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