RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 1

Study Title

Two-Generation Life-Cycle Toxicity Test with Mysids (Americamysis bahia) 

Inter-Laboratory Comparison

Wildlife International, Ltd. Authors

Henry O. Krueger, Ph.D.

Mary Beth Claude, B.S.

Timothy Z. Kendall, M.S.

Study Completion Date

December 21, 2012

Submitted To

U.S. Environmental Protection Agency

Office of Pesticide Programs (7507C)

401 M Street S.W.

Washington, D.C. 20460

Performing Laboratory

Wildlife International, Ltd.

8598 Commerce Drive

Easton, Maryland 21601

(410) 822-8600

Laboratory Project ID

Wildlife International, Ltd. Project No. 185A-102

RTI Project No. 0210114.030

EPA Contract No. EP-W-06-026

Page 1 of 419
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 2

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR: U.S. Environmental Protection Agency

TITLE: Two-Generation Life-Cycle Toxicity Test with Mysids (Americamysis bahia) 

Inter-Laboratory Comparison

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 185A-102

RTI PROJECT NUMBER: 0210114.030

EPA CONTRACT NUMBER: EP-W-06-026

STUDY COMPLETION: December 21, 2012

This study was conducted in compliance with Good Laboratory Practice Standards as published 

by the U.S. Environmental Protection Agency (40 CFR Parts 160 and 792); OECD Principles of Good 

Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF (11 NohSan, Notification No. 6283, 

Agricultural Production Bureau, 1 October 1999), with the following exceptions:

Periodic analyses of water for potential contaminants were not performed according to Good 

Laboratory Practice Standards, but were performed using a certified laboratory and standard US EPA 

analytical methods.

The characterization of the test and reference substances, and the stability of the test and 

reference substances under the conditions of storage at the test site, were not determined in accordance 

with Good Laboratory Practice Standards.

STUDY DIRECTOR:

Mary Beth Claude, B.S. Date

Senior Biologist

SPONSOR APPROVAL:

Sponsor Representative Date

Applicant/Submitter Date
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 3

QUALITY ASSURANCE STATEMENT

The study conduct, raw data and report for “Two-Generation Life-Cycle Toxicity Test with 

Mysids (Americamysis bahia) Inter-Laboratory Comparison” were inspected by the Quality 

Assurance Unit at Wildlife International, Ltd to determine adherence with the Quality Assurance 

Project Plan (QAPP) and laboratory standard operating procedures. Dates of study inspections, 

inspection types, and dates reported to the Study Director and to Management are given below.

Inspection Date Inspection Type

Reported to Study 

Director

Reported to 

Management

March 23, 2011 Protocol March 23, 2011 March 25, 2011

Trial 1 (185A-102-1)

March 24, 2011 Test Substance Preparation March 24, 2011 March 25, 2011

May 16, 2011 Assignment of F0 Pairs to May 16, 2011 May 25, 2011

Brood Chambers

June 16, 2011 Measurements of Mysids from June 16, 2011 June 21, 2011

F1 Growth Chambers

August 25-31 and Analytical Data and September 9, 2011 September 28, 2011

September 1, 2011 Draft Report

April 2 to 19, 2012 Biological Data and April 19, 2012 May 21, 2012

Draft Report

Trial 2 (185A-102-2)

January 4, 2012 Test Substance Preparation January 4, 2012 January 27, 2012

Trial 2 (185A-102-2A)

August 23, 2012 Observations August 23, 2012 August 24, 2012

September 21, 2012 F1 Generation Day 14 September 21, 2012 October 1, 2012

Length Measurements

October 26 to Data and Draft Report November 7, 2012 December 8, 2012

November 7, 2012

December 20, 2012 Final Report December 20, 2012 December 21, 2012

WILDLIFE INTERNATIONAL, LTD

________________________________________________ ________________________

Linda R. Mitchell Date

Director of Regulatory and Ecotox Operations
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 4

KEY STUDY PERSONNEL

The following Wildlife International, Ltd personnel were responsible for the conduct of the work 

and reporting of the study results.

Mary Beth Claude

Senior Biologist

Study Director

Date

Amanda L. Milligan

Principal Biologist Date

Timothy Ross

Laboratory Supervisor Date

Sean P. Gallagher

Manager of Aquatic 

Toxicology

Date

Timothy Z. Kendall

Principal Analyst Date

Henry O. Krueger

Director of Aquatic, Plant 

and Insect Toxicology

Date

Willard B. Nixon

Director of Chemistry Date

Jessica T. Anders Biologist

Blake A. Young Biologist

Jeremy M. Claude Biologist

Emily S. Burgess Biologist

Abul Siddiqui Chemist

David Leffingwell Chemist
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 5

TABLE OF CONTENTS

Page

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT................................... 2

QUALITY ASSURANCE STATEMENT ............................................................................... 3

KEY STUDY PERSONNEL.................................................................................................... 4

TABLE OF CONTENTS.......................................................................................................... 5

PROTOCOL ........................................................................................................................... 15

AMENDMENTS AND DEVIATIONS ................................................................................. 60

QAPP ...................................................................................................................................... 85

STUDY TRIAL 1 – 185A-102-1.......................................................................................... 121

SUMMARY.......................................................................................................................... 122

1.0 INTRODUCTION....................................................................................................... 125

2.0 DESCRIPTION OF THE METHOD.......................................................................... 127

2.1 QAPP/Protocol.............................................................................................. 127

2.2 Test Organism............................................................................................... 127

2.3 Feeding.......................................................................................................... 128

2.4 Handling........................................................................................................ 129

2.5 Dilution Water .............................................................................................. 129

2.6 Water Quality Measurements ....................................................................... 130

2.6.1 Temperature ...................................................................................... 130

2.6.2 Salinity.............................................................................................. 130

2.6.3 Lighting............................................................................................. 130

2.6.4 Dissolved Oxygen............................................................................. 131

2.6.5 pH...................................................................................................... 131

2.7 Assay System................................................................................................ 131

2.7.1 Test Vessels ...................................................................................... 131

2.7.2 Retention Chambers (Reproduction/Growth Chambers).................. 132

2.7.3 Brood Chambers ............................................................................... 132

2.8 Replication and Number of Test Organisms................................................. 132

2.8.1 Exposed First Generation Mysids (F0).............................................. 133

2.8.2 Exposed Second Generation Mysids (F1) ......................................... 134

2.9 Diluter ........................................................................................................... 134

2.10 Test Substance .............................................................................................. 135
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2.11 Test Substance Concentrations..................................................................... 136

2.12 Stock Preparation.......................................................................................... 136

2.13 Analytical Method and Verification ............................................................. 137

2.13.1 Analytical Sampling.......................................................................... 137

2.13.2 Analytical Determinations................................................................ 137

3.0 EXPOSURE INITIATION AND CONDUCT ........................................................... 139

3.1 Exposed First Generation Mysids (F0).......................................................... 139

3.1.1 F0 Exposure Initiation........................................................................ 139

3.1.2 F0 Growth Chambers......................................................................... 140

3.1.3 F0 Reproductive Chambers................................................................ 140

3.1.4 Feeding.............................................................................................. 141

3.2 Exposed Second Generation Mysids (F1) ..................................................... 141

3.2.1 Initiation of F1 Exposure................................................................... 141

3.2.2 F1 Growth Chambers......................................................................... 142

3.2.3 F1 Reproductive Chambers................................................................ 142

3.2.4 Feeding.............................................................................................. 143

3.3 Chamber Cleaning ........................................................................................ 143

3.4 Conditions of Exposure................................................................................. 144

4.0 OBSERVATION AND MEASUREMENTS ............................................................. 144

4.1 Biological Observations................................................................................ 144

5.0 STATISTICAL METHODS ....................................................................................... 145

5.1 Endpoints...................................................................................................... 145

5.2 Statistical Methods........................................................................................ 145

6.0 RESULTS.................................................................................................................... 146

6.1 In-life Analytical Results.............................................................................. 146

6.1.1 F0 Generation .................................................................................... 146

6.1.2 F1 Generation .................................................................................... 147

6.2 Biological Results......................................................................................... 148

6.3 Survival......................................................................................................... 148

6.3.1 Survival from Day 0 to Day 7........................................................... 148

6.3.2 Survival from Day 8 to Day 14......................................................... 148

6.3.3 Survival from Day 15 to Test Termination....................................... 149

6.4 Growth .......................................................................................................... 150

6.4.1 Day 0 (less than 24 Hours old) – Dry Weight and Total Length from 

F0 and F1 Generations ....................................................................... 150

6.4.2 Day 7 - Dry Weight and Total Length from F0 and F1 Generations. 150
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6.4.3 Day 14 – Male and Female Dry Weight and Total Length from F0 

and F1 Generations............................................................................ 151

6.4.3.1 Day 14 – Male Dry Weight and Total Length from F0 and 

F1 Generations.................................................................. 152

6.4.3.2 Day 14 – Female Dry Weight and Total Length from F0

and F1 Generations........................................................... 152

6.4.4 Test Termination– Dry Weight and Total Length from F0 (Day 44) 

and F1 (Day 35) Generations............................................................. 153

6.4.4.1 Males at Test Termination from F0 (Day 44) and F1 (Day 

35) Generations................................................................ 153

6.4.4.2 Females at Test Termination from F0 (Day 44) and F1

(Day 35) Generations....................................................... 154

6.5 Reproduction................................................................................................. 154

6.5.1 Time to Maturation from F0 and F1 Generations.............................. 155

6.5.2 Number of Offspring in the First Brood from F0 and F1 Generations155

6.5.3 Number of Offspring in the Second Brood from F0 and F1

Generations....................................................................................... 156

6.5.4 Total Number of Offspring from F0 and F1 Generations.................. 156

6.5.5 Sex Ratio (Male/Female) from F0 and F1 Generations..................... 157

6.5.6 Percent of Reproducing Females from F0 and F1 Generations......... 158

7.0 PERFORMANCE CRITERIA.................................................................................... 159

7.1 Water quality characteristics should remain within the tolerance limits 

illustrated in Tables 1 and 2 of the Protocol. ................................................ 159

7.2 There should be more than 70% survival of control animals in all test 

phases over the duration of the chemical exposure. ..................................... 159

7.3 At least 75% of the females in the F0 and F1 controls must produce 

young............................................................................................................. 159

7.4 The average number of young produced per control female in the first 

two broods is at least eight............................................................................ 159

8.0 CONCLUSIONS......................................................................................................... 160

REFERENCES AND LITERATURE.................................................................................. 161

Table 1. Means and Ranges of Water Quality Measurements Taken During the 

F0 Generation Exposure to Lindane.............................................................. 164

Table 2. Means and Ranges of Water Quality Measurements Taken During the 

F1 Generation Exposure to Lindane.............................................................. 165

Table 3. Measured Concentrations of Lindane in Pretest Solution Samples for 

the F0 Generation .......................................................................................... 166

Table 4. Measured Concentrations of Lindane for the F0 Generation in Stock 

Solution Samples .......................................................................................... 167
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Table 5. Measured Concentrations of Lindane in Saltwater Samples for the F0

Generation..................................................................................................... 168

Table 6. Measured Concentrations of Lindane in Pretest Diluter Verification 

Samples for the F1 Generation ...................................................................... 170

Table 7. Measured Concentration of Lindane in F1 Generation Stock Solution 

Samples......................................................................................................... 171

Table 8. Measured Concentrations of Lindane Saltwater Samples for the F1

Generation..................................................................................................... 172

Table 9. Summary of F0 Generation Survival of Saltwater Mysids Exposed to 

Lindane ......................................................................................................... 174

Table 10. Summary of F1 Generation Survival of Saltwater Mysids Exposed to 

Lindane ......................................................................................................... 175

Table 11. Summary of Day 0 Growth Measurements of Culture Saltwater 

Mysids........................................................................................................... 176

Table 12. Summary of F0 Generation Day 7 Growth Measurements of Saltwater 

Mysids Exposed to Lindane.......................................................................... 177

Table 13. Summary of F1 Generation Day 7 Growth Measurements of Saltwater 

Mysids Exposed to Lindane.......................................................................... 178

Table 14. Summary of F0 Generation Day 14 Growth Measurements of Saltwater 

Mysids Exposed to Lindane.......................................................................... 179

Table 15. Summary of F1 Generation Day 14 Growth Measurements of Saltwater 

Mysids Exposed to Lindane.......................................................................... 180

Table 16. Summary of F0 Generation Day 44 Growth Measurements of Saltwater 

Mysids Exposed to Lindane.......................................................................... 181

Table 17. Summary of F1 Generation Day 35 Growth Measurements of Saltwater 

Mysids Exposed to Lindane.......................................................................... 182

Table 18. Summary of F0 Generation Reproduction of Saltwater Mysids Exposed 

to Lindane ..................................................................................................... 183

Table 19. Summary of F1 Generation Reproduction of Saltwater Mysids Exposed 

to Lindane ..................................................................................................... 184

Figure 1. Mysid Two-Generation Test Method Schematic.......................................... 185

Figure 2. F0 Analytical Recoveries Over Time ............................................................ 186

Figure 3. F1 Analytical Recoveries Over Time ............................................................ 187

Appendix 1. Dilution Water Chemistry Parameters Measured During the 4-Week 

Period Immediately Preceding the Test ........................................................ 188

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, 

Ltd. Saltwater................................................................................................ 189

Appendix 3. Analyses of Pesticides, Organics and Metals in Brine (Artemia) Egg 

Sample........................................................................................................... 191

Appendix 4. The Analysis of Lindane in Saltwater.......................................................... 193
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Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0

Generation..................................................................................................... 208

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1

Generation..................................................................................................... 220

Appendix 7 Replicate Mean Growth Measurements for the F0 Generation Day 0 

Culture Mysids.............................................................................................. 232

Appendix 8 Replicate Mean Total Body Lengths for the F0 Generation Day 7 

Mysids........................................................................................................... 234

Appendix 9 Replicate Mean Dry Weights for the F0 Generation Day 7 Mysids ............. 237

Appendix 10 Replicate Mean Total Body Lengths for the F1 Generation Day 7 

Mysids........................................................................................................... 240

Appendix 11 Replicate Mean Dry Weights for the F1 Generation Day 7 Mysids ............. 243

Appendix 12 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 14 Mysids.............................................................................................. 246

Appendix 13 Replicate Mean Dry Weights for Male and Female F0 Generation Day 

14 Mysids...................................................................................................... 249

Appendix 14 Replicate Mean Body Lengths for Male and Female F1 Generation 

Day 14 Mysids.............................................................................................. 252

Appendix 15 Replicate Mean Dry Weights for Male and Female F1 Generation Day 

14 Mysids...................................................................................................... 253

Appendix 16 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 44 Mysids.............................................................................................. 254

Appendix 17 Replicate Mean Dry Weights for Male and Female F0 Generation Day 

44 Mysids...................................................................................................... 257

Appendix 18 Replicate Mean Body Lengths for Male and Female F1 Generation 

Day 35 Mysids.............................................................................................. 260

Appendix 19 Replicate Mean Dry Weights for Male and Female F1 Generation Day 

35 Mysids...................................................................................................... 263

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and 

Interbrood Duration F0 Generation ............................................................... 266

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and 

Interbrood Duration F1 Generation ............................................................... 272

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods 

and Total Produced F0 Generation................................................................ 278

Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods 

and Total Produced F1 Generation ................................................................ 284

Appendix 24 Replicate Mean Percent of Males Sex Ratio and Percent of Females 

Producing Offspring F0 Generation .............................................................. 289

Appendix 25 Replicate Mean Percent of Males Sex Ratio and Percent of Females 

Producing Offspring F1 Generation .............................................................. 290
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STUDY TRIAL 2 – 185A-102-2A....................................................................................... 291

SUMMARY.......................................................................................................................... 292

9.0 INTRODUCTION....................................................................................................... 295

10.0 DESCRIPTION OF THE METHOD.......................................................................... 296

10.1 QAPP/Protocol.............................................................................................. 296

10.2 Test Organism............................................................................................... 297

10.3 Feeding.......................................................................................................... 298

10.4 Handling........................................................................................................ 299

10.5 Dilution Water .............................................................................................. 299

10.6 Water Quality Measurements ....................................................................... 299

10.6.1 Temperature ...................................................................................... 299

10.6.2 Salinity.............................................................................................. 300

10.6.3 Lighting............................................................................................. 300

10.6.4 Dissolved Oxygen............................................................................. 300

10.6.5 pH...................................................................................................... 301

10.7 Assay System................................................................................................ 301

10.7.1 Test Vessels ...................................................................................... 301

10.7.2 Retention Chambers (Reproduction/Growth Chambers).................. 301

10.7.3 Brood Chambers ............................................................................... 302

10.8 Replication and Number of Test Organisms................................................. 302

10.8.1 Exposed First Generation Mysids (F0).............................................. 303

10.8.2 Exposed Second Generation Mysids (F1) ......................................... 303

10.9 Diluter ........................................................................................................... 304

10.10 Test Substance .............................................................................................. 305

10.11 Test Substance Concentrations..................................................................... 305

10.12 Saturation Column Preparation..................................................................... 305

10.13 Stock Preparation.......................................................................................... 306

10.14 Analytical Method and Verification ............................................................. 307

10.14.1 Analytical Sampling.................................................................. 307

10.14.2 Analytical Determinations......................................................... 307

11.0 EXPOSURE INITIATION AND CONDUCT ........................................................... 308

11.1 Exposed First Generation Mysids (F0).......................................................... 309

11.1.1 F0 Exposure Initiation........................................................................ 309

11.1.2 F0 Growth Chambers......................................................................... 309

11.1.3 F0 Reproductive Chambers................................................................ 310

11.1.4 Feeding.............................................................................................. 311
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11.2 Exposed Second Generation Mysids (F1) ..................................................... 311

11.2.1 F1 Exposure Initiation........................................................................ 311

11.2.2 F1 Growth Chambers......................................................................... 312

11.2.3 F1 Reproductive Chambers................................................................ 313

11.2.4 Feeding.............................................................................................. 313

11.3 Chamber Cleaning ........................................................................................ 314

11.4 Conditions of Exposure................................................................................. 314

12.0 OBSERVATION AND MEASUREMENTS ............................................................. 314

12.1 Biological Observations................................................................................ 314

13.0 STATISTICAL METHODS ....................................................................................... 315

13.1 Endpoints...................................................................................................... 315

13.2 Statistical Methods........................................................................................ 315

14.0 RESULTS.................................................................................................................... 316

14.1 In-life Analytical Results.............................................................................. 316

14.1.1 F0 Generation .................................................................................... 316

14.1.2 F1 Generation .................................................................................... 317

14.2 Biological Results......................................................................................... 318

14.3 Survival......................................................................................................... 318

14.3.1 Survival from Day 0 to Day 7........................................................... 318

14.3.2 Survival from Day 8 to Day 14......................................................... 318

14.3.3 Survival from Day 15 to Test Termination of F0 .............................. 319

14.4 Growth .......................................................................................................... 319

14.4.1 Day 0 (less than 24 Hours old) – Dry Weight and Total Length 

from F0 Generation ........................................................................... 320

14.4.2 Day 7 - Dry Weight and Total Length from F0 Generation.............. 320

14.4.3 Day 14 – Male and Female Dry Weight and Total Length from 

F0 Generation .................................................................................... 320

14.4.3.1 Day 14 – Male Dry Weight and Total Length from F0

Generation........................................................................ 321

14.4.3.2 Day 14 – Female Dry Weight and Total Length from 

F0 Generation ................................................................... 321

14.4.4 Test Termination – Dry Weight and Total Length from F0 (Day 

46) Generation .................................................................................. 321

14.4.4.1 Males at Test Termination from F0 (Day 46) 

Generation........................................................................ 322

14.4.4.2 Females at Test Termination from F0 (Day 46) 

Generation........................................................................ 322
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14.5 Reproduction................................................................................................. 322

14.5.1 Time to Maturation, First Brood, Second Brood and Interbrood 

Duration from F0 Generation ............................................................ 323

14.5.2 Number of Offspring in the First Brood from F0 Generation ........... 323

14.5.3 Number of Offspring in the Second Brood from F0 Generation....... 323

14.5.4 Total Number of Offspring from F0 Generation............................... 324

14.5.5 Sex Ratio (Male/Female) from F0 Generation.................................. 324

14.5.6 Percent of Reproducing Females from F0 Generation ...................... 325

14.6 Summary of Survival, Growth Measurements and Reproduction 

Endpoints for the F1 Generation ................................................................... 325

14.6.1 Summary of Survival for the F1 Generation ..................................... 325

14.6.2 Summary of Growth Measurements for the F1 Generation .............. 326

14.6.3 Summary of Reproduction for the F1 Generation............................. 326

15.0 PERFORMANCE CRITERIA.................................................................................... 327

15.1 Water quality characteristics should remain within the tolerance limits 

illustrated in Tables 1 and 2 of the Protocol. ................................................ 327

15.2 There should be more than 70% survival of control animals in all test 

phases over the duration of the chemical exposure. ..................................... 327

15.3 At least 75% of the females in the F0 and F1 controls must produce 

young............................................................................................................. 327

15.4 The average number of young produced per control female in the first 

two broods is at least eight............................................................................ 327

16.0 CONCLUSIONS......................................................................................................... 328

REFERENCES AND LITERATURE.................................................................................. 329

Table 20. Means and Ranges of Water Quality Measurements Taken During the 

F0 Generation Exposure to 4-tert-octylphenol .............................................. 332

Table 21. Means and Ranges of Water Quality Measurements Taken During the 

F1 Generation Exposure to 4-tert-octylphenol .............................................. 333

Table 22. Measured Concentrations of 4-tert-octylphenol in Pretest Solution 

Samples for the F0 Generation ...................................................................... 334

Table 23. Measured Concentrations of 4-tert-octylphenol for the F0 Generation 

in Stock Solution Samples............................................................................ 335

Table 24. Measured Concentrations of 4-tert-octylphenol in Saltwater Samples 

for the F0 Generation..................................................................................... 336

Table 25. Measured Concentrations of 4-tert-octylphenol in Pretest Solution 

Samples for the F1 Generation ...................................................................... 338

Table 26. Measured Concentrations of 4-tert-octylphenol in Saltwater Samples 

for the F1 Generation..................................................................................... 339
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Table 27. Summary of F0 Generation Survival of Saltwater Mysids Exposed to 

4-tert-octylphenol.......................................................................................... 340

Table 28. Summary of Day 0 Growth Measurements of Culture Saltwater 

Mysids........................................................................................................... 341

Table 29. Summary of F0 Generation Day 7 Growth Measurements of Saltwater 

Mysids Exposed to 4-tert-octylphenol.......................................................... 342

Table 30. Summary of F0 Generation Day 14 Growth Measurements of Saltwater 

Mysids Exposed to 4-tert-octylphenol.......................................................... 343

Table 31. Summary of F0 Generation Day 46 Growth Measurements of Saltwater 

Mysids Exposed to 4-tert-octylphenol.......................................................... 344

Table 32. Summary of F0 Generation Reproduction of Saltwater Mysids Exposed 

to 4-tert-octylphenol...................................................................................... 345

Figure 4. Mysid Two-Generation Test Method Schematic.......................................... 346

Figure 5. F0 Generation Analytical Recoveries Over Time ......................................... 347

Figure 6. F1 Generation Analytical Recoveries Over Time ......................................... 348

Appendix 26. Dilution Water Chemistry Parameters Measured During the 4-Week 

Period Immediately Preceding the Test ........................................................ 349

Appendix 27. Analyses of Pesticides, Organics and Metals in Wildlife International, 

Ltd. Saltwater................................................................................................ 350

Appendix 28. Analyses of Pesticides, Organics and Metals in Brine (Artemia) Egg 

Sample........................................................................................................... 352

Appendix 29. The Analysis of 4-Tert-Octylphenol in Saltwater......................................... 354

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-TertOctylphenol - F0 Generation ......................................................................... 368

Appendix 31 Replicate Mean Growth Measurements for the F0 Generation Day 0 

Culture Mysids.............................................................................................. 380

Appendix 32 Replicate Mean Total Body Lengths for the F0 Generation Day 7 

Mysids........................................................................................................... 382

Appendix 33 Replicate Mean Dry Weights for the F0 Generation Day 7 Mysids ............. 385

Appendix 34 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 14 Mysids.............................................................................................. 388

Appendix 35 Replicate Mean Dry Weights for Male and Female F0 Generation Day 

14 Mysids...................................................................................................... 391

Appendix 36 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 46 Mysids.............................................................................................. 394

Appendix 37 Replicate Mean Dry Weights for Male and Female F0 Generation Day 

46 Mysids...................................................................................................... 397

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and 

Interbrood Duration F0 Generation ............................................................... 400
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Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods 

and Total Produced F0 Generation................................................................ 406

Appendix 40 Replicate Mean Percent of Males Sex Ratio and Percent of Females 

Producing Offspring F0 Generation .............................................................. 412

Appendix 41 F1 Summary Tables....................................................................................... 413
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PROTOCOL
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AMENDMENTS AND DEVIATIONS
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QAPP
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STUDY TRIAL 1 – 185A-102-1
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SUMMARY

Two-Generation Life-Cycle Toxicity Test with Mysids (Americamysis bahia) 

Inter-Laboratory Comparison

SPONSOR: U.S. Environmental Protection Agency 

WILDLIFE INTERNATIONAL, LTD

STUDY NUMBERS: 185A-102-1

RTI

PROJECT NUMBER: 0210114.030

EPA CONTRACT

NUMBER: EP-W-06-026

TEST SUBSTANCE: Lindane, Lot No. 14419EB, CAS No. 58-89-9, reported to have a 

purity of 99.6% and was received from Battelle Marine Sciences 

Laboratory on 19 January 2011.

DEFINITIVE

TEST DATES: 2 May 2011 to 20 July 2011

TEST ORGANISM: The test was initiated with Americamysis bahia (newly released, 

≤ 24 hours old, F0 generation) that were grown to maturity. Their 

offspring (F1 generation) were collected, which in turn were 

grown to maturity, and their offspring (F2 generation) were 

collected.

Origin: Wildlife International, Ltd culture

DILUTION WATER: Natural seawater collected at Indian River Inlet, Delaware and 

diluted with laboratory well water to a salinity of approximately 

20 ‰

TEST CONDITIONS: Test duration: 77 days

Temperature ranges: 24.1 to 27.2oC

Salinity ranges: 19 to 21 ‰

Dissolved oxygen ranges: 6.0 to 7.2 mg/L

pH range: 7.9 to 8.6

Light intensity: 190 to 367 lux

Photoperiod: Illumination of 14 hours light: 

10 hours dark
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NOMINAL TEST

CONCENTRATIONS: Dilution water (negative) control (0.0), 32, 80, 200, 500 and 

1250 ng Lindane/L

ENDPOINTS:

Parental Generation (F0)

• Survival

• Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14 and termination)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14 and termination)

• Time to maturation, appearance of marsupial pouch

• Time to first brood release, number of days after test initiation to release of young 

in the first brood

• Time to second brood release, number of days after test initiation to release of 

young in the second brood

• Interbrood duration, time between release of first brood and release of second 

brood

• Number of young per female in each of the first two broods

• Total number of young in each replicate population

• Sex ratio

• Percentage of females that are reproductively active

First Filial Generation (F1)

• Survival

• Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14 and termination)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14 and termination)

• Time to maturation, appearance of marsupial pouch

• Time to first brood release, number of days after test initiation to release of young 

in the first brood

• Time to second brood release, number of days after test initiation to release of 

young in the second brood

• Interbrood duration, time between release of first brood and release of second 

brood

• Number of young per female in each of the first two broods

• Total number of young in each replicate population

• Sex ratio

• Percentage of females that are reproductively active
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RESULTS: Reproduction, in the form of mean number of offspring in the second 

brood per reproducing female, mean total number of offspring during 

the study and the mean percent of reproducing females, in the F0

generation was the most sensitive endpoint in the mysid twogeneration exposure to Lindane. Based on reproduction, the NOEC 

and LOEC was determined to be 500 and 1250 ng/L, respectively. 

The MATC was calculated to be 791 ng/L.
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1.0 INTRODUCTION

The Environmental Protection Agency’s EDSP has completed validation of a battery of Tier 1 

assays to identify chemicals that have the potential to interact with the endocrine system, and is 

pursuing validation of longer term Tier 2 assays to identify adverse endocrine effects and provide 

dose response information needed for risk assessment. One of the longer term Tier 2 assays 

being developed is the Mysid Two-generation Test with Americamysis bahia. This test contrasts 

with existing mysid life cycle methods (that represent a single generation life cycle) in that an 

additional generation is included to allow evaluation of potential trans-generational effects. The 

inclusion of the invertebrate assay in Tier 2 is important because invertebrates make up the 

majority of species and the endocrine systems of invertebrates are not always directly analogous 

to those of vertebrates. Additionally, a method for testing crustaceans for effects of EDCs is 

relevant to assessing the adverse consequences of chemicals indicated to be endocrine active in 

Tier 1 assays.

As part of the validation of the two generation test method, the value-added of this method must 

be demonstrated over the existing mysid life cycle test method. There is a theoretical basis that 

the two-generation test is more comprehensive for endocrine disrupting chemicals (EDCs) than 

the life cycle test, but the single generation test is less logistically complex and in practical 

aspects and general sensitivity may be sufficient. The value-added of the two-generation test 

over the existing life cycle test must be empirically demonstrated before the two-generation test 

can be accepted. An interlaboratory evaluation is needed to support a value-added comparison. 

In addition, a copepod development and reproduction test method with Amphiascus tenuiremis 

has been under development within the Organization for Economic Cooperation and 

Development (OECD). This method offers some logistical advantages in time and resources, but 

it is uncertain to what extent this may be at the expense of sensitivity. The OECD validation 

effort was led by Dr. G. Thomas Chandler (Dean, Arnold School of Public Health, University of 

South Carolina, Columbia, SC 29208) who developed this test method, a related ASTM method, 

and provided training to the OECD participants. An inter-laboratory exercise where both 

methods are compared for their practical transferability and quantitative reproducibility is needed 
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to support the ultimate selection of the most appropriate EDSP Tier 2 invertebrate test. Two 

chemicals (lindane and vinclozolin) will be tested in three laboratories for both the mysid twogeneration test method and the copepod development and reproduction test method for a total of 

12 experiments.

The main objective of this Task Order was to obtain data on the inter-laboratory reproducibility 

and comparability of the mysid two-generation test method and the copepod development and 

reproduction test method in support of the validation of the mysid two-generation test method 

and copepod development and reproduction test method being considered for the Endocrine 

Disruptor Screening Program (EDSP).

All of the work supporting these studies was conducted in accordance with EPA Good 

Laboratory Practice (GLP) regulations. RTI International was responsible for overall Task Order 

management, ensuring the work of all six independent laboratories was of high quality and 

delivered on schedule. The RTI Team for mysid work included Smithers Viscient, LLC (Lead 

Laboratory, SV), Wildlife International, Ltd. (WLI), and a Laboratory at the University of North 

Texas (UNT). The RTI team for the copepod work included Brixham Environmental Laboratory 

(Lead Laboratory, Brixham), Centre for Fisheries, Environment & Aquaculture (Cefas 

Weymouth) and Block Environmental Services, Inc. (BES). The lead laboratories’ roles 

included responsibility for coordinating the inter-laboratory efforts and ensuring that participants 

adhere to the standardized guidelines/protocols.

RTI arranged to have all original records (both its own and those from the participating 

laboratories) transferred to the EPA for storage, by the end of the period of performance for this 

task order. The records were organized and included an index in MS-Word as well as hard copy. 

Quantitative data were submitted in MS Excel spreadsheets. Both the draft and final reports 

were provided to the EPA TOPO in an editable format.
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The raw data generated at all laboratories and at RTI International were sent to the EPA TOPO. 

All raw data and records from each participating laboratory were sent to RTI, who submitted

them to EPA as one cohesive package. No records were disposed of without the authorization of 

RTI. Each facility had their own archivist, responsible for archiving and retrieving materials. 

An archive inventory was maintained and storage capability was provided for the expedient 

retrieval of materials. Specimens and samples were disposed of only after project management 

staff has determined and QA verified that they no longer afforded evaluation.

2.0 DESCRIPTION OF THE METHOD

2.1 QAPP/Protocol

This study was conducted according to the procedures outlined in the Quality Assurance Project 

Plan (QAPP) for Task Order 30 (QAPP). The study protocol (Protocol) describes a twogeneration test with an estuarine mysid that considers reproductive fitness in parents and 

offspring as an integrated measure of toxicant exposure. It also enables measurement of a suite 

of other endpoints that allow for diagnostic and definitive evaluation of endocrine disrupting 

chemicals (EDCs) or other types of reproductive toxicants (Verslycke et al., 2004). The twogeneration test described in the guideline is an extension of existing standard practice for 

conducting a mysid life-cycle test (ASTM, 1999; McKenney, 1986, 1998; and Nimmo et al., 

1977, 1978).

2.2 Test Organism

The saltwater mysid (Americamysis bahia) was selected as the test species for this study. The 

saltwater mysid is representative of an important group of marine invertebrates and was selected 

for use in the test based upon past use in the laboratory. Mysids used in the test were obtained as 

juveniles (<24 hours old) from cultures maintained by Wildlife International, Ltd., Easton, 

Maryland. The brood stock was originally obtained from two different commercial suppliers: 

Chesapeake Cultures, Inc. located in Hayes, Virginia and Reed Mariculture, Inc. located in 
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Campbell, California, and are refreshed periodically. The last time brood stock mysids were 

added to the cultures prior to this study was March 30, 2011. 

Adult mysids in the cultures were held in the laboratory for at least 14 days before juveniles were 

collected for testing. The culture was maintained in a recirculating saltwater system using water 

from the same source as used during the test. During the 2-week period immediately preceding 

the test, water temperatures in the cultures ranged from 23.0 to 27.5oC, measured with a liquidin-glass thermometer. The pH of the water ranged from 7.7 to 8.0, measured with a Fisher 

Scientific Accumet Model 4 Star Plus pH meter. Dissolved oxygen ranged from 6.1 to 7.0 mg/L 

(≥83% of saturation), measured with a Yellow Springs Instruments Model 3 Star Plus dissolved 

oxygen meter. Salinity of the filtered saltwater ranged from 20 to 21 parts per thousand (‰), 

measured with a VitalSine refractometer. The area in which the mysids were cultured received a 

regulated photoperiod of 16 hours of light and 8 hours of darkness. 

2.3 Feeding

During culturing and the test, mysids from both generations (F0 and F1) for all control and 

treatment groups were fed three to four times daily with brine shrimp (Artemia sp.) nauplii ≤ 24 

– 48 hours old (post-hydration) supplied by Brine Shrimp Direct, Ogden, Utah (CSP Numbers: 

11-13; 11-35, 11-40 and 11-58). For each generation, at least one feeding each day was with 

brine shrimp enriched with Algamac 3050®, a supplemental substance high in saturated fatty 

acids, supplied by Aquafauna Bio-Marine, Hawthorne, California (CSP Number 10-105). 

Densities (nauplii/mL) of concentrated collections of nauplii (a slurry of nauplii and dilution 

water) from Artemia hatched daily from hydrated cysts were determined by replicate counts of 

aliquots pipetted onto filter paper. Based on these densities, the volume of nauplii fed to mysids 

was adjusted daily in accordance with the following regime: rations for reproduction/growth 

chambers (containing up to 20 mysids each) were approximately 1800 nauplii/chamber on days 0 

to 3, 2250/chamber on days 4 to 6, 2700/chamber on days 7 to 9, 3150/chamber on days 10 

to 12, and 3600/chamber on days 12 through the end of the study, while brood chambers 

(containing individually paired mysids) were fed approximately 900 nauplii/chamber throughout 
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the test. All treatments and controls were fed using the same regime throughout the test. Excess 

brine shrimp and organic debris in the growth/reproductive chambers and brood chambers were 

removed periodically when observed. Periodic analyses of representative samples of the food 

was conducted using U.S. EPA standard methods (U.S. EPA, 1997) by Lancaster Laboratories, 

Lancaster, PA 17605-2425 to ensure the absence of potential toxicants, including pesticides, 

PCBs and selected toxic metals, at concentrations which may be harmful to mysids 

(Appendix 3). Specifications for acceptable levels of contaminants in mysid diets have not been 

established. However, there are no known levels of contaminants reasonably expected to be 

present in the diet that are considered to interfere with the purpose or conduct of the test.

2.4 Handling

Mysids were handled as gently as possible to avoid unnecessary stress. Great care was exercised 

to avoid injury to juvenile mysids during counting. All mysids were transferred using a smoothedged pipette.

2.5 Dilution Water

The water used for culturing and testing was natural seawater collected at Indian River Inlet, 

Delaware. The freshly-collected seawater was passed through a sand filter to remove particles 

greater than approximately 25 μm, and pumped into a 37,800-L storage tank. The filtered 

saltwater then was diluted to a salinity of approximately 20‰ with freshwater from a well on the 

Wildlife International, Ltd. site and was aerated with spray nozzles. Prior to use, the 20‰ water 

was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) 

sterilizer.

Salinity and pH measurements taken during the four-week period immediately preceding the test 

are presented in Appendix 1. The results of periodic analyses performed to measure the 

concentrations of selected organic and inorganic constituents in the saltwater are presented in 

Appendix 2.
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During the four-week period immediately preceding the test, the pH was 8.1 (range 8.0 to 8.1) 

and salinity was 20 ‰ (range of 19 to 21 ‰). Specific conductance and pH were measured 

using a VitalSine refractometer and a Thermo Orion Model 525Aplus pH Meter, respectively.

2.6 Water Quality Measurements

2.6.1 Temperature

The test vessels were indiscriminately positioned by treatment group in an environmental 

chamber designed to maintain the desired test temperature throughout the test period. The target 

test temperature during the study was 26 ± 1°C. Temperature was measured in each test vessel at 

the beginning of the F0 generation, and in alternating replicates daily thereafter in both the F0 and 

F1 generations, using a hand-held liquid-in-glass thermometer. Temperature also was monitored 

continuously in one negative control test vessel using a Fulscope ER/C Recorder, which was 

verified prior to test initiation and verified or calibrated approximately weekly during the test 

with a hand-held liquid-in-glass thermometer.

2.6.2 Salinity

The salinity of the test solutions was maintained at 20 ± 2‰ as described in the dilution water 

section above. Salinity was measured in each test vessel at the beginning test, and in alternating 

replicates daily thereafter. Salinity was measured using a VitalSine refractometer. 

2.6.3 Lighting

Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that 

emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a 

photoperiod of 14 hours of light and 10 hours of darkness. A 120-minute transition period of 

low light intensity was provided when lights went on and off to avoid sudden changes in 
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lighting. Light intensity was measured at the water surface of each test vessel once during each 

generation using a SPER Scientific Model 840006C light meter.

2.6.4 Dissolved Oxygen

Total dissolved oxygen was measured to be 100% of saturation at the start of the test for the F0

generation and was not allowed to drop below 60% saturation (4.3 mg/L at 26oC and 20‰ 

salinity) throughout the test. The dilution water was aerated prior to the addition of organisms to 

the test system and continually through the test to maintain dissolved oxygen levels above 60% 

saturation. Dissolved oxygen was measured in each test vessel at the beginning of the test for the 

F0 generation, and in alternating replicates daily thereafter for both generations during the test. 

Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen 

meter.

2.6.5 pH

Measurements of pH were made in each test vessel at the beginning of the test for the F0

generation, and in alternating replicates daily thereafter during the test. Measurements of pH 

were made using a Thermo Orion Model 525Aplus meter. 

2.7 Assay System

2.7.1 Test Vessels

The test vessels were 38-L glass aquaria measuring 50 x 25 x 30 cm equipped with self-starting 

siphon systems to ensure that test solution flowed through the vessels. The total solution volume 

maintained in each vessel was approximately 21 L. The aquaria were indiscriminately 

distributed by concentration within the environmental chamber, and labeled to identify the 

project number, treatment/control group, and the replicate designation. The depth of the water in 

a representative test vessel was 14.7 – 14.9 cm. 
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2.7.2 Retention Chambers (Reproduction/Growth Chambers)

Mysid juveniles were held in retention chambers within test chambers to facilitate observations 

and eliminate loss through outflow water. These retention chambers were constructed of 15-cm 

glass Petri dishes to which an approximately 15-cm high cylinder of nylon mesh screen (210-μm 

mesh) had been attached with aquarium grade (without fungicides) silicone adhesive. The depth 

of the water in a representative test retention compartment was 14.1 – 14.2 cm.

For the individual mysid reproduction or growth chambers, the following code was used to 

identify retention chambers and was marked on each Reproduction (R) or Growth chamber (G): 

Treatment Level – Replicate Vessel – Chamber Type (R or G) – Generation (F0 or F1) – date 

chamber was initiated.

2.7.3 Brood Chambers

Upon maturing, gravid females were paired with males and isolated in brood chambers

constructed of 10-cm glass Petri dishes to which an approximately 15-cm high cylinder of nylon 

mesh screen (210-μm mesh) was attached. The depth of the water in a representative test brood 

chamber was 14.2 cm, respectively.

For the individual mysid brood chambers, the following code was used to identify brood 

chambers and was marked on each Brood Chamber (BC): Treatment Level – Replicate – Brood 

Chamber (BC) – Brood Chamber Number (1-9) – Generation (F0 or F1) – date brood chamber 

was initiated.

2.8 Replication and Number of Test Organisms

For each mysid, the time to maturation was defined as the day of the study in which the mysid 

could be sexually identified as being male or female. For this study, both the F0 and F1 mysids 

were all first examined for reproductive characteristics starting on Day 14. In both generations, 

sex could be assigned to the majority of the mysids in each treatment group on Day 14 . Those 
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mysids that were not able to be sexually identified (this only occurred in the F0 generation) were 

maintained in a separate brood chamber until identification was possible (Appendix 5). For each 

mysid, the time to first brood release was defined as the first day of the study in which the mysid 

released young. For each mysid, the time to second brood release was defined as the day of the 

study in which the mysid released its second brood. The typical gestational interval for mysids is 

between 5 to 9 days. Therefore, any mysids released within four days of the first brood were still 

considered part of that first brood. Any young released 5 days or more out from the first brood 

release were considered part of the second brood. For each mysid, the time of interbrood 

duration was defined as the time between release of the first brood and the release of the second 

brood.

2.8.1 Exposed First Generation Mysids (F0)

Three replicate vessels per treatment were used in the study. The exposure was initiated with 

20 newly released mysid juveniles (< 24 hours old) assigned to reproduction chambers within 

each replicate tank. Within the three replicate vessels, one additional growth chamber was also 

initiated with 20 newly released mysid juveniles (< 24 hours old) to be sub-sampled weekly for 

growth measurements. Upon reaching maturity (Day 14 in this study), one male and one gravid 

female were indiscriminately assigned to separate brood chambers within each replicate (with a 

maximum of seven male/female pairs possible per replicate). Individuals in the reproduction 

chambers not paired in brood chambers were maintained and observed for survival and sex 

determination within the retention chambers until they were paired or until the test was 

terminated (one week after the mean day of release of the second brood). Unused mysids from 

the growth retention chambers were used to complete male: female pairings in the event that sex 

ratio in the reproduction chamber was skewed preventing obtaining seven pairs. The F0

exposure continued until Day 44, which was one week after the median day of release of the 

second brood (Day 37) in the negative control group. 
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2.8.2 Exposed Second Generation Mysids (F1)

The F1 generation exposure was initiated with up to 15 newly released mysid juveniles 

(< 24 hours old) per day produced by the F0 generation. F1 generation mysids were assigned to 

reproduction chambers within the same treatment group, for each of the F0 exposure conditions 

(e.g. juveniles from Replicate A of the F0 generation vessel were assigned to Replicate A of the 

F1 generation vessel). Mixing of young released on the same day and across replicate chambers 

from the same treatment or control group, was conducted to minimize reproduction between 

siblings. An additional F1 generation growth chamber was initiated with 15 newly released 

mysid juveniles (when possible) within each replicate treatment level to be sub-sampled weekly 

for growth measurements. Priority was given to filling the reproduction retention chambers first, 

followed by the growth chambers. If possible, F1 offspring was indiscriminately placed into 

chambers designated as reproduction or growth. The ages of offspring in a given chamber were 

tracked to ensure growth measurements were collected on the appropriate day (e.g. Day 7 and 

Day 14). Upon reaching maturity (Day 14 in this study), one male and one gravid female were 

indiscriminately assigned to separate F1 generation brood chambers within each replicate (with a 

maximum of 7 m/f pairs possible per replicate). Individuals in the F1 generation reproduction 

chambers not paired in brood chambers were maintained and observed for survival and sex 

determination within the F1 generation retention chambers until they were paired or until the test 

was terminated. Unused mysids from the growth retention chambers were used to complete 

male: female pairings in the event that sex ratio in the reproduction chamber was skewed 

preventing obtaining seven pairs. The F1 exposure continued until chamber Day 35 (i.e., the 

organisms in each reproductive chamber reached an age of 35 days).

2.9 Diluter

The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter 

used to deliver each concentration of the test substance and a negative control (dilution water) to 

test vessels. A peristaltic pump (Rainin Instrument Company, Inc., Woburn, Massachusetts) was 

used to deliver test substance stock solutions to impartially assigned mixing chambers where the 
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stocks were mixed with dilution water prior to delivery to the test vessels. The flow of dilution 

water into each mixing chamber was controlled using rotameters, and was adjusted to provide 

each test vessel with at least 5 volume additions of test water per day (approximately 83 mL/min 

solution exchange for each test vessel). After mixing, the flow from each mixing chamber was 

split to deliver test water to three replicate test vessels.

The peristaltic pump used to deliver stock solutions to the mixing chambers was calibrated prior 

to the test and verified or recalibrated at least weekly during the test. The rotameters used to 

control the flow of dilution water to the mixing chambers were calibrated prior to the test and 

verified or recalibrated approximately weekly during the test. The proportion of the test water 

that was split into each replicate test vessel was checked prior to the test and approximately 

weekly during the test to ensure that flow rates varied by no more than ± 10% of the mean flow 

rate for the three replicates. Delivery of test solutions to the F0 and F1 generation test vessels 

was initiated 11 and 6 days, respectively, prior to the introduction of the test organisms to the test 

water in order to achieve equilibrium of the test substance. The general operation of the 

exposure system was checked visually at least once on the first and last days of the test and at 

least two times per day during the test, except on Day 23 of the F0 generation when the checklist 

was only done once.

2.10 Test Substance

The test substance, Lindane, was received from Battelle Marine Sciences Laboratory, Sequim, 

Washington. The following information was provided:

Name: Lindane

Synonyms (per MSDS): gamma-1,2,3,4,5,6-Hexachlorocyclohexane

Lot No.: 14419eb

CAS No.: 58-89-9

Purity: 99.6%

Description: Solid

Date Received: January 19, 2011

Expiration Date: December, 2012
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Lindane was assigned Wildlife International, Ltd. identification number 9859 upon receipt and 

was stored under ambient conditions.

2.11 Test Substance Concentrations

Test substance concentrations for the study were selected by the EPA to be dilution water 

(negative) control (0), 32, 80, 200, 500 and 1250 ng Lindane/L. 

2.12 Stock Preparation

Individual stock solutions were prepared for each of the concentrations tested, and were prepared 

24 times during the test for the F0 and F1 generations combined. A primary stock solution was 

prepared by mixing a calculated amount of test substance into dilution water (filtered saltwater) 

at a nominal concentration of 80 ng/mL. The primary stock solution was mixed by combining 

the test material with a portion of the dilution water into a volumetric flask and then sonicating 

the solution in the flask for approximately two hours. Next, the solution in the flask was 

combined with the rest of the pre-measured dilution water and the primary stock solution was 

stirred for approximately 24 hours. Four secondary stock solutions were prepared in dilution 

water at nominal concentrations of 2.1, 5.1, 13 and 32 ng/mL by proportional dilution of the 

primary stock solution. The stock solutions were mixed by stirring with a stainless steel whisk 

for approximately one to two minutes, and all appeared clear and colorless. Stock solutions were 

stored ambiently in 38 L glass tanks covered with dark plastic to shield from light. Fresh stock 

solutions were prepared and replaced in the delivery system every two to three days during the 

study.

The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 

3.90 or 3.91 mL/minute where they were mixed with dilution water delivered at a rate of 246 to 

250 mL/minute to achieve the desired test concentrations for both the F0 and F1 generation test 

systems. The negative control received dilution water only for both test systems. 
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2.13 Analytical Method and Verification

2.13.1 Analytical Sampling

Samples were collected from one test vessel of each treatment and control group seven and six 

days prior to the start of the test after conditioning the diluter for approximately four days. 

Water samples were collected from alternating replicate test vessels at test initiation, at 

approximately weekly intervals during the study and at test termination to measure 

concentrations of the test substance. Additional water samples were collected on Day 15 in both 

the F0 and F1 generations as well as on Day 2 of the F1 generation. The stock solutions used to 

dose the test system and prepare the test solutions were also sampled prior to test initiation. All 

samples were collected from mid-depth in the test vessel, placed in glass separatory funnels, 

immediately acidified with 10% phosphoric acid (H3PO4) and processed immediately for 

analysis. 

2.13.2 Analytical Determinations

The method used for the analysis of Lindane samples in saltwater was developed by Wildlife 

International, Ltd. The analytical method consisted of acidifying the saltwater samples with 10% 

phosphoric acid. Samples were extracted with hexane and sodium sulfate was added. Samples 

were analyzed by gas chromatography (GC) with electronic capture detection (ECD). 

Concentrations of Lindane in the samples were determined using an Agilent Model 5890 Gas 

Chromatograph (GC) equipped with an Electron Capture Detector (ECD). Chromatographic 

separations were achieved using a Zebron-DB-5 column (0.53 mm x 30 m, 0.5 μm film 

thickness). A method flowchart is provided in Appendix 4.1 and typical instrumental parameters 

for Lindane are summarized in Appendix 4.2.

Five calibration standards of Lindane, ranging in concentration from 0.500 to 5.00 μg a.i./L, were 

prepared in hexane using a stock solution of Lindane test substance in acetone (Appendix 4.3). 

Calibration standards were analyzed with each sample set. Linear regression equations were 

generated using the peak area responses versus the respective concentrations of the calibration 
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standards. The concentration of Lindane in the samples was determined by substituting the peak 

area responses of the samples into the applicable linear regression equation. An example of the 

calculations for a representative sample is included in Appendix 4.4.

A stock analysis for the F0 generation was conducted at nominal concentrations of 2.1, 5.1, 13, 

32 and 80 μg/L. The measured concentrations for the stock solution samples were 83.2, 87.4, 

75.3, 86.8 and 84.3% of nominal concentrations (Table 4). A stock analysis was conducted for 

the F1 generation at a nominal concentration of 2.1 μg/L. The measured concentration for the 

stock solution was 81.4% of the nominal concentration. (Table 7). 

The limit of quantitation (LOQ) for the analysis of Lindane in saltwater study samples was set at 

0.0151 μg/L, calculated as the product of the lowest calibration standard analyzed (0.500 μg 

a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test substance 

purity (99.6%). Eight matrix blank samples were analyzed to determine possible interferences in 

the F0 generation (Appendix 4.5). Nine matrix blank samples were analyzed to determine 

possible interferences in the F1 generation (Appendix 4.6). 

Acidified saltwater samples were fortified at 0.0320, 0.400 and 1.25 μg a.i./L using a stock 

solution of Lindane test substance prepared in acetone and were analyzed concurrently with the 

samples. The measured concentrations for the matrix fortification samples for the F0 generation 

ranged from 77.1 to 92.3% of nominal concentrations (Appendix 4.5). The measured 

concentrations for the matrix fortification samples for the F1 generation ranged from 70.6 to 

141% of nominal concentrations (Appendix 4.6)

A representative calibration curve is presented in Appendix 4.7. Representative chromatograms 

of low and high-level calibration standards are presented in Appendices 4.8 and 4.9, respectively. 

A representative chromatogram of a matrix blank sample is presented in Appendix 4.10, and a 

representative chromatogram of a matrix fortification sample is presented in Appendix 4.11. A 

representative chromatogram of a test sample is presented in Appendix 4.12.
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3.0 EXPOSURE INITIATION AND CONDUCT

The test substance was introduced into the test vessels approximately 11 days prior to test 

initiation in order to allow the test solutions to reach an equilibrium concentration of test 

substance in the exposure system. Exposure for the F0 mysids began when all of the <24-hour 

old individuals were indiscriminately transferred into the test vessels from the stock cultures. 

3.1 Exposed First Generation Mysids (F0)

3.1.1 F0 Exposure Initiation

At exposure initiation, 20 < 24-hour-old individuals were impartially transferred into 

reproduction and growth chambers. For each treatment level, triplicate exposure tanks included 

one growth and one retention chamber in each tank (Figure 1). Mysids were distributed among 

growth and reproduction chambers in the following manner: A pool of ~ 720 mysids (< 24 hours 

old) were removed from the cultures using siphon collectors. Mysids (< 24 hours old) were 

distributed, one to four at a time, to each of the unlabeled intermediate beakers (two per 

replicate) containing dilution water until each beaker contained four mysids. This process was 

then repeated five more times until each intermediate beaker contained 20 mysids. Following 

distribution, the mysids in each intermediate beaker were then transferred to a labeled retention 

chamber. The exposure was initiated when each growth and reproduction chamber was added to 

the respective exposure tank.

In addition on Day 0, forty-five mysids were collected from the population used to initiate the 

exposure. These mysids were separated into groups of five, euthanized in ice-cold de-ionized 

water, blotted dry and measured for total length to the nearest 0.1 mm. Following length 

measurements, each group was placed on pre-weighed aluminum tins and dried in an oven at 

approximately 100 oC for approximately 39 hours. Dried mysid samples were kept in a 

desiccator for at least 30 minutes (to allow for cooling of the sample) prior to weighing to the 

nearest 0.01 mg on an analytical balance.
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3.1.2 F0 Growth Chambers

At two time intervals (Day 7 and Day 14), mysids were sampled from each growth chamber. On 

Day 7, six mysids were removed from the growth chamber, separated into three groups of two 

mysids, euthanized in ice-cold reverse-osmosis water, blotted dry and measured for total length 

to the nearest 0.1 mm. Following length measurements, each group was transferred to a preweighed aluminum tin and dried in an oven at approximately 100 oC for approximately 24 hours. 

On Day 14, the remaining mysids – those not sampled at Day 7 and those not used to complete 

reproduction pairings – were removed, euthanized in ice-cold reverse-osmosis water, blotted dry 

and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 

100 oC for approximately 24 hours. Mysids on Day 14 were identified as male, female or 

immature organisms. Dried mysid samples were kept in a desiccator for at least 30 minutes prior 

to weighing to the nearest 0.01 mg on an analytical balance.

In addition, total length and dried body weight were measured from mature mysids housed 

initially in reproductive chambers then transferred to brood chambers at termination.

3.1.3 F0 Reproductive Chambers

When developing embryos were discerned within the majority of females in the reproduction 

chambers (Day 14 in this study), an ovigerous female was paired with one male and the pair was 

isolated in a separate brood chamber within that replicate chamber for that specific exposure 

condition (Figure 1). Mysids that were not paired were transferred to a separate brood chamber 

within the respective replicate test vessel. Remaining male mysids (not used in the initial pairing 

of mysids) were held in a separate brood chamber and used to replace male mysids that died in 

brood chambers during the reproduction phase of the exposure. Unused mysids from the growth 

retention chambers were used to complete the male: female pairings in the event that the sex 
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ratio in the reproduction chamber was skewed preventing obtaining seven pairs. The F0

exposure terminated at least seven days after the median day of release of the second brood (F0”) 

in the negative control treatment group. 

Upon termination, each surviving F0 mysid was removed from the reproduction chambers, 

euthanized in ice-cold reverse-osmosis water, blotted dry, sexually identified as male or female 

and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 

100 oC for approximately 24 hours. Dried mysid samples were kept in a desiccator for at least 

30 minutes prior to weighing to the nearest 0.01 mg on an analytical balance.

Offspring of the F0 mysids (< 24 hours old) were collected and transferred to nylon screen 

chambers in a separate test system at the same test substance concentrations to which they were 

exposed during the F0 generation. 

3.1.4 Feeding

The mysids were fed daily hatched (24 or 48 hour old) brine shrimp (Artemia sp.) nauplii at the 

recommended densities described in section 2.3.

3.2 Exposed Second Generation Mysids (F1)

3.2.1 Initiation of F1 Exposure

Groups of < 24-hour old offspring (F1) from either the first (F1’) or second brood (F1”) of the F0

mysids were isolated in reproduction chambers (as previously described), and placed in the same 

treatment group for the respective exposure conditions from which they originated. Mixing of 

young released on the same day and across replicate chambers from the same treatment or 

control group was conducted to minimize reproduction between siblings. In addition, groups of 

Day 0 F1 mysids were indiscriminately assigned to growth chambers within the exposure vessels 
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of the same exposure treatment from which they originated, if available. Priority was given to 

filling the reproduction retention chambers first, and then the growth chambers.

There were not enough of F1 mysids available to conduct the Day 0 mysid growth measurements 

for the F1 generation.

3.2.2 F1 Growth Chambers

At two time intervals (Day 7 and Day 14), mysids were sampled from each growth chamber, 

when available. On Day 7, up to six mysids were removed from the growth chamber, separated 

into three groups of two mysids, euthanized in ice-cold reverse-osmosis water, blotted dry and

measured for total length to the nearest 0.1 mm. Following length measurements, each group 

was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 100 oC for 

approximately 24 hours. 

On Day 14, the remaining mysids – those not sampled at Day 7 and those not used to complete 

reproduction pairings – when available, were removed euthanized in ice-cold reverse-osmosis 

water, blotted dry and measured for total length to the nearest 0.1 mm. Following length 

measurements, each mysid was transferred to a pre-weighed aluminum tin and dried in an oven 

at 100 oC for 24 hours. Mysids on Day 14 were identified as male, female or immature 

organisms. Dried mysid samples were kept in a desiccator for at least 30 minutes prior to 

weighing to the nearest 0.01 mg on an analytical balance.

In addition, total length and dried body weight were measured from mature mysids housed 

initially in reproductive chambers then transferred to brood chambers at termination.

3.2.3 F1 Reproductive Chambers

When developing embryos were discerned within the majority of individual females in the 

reproduction chambers (Day 14 in this study), an ovigerous female was paired with one male and 
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the pair was isolated in a separate brood chamber within that replicate chamber for that specific 

exposure condition. Mysids that were not paired were transferred to a separate brood chamber 

within the respective replicate test vessel. Remaining male mysids (not used in the initial pairing 

of mysids) were held in a separate brood chamber and used to replace male mysids that died in 

brood chambers during the reproduction phase of the exposure. Unused mysids from the growth 

retention chambers were used to complete the male: female pairings in the event that the sex 

ratio in the reproduction chamber was skewed preventing obtaining seven pairs. The F1

exposure terminated on Reproductive Chamber Day 35 or 36. 

Upon termination, each surviving F1 mysid was removed from the reproduction chambers, 

euthanized in ice-cold reverse-osmosis water, blotted dry, sexually identified as male or female 

and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 

100 oC for approximately 24 hours. Dried mysid samples were kept in a desiccator for at least 

30 minutes prior to weighing to the nearest 0.01 mg on an analytical balance.

3.2.4 Feeding

The mysids were fed newly hatched (24 or 48 hour old) brine shrimp (Artemia sp.) nauplii at the

recommended densities described in section 2.3.

3.3 Chamber Cleaning

Dead brine shrimp and other debris in the retention chambers and brood chambers were removed 

daily. Based on daily observations of water flow through the screens of these containers, mysids 

were transferred to clean retention chambers or brood chambers twice a week to prevent water 

flow restriction.
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3.4 Conditions of Exposure 

Offspring of the F1 mysids were enumerated daily until 7 days after the median day of release of 

the second brood, at which time the test was terminated. The study length, measured from the F0

initiation to the F1 termination, was 77 days.

4.0 OBSERVATION AND MEASUREMENTS

4.1 Biological Observations

At the start of the test and daily thereafter, observations of stress, abnormal behavior (including 

discoloration, lethargy, immobilization, erratic swimming, and inability to maintain position in 

the water column), differences in food consumption, and mortality were made. Dead mysids 

were recorded and removed from the test vessels at these intervals. Mysids which were missing 

were considered dead. The criterion for death was absence of mobility and failure to respond to 

gentle prodding. Survival was based on the number of mysids initially present in each test 

vessel. Daily assessment of survival was necessary to provide a basis for expression and 

interpretation of reproductive output, that is, number of offspring/female/day.

The primary observations made during the test included:

• Survival (F0, F1) 

• Growth, weight to the nearest 0.01 mg and length to the nearest 0.1 mm (Days 0, 

7 and 14 for F0 and F1, when possible)

• Time to first brood release, number of days after exposure initiation to release of 

young in the first brood (F0, F1, when possible)

• Time to second brood release, number of days after exposure initiation to release 

of young in the second brood (F0, F1, when possible)

• Interbrood duration, time between release of first brood and release of second 

brood (F0, F1, when possible)

• Number of young per female in each of the first two broods (F0, F1, when 

possible)

• Total number of young in the first two broods in each replicate population (F0, F1, 

when possible)

• Sex ratio (F0, F1)

• Percentage of females that are reproductively active (F0, F1)
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5.0 STATISTICAL METHODS

5.1 Endpoints

The endpoints used for determination of significant effects by statistical evaluation are outlined 

below: 

• Survival, (F0, F1)

• Growth - length and dry weight, (Days 0, 7 and 14 for F0 and F1)

• Time to maturation, (F0, F1)

• Time to first brood release, (F0, F1)

• Time to second brood release, (F0, F1)

• Interbrood duration, (F0, F1)

• Number of young per female in each of the first two broods, (F0, F1)

• Total number of young in the first two broods in each replicate population (F0, F1)

• Sex ratio, (F0, F1)

• Percentage of females that are reproductively active, (F0, F1)

5.2 Statistical Methods

Survival data, sex ratio and females that were reproductively active were considered to be 

discrete-variable data, while the remaining reproduction endpoints and growth data were 

considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and 

Fisher’s Exact tests to identify treatment groups that showed a statistically significant difference 

from the negative control (p ≤ 0.05). All continuous-variable data were evaluated for normality 

using the Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (p = 0.01). 

The data for all parameters passed the assumptions of normality and homogeneity of variance, 

with the exception of F1 generation time to maturation, F1 generation time to second brood 

release, and F1 generation interbrood duration. The data was not conducive to transformation to 

correct the condition. Therefore, the Nonparametric Wilcoxon rank-sum test was used for 

comparisons to the control for data that did not conform to assumptions of parametric tests. For 

the parameters that passed the assumptions of normality and homogeneity of variances, those 

treatment means that were significantly different from the negative control means were identified 

using Dunnett’s test (p ≤ 0.05). Survival and percent of reproducing females were tested using 

Fisher’s Exact test (p ≤ 0.05). Statistically significant treatment effects were used to define the 
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Lowest-Observed-Effect-Concentration (LOEC; the lowest test concentration with statistically 

significant differences from controls) and the No-Observed-Effect-Concentration (NOEC; and 

the highest test concentration that showed no statistically significant difference from the control). 

All statistical analyses were conducted using SAS software and a personal computer.

6.0 RESULTS 

Measurements of temperature, dissolved oxygen, pH, light intensity and salinity in the test 

chambers are summarized for F0 in Table 1 and for F1 in Table 2. Water temperatures were within 

the 26 ± 1°C range established for the test, except for one instance on Day 47 during the F1

generation when the temperature fell to 24.1°C in replicate B in the 32 ng/L treatment group. 

Dissolved oxygen concentrations were at 100% of air saturation at test initiation and remained 

≥85% of saturation (6.1 mg/L) throughout the exposure period for both the F0 and F1 generations 

tested. Measurements of pH ranged from 7.9 to 8.6, and salinity ranged from 19 to 21‰ during 

the test. Light intensity at test initiation was 394 lux for the F0 generation and 326 lux for the F1

generation, measured at the surface of the water of one representative test chamber. Also, on 

Day 0 of the study, light intensity measured at the surface of each test vessel ranged from 190 

to 308 lux for the F0 generation and 238 to 367 lux for the F1 generation.

6.1 In-life Analytical Results

6.1.1 F0 Generation

Test solutions were premixed in mixing chambers and then delivered into tanks. Samples used 

for analytical measurements to determine mean measured concentrations were only collected 

from tanks. The appearance of test solutions in the mixing chambers and test chambers appeared 

clear and colorless during the test, with no evidence of precipitation observed in any control or 

treatment solution. 
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Lindane concentrations measured in pretest diluter verification samples ranged from 

approximately 81.0 to 123% of nominal (Table 3). Lindane concentrations measured in stock 

solutions prior to test initiation ranged from approximately 75.3 to 87.4% of nominal (Table 4). 

Lindane concentrations in the test solution samples collected during the test ranged from 

approximately 68 to 102% in the four highest test concentrations and from 93 to 137% of 

nominal in the lowest test concentration, which was near the limit of the analytical method 

(Table 5). When measured concentrations of the samples collected during the test were 

averaged, the mean measured test concentrations for this study were 36, 73, 170, 400 and 

930 ng/L, representing 113, 91, 85, 80 and 74% of nominal concentrations, respectively 

(Figure 2). The results of the F0 generation were based on nominal concentrations.

6.1.2 F1 Generation

Test solutions were premixed in mixing chambers and then delivered into tanks. Samples used 

for analytical measurements to determine mean measured concentrations were only collected 

from tanks. The appearance of test solutions in the mixing chambers and test chambers appeared 

clear and colorless during the test, with no evidence of precipitation observed in any control or 

treatment solution. 

Results of analyses to measure concentrations of Lindane in the pretest diluter verification

samples are presented in Table 6. Measured concentrations of the samples ranged from 

approximately 73.9 to 108% of nominal. Results of analyses to measure concentrations of 

Lindane in the stock solution samples collected prior to test initiation for the highest test 

concentration to confirm mixing procedures are presented in Table 7. Results of analyses to 

measure concentrations of Lindane in the test solution samples collected during the test are 

presented in Table 8. Lindane concentrations ranged from approximately 67 to 107% in the four 

highest test concentrations and from 108 to 211% of nominal in the lowest test concentration,

which was near the limit of the analytical method. When measured concentrations of the 

samples collected during the test were averaged, the mean measured test concentrations for this 
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study were 47, 76, 170, 380 and 910 ng/L, representing 147, 95, 85, 76 and 73% of nominal 

concentrations, respectively (Figure 3). The results of the F1 generation also were based on the 

nominal concentrations.

6.2 Biological Results

Table 9 through Table 19 present endpoint summaries of the total means, standard deviations 

(SD) and coefficients of variability (CV) for the data collected during the study. Data from 

individual animals and replicates are summarized in Appendix 5 to Appendix 25.

6.3 Survival

6.3.1 Survival from Day 0 to Day 7

A summary of survival of F0 juvenile mysids from test initiation to the Day 7 growth subsampling interval is located in Table 9. After 7 days of exposure, survival in the negative control 

(untreated filtered saltwater) and in the 32, 80, 200, 500 and 1250 ng/L treatment groups was 

96.7, 100, 95.8, 91.7, 95.8 and 99.2%, respectively. Fisher’s Exact test indicated there were no 

statistically significant decreases in survival in any treatment group when compared to the 

negative control (p > 0.05). 

A summary of survival of F1 juvenile mysids from test initiation to the Day 7 growth subsampling interval is located in Table 10. After 7 days of exposure, survival in the negative 

control and in the 32, 80, 200, 500 and 1250 ng/L treatment groups was 85.0, 95.2, 89.0, 73.9, 

92.0 and 83.3%, respectively. Fisher’s Exact test indicated there were no statistically significant 

decreases in survival in any treatment group when compared to the negative control (p > 0.05). 

6.3.2 Survival from Day 8 to Day 14

A summary of survival of F0 juvenile mysids from Day 8 to the Day 14 growth sub-sampling 

interval is located in Table 9. Survival in the negative control and in the 32, 80, 200, 500 and 
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1250 ng/L treatment groups was 96.0, 98.8, 92.7, 92.2, 78.8 and 98.0%, respectively. Fisher’s 

Exact test indicated there were no statistically significant decreases in survival in any of the 

treatment groups when compared to the negative control (p > 0.05), except in the 500 ng/L 

treatment group. While statistically significant (p ≤ 0.05), the 18% decrease in survival in the 

500 ng/L treatment group was not considered to be treatment related, since the decrease was within 

control performance criteria and not dose-responsive. 

A summary of survival of F1 juvenile mysids from test Day 8 to Day 14 growth sub-sampling 

interval is located in Table 10. Survival in the negative control and in the 32, 80, 200, 500 and 

1250 ng/L treatment groups was 88.9, 92.8, 96.3, 100, 82.2 and 100%, respectively. Fisher’s 

Exact test indicated there were no statistically significant decreases in survival between any of the 

treatment groups when compared to the negative control (p > 0.05).

6.3.3 Survival from Day 15 to Test Termination

A summary of survival of F0 adult mysids after pairing (from Day 15 to test termination on 

Day 44) is presented in Table 9. Survival in the negative control and in the 32, 80, 200, 500 and 

1250 ng/L treatment groups was 84.7, 79.0, 73.4, 81.8, 80.2 and 81.3%, respectively. Fisher’s 

Exact test indicated there were no statistically significant decreases in survival in any treatment 

group when compared to the negative control (p > 0.05). Daily survival by gender and as a 

percentage is presented in Appendix 5.

A summary of survival of F1 adult mysids after pairing (from Day 15 to test termination on 

Day 35) is presented in Table 10. Survival in the negative control and in the 32, 80, 200, 500 

and 1250 ng/L treatment groups was 100, 76.7, 80.0, 88.0, 50.0 and 100%, respectively. The 

variability in survival values were attributed to small numbers of live young that could be used in 

F1 generation. Fisher’s Exact test indicated there were no statistically significant decreases in 

survival in any treatment group when compared to the negative control (p > 0.05). Daily survival 

by gender and as a percentage is presented in Appendix 6.
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6.4 Growth

Mysid size was measured at four time points (Days 0, 7, 14 and 44) during the F0 generation and 

three time points (Days 7, 14 and 35) for the F1 generation from the three test replicates. Mysid 

dry weight and mysid total body length were measured on days 0, 7 and 14 from the additional 

growth chamber that was subsampled for growth measurements. This was done for the F0

generation and the F1 generation when possible. There were not always enough mysids available 

in the F1 to fill the reproductive retention chambers and the retention chamber used to monitor 

growth. Priority was given to reproduction chambers. There were insufficient numbers of 

mysids in growth replicates to collect growth data. For those started, there were only enough 

mysids to collect growth data at termination and not on Days 0, 7, and 14.

Summaries of the total lengths and dry weights of mysids throughout the study are presented in 

Table 11 to Table 17. Individual animals and their replicate mean measurements of total lengths 

and dry weights are provided in Appendix 7 to Appendix 19.

6.4.1 Day 0 (less than 24 Hours old) – Dry Weight and Total Length from F0 and F1

Generations

A representative sample of 45 Day 0 mysids was collected from the same pool of organisms used 

to initiate the test. The mean total lengths and dry weights of Day 0 mysids from the F0

generation are presented in Table 11, with measurements for individual test replicates presented 

in Appendix 7. The mean total length and dry weight of 45 Day 0 mysids for the F0 generation 

were 2.20 mm and 0.017 mg, respectively. There were insufficient mysids produced on any 

given day to initiate a separate test replicate to monitor growth and determine Day 0 total length 

and dry weight measurements in the F1 generation.

6.4.2 Day 7 - Dry Weight and Total Length from F0 and F1 Generations

Summaries of the Day 7 total lengths and dry weights of surviving juvenile mysids in the 

treatment and control groups for the F0 and F1 generations are presented in Table 12 and 
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Table 13, with replicate mean measurements of total lengths and dry weights presented in 

Appendix 8 to Appendix 11, respectively. 

Sex could not be determined for Day 7 mysids so the data represent all juveniles. The mean total 

length of the Day 7 mysids in the negative control, 32, 80, 200, 500 and 1250 ng/L treatment 

groups from the F0 generation was 4.44, 5.10, 4.75, 4.61, 5.18 and 4.76 mm, respectively, while 

the corresponding mean dry weights were 0.130, 0.213, 0.193, 0.191, 0.216 and 0.194 mg, 

respectively. Dunnett’s test indicated there were no statistically significant decreases in mean total 

length or dry weight for the Day 7 mysids from the F0 generation in any of the treatment groups 

(p > 0.05) (Table 12). 

Results for the F1 generation were similar to the F0 generation. The mean total length of the 

Day 7 mysids in the F1 generation in the negative control, 32, 80, 200 and 500 ng/L treatment 

groups was 5.98, 5.72, 5.46, 5.70 and 5.70 mm, respectively, while the corresponding mean dry 

weights were 0.308, 0.437, 0.291, 0.515 and 0.336 mg, respectively. Dunnett’s test indicated 

there were no statistically significant decreases in mean total length or dry weight for the Day 7 

mysids from the F1 generation in any of the treatment groups (p > 0.05) (Table 13). Because of 

low production of offspring in the two highest treatment levels in the F0 generation, there were 

not enough F1 mysids to set aside mysids for growth determinations.

6.4.3 Day 14 – Male and Female Dry Weight and Total Length from F0 and F1

Generations

Summaries of the Day 14 total lengths and dry weights of surviving male and female mysids in 

the treatment and control groups for the F0 and F1 generations are presented in Table 14 and 

Table 15, with replicate mean measurements of total lengths and dry weights presented in 

Appendix 12 to Appendix 15. Immature mysids present in the growth chambers were measured 

for length and weight and are presented in Appendix 12 to Appendix 15, but excluded from the 

statistical analysis of male and female growth measurements. 
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6.4.3.1 Day 14 – Male Dry Weight and Total Length from F0 and F1 Generations

The mean total length of the Day 14 male mysids in the F0 generation in the negative control, 32, 

80, 200, 500 and 1250 ng/L treatment groups was 6.05, 6.80, 6.57, 6.47, 7.33 and 6.37 mm, 

respectively, while the corresponding mean dry weights of males were 0.403, 0.498, 0.474, 0.457, 

0.600 and 0.483 mg, respectively. Dunnett’s test indicated there were no statistically significant

decreases in mean total length or dry weight for the Day 14 mysids from the F0 generation in any 

of the treatment groups (p > 0.05) (Table 14). 

The mean total length of the Day 14 male mysids in the F1 generation in the 32 and 80 ng/L 

treatment groups was 7.28 and 7.23 mm, respectively. The mean dry weight of males in the 32

and 80 ng/L treatment groups was 0.699 and 0.698 mg, respectively. These results were 

consistent with the Day 14 growth observed in the F0 generation. F0 control mysids did not 

produce enough live mysids to initiate as many F1 generation brood chambers as was needed. 

Due to low numbers of live young in the negative control and the 200, 500 and 1250 ng/L 

treatment groups in the F0 generation, there were not enough F1 mysids to set aside mysids for 

growth determinations (Table 15). 

6.4.3.2 Day 14 – Female Dry Weight and Total Length from F0 and F1 Generations

The mean total length of the Day 14 female mysids in the F0 generation in the negative control, 32, 

80, 200, 500 and 1250 ng/L treatment groups was 6.22, 6.73, 6.53, 6.60, 6.79 and 6.64 mm, 

respectively, while the corresponding mean dry weights of females in the 32, 80, 200, 500 and 

1250 ng/L treatment groups was 0.488, 0.562, 0.503, 0.527, 0.533 and 0.593 mg, respectively. 

Dunnett’s test indicated there were no statistically significant decreases in mean total length or dry 

weight for the Day 14 mysids from the F0 generation in any of the treatment groups (p > 0.05)

(Table 14). 

The mean total length of the Day 14 female mysids in the F1 generation in the 32 and 80 ng/L 

treatment groups was 7.06 and 7.55 mm, respectively. The mean dry weight of males in the 32 
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and 80 ng/L treatment groups was 0.680 and 1.095 mg, respectively. Because of low production 

of offspring in the negative control and the 200, 500 and 1250 ng/L treatment groups in the F0

generation, there were not enough F1 mysids to set aside mysids for growth determinations. 

Male and female mysids from the F1 generation on Day 14 were, overall, larger than the F0

generation in both total length and dry weight (Table 15).

6.4.4 Test Termination– Dry Weight and Total Length from F0 (Day 44) and F1 (Day 35) 

Generations

Summaries of total lengths and dry weights of surviving male and female mysids in the treatment 

and control groups at test termination for the F0 (Day 44) and F1 (Day 35) generations are 

presented in Table 16 and Table 17, with replicate mean measurements of total lengths and dry 

weights presented in Appendix 16 to Appendix 19. 

6.4.4.1 Males at Test Termination from F0 (Day 44) and F1 (Day 35) Generations

The mean total length of the Day 44 male mysids in the F0 generation in the negative control, 32, 

80, 200, 500 and 1250 ng/L treatment groups at test termination was 8.96, 8.95, 8.97, 8.93, 9.09 

and 8.91 mm, respectively, while the corresponding mean dry weights were 1.11, 1.24, 1.27, 1.26, 

1.28 and 1.19 mg, respectively. Dunnett’s test indicated there were no statistically significant

decreases in mean total length or dry weight in males from the F0 generation in any of the 

treatment groups at test termination (Day 44) (p > 0.05) presented in Table 16. 

The mean total length of the Day 44 male mysids in the F1 generation in the negative control, 32, 

80, 200, 500 and 1250 ng/L treatment groups at test termination was 8.93, 8.78, 9.02, 9.22, 9.50 

and 8.40 mm, respectively, while the corresponding mean dry weights were 1.06, 1.14, 1.15, 1.15, 

1.19 and 0.830 mg, respectively. The lower value in the high treatment group may have been the 

result of the reduced number of replicates for that group. Dunnett’s test indicated there were no 

statistically significant decreases in mean total length or dry weight in males from the F1

generation in any of the treatment groups at test termination (Day 35) (p > 0.05) presented in 
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Table 17. In general, the male mysids were similar in length and weight between the F0 and F1

generations, even though there was a 12-day difference in age at termination of the two 

generations.

6.4.4.2 Females at Test Termination from F0 (Day 44) and F1 (Day 35) Generations

The mean total length of the Day 44 female mysids in the F0 generation in the negative control, 32, 

80, 200, 500 and 1250 ng/L treatment groups was 9.29, 9.29, 9.28, 9.35, 9.30 and 9.20 mm, 

respectively, while the corresponding mean dry weights were 1.32, 1.46, 1.54, 1.58, 1.45 and 

1.50 mg, respectively. Dunnett’s test indicated there were no statistically significant decreases in 

mean total length or dry weight in females for the Day 44 mysids from the F0 generation in any of 

the treatment groups (p > 0.05) presented in Table 16. 

The mean total length of the Day 44 female mysids in the F1 generation in the negative control, 32, 

80, 200, 500 and 1250 ng/L treatment groups was 9.28, 8.87, 9.13, 9.47, 9.92 and 9.40 mm, 

respectively, while the corresponding mean dry weights were 1.18, 1.23, 1.37, 1.28, 1.30 and 

1.40 mg, respectively. Dunnett’s test indicated there were no statistically significant decreases in 

mean total length or dry weight in males from the F1 generation in any of the treatment groups at 

test termination (Day 35) (p > 0.05) presented in Table 17. In general, at test termination female 

mysids in the F0 generation were slightly heavier than females from the F1 generation, but they 

were also 12-days older than the F1 females.

6.5 Reproduction

Mysids in both generations were evaluated for several reproductive endpoints, including time to 

maturation, time to first brood and second brood, interbrood duration, number of offspring per 

reproducing female in the first and second broods, total offspring produced, sex ratio and the 

percent of females that produced offspring. 
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Summaries of the reproductive endpoints for the mysids throughout the study are presented in 

Table 18 and Table 19. Individual animals and their replicate means for each endpoint are 

provided in Appendix 20 to Appendix 25.

6.5.1 Time to Maturation from F0 and F1 Generations

There were no significant differences in mean number of days to maturation, days to 1st brood, 

days to second brood, or mean interbrood duration for any treatment group in the F0 and F1

generations (Table 18 and Table 19). Summaries of the mean time to maturation, number of 

days to first and second brood, and interbrood duration in the negative control and treatment 

groups for the F0 and F1 generations are presented in Table 18 and Table 19, respectively. The 

replicate mean time to maturation is presented in Appendix 20 and Appendix 21. There were no 

young in the 1250 ng/L treatment group of the F1 generation, therefore this level was excluded 

from statistical analysis of the F1 generation reproduction data.

6.5.2 Number of Offspring in the First Brood from F0 and F1 Generations

Summaries of the mean number of offspring in the first brood per reproductive female in the 

negative control and treatment groups for the F0 and F1 generations are presented in Table 18 and 

Table 19, respectively. The replicate mean number of offspring in the first brood is presented in 

Appendix 22 and Appendix 23. 

The mean number of offspring in the first brood for the F0 generation in the negative control and 

the 32, 80, 200, 500 and 1250 ng/L treatment groups was 4.0, 3.1, 3.2, 2.4, 2.4 and 2.4 young per 

reproducing female, respectively. Dunnett’s test indicated there were no statistically significant

decreases in mean number of offspring in the first brood for the mysids from the F0 generation in 

any of the treatment groups (p > 0.05) (Table 18). 

The mean number of offspring for the F1 generation in the negative control and the 32, 80, 200 

and 500 ng/L treatment groups was 3.5, 2.2, 6.0, 1.9 and 2.5 young per reproductive female, 
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respectively. The 1250 ng/L treatment group never produced young, therefore this level was 

excluded from statistical analysis. Dunnett’s test indicated there were no statistically significant

decreases in mean number of offspring in the first brood for the mysids from the F1 generation in 

any of the treatment groups (p > 0.05) (Table 19). 

6.5.3 Number of Offspring in the Second Brood from F0 and F1 Generations

Summaries of the mean number of offspring in the second brood per reproductive female in the 

negative control and treatment groups for the F0 and F1 generations are presented in Table 18 and 

Table 19, respectively. The replicate mean number of offspring in the second brood is presented 

in Appendix 22 and Appendix 23.

The mean number of offspring in the second brood for the F0 generation in the negative control 

and the 32, 80, 200, 500 and 1250 ng/L treatment groups was 4.2, 3.5, 2.1, 2.4, 3.5 and 1.3 

young per reproductive female, respectively. Dunnett’s test indicated there were no statistically 

significant decreases in the number of offspring in the second brood per reproductive female in 

any of the treatment groups with the exception of the decrease in the 1250 ng/L treatment group

(p ≤ 0.05) (Table 18), which appeared to be treatment related. 

The mean number of offspring in the second brood for the F1 generation in the negative control 

and the 32, 80, 200 and 500 ng/L treatment groups was 3.5, 1.4, 0.2, 1.0 and 1.0, respectively. 

The 1250 ng/L treatment group never produced young, therefore this level was excluded from 

statistical analysis. Dunnett’s test indicated there were no statistically significant decreases in 

mean number of offspring in the second brood for the mysids from the F1 generation in any of the 

treatment groups (p > 0.05) (Table 19). 

6.5.4 Total Number of Offspring from F0 and F1 Generations

Summaries of the mean total number of offspring produced in the study in the negative control 

and treatment groups for the F0 and F1 generations are presented in Table 18 and Table 19, 
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respectively. The replicate mean total number of offspring produced in the study for both 

generations is presented in Appendix 22 and Appendix 23.

The mean total number of offspring produced in the study for the F0 generation in the negative 

control and the 32, 80, 200, 500 and 1250 ng/L treatment groups was 71.7, 71.7, 61.3, 58.7, 45.3 

and 16.0, respectively. Although highly variable, Dunnett’s test indicated there were no 

statistically significant decreases in the mean total number of offspring produced in any of the 

treatment groups with the exception of the decrease in the 1250 ng/L treatment group (p ≤ 0.05) 

(Table 18), which was considered to be treatment related. 

The mean total number of offspring produced in the study for the F1 generation in the negative 

control and the 32, 80, 200 and 500 ng/L treatment groups was 11.0, 9.7, 16.3, 8.3 and 3.5, 

respectively. The 1250 ng/L treatment group never produced young, therefore this level was 

excluded from statistical analysis. Dunnett’s test indicated there were no statistically significant

decreases in mean total number of offspring produced in the study for the mysids from the F1

generation in any of the treatment groups (p > 0.05) (Table 19). However, the dramatic reduction 

in the 500 ng/L treatment group that may have been treatment related, was attributed to a small 

sample size and therefore was not considered to be treatment related.

6.5.5 Sex Ratio (Male/Female) from F0 and F1 Generations

Summaries of the mean percent of males present (used to determine the sex ratio in the study) in 

the negative control and treatment groups for the F0 and F1 generations are presented in Table 18

and Table 19, respectively. The replicate mean percent male sex ratio in the study for both 

generations is presented in Appendix 24 and Appendix 25.

The mean percent of males present in the study for the F0 generation in the negative control and 

the 32, 80, 200, 500 and 1250 ng/L treatment groups was 50.3, 59.9, 43.9, 43.6, 52.2 and 44.1%, 

respectively. Dunnett’s test indicated there were no statistically significant decreases in the mean 
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percent male sex ratio in the study for the mysids from the F0 generation in any of the treatment 

groups (p > 0.05) (Table 18). 

The mean percent of males present for the F1 generation in the negative control and the 32, 80, 

200, 500 and 1250 ng/L treatment groups was 30.0, 40.5, 62.3, 45.8, 12.5 and 50.0%, 

respectively. There were inadequate sample sizes in the F1 generation to generate reliable 

percentages of males to perform statistical analyses (Table 19).

6.5.6 Percent of Reproducing Females from F0 and F1 Generations

Summaries of the mean percent of reproducing females in the study in the negative control and 

treatment groups for the F0 and F1 generations is presented in Table 18 to Table 19, respectively. 

The replicate mean percent of reproducing females in the study for both generations is presented 

in Appendix 24 and Appendix 25.

The mean percent of reproducing females in the study for the F0 generation in the negative 

control and the 32, 80, 200, 500 and 1250 ng/L treatment groups was 90.5, 90.5, 95.2, 100, 85.7 

and 47.6, respectively. Dunnett’s test indicated there was a statistically significant decrease in the 

percent of females producing young in the 1250 ng/L treatment group when compared to the 

negative control (p ≤ 0.05), which was considered to be treatment related (p > 0.05) (Table 18).

The mean percent of reproducing females in the study for the F1 generation in the negative 

control and the 32, 80, 200 and 500 ng/L treatment groups was 50.0, 53.3, 68.3, 77.8 and 100%, 

respectively. The 1250 ng/L treatment group never produced young, therefore this level was 

excluded from statistical analysis. Dunnett’s test indicated there were no statistically significant

decreases in the mean percent of reproducing females for the mysids from the F1 generation in any 

of the treatment groups (p > 0.05) (Table 19). 
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7.0 PERFORMANCE CRITERIA

The following criteria are for judging test data acceptability:

7.1 Water quality characteristics should remain within the tolerance limits 

illustrated in Tables 1 and 2 of the Protocol.

The water quality characteristics remained within the tolerance limits defined in the protocol 

throughout the study. The only exception was one instance on Day 47 during the F1 generation 

when the temperature fell to 24.1°C in replicate B in the 32 ng/L treatment group.

7.2 There should be more than 70% survival of control animals in all test phases 

over the duration of the chemical exposure.

Survival exceeded 70% of control animals in all test phases over the duration of the chemical 

exposure.

7.3 At least 75% of the females in the F0 and F1 controls must produce young. 

More than 75% of females in the F0 generation produced young. For the F1 generation, 50% of 

females produced young, however, in two of the treatment groups there was more than 75% of 

the females that produced young.

7.4 The average number of young produced per control female in the first two 

broods is at least eight. 

The average combined total number of young in the first two broods was greater than eight per 

control female in the F0 generation. While the F1 generation did not produce at least eight per 

female, seven were produced per female, and so only missed this criterion by 13%.
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8.0 CONCLUSIONS

Saltwater mysids (Americamysis bahia) were exposed to Lindane at nominal concentrations of 

32 to 1250 ng/L for the F0 and F1 generations. The test was conducted under flow-through 

conditions for 44 and 35 days for the F0 and F1 generations, respectively, and the mysids were 

evaluated for survival, growth and reproductive endpoints. The most sensitive biological 

endpoints were reproduction variables in the F0 generation. There was a treatment related and 

statistically significant decrease in mean number of offspring in the second brood per 

reproductive female, mean total number of offspring and mean percent of females reproducing in 

the 1250 ng/L treatment group in the F0 generation. Consequently, the NOEC, based on 

reproduction for the F0 generation, was 500 ng/L. The LOEC was 1250 ng/L and the MATC was 

calculated to be 791 ng/L.
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109-116. 
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Nimmo DR, Hamaker TL and Sommers CA. 1978. Entire life cycle toxicity test using mysids 

(Mysidopsis bahia) in flowing water. In: Bioassay Procedures for the Ocean Disposal 

Permit Program, EPA-600/9-78-010, U.S. Environmental Protection Agency, 

Environmental Research Laboratory, Gulf Breeze, FL, pp. 64-68.

OECD. 1998. OECD Series on Principles of Good Laboratory Practice and Compliance 

Monitoring. Number 1. OECD Principles on Good Laboratory Practice (as revised in 

1997). Environment Directorate Chemicals Group and Management Committee. 

ENV/MC/CHEM(98)17. OECD Paris. France. 41 pp.

OECD. 2000. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

Mixtures. Environmental Health and Safety Publications. Series on Testing and 

Assessment. No. 23. 

Reitsema, L.A. and J.M. Neff. 1980. A recirculating seawater system for the laboratory culture 

of Mysidopsis bahia Crustacea: Pericaridae). Estuaries 3(4):321-323.

OECD. 2004. Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance 

to Application. Environmental Health and Safety Publications. Series on Testing and 

Assessment No. 54. 

OECD. 2005. Detailed Review Paper on Aquatic Arthropods in Life-Cycle and TwoGeneration Toxicity Tests. OECD Environmental Health and Safety Publications. Series 

on Testing and Assessment No. 50. 

U.S. EPA. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good 

Laboratory Practices Standards; Final Rule. Office of the Federal Register, National 

Archives and Records Administration. U.S. Government Printing Office, Washington, 

D.C.

U.S. EPA. 1996b. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects 

Guideline, OPPTS 850.1000. Special Consideration for Conducting Aquatic Laboratory 

Studies. "Public Draft". EPA 712-C-96-113. April 1996. U.S. Environmental Protection 

Agency, Washington, D.C.

U.S. EPA. 1997. Office of Waste. Test Methods for Evaluating Solid Waste, 

Physical/Chemical Methods (SW-846). February 2007. U.S. Environmental Protection 

Agency, Washington, D.C.

U.S.EPA. 2002. Short-term methods for estimating the chronic toxicity of effluents and 

receiving waters to marine and estuarine organisms. Third edition. Office of Water, U. S. 

Environmental Protection Agency, Washington, DC 20460. EPA/821/R-02/014.

Williams, D.A. 1971. A test for differences between treatment means when several dose levels 

are compared with a zero dose control. Biometrics 27: 103-117.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 163

Williams, D.A. 1972. A comparison of several dose levels with a zero control. Biometrics 28: 

519-531.

Verslycke TA, Fockedey N, McKenney CL, Jr., Roast, SD, Jones, MB, Mees, J and Janssen, CR. 

2004. Mysid crustaceans as potential test organisms for the evaluation of environmental 

endocrine disruption: a review. Environ Tox Chem 23 (5): IN PRESS.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 164

Table 1. Means and Ranges of Water Quality Measurements Taken During 

the F0 Generation Exposure to Lindane 

Nominal

Concentration

(ng/L)

Mean ± SD and Range of Measured Parameters

Temperature1

(°C)

DO2

(mg/L) pH

Salinity

(‰)

Negative Control 26.0 ± 0.36 7.0 ± 0.20 8.1 ± 0.10 20 ± 0.51

(25.1 – 26.5) (6.4 – 7.2) (7.9 – 8.3) (19 – 21)

32 26.2 ± 0.33 7.0 ± 0.18 8.1 ± 0.09 20 ± 0.41

(25.1 – 26.8) (6.6 – 7.2) (7.9 – 8.3) (19 – 21)

80 26.4 ± 0.34 6.9 ± 0.21 8.1 ± 0.08 20 ± 0.46

(25.5 – 27.0) (6.2 – 7.2) (7.9 – 8.2) (19 – 21)

200 26.2 ± 0.30 6.9 ± 0.20 8.1 ± 0.09 20 ± 0.46

(25.4 – 26.9) (6.3 – 7.2) (7.9 – 8.3) (19 – 21)

500 26.2 ± 0.34 6.9 ± 0.18 8.1 ± 0.10 20 ± 0.46

(25.1 – 26.9) (6.4 – 7.2) (7.9 – 8.3) (19 – 21)

1250 26.4 ± 0.32 6.9 ± 0.19 8.1 ± 0.08 20 ± 0.44

(25.6 – 27.0) (6.4 – 7.2) (7.9 – 8.2) (19 – 21)

1 Temperature monitored continuously during the test ranged from approximately 25 to 27oC, measured to the nearest 1oC. 2 A dissolved oxygen concentration of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 165

Table 2. Means and Ranges of Water Quality Measurements Taken During 

the F1 Generation Exposure to Lindane 

Nominal

Concentration

(ng/L)

Mean ± SD and Range of Measured Parameters

Temperature1

(°C)

DO2

(mg/L) pH

Salinity

(‰)

Negative Control 26.3 ± 0.41 6.9 ± 0.20 8.1 ± 0.08 20 ± 0.30

(24.7 – 27.0) (6.5 – 7.2) (8.0 – 8.3) (20 – 21)

32 26.2 ± 0.57 7.0 ± 0.18 8.1 ± 0.14 20 ± 0.24

(24.1 – 27.1) (6.5 – 7.2) (8.0 – 8.6) (20 – 21)

80 26.4 ± 0.46 6.9 ± 0.19 8.1 ± 0.10 20 ± 0.27

(24.8 – 27.2) (6.5 – 7.2) (8.0 – 8.3) (20 – 21)

200 26.5 ± 0.42 6.9 ± 0.22 8.1 ± 0.09 20 ± 0.33

(24.6 – 27.2) (6.0 – 7.2) (8.0 – 8.3) (20 – 21)

500 26.4 ± 0.38 6.9 ± 0.19 8.1 ± 0.09 20 ± 0.34

(24.7 – 27.1) (6.1 – 7.2) (7.9 – 8.3) (19 – 21)

1250 26.5 ± 0.39 6.9 ± 0.13 8.1 ± 0.09 20 ± 0.33

(25.0 – 27.1) (6.6 – 7.2) (8.0 – 8.3) (20 – 21)

1 Temperature monitored continuously during the test ranged from approximately 25 to 27oC, measured to the nearest 1oC. 2 A dissolved oxygen concentration of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 166

Table 3. Measured Concentrations of Lindane in Pretest Solution Samples 

for the F0 Generation 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-)

Sampling

Time

(Day)

Measured 

Concentration

(μg/L) 1

Percent

of

Nominal2

0.0

(Negative Control)

PT-13

PT-19

-7

-6

< LOQ

< LOQ

--

--

0.032 PT-14

PT-20

-7

-6

0.0372

0.0340

116

106

0.080 PT-15

PT-21

-7

-6

0.0732

0.0724

91.5

90.5

0.200 PT-16

PT-22

-7

-6

0.198

0.200

99.1

99.9

0.500 PT-17

PT-23

-7

-6

0.421

0.405

84.3

81.0

1.25 PT-18

PT-24

-7

-6

1.12

1.54

89.3

123 1 The limit of quantitation (LOQ) was 0.0151 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test 

substance purity (99.6%). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 167

Table 4. Measured Concentrations of Lindane for the F0 Generation in Stock 

Solution Samples 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-)

Sampling

Time

(Day)

Measured 

Concentration

(μg/L) 1

Percent

of

Nominal1

2.1 S-13 -7 1.75 83.2

5.1 S-14 -7 4.46 87.4

13 S-15 -7 9.79 75.3

32 S-16 -7 27.8 86.8

80 S-17 -7 67.5 84.3 1 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 168

Table 5. Measured Concentrations of Lindane in Saltwater Samples for the 

F0 Generation

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-)

Sampling

Time

(Days)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Percent of

Nominal

0.0

(Negative Control)

1

7

13

19

25

31

37

43

0

7

14

15

22

29

36

44

< LOQ

< 0.0442

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

--

--

--

--

--

--

--

--

-- --

0.032 2

8

14

20

26

32

38

44

0

7

14

15

22

29

36

44

0.0340

0.0402

0.0297

0.0317

0.0394

0.0347

0.0363

0.0439

106

126

92.9

99.1

123

108

113

137

0.036 113

0.080 3

9

15

21

27

33

39

45

0

7

14

15

22

29

36

44

0.0739

0.0762

0.0762

0.0671

0.0740

0.0708

0.0642

0.0817

92.0

95.3

95.2

83.9

92.5

88.5

80.3

102

0.073 91

1 The limit of quantitation (LOQ) was 0.0151 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test substance purity 

(99.6%). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 169

Table 5. Measured Concentrations of Lindane in Saltwater Samples for the F0

Generation (Continued)

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-)

Sampling

Time

(Days)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Percent of

Nominal

0.20 4

10

16

22

28

34

40

46

0

7

14

15

22

29

36

44

0.159

0.147

0.194

0.153

0.161

0.156

0.168

0.193

79.5

73.5

96.9

76.6

80.4

77.9

83.8

96.6

0.17 85

0.50 5

11

17

23

29

35

41

47

0

7

14

15

22

29

36

44

0.421

0.442

0.448

0.366

0.385

0.408

0.370

0.387

84.3

88.4

89.5

73.2

77.0

81.6

74.0

77.4

0.40 80

1.25 6

12

18

24

30

36

42

48

0

7

14

15

22

29

36

44

0.992

0.916

1.11

0.847

0.883

0.852

0.863

1.01

79.4

73.3

88.4

67.8

70.7

68.2

69.1

80.9

0.93 74

1 The limit of quantitation (LOQ) was 0.0151 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test substance purity 

(99.6%). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 170

Table 6. Measured Concentrations of Lindane in Pretest Diluter Verification 

Samples for the F1 Generation 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-F1-)

Sampling

Time

(Day)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

0.0

(Negative Control)

PT-1 -2 < LOQ --

0.032 PT-2 -2 0.0347 108

0.080 PT-3 -2 0.0646 80.7

0.20 PT-4 -2 0.161 80.6

0.50 PT-5 -2 0.369 73.9

1.25 PT-6 -2 1.06 84.8

1 The limit of quantitation (LOQ) was 0.0151 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test substance purity 

(99.6%). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 171

Table 7. Measured Concentration of Lindane in F1 Generation Stock 

Solution Samples 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-)

Sampling

Time

(Day)

Measured 

Concentration

(μg/L) 1

Percent

of

Nominal1

2.1 S-F1-1-1 15 1.71 81.4

1 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 172

Table 8. Measured Concentrations of Lindane Saltwater Samples for the F1

Generation 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-F1-)

Sampling

Time

(Day)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Measured

Percent of

Nominal

0.0

(Negative Control)

2

8

14

21

27

33

39

45

2

7

14

23

28

35

42

49

< LOQ

< LOQ

0.0244

0.0340

0.0178

0.0400

0.0186

0.0178

--

--

--

--

--

--

--

--

-- --

0.032 3

9

15

20

22

28

34

40

46

48

2

7

14

15

23

28

35

42

49

54

0.0436

0.0374

0.0675

0.0370

0.0500

0.0346

0.0614

0.0479

0.0419

0.0455

136

117

211

116

156

108

192

150

131

142

0.047 147

0.080 4

10

16

23

29

35

41

47

49

2

7

14

23

28

35

42

49

54

0.0698

0.0643

0.0753

0.0846

0.0707

0.0859

0.0783

0.0759

0.0770

87.2

80.4

94.1

106

88.3

107

97.9

94.8

96.3

0.076 95

0.20 5

11

17

24

30

36

42

2

7

14

23

28

35

42

0.181

0.157

0.172

0.172

0.152

0.201

0.188

90.6

78.3

86.0

86.1

76.1

100

93.9

0.17 85

1 The limit of quantitation (LOQ) was 0.0151 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test substance purity 

(99.6%). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 173

Table 8. Measured Concentrations of Lindane Saltwater Samples for the F1

Generation (Continued)

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-1-F1-)

Sampling

Time

(Day)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Measured

Percent of

Nominal

0.50 1

6

12

18

25

31

37

43

0

2

7

14

23

28

35

42

0.391

0.389

0.351

0.345

0.387

0.350

0.399

0.444

78.2

77.9

70.3

69.0

77.4

70.0

79.7

88.8

0.38 76

1.25 7

13

19

26

32

38

44

2

7

14

23

28

35

42

0.998

0.833

0.855

0.882

0.862

0.911

1.01

79.9

66.7

68.4

70.5

68.9

72.9

80.6

0.91 73

1 The limit of quantitation (LOQ) was 0.0151 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test 

substance purity (99.6%). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 174

Table 9. Summary of F0 Generation Survival of Saltwater Mysids Exposed to 

Lindane

Nominal

Concentration

(ng/L)

Mean Percent Juvenile

Survival to

Lengths

On Day 7 ± SD

(CV%) 1

Mean Percent Juvenile

Survival to

Pairing

On Day 14 ± SD

(CV%) 1

Mean Percent Adult

Survival to

Test Termination

On Day 44 ± SD

(CV%) 1

Negative Control

96.7 ± 5.16

(5.34)

n = 116

96.0 ± 3.32

(3.46)

n = 94

84.7 ± 18.1

(21.4)

n = 47

32

100 ± 0.0

(0.0)

n = 120

98.8 ± 2.92

(2.95)

n = 101

79.0 ± 10.1

(12.8)

n = 49

80

95.8 ± 6.65

(6.93)

n = 115

92.7 ± 7.65

(8.25)

n = 90

73.4 ± 7.61

(10.4)

n = 39

200

91.7 ± 5.16

(5.63)

n = 110

92.2 ± 10.6

(11.5)

n = 86

81.8 ± 18.1

(22.1)

n = 46

500

95.8 ± 4.92

(5.13)

n = 115

78.8 ± 24.5*2

(31.0)

n = 77

80.2 ± 5.77

(7.19)

n = 39

1250

99.2 ± 2.04

(2.06)

n = 119

98.0 ± 3.21

(3.27)

n = 99

81.3 ± 12.7

(15.6)

n = 48

* Statistically significant decrease in survival in comparison to the negative control using Fisher’s Exact test (p ≤ 0.05). 1 For all treatment and control groups, n = number of surviving organisms. 2 While the decrease in survival was statistically significant in comparison to the negative control, it was not considered to be 

treatment-related since the difference was slight, was within control acceptability and was not dose-responsive.
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Wildlife International, Ltd. Study No. 185A-102 Page 175

Table 10. Summary of F1 Generation Survival of Saltwater Mysids Exposed to 

Lindane

Nominal

Concentration

(ng/L)

Mean Percent Juvenile

Survival to

Lengths

On Day 7 ± SD

(CV%) 1,2

Mean Percent Juvenile

Survival to

Pairing

On Day 14 ± SD

(CV%) 1,2

Mean Percent Adult

Survival to

Test Termination

On Day 35 ± SD

(CV%) 1,2

Negative Control

85.0 ± 22.4

(26.3)

n = 14

88.9 ± 19.2

(21.7) 

n = 8

100 ± 0.0

(0.0) 

n = 8

32

95.2 ± 7.2

(7.6) 

n = 60

92.8 ± 7.2

(7.8) 

n = 42

76.7 ± 9.42

(12.3) 

n = 22

80

89.0 ± 15.7

(17.7) 

n = 48

96.3 ± 5.6

(5.8) 

n = 35

80.0 ± 17.6

(22.0) 

n = 22

200

73.9 ± 19.2

(26.0) 

n = 21

100 ± 0.0

(0.0) 

n = 19

88.0 ± 12.5

(14.2) 

n = 16

500

92.0 ± 17.9

(19.4) 

n = 13

82.2 ± 16.8

(20.4) 

n = 7

50.0 ± 50.0

(100) 

n = 5

1250

83.3 ± 23.6

(28.3)

n = 3

100 ± 0.0

(0.0)

n = 3

100 ± 0.0

(0.0)

n = 3

1 For all treatment and control groups, n = number of surviving organisms. 2 There were no statistically significant decreases in survival in comparison to the negative control using Fisher’s Exact test 

(p > 0.05).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 176

Table 11. Summary of Day 0 Growth Measurements of Culture Saltwater 

Mysids

Mean Total Body Length (mm)

± SD (CV%)

Mean Dry Weight (mg)

± SD (CV%)

2.20 ± 0.159

(7.26)

n = 45

0.017 ± 0.039

(22.3)

n = 9
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Wildlife International, Ltd. Study No. 185A-102 Page 177

Table 12. Summary of F0 Generation Day 7 Growth Measurements of 

Saltwater Mysids Exposed to Lindane

Nominal

Concentration

(ng/L)

Growth Parameters on Day 7 1, 2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Negative Control 4.44 ± 0.475

(10.7)

0.130 ± 0.050

(38.5)

32 5.10 ± 0.067

(1.31)

0.213 ± 0.050

(23.5)

80 4.75 ± 0.524

(11.0)

0.193 ± 0.040

(20.7)

200 4.61 ± 0.942

(20.4)

0.191 ± 0.077

(40.3)

500 5.18 ± 0.377

(7.28)

0.216 ± 0.043

(19.9)

1250 4.76 ± 0.211

(4.43)

0.194 ± 0.012

(6.19)

1 For all treatment and control groups tested, statistics were based on replicate means. Therefore, n = 3 for all treatment and 

control groups presented. 2 There were no statistically significant decreases in growth in comparison to the negative control using Dunnett’s test 

(p > 0.05).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 178

Table 13. Summary of F1 Generation Day 7 Growth Measurements of 

Saltwater Mysids Exposed to Lindane

Nominal

Concentration

(ng/L)

Growth Parameters on Day 7 1,2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Negative Control

5.98 ± 0.218

(3.65)

n = 2

0.308 ± 0.081

(26.4) 

n = 2

32

5.72 ± 0.241

(4.21)

n = 3

0.437 ± 0.085

(19.2) 

n = 3

80

5.46 ± 0.802

(14.7)

n = 3

0.291 ± 0.068

(23.4) 

n = 3

200 5.70 3

n = 1

 0.515 3 

n = 1

500

5.70 ± 0.141

(2.48)

n = 2

0.336 ± 0.090

(26.8) 

n = 2

1250 --4 --4

1 For all treatment and control groups tested, statistics were based on replicate means. Therefore, n = the number of 

replicates available. 2 There were no statistically significant decreases in growth in comparison to the negative control using Dunnett’s 

test (p > 0.05). 3 Standard deviation and CV were not determined as only one of the three replicates were available for the treatment 

group. 4 No mysids were available for Day 7 growth measurements due to insufficient numbers of offspring produced by the 

F0 adults.
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Wildlife International, Ltd. Study No. 185A-102 Page 179

Table 14. Summary of F0 Generation Day 14 Growth Measurements of 

Saltwater Mysids Exposed to Lindane

Nominal

Concentration

(ng/L)

Growth Parameters on Day 14 1, 2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Males Females Males Females

Negative Control 6.05 ± 0.519

(8.58)

6.22 ± 0.041

(0.66)

0.403 ± 0.068

(16.9)

0.488 ± 0.017

(3.48)

32 6.80 ± 0.242

(3.56)

6.73 ± 0.121

(1.80)

0.498 ± 0.063

(12.7)

0.562 ± 0.162

(28.8)

80 6.57 ± 0.100

(1.52)

6.53 ± 0.127

(1.94)

0.474 ± 0.029

(6.12)

0.503 ± 0.037

(7.36)

200 6.47 ± 0.865

(13.4)

6.60 ± 0.567

(8.59)

0.457 ± 0.116

(25.4)

0.527 ± 0.165

(31.3)

500 7.33 ± 0.327

(4.46)

6.79 ± 0.497

(7.32)

0.600 ± 0.117

(19.5)

0.533 ± 0.147

(27.6)

1250 6.37 ± 0.403

(6.33)

6.64 ± 0.214

(3.22)

0.483 ± 0.085

(17.6)

0.593 ± 0.093

(15.7)

1 For all treatment and control groups tested, statistics were all based on replicate means. Therefore, n = 3 for all treatment 

and control groups presented. 2 There were no statistically significant decreases in growth in comparison to the negative control using Dunnett’s test 

(p > 0.05).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 180

Table 15. Summary of F1 Generation Day 14 Growth Measurements of 

Saltwater Mysids Exposed to Lindane

Nominal

Concentration

(ng/L)

Growth Parameters on Day 14 1,2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Males Females Males Females

Negative Control --4 --4 --4 --4

32

7.28 ± 0.460

(6.32)

n = 2

7.06 ± 0.230

(3.26) 

n = 2

0.699 ± 0.020

(2.86) 

n = 2

0.680 ± 0.074

(10.9) 

n = 2

80

7.23 ± 0.460

(6.36)

n = 2

 7.55 3

n = 1

0.698 ± 0.180

(25.8) 

n = 2

 1.095 3

n = 1

200 --4 --4 --4 --4

500 --4 --4 --4 --4

1250 --4 --4 --4 --4

1 For all treatment and control groups tested, n = the number of replicates available. 2 Statistics were not performed on the Day 14 growth measurements due to a lack of organisms in the negative control 

group. 3 Standard deviation and CV were not determined as only one of the three replicates were available for the treatment 

group. 4 No mysids were available for Day 14 growth measurements due to insufficient numbers of offspring produced by the F0 

adults.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 181

Table 16. Summary of F0 Generation Day 44 Growth Measurements of 

Saltwater Mysids Exposed to Lindane

Nominal

Concentration

(ng/L)

Growth Parameters on Day 44 1,2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Males Females Males Females

Negative Control 8.96 ± 0.141

(1.57)

9.29 ± 0.201

(2.16)

1.11 ± 0.013

(1.17)

1.32 ± 0.092

(6.97)

32 8.95 ± 0.299

(3.34)

9.29 ± 0.080

(0.86)

1.24 ± 0.028

(2.26)

1.46 ± 0.183

(12.5)

80 8.97 ± 0.243

(2.71)

9.28 ± 0.068

(0.73)

1.27 ± 0.082

(6.46)

1.54 ± 0.034

(2.21)

200 8.93 ± 0.065

(0.73)

9.35 ± 0.180

(1.93)

1.26 ± 0.079

(6.27)

1.58 ± 0.151

(9.56)

500 9.09 ± 0.235

(2.59)

9.30 ± 0.259

(2.78)

1.28 ± 0.054

(4.22)

1.45 ± 0.075

(5.17)

1250 8.91 ± 0.193

(2.17)

9.20 ± 0.081

(0.88)

1.19 ± 0.029

(2.44)

1.50 ± 0.065

(4.33)

1 For all treatment and control groups tested, statistics were all based on replicate means. Therefore, n = 3 for all treatment 

and control groups presented. 2 There were no statistically significant decreases in growth in comparison to the negative control using Dunnett’s test 

(p > 0.05).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 182

Table 17. Summary of F1 Generation Day 35 Growth Measurements of 

Saltwater Mysids Exposed to Lindane

Nominal

Concentration

(ng/L)

Growth Parameters on Day 35 1,2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Males Females Males Females

Negative Control

8.93 ± 0.460

(5.15)

n = 2

9.28 ± 0.255

(2.75)

n = 3

1.06 ± 0.060

(5.66) 

n = 2

1.18 ± 0.266

(22.5) 

n = 3

32

8.78 ± 0.214

(2.44)

n = 3

8.87 ± 0.498

(5.61) 

n = 3

1.14 ± 0.062

(5.44) 

n = 3

1.23 ± 0.187

(15.2) 

n = 3

80

9.02 ± 0.530

(5.88) 

n = 3

9.13 ± 0.435

(4.76) 

n = 3

1.15 ± 0.126

(11.0) 

n = 3

1.37 ± 0.252

(18.4) 

n = 3

200

9.22 ± 0.653

(7.08) 

n = 3

9.47 ± 0.586

(6.19) 

n = 3

1.15 ± 0.211

(18.4) 

n = 3

1.28 ± 0.189

(14.8) 

n = 3

500 9.50 3

n = 1

9.92 ± 0.825

(8.32)

n = 2

 1.19 3

n = 1

1.30 ± 0.167

(12.8) 

n = 2

1250 8.40 3

n = 1

 9.40 3

n = 1

 0.830 3

n = 1

 1.40 3

n = 1

1 For all treatment and control groups tested, statistics were based on replicate means. Therefore, n = the number of replicates 

available. 2 There were no statistically significant decreases in growth in comparison to the negative control using Dunnett’s test 

(p > 0.05). 3 Standard deviation and CV were not determined as only one of the three replicates were available for the treatment group.
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Wildlife International, Ltd. Study No. 185A-102 Page 183

Table 18. Summary of F0 Generation Reproduction of Saltwater Mysids Exposed to Lindane

Nominal

Conc.

(ng/L)

Mean Time 

to 

Maturation

Days ± SD

(CV%)1

Mean 

Time to 1st

Brood

Days ± SD

(CV%)1

Mean 

Time to 2nd

Brood

Days ± SD

(CV%)1

Mean

Interbrood 

Duration

Days ± SD

(CV%)1

Mean Number 

of Offspring in 

1st Brood Per 

Female ± SD

(CV%)1

Mean Number 

of Offspring in 

2nd Brood Per 

Female ± SD

(CV%)1

Mean

Total 

Number of

Offspring ± SD

(CV%)1

Mean

Percent of 

Males Sex

Ratio ± SD

(CV%)1

Mean

Percent of

Females

Producing 

Offspring ± SD

(CV%)1

Negative 

Control

14.5 ± 0.478

(3.29)

26.2 ± 0.721

(2.76)

33.2 ± 1.37

(4.12)

7.6 ± 2.71

(35.6)

4.0 ± 1.15

(28.8)

4.2 ± 1.10

(26.2)

71.7 ± 15.6

(21.8)

50.3 ± 8.61

(17.1)

n = 92

90.5 ± 8.25

(9.12) 

n = 19

32 14.0 ± 0.000

(0.00)

29.6 ± 1.49

(5.02)

35.2 ± 3.16

(8.98)

7.8 ± 1.38

(17.6)

3.1 ± 0.081

(25.3)

3.5 ± 1.80

(51.4)

71.7 ± 35.4

(49.4)

59.9 ± 11.9

(19.9) 

n = 100

90.5 ± 8.25

(9.12) 

n = 19

80 14.0 ± 0.000

(0.00)

26.7 ± 3.25

(12.2)

30.6 ± 0.625

(2.04)

5.3 ± 0.265

(4.99)

3.2 ± 0.351

(11.0)

2.1 ± 0.907

(43.2)

61.3 ± 19.1

(31.2)

43.9 ± 8.61

(19.6) 

n = 90

95.2 ± 8.25

(8.66) 

n = 20

200 14.1 ± 0.217

(1.53)

26.1 ± 3.15

(12.1)

31.0 ± 6.06

(19.6)

7.1 ± 1.17

(16.5)

2.4 ± 0.751

(31.3)

2.4 ± 1.00

(41.7)

58.7 ± 18.0

(30.7)

43.6 ± 5.68

(13.0) 

n = 85

100 ± 0.0

(0.0) 

n = 21

500 14.3 ± 0.577

(4.03)

24.2 ± 2.99

(12.3)

33.5 ± 3.38

(10.1)

9.1 ± 2.36

(26.1)

2.4 ± 0.608

(25.3)

3.5 ± 1.39

(39.7)

45.3 ± 18.3

(40.4)

52.2 ± 3.46

(6.63) 

n = 77

85.7 ± 14.3

(16.7) 

n = 18

1250 14.0 ± 0.000

(0.00)

23.3 ± 2.08

(8.92)

30.7 ± 1.86

(6.05)

6.7 ± 1.45

(21.8)

2.4 ± 1.50

(62.5)

 1.3 ± 1.23*

(94.6)

 16.0 ± 10.4*

(65.0)

44.1 ± 7.59

(17.2) 

n = 98

 47.6 ± 8.25**

(17.2) 

n = 10

1 Unless otherwise noted, n = 3, the number of replicates, for all treatment and control groups. For male sex ratio n = the total number of organisms that were able to be sexually 

identified throughout the test. For percent females producing offspring, n = the number of females that produced offspring.

* Statistically significant decrease in reproduction in comparison to the negative control using Dunnett’s test (p ≤ 0.05).

** Statistically significant decrease in reproduction in comparison to the negative control using Fisher’s Exact test (p ≤ 0.05).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 184

Table 19. Summary of F1 Generation Reproduction of Saltwater Mysids Exposed to Lindane

Nominal

Conc.

(ng/L)

Mean 

Time to 

Maturation

Days ± SD

(CV%)1,2

Mean 

Time to 1st

Brood

Days ± SD

(CV%)1,2

Mean 

Time to 2nd

Brood

Days ± SD

(CV%)1,2

Mean

Interbrood 

Duration

Days ± SD

(CV%)1,2

Mean Number 

of Offspring in 

1st Brood Per 

Female ± SD

(CV%)1,2

Mean Number 

of Offspring in 

2nd Brood Per 

Female ± SD

(CV%)1,2

Mean

Total Number of

Offspring ± SD

(CV%)1,2

Mean

Percent of 

Males Sex

Ratio ± SD

(CV%)1,2

Mean

Percent of

Females

Producing 

Offspring ± SD

(CV%)1,2

Negative 

Control

14.0 ± 0.0

(0.0)

n = 3

 17.0 3

n = 1

 23.03

n = 1

 6.0 3

n = 1

3.5

n = 1

3.5

n = 1

11.0 ± 15.6

(141.8)

n = 2

30.0 ± 26.5

(88.3)

n = 8

50.0 ± 70.7

(141.4) 

n = 2

32

13.7 ± 0.577

(4.21) 

n = 3

21.9 ± 6.15

(28.0)

n = 3

30.0 ± 1.41

(4.71)

n = 2

7.3 ± 3.18

(43.9)

n = 2

2.2 ± 1.61

(73.2) 

n = 3

1.4 ± 1.83

(130.7)

n = 3

9.7 ± 9.02

(93.0)

n = 3

40.5 ± 4.12

(10.2) 

n = 35

53.3 ± 41.6

(78.1) 

n = 7

80

14.0 ± 0.0

(0.0) 

n = 3

23.6 ± 5.68

(24.1)

n = 3

 33.0 3

n = 1

 6.0 3

n = 1

6.0 ± 1.91

(31.8) 

n = 3

0.2 ± 0.346

(173.0)

n = 3

16.3 ± 8.02

(49.2)

n = 3

62.3 ± 17.3

(27.8) 

n = 29

68.3 ± 33.4

(49.0) 

n = 9

200

14.0 ± 0.0

(0.0) 

n = 3

20.0 ± 3.46

(17.3)

n = 3

27.2 ± 8.72

(32.1)

n = 2

7.2 ± 3.06

(42.8)

n = 2

1.9 ± 0.902

(47.5) 

n = 3

1.0 ± 1.00

(100.0)

n = 3

8.3 ± 9.29

(111.9)

n = 3

45.8 ± 7.22

(15.8) 

n = 16

77.8 ± 38.5

(49.5) 

n = 6

500

14.0 ± 0.0

(0.0) 

n = 3

25.5 ± 13.4

(52.7)

n = 2

 26.0 3

n = 1

 10.0 3

n = 1

2.5 ± 0.707

(28.3) 

n = 2

1.0 ± 1.41

(141.0)

n = 2

3.5 ± 0.707

(20.2)

n = 2

12.5 ± 17.7

(141.6) 

n = 5

100 ± 0.0

(0.0) 

n = 2

1250

14.0 ± 0.0

(0.0) 

n = 3

-- 4 -- 4 -- 4 -- 4 -- 4 -- 4

50.0 ± 70.7

(141.4) 

n = 3

-- 4

1 Unless otherwise noted, n = the number of replicates available for all treatment and control groups. For male sex ratio n = the total number of organisms that were able to be

sexually identified throughout the test. For percent females producing offspring, n = the number of females that produced offspring. 2 There were no statistically significant decreases in reproduction in comparison to the negative control using Dunnett’s test, Wilcoxon Rank-Sum test, Fisher’s Exact test or 

Chi-Square test (p > 0.05). 3 Standard deviation and CV were not determined as only one replicate was available for the treatment group 4 No offspring were produced by the F1 adults in the 1250 ng/L treatment group.
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Wildlife International, Ltd. Study No. 185A-102 Page 185

Figure 1. Mysid Two-Generation Test Method Schematic

Source: Wildlife International, Ltd. Protocol No.: 185/021511/MYS-2GeN/SUB185; Page 44.
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Wildlife International, Ltd. Study No. 185A-102 Page 186

Figure 2. F0 Analytical Recoveries Over Time
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Wildlife International, Ltd. Study No. 185A-102 Page 187

Figure 3. F1 Analytical Recoveries Over Time
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Wildlife International, Ltd. Study No. 185A-102 Page 188

Appendix 1. Dilution Water Chemistry Parameters Measured During the 4-

Week Period Immediately Preceding the Test

Parameter Mean Range

Salinity (‰) 20

(N = 4)

19 – 21

pH 8.1

(N = 4)

8.0 – 8.1
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Wildlife International, Ltd. Study No. 185A-102 Page 189

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife 

International, Ltd. Saltwater

Pesticides and Organics 1

Component

Measured Concentration

(μg/L) Component

Measured Concentration

(μg/L)

Aldrin < 0.010 Gamma BHC – Lindane < 0.010

Alpha BHC < 0.010 Gamma Chlordane < 0.020

Alpha Chlordane < 0.010 Guthion (Azinphos-methyl) < 6.0

Beta BHC < 0.010 HCB < 0.010

Bolstar < 2.0 Heptachlor < 0.010

Chlordane < 0.50 Heptachlor Epoxide < 0.010

Coumaphos < 3.0 Kepone < 0.20

Delta BHC < 0.010 Malathion < 3.0

Demeton-O < 2.0 Merphos < 2.0

Demeton-S < 3.0 Methoxychlor < 0.10

Diazinon < 6.0 Methyl Parathion < 2.0

Dichlorvos < 3.0 Mevinphos < 5.0

Dieldrin < 0.020 Mirex < 0.25

Disulfoton < 2.0 Naled < 6.0

Dursban (Chlorpyrifos) < 3.0 o,p-DDD < 0.020

Endosulfan I < 0.010 o,p-DDE < 0.75

Endosulfan II < 0.020 o,p-DDT < 0.020

Endosulfan Sulfate < 0.020 p,p-DDD < 0.020

Endrin < 0.020 p,p-DDE < 0.020

Endrin Aldehyde < 0.10 p,p-DDT < 0.020

Endrin Ketone < 0.020 Phorate < 2.0

EPN < 2.0 Ronnel < 2.0

Ethion < 2.0 Stirophos < 2.0

Ethoprop < 3.0 Telodrin < 0.010

Ethyl Parathion < 2.0 Tokuthion < 2.0

Famphur < 3.0 Toxaphene < 3.0

Fensulfothion < 9.0 Trichloronate < 2.0

Fenthion < 2.0 Trithion < 2.0

1 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 190

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife 

International, Ltd. Saltwater (Continued)

Metals 1

Component

Measured Concentration

(mg/L) Component

Measured Concentration

(mg/L)

Aluminum < 0.200 Magnesium 531

Antimony < 0.0200 Manganese < 0.0050

Arsenic < 0.0200 Mercury < 0.00020

Barium 0.0063 Nickel < 0.0100

Beryllium < 0.0050 Nitrate Nitrogen < 0.50

Bromide 34.7 Nitrite Nitrogen < 20.0

Cadmium < 0.0050 Potassium 290

Calcium 235 Selenium < 0.0200

Chloride 12,200 Silver < 0.0050

Chromium < 0.0150 Sodium 5850

Cobalt < 0.0050 Sulfate 1430

Copper < 0.0100 Thallium < 0.0300

Fluoride 2.8 Vanadium < 0.0050

Iron < 0.200 Zinc < 0.0200

Lead < 0.0150

1 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 191

Appendix 3. Analyses of Pesticides, Organics and Metals in Brine (Artemia) 

Egg Sample

Pesticides and Organics 1

Component Measured Concentration

(μg/kg) Component Measured Concentration(μg/kg)

Aldrin < 2.7 Heptachlor Epoxide < 2.7

Alpha BHC < 2.7 Kepone < 23

Alpha Chlordane < 2.7 Malathion < 74

Beta BHC < 6.3 Merphos < 74

Bolstar < 74 Methoxychlor < 27

Chlordane < 56 Methyl Parathion < 74

Coumaphos < 74 Mevinphos < 74

Delta BHC < 2.7 Mirex < 5.6

Demeton-O < 74 Naled < 74

Demeton-S < 74 o,p-DDD < 5.6

Diazinon < 74 o,p-DDE < 5.6

Dichlorvos < 74 o,p-DDT < 5.6

Dieldrin < 5.6 p,p-DDD < 5.6

Disulfoton < 83 p,p-DDE < 5.6

Dursban (Chlorpyrifos) < 74 p,p-DDT < 5.6

Endosulfan I < 2.7 PCB-1016 < 19

Endosulfan II < 5.6 PCB-1221 < 19

Endosulfan Sulfate < 5.6 PCB-1232 < 19

Endrin < 5.6 PCB-1242 < 19

Endrin Aldehyde < 5.6 PCB-1248 < 19

Endrin Ketone < 5.9 PCB-1254 < 19

EPN < 74 PCB-1260 < 19

Ethion < 74 PCB-1262 < 19

Ethoprop < 74 PCB-1268 < 19

Ethyl Parathion < 74 TOTAL PCBs < 19

Famphur < 74 Phorate < 74

Fensulfothion < 220 Ronnel < 74

Fenthion < 74 Stirophos < 74

Gamma BHC – Lindane < 2.7 Telodrin < 4.0

Gamma Chlordane < 2.7 Tokuthion < 74

Guthion (Azinphos-methyl) < 74 Toxaphene < 110

HCB < 2.7 Trichloronate < 74

Heptachlor < 2.7 Trithion < 74

1 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 192

Appendix 3. Analyses of Pesticides, Organics and Metals in Brine (Artemia) 

Egg Sample (Continued)

Metals 1

Component Measured Concentration

(mg/kg) Component Measured Concentration(mg/kg)

Aluminum < 21.6 Magnesium 2180

Antimony < 2.16 Manganese 7.61

Arsenic 17.8 Mercury 0.192

Barium 0.921 Nickel < 1.08

Beryllium < 0.539 Nitrate by IC (solid) 1.7

Bromide by IC (solid) 7.6 Nitrite by IC (solid) < 1.1

Cadmium < 0.539 Potassium 7410

Calcium 559 Selenium 3.63

Chloride by IC (solid) 1530 Silver < 0.539

Chromium < 1.62 Sodium 2210

Cobalt < 0.539 Sulfate by IC (solid) 206

Copper 6.97 Thallium < 3.24

Fluoride by IC (solid) < 1.1 Vanadium < 0.539

Iron 596 Zinc 76.6

Lead < 1.62

1 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 193

Appendix 4. The Analysis of Lindane in Saltwater
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Wildlife International, Ltd. Study No. 185A-102 Page 194

Appendix 4.1 Method Flowchart for the Analysis of Lindane in 

Saltwater

METHOD FLOWCHART FOR THE ANALYSIS OF LINDANE IN SALTWATER

Rinse all separatory funnels with hexane.

↓

Add the requisite volume of saltwater to each separatory funnel. Acidify samples using 

10% phosphoric acid.

↓

Fortify recovery samples in acidified saltwater (pH ~5) with the appropriate Lindane stock solution. 

Unfortified saltwater will serve as the matrix blank.

↓

Add the requisite volume of hexane to each sample. Shake each sample for ~1 minute. 

Drain the bottom layer of water into beaker. Transfer the remaining hexane into a glass 

scintillation vial, and add a small amount of sodium sulfate. Transfer calibration 

standards and an aliquot of the hexane extracts to autosampler vials.

↓

Analyze by GC/ECD.
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Wildlife International, Ltd. Study No. 185A-102 Page 195

Appendix 4.2 Typical Gas Chromatograph (GC) Operational 

Parameters

INSTRUMENT: Agilent Model 5890 Gas Chromatograph (GC) 

DETECTOR: Hewlett-Packard Electron Capture Detector (ECD)

ANALYTICAL COLUMN: Zebron-DB-5 (0.53 mm x 30 m, 0.5 μm film thickness)

INJECTOR TEMPERATURE: 250°C

OVEN: Initial temperature: 150°C

Initial hold time: 1.00 minute

Ramp: 15.0°C/minute

Final temperature: 320°C

Final hold time: 3.00 minutes

DETECTOR TEMPERATURE: 300°C

INJECTION VOLUME: 2.00 μL

CARRIER GAS: Helium

HEAD PRESSURE: 4 psi

MAKE-UP GAS: Argon / Methane, 60 psi.

APPROXIMATE LINDANE PEAK 

RETENTION TIME: 8.9 minutes
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Wildlife International, Ltd. Study No. 185A-102 Page 196

Appendix 4.3 Analytical Stocks and Standards Preparation

A stock solution of Lindane test substance was prepared by weighing 0.1005 g (weight corrected 

for purity) of the test substance on an analytical balance. The test substance was transferred to a 100-mL 

class-A volumetric flask brought to volume using acetone. This primary stock solution contained 1.00 

mg a.i./mL of Lindane and was diluted in acetone to prepare 0.100, 0.0100, 0.00100 and 0.000100 mg 

a.i./mL stock solutions. The 0.000100 mg a.i./mL stock solution was used to fortify the quality control 

samples. Calibration standards were prepared in hexane using the 0.00100 mg a.i./mL stock solution. 

The following shows the dilution scheme for the set of calibration standards:

Stock

Concentration

(mg a.i./mL)

Aliquot

(μL)

Final

Volume

(mL)

Standard

Concentration

(μg a.i./L)

0.00100

0.00100

0.00100

0.00100

0.00100

50.0

150

250

350

500

100

100

100

100

100

0.500

1.50

2.50

3.50

5.00
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 197

Appendix 4.4 Example Calculations for a Representative Test Sample

The analytical result and percent recovery for sample number 185A-102-1-3, with a nominal 

concentration of 0.080 μg/L, were calculated using the following equations:

Concentration of Lindane in sample (μg/L) = Peak Area - Y-intercept

Slope X Dilution Factor ÷ Purity

Percent of nominal concentration = measured concentration in sample (μg/L)

nominal concentration in sample (μg/L) X 100 

Peak Area = 9287.33301

Y-intercept = -701.0670

Slope = 5429.6864

Dilution factor = 0.0400

Purity = 99.6%

Concentration of Lindane in sample (μg/L) = 9287.33301 + 701.0670

5429.6864 X 0.0400 ÷ 0.996

Concentration of Lindane in sample (μg/L) = 0.0739

Percent of nominal concentration = 0.0739 μg/L

0.080 μg/L X 100

Percent of nominal concentration = 92.3%*

* Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ 

slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 198

Appendix 4.5 Quality Control Samples of Lindane in Saltwater for the 

F0 Generation

Sample Number

(185A-102-1-) Sample

Type

Concentration of Lindane

Percent

Recovery2

Fortified

(μg a.i./L)

Measured

(μg a.i./L) 1

Pre-Test

PT-MAB-3

PT-MAB-4

PT-MAS-7

PT-MAS-8

PT-MAS-9

PT-MAS-10

PT-MAS-11

PT-MAS-12

Matrix Blank

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.0

0.0320

0.400

1.25

0.0320

0.400

1.25

< LOQ

< LOQ

0.320

0.359

0.195

0.0298

0.356

1.11

--

--

999

89.7

15.6

93.2

89.0

88.6

Exposure

MAB-1

MAB-2

MAB-3

MAB-4

MAB-5

MAB-6

MAB-7

MAB-8

MAS-1

MAS-2

MAS-3

MAS-4

MAS-5

MAS-6

MAS-7

MAS-8

MAS-9

MAS-10

MAS-11

MAS-12

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

0.0295

0.348

1.08

0.0287

0.336

1.05

0.0286

0.331

1.04

0.0286

0.329

1.02

--

--

--

--

--

--

--

--

92.3

87.1

86.0

89.5

84.1

84.0

89.3

82.8

83.1

89.4

82.3

81.6 1 The limit of quantitation (LOQ) was 0.0150 μg a.i./L, calculated as the product of the concentration of the lowest 

calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300). 2 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 199

Appendix 4.5. Quality Control Samples of Lindane in Saltwater for the 

F0 Generation (Continued)

Sample Number

(185A-102-1-) Sample

Type

Concentration of Lindane

Percent

Recovery2

Fortified

(μg a.i./L)

Measured

(μg a.i./L) 1

MAS-13

MAS-14

MAS-15

MAS-16

MAS-17

MAS-18

MAS-19

MAS-20

MAS-21

MAS-22

MAS-23

MAS-24

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0282

0.330

1.01

0.0277

0.323

0.998

0.0272

0.310

0.976

0.0267

0.309

0.964

88.0

82.6

80.7

86.5

80.6

79.8

85.0

77.5

78.1

83.6

77.2

77.1

Mean3 =

Standard Deviation =

CV =

83.7

4.25

 5.08% 1 The limit of quantitation (LOQ) was 0.0150 μg a.i./L, calculated as the product of the concentration of the lowest 

calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300). 2 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly. 3 Based on exposure samples only.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 200

Appendix 4.6. Quality Control Samples of Lindane in Saltwater for the 

F1 Generation

Sample Number

(185A-102-1-) Sample

Type

Concentration of Lindane

Percent

Recovery2

Fortified

(μg a.i./L)

Measured

(μg a.i./L) 1

Pre-Test

F1PTMAB-1

F1PTMAS-1

F1PTMAS-2

F1PTMAS-3

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.0320

0.400

1.25

< LOQ

0.0282

0.329

1.02

--

88.1

82.2

81.7

Exposure

F1-MAB-1

F1-MAB-2

F1-MAB-3

F1-MAB-4

F1-MAB-5

F1-MAB-6

F1-MAB-7

F1-MAB-8

F1-MAB-9

F1-MAS-1

F1-MAS-2

F1-MAS-3

F1-MAS-4

F1-MAS-5

F1-MAS-6

F1-MAS-7

F1-MAS-8

F1-MAS-9

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

0.0271

0.329

1.02

0.0289

0.327

1.01

0.0274

0.314

0.977

--

--

--

--

--

--

--

--

--

84.7

82.2

81.3

90.3

81.7

80.8

85.5

78.5

78.2 1 The limit of quantitation (LOQ) was 0.0150 μg a.i./L, calculated as the product of the concentration of the lowest 

calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300). 2 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 201

Appendix 4.6. Quality Control Samples of Lindane in Saltwater for the 

F1 Generation (Continued)

Sample Number

(185A-102-1-) Sample

Type

Concentration of Lindane

Percent

Recovery2

Fortified

(μg a.i./L)

Measured

(μg a.i./L) 1

Exposure

F1-MAS-10

F1-MAS-11

F1-MAS-12

F1-MAS-13

F1-MAS-14

F1-MAS-15

F1-MAS-16

F1-MAS-17

F1-MAS-18

F1-MAS-19

F1-MAS-20

F1-MAS-21

F1-MAS-22

F1-MAS-23

F1-MAS-24

F1-MAS-25

F1-MAS-26

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

1.25

0.0320

0.400

0.0268

0.305

0.940

0.0263

0.301

0.938

0.0261

0.303

0.941

0.0451

0.291

1.12

0.0250

0.283

0.894

0.0252

0.289

83.8

76.3

75.2

82.1

75.2

75.0

81.5

75.9

75.2

141

72.7

89.8

78.2

70.6

71.5

78.9

72.2

Mean =

Standard Deviation =

CV =

81.5

13.2

 16.2% 1 The limit of quantitation (LOQ) was 0.0150 μg a.i./L, calculated as the product of the concentration of the lowest 

calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (0.0300). 2 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 202

Appendix 4.7. Representative Calibration Curve for Lindane

0

5000

10000

15000

20000

25000

30000

0.000 1.000 2.000 3.000 4.000 5.000 6.000

(ug a.i./L)

Peak area counts

Slope = 5429.6864; Y-Intercept = -701.0670; R2 = 0.99779
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 203

Appendix 4.8. Representative Chromatogram of a Low-Level Lindane 

Calibration Standard

Nominal concentration: 0.500 μg a.i./L. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 204

Appendix 4.9. Representative Chromatogram of a High-Level Lindane 

Calibration Standard

Nominal concentration: 5.00 μg a.i./L. The arrow indicates the approximate retention time of Lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 205

Appendix 4.10. Representative Chromatogram of a Matrix Blank

Sample number 185A-102-1-MAB-1. The arrow indicates the approximate retention time of Lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 206

Appendix 4.11. Representative Chromatogram of a Matrix Fortification

Sample number 185A-102-1-MAS-1; 0.0320 μg a.i./L, nominal concentration. The arrow indicates the 

approximate retention time of Lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 207

Appendix 4.12. Representative Chromatogram of a Test Sample

Sample number 185A-102-1-4; 0.20 μg/L, nominal concentration. The arrow indicates the approximate 

retention time of Lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 208

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation

Day of

Test

Negative Control – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 12 0 7 0 9 100

16 0 12 0 7 0 9 100

17 0 12 0 7 0 9 100

18 0 12 0 7 1 8 96

19 0 12 0 7 0 8 96

20 0 12 0 7 0 8 96

21 0 12 0 7 0 8 96

22 0 12 0 7 1 7 93

23 0 12 0 7 0 7 93

24 1 11 0 7 0 7 89

25 0 11 0 7 0 7 89

26 0 11 0 7 0 7 89

27 0 11 0 7 0 7 89

28 0 11 0 7 0 7 89

29 0 11 0 7 0 7 89

30 0 11 0 7 0 7 89

31 0 11 0 7 0 7 89

32 0 10 2 0 7 0 7 86

33 0 10 0 7 0 7 86

34 0 10 0 7 0 7 86

35 0 10 0 7 0 7 86

36 0 10 0 7 1 6 82

37 0 10 0 7 0 6 82

38 0 10 0 8 3 0 6 86

39 0 10 0 8 0 6 86

40 0 10 0 8 0 6 86

41 0 10 0 8 0 6 86

42 0 10 0 8 0 6 86

43 0 10 0 8 0 6 86

44 0 10 0 8 0 6 86 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 One male mysid was identified as female on Day 32. 3 One female mysid was identified as male on Day 38.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 209

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

Negative Control – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 7 0 12 0 7 (1 I) 100

16 1 6 0 12 0 7 (1 I) 96

17 0 6 0 12 0 7 (1 I) 96

18 0 6 0 12 0 7 (1 I) 96

19 0 6 0 12 0 7 (1 I) 96

20 0 6 0 12 0 7 (1 I) 96

21 0 6 0 12 0 7 (1 I) 96

22 0 6 0 12 1 72 93

23 0 6 0 12 0 7 93

24 0 6 0 12 0 7 93

25 0 6 0 12 0 7 93

26 0 6 0 12 0 7 93

27 0 6 0 12 0 7 93

28 0 6 0 12 0 7 93

29 0 6 0 12 0 7 93

30 0 6 0 12 0 7 93

31 0 6 0 12 0 7 93

32 0 7 3 0 12 0 7 96

33 0 7 0 12 0 7 96

34 0 7 0 12 0 7 96

35 0 7 0 12 0 7 96

36 0 7 0 12 1 6 93

37 0 7 0 12 0 6 93

38 0 7 0 11 4 0 6 89

39 0 7 0 11 0 6 89

40 0 7 0 11 1 5 85

41 0 7 0 11 0 5 85

42 0 7 0 11 0 5 85

43 0 7 0 11 0 5 85

44 0 7 0 11 0 5 85 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 I = Mysid was not sexually identified (immature) during the Day 14 pairing interval. Upon maturation, it was identified as a female. Percent survival includes the immature mysid(s). 3 One male mysid was identified as female on Day 32. 4 One female mysid was identified as male on Day 38.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 210

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

32 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 11 0 7 0 14 100

16 0 11 0 7 0 14 100

17 0 11 0 7 0 14 100

18 0 11 0 7 0 14 100

19 1 10 0 7 0 14 97

20 0 10 0 7 0 14 97

21 0 10 0 7 1 13 94

22 1 9 0 7 0 13 91

23 0 9 0 7 0 13 91

24 1 8 0 7 0 13 88

25 0 8 0 7 0 13 88

26 0 8 1 6 0 13 84

27 0 8 0 6 0 13 84

28 0 8 0 6 0 13 84

29 0 8 0 6 0 13 84

30 1 7 0 6 1 12 78

31 0 7 0 6 0 12 78

32 0 7 0 6 0 12 78

33 0 7 0 6 0 12 78

34 0 7 1 5 0 12 75

35 0 7 0 5 0 12 75

36 0 7 0 5 0 12 75

37 0 7 0 5 0 12 75

38 0 7 0 5 0 12 75

39 0 7 0 5 0 12 75

40 0 7 0 5 0 12 75

41 0 7 0 5 0 12 75

42 0 7 1 4 0 12 72

43 0 7 1 3 0 12 69

44 0 7 0 3 0 12 69 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 211

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

32 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 9 0 14 0 7 100

16 0 9 0 14 0 7 100

17 0 9 0 14 0 7 100

18 0 9 0 14 0 7 100

19 0 9 0 14 0 7 100

20 0 9 0 14 0 7 100

21 0 9 0 14 0 7 100

22 0 9 0 14 0 7 100

23 0 9 0 14 0 7 100

24 0 9 0 14 0 7 100

25 1 8 0 14 0 7 97

26 0 8 0 14 0 7 97

27 0 8 0 14 0 7 97

28 0 8 0 14 0 7 97

29 0 8 0 14 0 7 97

30 0 8 0 14 0 7 97

31 0 8 0 14 0 7 97

32 0 8 0 14 0 7 97

33 0 8 1 13 0 7 93

34 0 8 1 12 0 7 90

35 0 8 0 12 0 7 90

36 0 8 0 12 0 7 90

37 0 8 0 12 0 7 90

38 0 8 0 12 0 7 90

39 0 8 0 12 0 7 90

40 0 8 0 12 0 7 90

41 0 8 0 12 0 7 90

42 0 8 0 12 0 7 90

43 0 8 0 12 0 7 90

44 0 8 0 12 0 7 90 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 212

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

80 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 9 0 11 0 7 100

16 0 9 0 11 0 7 100

17 0 9 0 11 0 7 100

18 0 9 0 11 0 7 100

19 0 9 0 11 0 7 100

20 0 9 0 11 0 7 100

21 0 9 0 11 0 7 100

22 0 9 0 11 1 6 96

23 0 9 0 11 0 6 96

24 0 9 1 10 0 6 93

25 0 9 0 10 0 6 93

26 0 9 0 10 0 6 93

27 0 9 0 10 0 6 93

28 0 9 0 10 0 6 93

29 0 9 0 10 0 6 93

30 0 9 0 10 0 6 93

31 0 9 0 10 0 6 93

32 0 9 0 10 0 6 93

33 0 9 1 9 0 6 89

34 0 9 0 9 0 6 89

35 0 9 0 9 0 6 89

36 1 8 0 9 0 6 85

37 0 8 0 9 1 5 81

38 0 8 0 9 0 5 81

39 0 8 1 8 0 5 78

40 0 8 0 8 0 5 78

41 1 7 0 8 0 5 74

42 0 7 0 8 0 5 74

43 0 7 0 8 0 5 74

44 0 7 0 8 1 4 70 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 213

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

80 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 8 0 8 0 10 100

16 0 8 0 8 0 10 100

17 0 8 0 8 0 10 100

18 0 8 0 8 0 10 100

19 0 8 0 8 0 10 100

20 0 8 0 8 0 10 100

21 0 8 0 8 0 10 100

22 0 8 0 8 0 10 100

23 0 8 0 8 0 10 100

24 0 8 0 8 0 10 100

25 0 8 0 8 0 10 100

26 0 8 0 8 0 10 100

27 0 8 0 8 0 10 100

28 0 8 1 7 0 10 96

29 0 8 0 7 0 10 96

30 0 8 0 7 1 9 92

31 0 8 0 7 0 9 92

32 0 8 0 7 0 9 92

33 1 7 0 7 0 9 88

34 0 7 0 7 1 8 85

35 0 7 0 7 0 8 85

36 0 7 0 7 0 8 85

37 0 7 0 7 0 8 85

38 0 7 0 7 0 8 85

39 0 7 0 7 0 8 85

40 0 7 0 7 0 8 85

41 0 7 0 7 1 7 81

42 1 6 0 7 0 7 77

43 0 6 0 7 0 7 77

44 0 6 0 7 0 7 77 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 214

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

200 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 9 0 7 0 8 100

16 0 9 0 7 0 8 100

17 0 9 0 7 0 8 100

18 0 9 0 7 0 8 100

19 0 9 0 7 0 8 100

20 0 9 0 7 0 8 100

21 0 9 0 7 1 7 96

22 0 9 0 7 0 7 96

23 0 9 0 7 0 7 96

24 1 8 0 7 0 7 92

25 0 8 0 7 0 7 92

26 1 7 0 7 0 7 88

27 0 7 0 7 0 7 88

28 0 7 0 7 0 7 88

29 0 7 0 7 0 7 88

30 0 7 0 7 0 7 88

31 2 5 0 7 0 7 79

32 0 5 0 7 0 7 79

33 0 5 0 7 0 7 79

34 0 5 0 7 0 7 79

35 0 5 0 7 0 7 79

36 0 5 0 7 0 7 79

37 1 4 0 7 0 7 75

38 0 4 0 7 0 7 75

39 1 3 0 7 0 7 71

40 0 3 0 7 0 7 71

41 0 3 0 7 0 7 71

42 0 3 0 7 0 7 71

43 0 3 0 7 0 7 71

44 0 3 0 7 0 7 71 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 215

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

200 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 9 0 10 (2 I) 2 0 11 100

16 0 9 0 10 (2 I) 2 0 11 100

17 0 9 0 10 (2 I) 2 0 11 100

18 0 9 0 11 (1 I) 2 0 11 100

19 0 9 0 11 (1 I) 2 0 11 100

20 1 8 0 11 (1 I) 2 0 11 97

21 0 8 0 11 (1 I) 2 0 11 97

22 0 8 0 11 (1 I) 2 1 10 94

23 0 8 0 11 (1 I) 2 0 10 94

24 0 8 0 11 (1 I) 2 0 10 94

25 0 8 0 11 (1 I) 2 0 10 94

26 0 8 0 11 (1 I) 2 0 10 94

27 0 8 1 3 11 0 10 91

28 0 8 0 11 0 10 91

29 0 8 0 11 0 10 91

30 0 8 0 11 0 10 91

31 0 8 0 11 0 10 91

32 0 8 0 11 0 10 91

33 0 8 0 11 0 10 91

34 0 8 0 11 0 10 91

35 0 8 0 11 0 10 91

36 0 8 0 11 0 10 91

37 0 8 0 11 0 10 91

38 0 8 0 11 0 10 91

39 0 8 0 11 0 10 91

40 0 8 0 11 0 10 91

41 0 8 0 11 0 10 91

42 0 8 0 11 0 10 91

43 0 8 0 11 0 10 91

44 0 8 0 11 0 10 91 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 I = Mysid was not sexually identified (immature) during the Day 14 pairing interval. Upon maturation, it was identified as a female. Percent survival includes the immature mysid(s). 3 One mysid died on Day 27, before it was sexually identified (immature).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 216

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

500 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 8 1 6 0 9 96

16 0 8 0 6 0 9 96

17 0 8 0 6 0 9 96

18 2 6 0 6 0 9 88

19 0 6 0 6 0 9 88

20 0 6 0 6 0 9 88

21 0 6 0 6 0 9 88

22 0 6 0 6 0 9 88

23 1 5 1 5 0 9 79

24 0 5 0 5 0 9 79

25 0 5 0 5 0 9 79

26 0 5 0 5 1 8 75

27 0 5 0 5 0 8 75

28 0 5 0 5 0 8 75

29 1 4 0 5 0 8 71

30 0 4 0 5 0 8 71

31 0 4 0 5 0 8 71

32 0 4 0 5 1 7 67

33 0 4 0 5 0 7 67

34 0 4 0 5 0 7 67

35 0 4 0 5 0 7 67

36 0 4 0 5 0 7 67

37 0 4 0 5 0 7 67

38 0 4 0 5 0 7 67

39 0 4 0 5 0 7 67

40 0 4 0 5 0 7 67

41 0 4 0 5 0 7 67

42 0 4 0 5 0 7 67

43 0 4 0 5 0 7 67

44 0 4 0 5 0 7 67 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 217

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

500 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 11 0 7 0 7 (1 I) 100

16 0 11 0 7 0 7 (1 I) 100

17 0 11 0 7 0 7 (1 I) 100

18 0 11 0 7 0 7 (1 I) 100

19 0 11 0 7 0 7 (1 I) 100

20 0 11 0 7 0 7 (1 I) 100

21 0 11 0 7 0 7 (1 I) 100

22 0 11 0 7 0 7 (1 I) 100

23 0 11 0 7 0 82 100

24 0 11 0 7 0 8 100

25 0 11 0 7 0 8 100

26 0 11 0 7 1 7 96

27 0 11 0 7 0 7 96

28 0 11 0 7 0 7 96

29 0 11 0 7 0 7 96

30 0 11 0 7 0 7 96

31 1 10 0 7 0 7 92

32 0 10 0 7 0 7 92

33 0 10 0 7 0 7 92

34 0 10 0 7 0 7 92

35 0 10 0 7 0 7 92

36 0 10 0 7 0 7 92

37 0 10 0 7 0 7 92

38 0 10 0 7 0 7 92

39 0 10 0 7 0 7 92

40 0 10 0 7 0 7 92

41 0 10 0 7 0 7 92

42 0 10 1 6 0 7 88

43 0 10 0 6 0 7 88

44 0 10 0 6 0 7 88 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 I = Mysid was not sexually identified (immature) during the Day 14 pairing interval. Upon maturation, it was identified as a female. Percent survival includes the immature mysid(s).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 218

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

1250 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. A (%) live

 151 0 8 0 10 0 10 100

16 0 8 0 10 0 10 100

17 0 8 0 10 0 10 100

18 0 8 0 10 0 10 100

19 0 8 0 10 0 10 100

20 0 8 0 10 0 10 100

21 0 8 0 10 0 10 100

22 0 8 0 10 0 10 100

23 0 8 0 10 0 10 100

24 0 8 0 10 0 10 100

25 0 8 0 10 0 10 100

26 0 8 0 10 0 10 100

27 0 8 0 10 2 8 93

28 0 8 0 10 0 8 93

29 0 8 0 10 0 8 93

30 0 8 0 10 0 8 93

31 0 8 0 10 0 8 93

32 0 8 1 9 0 8 89

33 0 8 0 9 1 7 86

34 0 8 0 9 0 7 86

35 0 8 1 8 0 7 82

36 0 8 0 8 0 7 82

37 0 8 0 8 0 7 82

38 0 8 0 8 0 7 82

39 0 8 0 8 0 7 82

40 0 8 1 7 0 7 79

41 0 8 0 7 0 7 79

42 0 8 0 7 0 7 79

43 0 8 0 7 0 7 79

44 0 8 0 7 0 7 79 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 219

Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane - F0 Generation (Continued)

Day of

Test

1250 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 12 0 8 (2 I)2 0 9 100

16 0 12 0 8 (2 I)2 0 9 100

17 0 12 0 8 (2 I)2 0 9 100

18 0 12 1 3 8 (1 I)2 0 9 97

19 0 12 1 3 8 0 9 94

20 0 12 0 8 0 9 94

21 0 12 0 8 0 9 94

22 0 12 0 8 0 9 94

23 0 12 0 8 0 9 94

24 0 12 0 8 0 9 94

25 0 12 0 8 0 9 94

26 0 12 0 8 0 9 94

27 0 12 0 8 0 9 94

28 0 12 0 8 0 9 94

29 0 12 0 8 0 9 94

30 0 12 0 8 0 9 94

31 0 12 0 8 0 9 94

32 0 12 0 8 0 9 94

33 0 12 0 8 0 9 94

34 1 11 0 8 0 9 90

35 0 11 0 8 0 9 90

36 0 11 0 8 0 9 90

37 0 11 0 8 0 9 90

38 0 11 0 8 0 9 90

39 0 11 0 8 0 9 90

40 0 11 0 8 0 9 90

41 0 11 0 8 1 8 87

42 0 11 0 8 0 8 87

43 0 11 1 7 0 8 84

44 0 11 0 7 0 8 84 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 I = Mysid was not sexually identified (immature) during the Day 14 pairing interval. Percent survival includes the immature mysid(s). 3 One mysid died on Day 18; one mysid died on Day 19. Both mysids died before they were sexually identified (immature).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 220

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation

Day of

Test

Negative Control – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 1 0 2 0 0 100

16 0 1 0 2 0 0 100

17 0 1 0 2 0 0 100

18 0 1 0 2 0 0 100

19 0 1 0 2 0 0 100

20 0 1 0 2 0 0 100

21 0 1 0 2 0 0 100

22 0 1 0 2 0 0 100

23 0 1 0 2 0 0 100

24 0 1 0 2 0 0 100

25 0 1 0 2 0 0 100

26 0 1 0 2 0 0 100

27 0 1 0 2 0 0 100

28 0 1 0 2 0 0 100

29 0 1 0 2 0 0 100

30 0 1 0 2 0 0 100

31 0 1 0 2 0 0 100

32 0 1 0 2 0 0 100

33 0 1 0 2 0 0 100

34 0 1 0 2 0 0 100

35 0 1 0 2 0 0 100

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 221

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

Negative Control – Females

Replicate A Replicate B Replicate C Percent Survival

No. D (%) ead No. Alive No. Dead No. Alive No. Dead No. Alive

 151 0 1 0 3 0 1 100

16 0 1 0 3 0 1 100

17 0 1 0 3 0 1 100

18 0 1 0 3 0 1 100

19 0 1 0 3 0 1 100

20 0 1 0 3 0 1 100

21 0 1 0 3 0 1 100

22 0 1 0 3 0 1 100

23 0 1 0 3 0 1 100

24 0 1 0 3 0 1 100

25 0 1 0 3 0 1 100

26 0 1 0 3 0 1 100

27 0 1 0 3 0 1 100

28 0 1 0 3 0 1 100

29 0 1 0 3 0 1 100

30 0 1 0 3 0 1 100

31 0 1 0 3 0 1 100

32 0 1 0 3 0 1 100

33 0 1 0 3 0 1 100

34 0 1 0 3 0 1 100

35 0 1 0 3 0 1 100

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 222

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

32 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 5 0 4 0 5 100

16 0 5 0 4 0 5 100

17 0 5 0 4 0 5 100

18 0 5 0 4 0 5 100

19 0 5 0 4 0 5 100

20 1 4 0 4 1 4 86

21 0 4 0 4 0 4 86

22 0 4 0 4 0 4 86

23 0 4 1 3 0 4 79

24 0 4 0 3 0 4 79

25 0 4 0 3 0 4 79

26 0 4 0 3 0 4 79

27 0 4 0 3 0 4 79

28 0 4 0 3 0 4 79

29 0 4 0 3 1 3 71

30 0 4 0 3 0 3 71

31 1 3 0 3 0 3 64

32 0 3 0 3 0 3 64

33 0 3 0 3 0 3 64

34 0 3 0 3 0 3 64

35 0 3 0 3 0 3 64 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 223

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

32 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 6 0 4 0 5 100

16 0 6 0 4 0 5 100

17 0 6 0 4 0 5 100

18 1 5 0 4 0 5 93

19 0 5 0 4 0 5 93

20 0 5 0 4 0 5 93

21 0 5 0 4 0 5 93

22 0 5 0 4 0 5 93

23 0 5 0 4 0 5 93

24 0 5 0 4 0 5 93

25 0 5 0 4 0 5 93

26 0 5 0 4 0 5 93

27 0 5 0 4 0 5 93

28 0 5 0 4 0 5 93

29 0 5 0 4 0 5 93

30 0 5 0 4 0 5 93

31 0 5 0 4 0 5 93

32 0 5 0 4 0 5 93

33 0 5 0 4 0 5 93

34 0 5 0 4 1 4 87

35 0 5 0 4 0 4 87

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 224

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

80 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 4 1 7 0 3 93

16 0 4 0 7 0 3 93

17 0 4 0 7 0 3 93

18 0 4 0 7 0 3 93

19 0 4 0 7 0 3 93

20 0 4 0 7 0 3 93

21 0 4 0 7 0 3 93

22 0 4 0 7 0 3 93

23 0 4 1 6 0 3 87

24 0 4 0 6 0 3 87

25 0 4 0 6 0 3 87

26 0 4 0 6 0 3 87

27 0 4 0 6 0 3 87

28 0 4 0 6 0 3 87

29 0 4 0 6 0 3 87

30 0 4 0 6 0 3 87

31 0 4 0 6 0 3 87

32 0 4 0 6 0 3 87

33 0 4 1 5 0 3 80

34 0 4 0 5 0 3 80

35 0 4 0 5 0 3 80 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 225

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

80 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 3 0 7 0 3 100

16 0 3 0 7 0 3 100

17 0 3 0 7 0 3 100

18 0 3 0 7 0 3 100

19 0 3 0 7 1 2 92

20 0 3 0 7 1 1 85

21 0 3 0 7 0 1 85

22 0 3 0 7 0 1 85

23 0 3 0 7 0 1 85

24 0 3 0 7 0 1 85

25 0 3 0 7 0 1 85

26 0 3 0 7 0 1 85

27 0 3 0 7 0 1 85

28 0 3 0 7 0 1 85

29 0 3 0 7 0 1 85

30 0 3 1 6 0 1 77

31 0 3 0 6 0 1 77

32 0 3 0 6 0 1 77

33 0 3 0 6 0 1 77

34 0 3 0 6 0 1 77

35 0 3 0 6 0 1 77 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 226

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

200 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 4 0 5 0 1 100

16 0 4 0 5 0 1 100

17 0 4 0 5 0 1 100

18 0 4 0 5 0 1 100

19 0 4 0 5 0 1 100

20 0 4 0 5 0 1 100

21 0 4 0 5 0 1 100

22 0 4 1 4 0 1 90

23 0 4 0 4 0 1 90

24 0 4 0 4 0 1 90

25 0 4 0 4 0 1 90

26 0 4 0 4 0 1 90

27 0 4 1 3 0 1 80

28 0 4 0 3 0 1 80

29 0 4 0 3 0 1 80

30 1 3 0 3 0 1 70

31 0 3 0 3 0 1 70

32 0 3 0 3 0 1 70

33 0 3 0 3 0 1 70

34 0 3 0 3 0 1 70

35 0 3 0 3 0 1 70 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 227

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

200 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 5 0 3 0 1 100

16 0 5 0 3 0 1 100

17 0 5 0 3 0 1 100

18 0 5 0 3 0 1 100

19 0 5 0 3 0 1 100

20 0 5 0 3 0 1 100

21 0 5 0 3 0 1 100

22 0 5 0 3 0 1 100

23 0 5 0 3 0 1 100

24 0 5 0 3 0 1 100

25 0 5 0 3 0 1 100

26 0 5 0 3 0 1 100

27 0 5 0 3 0 1 100

28 0 5 0 3 0 1 100

29 0 5 0 3 0 1 100

30 0 5 0 3 0 1 100

31 0 5 0 3 0 1 100

32 0 5 0 3 0 1 100

33 0 5 0 3 0 1 100

34 0 5 0 3 0 1 100

35 0 5 0 3 0 1 100 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 228

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

500 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 1 0 1 0 0 100

16 0 1 0 1 0 0 100

17 0 1 0 1 0 0 100

18 0 1 0 1 0 0 100

19 0 1 0 1 0 0 100

20 0 1 0 1 0 0 100

21 0 1 0 1 0 0 100

22 0 1 0 1 0 0 100

23 0 1 0 1 0 0 100

24 0 1 0 1 0 0 100

25 0 1 0 1 0 0 100

26 1 0 0 1 0 0 50

27 0 0 0 1 0 0 50

28 0 0 0 1 0 0 50

29 0 0 0 1 0 0 50

30 0 0 0 1 0 0 50

31 0 0 0 1 0 0 50

32 0 0 0 1 0 0 50

33 0 0 0 1 0 0 50

34 0 0 0 1 0 0 50

35 0 0 0 1 0 0 50 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 229

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

500 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 1 0 3 0 1 100

16 0 1 0 3 0 1 100

17 0 1 0 3 0 1 100

18 0 1 0 3 0 1 100

19 0 1 0 3 0 1 100

20 0 1 0 3 0 1 100

21 0 1 0 3 0 1 100

22 0 1 0 3 0 1 100

23 0 1 0 3 0 1 100

24 0 1 0 3 0 1 100

25 0 1 0 3 1 0 80

26 0 1 0 3 0 0 80

27 0 1 0 3 0 0 80

28 0 1 0 3 0 0 80

29 0 1 0 3 0 0 80

30 0 1 0 3 0 0 80

31 0 1 0 3 0 0 80

32 0 1 0 3 0 0 80

33 0 1 0 3 0 0 80

34 0 1 0 3 0 0 80

35 0 1 0 3 0 0 80 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 230

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

1250 ng/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 0 0 1 0 0 100

16 0 0 0 1 0 0 100

17 0 0 0 1 0 0 100

18 0 0 0 1 0 0 100

19 0 0 0 1 0 0 100

20 0 0 0 1 0 0 100

21 0 0 0 1 0 0 100

22 0 0 0 1 0 0 100

23 0 0 0 1 0 0 100

24 0 0 0 1 0 0 100

25 0 0 0 1 0 0 100

26 0 0 0 1 0 0 100

27 0 0 0 1 0 0 100

28 0 0 0 1 0 0 100

29 0 0 0 1 0 0 100

30 0 0 0 1 0 0 100

31 0 0 0 1 0 0 100

32 0 0 0 1 0 0 100

33 0 0 0 1 0 0 100

34 0 0 0 1 0 0 100

35 0 0 0 1 0 0 100 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 231

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane – F1 Generation (Continued)

Day of

Test

1250 ng/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 2 0 0 0 0 100

16 0 2 0 0 0 0 100

17 0 2 0 0 0 0 100

18 0 2 0 0 0 0 100

19 0 2 0 0 0 0 100

20 0 2 0 0 0 0 100

21 0 2 0 0 0 0 100

22 0 2 0 0 0 0 100

23 0 2 0 0 0 0 100

24 0 2 0 0 0 0 100

25 0 2 0 0 0 0 100

26 0 2 0 0 0 0 100

27 0 2 0 0 0 0 100

28 0 2 0 0 0 0 100

29 0 2 0 0 0 0 100

30 0 2 0 0 0 0 100

31 0 2 0 0 0 0 100

32 0 2 0 0 0 0 100

33 0 2 0 0 0 0 100

34 0 2 0 0 0 0 100

35 0 2 0 0 0 0 100 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 232

Appendix 7 Replicate Mean Growth Measurements for the F0 Generation 

Day 0 Culture Mysids

Pan# Mysid#

Total Body 

Length (mm)

Dry Weight of 

Mysids Per Pan

(mg)

Avg. Dry 

Weight Per 

Mysid (mg) 

1

1-1 2.10

0.10

0.020

1-2 2.40

1-3 2.30

1-4 1.80

1-5 2.10

2

2-1 2.20

0.07

0.014

2-2 2.00

2-3 2.50

2-4 2.10

2-5 2.40

3

3-1 1.80

0.10

0.020

3-2 2.00

3-3 2.30

3-4 1.80

3-5 2.30

4

4-1 2.20

0.12

0.024

4-2 2.10

4-3 2.30

4-4 2.10

4-5 2.30

5

5-1 2.10

0.06

0.012

5-2 2.20

5-3 2.10

5-4 2.20

5-5 2.20
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 233

Appendix 7 Replicate Mean Growth Measurements for the F0

Generation Day 0 Culture Mysids (Continued)

Pan# Mysid#

Total Body 

Length (mm)

Dry Weight of 

Mysids Per Pan

(mg)

Avg. Dry 

Weight Per 

Mysid (mg) 

6

6-1 2.40

0.08

0.016

6-2 2.30

6-3 2.20

6-4 2.10

6-5 2.30

7

7-1 2.20

0.08

0.016

7-2 2.30

7-3 2.30

7-4 2.40

7-5 2.00

8

8-1 2.30

0.07

0.014

8-2 2.20

8-3 2.20

8-4 2.30

8-5 2.10

9

9-1 2.30

0.10

0.020

9-2 2.10

9-3 2.40

9-4 2.30

9-5 2.20

Average 2.20 0.09 0.017

N 45 9 --

S.D. 0.159 0.019 0.0039

CV 7.26 22.3 22.3
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 234

Appendix 8 Replicate Mean Total Body Lengths for the F0 Generation Day 7 

Mysids

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Negative Control 4.50 4.50 4.60

4.20 3.20 4.50

6.20 4.30 5.20

5.60 4.80 4.20

4.20 3.10 3.80

4.80 3.90 4.40

Replicate Mean 1: 4.92 3.97 4.45

Number 6 6 6

Standard Dev.: 0.816 0.698 0.464

CV (%): 16.6 17.6 10.4

32 5.10 6.30 5.20

4.80 6.10 5.80

5.10 5.00 4.60

4.50 4.50 4.90

5.20 3.70 5.80

5.90 4.60 4.70

Replicate Mean 1: 5.10 5.03 5.17

Number 6 6 6

Standard Dev.: 0.469 0.999 0.532

CV (%): 9.2 19.9 10.3

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 235

Appendix 8 Replicate Mean Total Body Lengths for the F0

Generation Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

80 5.10 4.70 4.60

4.90 5.30 4.30

5.80 5.20 4.30

4.00 6.10 3.50

4.30 4.40 4.70

3.80 6.20 4.30

Replicate Mean 1: 4.65 5.32 4.28

Number 6 6 6

Standard Dev.: 0.756 0.725 0.422

CV (%): 16.3 13.6 9.8

200 4.30 4.50 5.60

4.20 4.10 5.60

4.30 4.20 5.50

3.40 4.90 6.30

3.50 3.80 5.80

3.50 4.20 5.20

Replicate Mean 1: 3.87 4.28 5.67

Number 6 6 6

Standard Dev.: 0.441 0.376 0.367

CV (%): 11.4 8.8 6.5

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 236

Appendix 8 Replicate Mean Total Body Lengths for the F0

Generation Day 7 Mysids (Continued)

Nominal Test

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

500 6.30 5.60 5.20

4.80 6.30 4.80

5.20 5.30 5.60

3.90 5.30 4.60

4.60 5.80 5.10

4.40 5.30 5.20

Replicate Mean 1: 4.87 5.60 5.08

Number 6 6 6

Standard Dev.: 0.824 0.400 0.349

CV (%): 16.9 7.1 6.9

1250 3.80 5.20 4.30

3.90 5.20 5.20

5.80 5.20 3.90

5.20 3.90 5.20

4.30 5.60 5.20

4.60 4.90 4.30

Replicate Mean 1: 4.60 5.00 4.68

Number 6 6 6

Standard Dev.: 0.777 0.583 0.585

CV (%): 16.9 11.7 12.5

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 237

Appendix 9 Replicate Mean Dry Weights for the F0 Generation Day 7 

Mysids

Nominal Test 

Concentration

(ng/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

Negative Control

Pan 1 0.05 0.14 0.22

Pan 2 0.12 0.08 0.18

Pan 3 0.08 0.15 0.15

Replicate Mean 2: 0.08 0.12 0.18

Number 3 3 3

Standard Dev.: 0.035 0.038 0.033

CV (%): 42.1 30.7 17.7

32

Pan 1 0.21 0.18 0.27

Pan 2 0.28 0.19 0.23

Pan 3 0.24 0.09 0.23

Replicate Mean 2: 0.24 0.15 0.24

Number 3 3 3

Standard Dev.: 0.038 0.056 0.025

CV (%): 15.5 36.4 10.2

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two. 2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 238

Appendix 9 Replicate Mean Dry Weights for the F0 Generation 

Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

80

Pan 1 0.23 0.24 0.13

Pan 2 0.21 0.23 0.14

Pan 3 0.13 0.23 0.19

Replicate Mean 2: 0.19 0.23 0.15

Number 3 3 3

Standard Dev.: 0.056 0.008 0.032

CV (%): 28.8 3.3 21.0

200

Pan 1 0.11 0.16 0.24

Pan 2 0.15 0.14 0.30

Pan 3 0.18 0.13 0.31

Replicate Mean 2: 0.15 0.15 0.28

Number 3 3 3

Standard Dev.: 0.038 0.016 0.035

CV (%): 25.6 11.0 12.5

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two. 2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 239

Appendix 9 Replicate Mean Dry Weights for the F0 Generation 

Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

500

Pan 1 0.29 0.26 0.18

Pan 2 0.17 0.28 0.23

Pan 3 0.09 0.26 0.21

Replicate Mean 2: 0.18 0.26 0.20

Number 3 3 3

Standard Dev.: 0.100 0.014 0.026

CV (%): 55.8 5.5 12.6

1250

Pan 1 0.27 0.18 0.21

Pan 2 0.21 0.13 0.18

Pan 3 0.14 0.24 0.18

Replicate Mean 2: 0.21 0.18 0.19

Number 3 3 3

Standard Dev.: 0.068 0.055 0.013

CV (%): 32.4 29.7 7.0

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two. 2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 240

Appendix 10 Replicate Mean Total Body Lengths for the F1 Generation Day 7 

Mysids

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Negative Control 6.10 6.05 --

6.30 5.60 --

6.00 -- --

-- -- --

-- -- --

-- -- --

Replicate Mean 1: 6.133 5.825 --

Number 3 2 --

Standard Dev.: 0.153 0.318 --

CV (%): 2.49 5.46 --

32 5.80 5.90 6.50

5.50 5.20 5.50

5.50 5.60 7.00

5.90 -- 5.50

5.60 -- 5.50

5.30 -- --

Replicate Mean 1: 5.600 5.567 6.000

Number 6 3 5

Standard Dev.: 0.219 0.351 0.707

CV (%): 3.91 6.31 11.79

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 241

Appendix 10 Replicate Mean Total Body Lengths for the F1

Generation Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

80 5.30 4.50 6.35

5.60 5.00 6.15

5.50 4.80 7.25

4.70 -- 5.60

-- -- --

-- -- --

Replicate Mean 1: 5.275 4.767 6.338

Number 4 3 4

Standard Dev.: 0.403 0.252 0.686

CV (%): 7.64 5.28 10.82

200 6.00 -- --

5.40 -- --

-- -- --

-- -- --

-- -- --

-- -- --

Replicate Mean 1: 5.700 -- --

Number 2 -- --

Standard Dev.: 0.424 -- --

CV (%): 7.44 -- --

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 242

Appendix 10 Replicate Mean Total Body Lengths for the F1

Generation Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

500 5.80 5.60 --

-- 5.80 --

-- 5.40 --

-- -- --

-- -- --

-- -- --

Replicate Mean 1: 5.800 5.600 --

Number 1 3 --

Standard Dev.: 2 0.200 --

CV (%): 2 3.57 --

1250 -- -- --

-- -- --

-- -- --

-- -- --

-- -- --

-- -- --

Replicate Mean 1: -- -- --

Standard Dev.: -- -- --

CV (%): -- -- --

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 243

Appendix 11 Replicate Mean Dry Weights for the F1 Generation Day 7 Mysids

Nominal Test 

Concentration

(ng/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

Negative Control

Pan 1 0.38 0.25 --

Pan 2 0.35 -- --

Pan 3 -- -- --

Replicate Mean 2: 0.365 0.250 --

Number 2 1 3

Standard Dev.: 0.021 3 --

CV (%): 5.81 3 --

32

Pan 1 0.40 0.35 0.43

Pan 2 0.37 0.48 0.43

Pan 3 0.34 -- 0.73

Replicate Mean 2: 0.367 0.415 0.530

Number 3 2 3

Standard Dev.: 0.028 0.092 0.173

CV (%): 7.51 22.15 32.68

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two (unless the pan contained 

only one mysid). 2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 3 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 244

Appendix 11 Replicate Mean Dry Weights for the F1 

Generation Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

80

Pan 1 0.26 0.28 0.39

Pan 2 0.22 0.26 0.35

Pan 3 -- -- --

Replicate Mean 2: 0.238 0.268 0.368

Number 2 2 2

Standard Dev.: 0.032 0.011 0.025

CV (%): 13.40 3.97 6.73

200

Pan 1 0.52 -- --

Pan 2 -- -- --

Pan 3 -- -- --

Replicate Mean 2: 0.515 -- --

Number 1 -- --

Standard Dev.: 3 -- --

CV (%): 3 -- --

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two (unless the pan contained 

only one mysid). 2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 3 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 245

Appendix 11 Replicate Mean Dry Weights for the F1 

Generation Day 7 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

500

Pan 1 0.40 0.31 --

Pan 2 -- 0.24 --

Pan 3 -- -- --

Replicate Mean 2: 0.400 0.273 --

Number 1 2 --

Standard Dev.: 3 0.046 --

CV (%): 3 16.87 --

1250

Pan 1 -- -- --

Pan 2 -- -- --

Pan 3 -- -- --

Replicate Mean 2: -- -- --

Standard Dev.: -- -- --

CV (%): -- -- --

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two (unless the pan contained 

only one mysid). 2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 3 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 246

Appendix 12 Replicate Mean Body Lengths for Male and Female F0

Generation Day 14 Mysids

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 6.3 7.3 5.6 6.8 5.6 6.5

6.4 5.2 6.1 6.5 6.7 6.2

5.5 6.1 6.1 5.4 7.4 6.1

4.9 6.4 6.5 6.1 6.4 6.2

4.5 5.7 6.4 6.2 6.9

5.9 5.8 5.9 6.8

6.3 6.8 6.1

6.5

Immature2 Immature2

4.5 4.3

Replicate Mean 1: 5.52 6.18 6.08 6.24 6.56 6.25

Number 5 8 6 7 7 4

Standard Dev.: 0.838 0.620 0.343 0.506 0.586 0.173

CV (%): 15.2 10.0 5.6 8.1 8.9 2.8

32 6.5 6.7 6.7 6.7 6.6 6.6

6.4 5.9 7.2 6.5 6.4 6.9

6.5 7.2 7.1 6.7

6.1 7.2 5.8 7.3

6.4 7.2 7.9 6.3

6.7 6.6 6.2

6.8 6.7 7.4

6.8 7.9 6.7

7.3 7.2

6.4 7.8

7.6

Replicate Mean 1: 6.53 6.84 6.96 6.60 6.93 6.75

Number 8 5 10 2 11 2

Standard Dev.: 0.238 0.568 0.657 0.141 0.553 0.212

CV (%): 3.6 8.3 9.4 2.1 8.0 3.1

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Immature mysids were measured for total length and dry weight. They were not, however, included in the replicate 

mean, standard deviation or CV calculations or in the statistical analyses.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 247

Appendix 12 Replicate Mean Body Lengths for Male and Female F0

Generation Day 14 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

80 6.5 6.4 6.6 6.4 7.0 6.0

6.1 7.4 6.3 6.8 6.2 6.9

6.3 6.4 6.9 7.1 6.9

5.4 6.3 6.8 6.1 6.4

7.3 6.8 6.2 6.3

6.8 6.2 6.3 7.1

6.8 6.9 6.6 6.5

5.2

6.6

6.6

Replicate Mean 1: 6.46 6.63 6.65 6.39 6.60 6.59

Number 7 7 4 10 2 7

Standard Dev.: 0.608 0.427 0.265 0.511 0.566 0.393

CV (%): 9.4 6.4 4.0 8.0 8.6 6.0

200 6.3 6.7 6.4 6.5 8.0 7.4

5.8 5.7 6.2 5.7 7.3 6.5

5.5 6.9 4.9 5.9 7.1 7.3

6.7 6.8 6.0 6.6 7.2

5.7 6.8 6.2 5.7 7.5

5.9 7.2

Replicate Mean 1: 6.00 6.58 5.94 6.05 7.47 7.18

Number 5 5 5 6 3 6

Standard Dev.: 0.490 0.497 0.598 0.399 0.473 0.354

CV (%): 8.2 7.6 10.1 6.6 6.3 4.9

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 248

Appendix 12 Replicate Mean Body Lengths for Male and Female F0

Generation Day 14 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

500 6.5 7.3 7.6 7.1 7.0 6.4

6.6 7.4 7.8 7.7 6.5

7.6 6.9 6.5 6.0

7.5 6.5 7.8 5.9

8.1 7.2 7.8 6.3

7.3 6.6

7.0 6.1

Replicate Mean 1: 7.23 7.06 7.70 7.10 7.07 6.22

Number 7 5 2 1 7 5

Standard Dev.: 0.571 0.365 0.141 2 0.702 0.259

CV (%): 7.9 5.2 1.8 2 9.9 4.2

1250 6.1 6.6 6.5 6.5 6.5 6.1

5.6 6.3 6.3 7.2 6.7 7.0

5.9 6.2 7.4 6.3 6.6 6.7

6.3 6.2 6.5 7.1 7.1 6.9

6.9 7.2 7.0 5.5 7.4

7.0 6.9 5.7 6.7

6.3 7.0 6.5

6.2 6.1

5.4 Immature3

6.8 4.9

Replicate Mean 1: 5.98 6.39 6.78 6.76 6.35 6.76

Number 4 10 5 8 6 7

Standard Dev.: 0.299 0.465 0.487 0.407 0.619 0.408

CV (%): 5.0 7.3 7.2 6.0 9.7 6.0

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate. 3 Immature mysids were measured for total length and dry weight. They were not, however, included in the replicate 

mean, standard deviation or CV calculations or in the statistical analyses.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 249

Appendix 13 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 14 Mysids

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 0.48 0.49 0.32 0.52 0.29 0.41

0.44 0.38 0.31 0.54 0.56 0.48

0.35 0.34 0.35 0.25 0.60 0.50

0.22 0.71 0.60 0.55 0.40 0.60

0.20 0.45 0.42 0.45 0.55

0.44 0.39 0.46 0.55

0.57 0.51 0.36

0.60

Immature2 Immature2

0.11 0.15

Replicate Mean 1: 0.34 0.50 0.40 0.47 0.47 0.50

Number 5 8 6 7 7 4

Standard Dev.: 0.126 0.123 0.107 0.104 0.121 0.078

CV (%): 37.3 24.6 26.9 22.1 25.5 15.8

32 0.68 0.63 0.50 0.42 0.49 0.58

0.48 0.48 0.42 0.33 0.47 0.74

0.40 0.67 0.44 0.49

0.41 0.69 0.25 0.69

0.60 0.78 0.52 0.46

0.44 0.41 0.39

0.41 0.52 0.49

0.58 0.64 0.55

0.27 0.58

0.37 0.68

0.86

Replicate Mean 1: 0.50 0.65 0.43 0.38 0.56 0.66

Number 8 5 10 2 11 2

Standard Dev.: 0.106 0.110 0.119 0.064 0.135 0.113

CV (%): 21.2 16.9 27.4 17.0 24.2 17.1

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Immature mysids were measured for total length and dry weight. They were not, however, included in the replicate 

mean, standard deviation or CV calculations or in the statistical analyses.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 250

Appendix 13 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 14 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

80 0.35 0.31 0.56 0.74 0.60 0.38

0.47 0.57 0.51 0.53 0.39 0.67

0.52 0.61 0.48 0.77 0.62

0.23 0.66 0.39 0.31 0.46

0.70 0.35 0.40 0.44

0.37 0.31 0.56 0.40

0.45 0.65 0.51 0.33

0.26

0.66

0.69

Replicate Mean 1: 0.44 0.49 0.49 0.54 0.50 0.47

Number 7 7 4 10 2 7

Standard Dev.: 0.148 0.163 0.071 0.177 0.148 0.127

CV (%): 33.6 33.0 14.7 32.6 30.0 26.8

200 0.45 0.72 0.38 0.41 0.68 0.86

0.36 0.40 0.47 0.31 0.49 0.66

0.40 0.41 0.18 0.31 0.60 0.64

0.49 0.35 0.35 0.64 0.73

0.31 0.48 0.51 0.36 0.81

0.35 0.58

Replicate Mean 1: 0.40 0.47 0.38 0.40 0.59 0.71

Number 5 5 5 6 3 6

Standard Dev.: 0.071 0.146 0.128 0.125 0.095 0.107

CV (%): 17.7 31.0 34.0 31.5 16.2 15.0

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 251

Appendix 13 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 14 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

500 0.48 0.93 0.76 0.44 0.55 0.47

0.57 0.77 0.71 0.64 0.45

0.59 0.60 0.45 0.52

0.47 0.57 0.67 0.45

0.70 0.64 0.67 0.39

0.53 0.44

0.46 0.24

Replicate Mean 1: 0.54 0.70 0.73 0.44 0.52 0.46

Number 7 5 2 1 7 5

Standard Dev.: 0.086 0.149 0.035 2 0.158 0.047

CV (%): 15.8 21.2 4.8 2 30.2 10.2

1250 0.49 0.57 0.54 0.88 0.52 0.71

0.18 0.60 0.50 0.65 0.66 0.58

0.46 0.45 0.58 0.44 0.60 0.76

0.47 0.49 0.58 0.63 0.52 0.58

0.54 0.65 0.81 0.34 0.67

0.46 0.70 0.23 0.44

0.52 0.53 0.65

0.40 0.68

0.37 Immature3

0.48 0.10

Replicate Mean 1: 0.40 0.49 0.57 0.66 0.48 0.63

Number 4 10 5 8 6 7

Standard Dev.: 0.147 0.072 0.056 0.141 0.163 0.105

CV (%): 36.8 14.8 9.8 21.2 34.0 16.8

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate. 3 Immature mysids were measured for total length and dry weight. They were not, however, included in the replicate 

mean, standard deviation or CV calculations or in the statistical analyses.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 252

Appendix 14 Replicate Mean Body Lengths for Male and Female F1 Generation 

Day 14 Mysids

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

32 6.7 6.9 7.8 7.8 -- --

7.2 7.3 7.6 6.5

6.4 7.4 6.9

7.0 7.7

Replicate Mean 1: 7.0 6.9 7.6 7.2 -- --

Number 2 4 3 4 -- --

Standard Dev.: 0.354 0.374 0.200 0.629

CV (%): 5.1 5.4 2.6 8.7

80 7.8 7.6 -- -- 6.9 --

7.6 7.5

7.5

7.3

Replicate Mean 1: 7.6 7.6 -- -- 6.9 --

Number 4 2 -- -- 1 --

Standard Dev.: 0.208 0.071 2

CV (%): 2.8 0.9 2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 253

Appendix 15 Replicate Mean Dry Weights for Male and Female F1 Generation 

Day 14 Mysids

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

32 0.70 0.85 0.71 0.81 -- --

0.67 0.53 0.66 0.59

0.68 0.77 0.72

0.45 0.81

Replicate Mean 1: 0.68 0.63 0.71 0.73 -- --

Number 2 4 3 4 -- --

Standard Dev.: 0.021 0.176 0.055 0.104

CV (%): 3.1 28.1 7.7 14.2

80 0.82 1.28 -- -- 0.57 --

0.84 0.91

0.88

0.76

Replicate Mean 1: 0.83 1.09 -- -- 0.57 --

Number 4 2 -- -- 1 --

Standard Dev.: 0.050 0.262 2

CV (%): 6.1 23.9 2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 254

Appendix 16 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 44 Mysids

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 9.3 10.1 9.4 8.8 8.9 9.0

9.4 9.9 9.4 9.2 9.4 8.6

9.4 9.6 9.0 9.0 9.1 9.0

9.5 9.5 8.8 9.9 8.9 9.5

9.0 8.5 8.9 9.5 8.9 9.3

8.7 9.1 8.7 10.3 8.8

8.7 9.2 8.5 8.9

9.4 8.2 9.8

9.0 9.4

8.9 9.3

9.8

Replicate Mean 1: 9.1 9.4 8.8 9.4 9.0 9.1

Number 10 7 8 11 6 5

Standard Dev.: 0.312 0.540 0.415 0.457 0.221 0.352

CV (%): 3.4 5.7 4.7 4.8 2.5 3.9

32 8.6 9.6 8.7 9.3 9.3 9.3

9.7 9.1 8.6 9.3 9.3 9.6

9.3 9.5 8.6 9.4 8.3 9.3

9.1 9.9 9.5 9.0 9.5

9.0 9.1 9.1 8.3 9.3

9.1 9.1 8.5 9.7 9.0

9.7 9.5 9.4 9.1 9.3

9.3 9.7 9.5

9.2 8.9

8.8 8.7

9.2 9.2

9.3 9.7

Replicate Mean 1: 9.2 9.4 8.6 9.2 9.0 9.3

Number 7 8 3 12 12 7

Standard Dev.: 0.412 0.290 0.076 0.308 0.460 0.186

CV (%): 4.5 3.1 0.9 3.4 5.1 2.0

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 255

Appendix 16 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 44 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

80 8.8 9.6 8.5 8.9 9.0 9.7

8.9 8.9 8.5 9.1 9.4 9.1

9.3 9.1 8.9 9.3 9.6 9.3

8.7 9.1 8.9 8.8 8.6 9.6

9.4 9.0 8.6 8.9 8.9

9.2 10.1 8.6 9.5 9.1

9.5 8.5 10.1 9.9

9.3

Replicate Mean 1: 9.1 9.3 8.7 9.2 9.1 9.3

Number 7 6 8 7 4 7

Standard Dev.: 0.316 0.437 0.286 0.454 0.464 0.376

CV (%): 3.5 4.7 3.3 4.9 5.1 4.0

200 9.4 8.6 8.6 9.2 8.6 9.5

8.6 9.4 8.2 9.5 9.0 9.5

9.0 9.1 9.8 9.8 8.8 9.7

8.5 9.3 9.9 9.2 10.0

9.7 8.6 9.1 8.4 9.1

9.5 9.2 9.8 8.9 9.4

9.5 9.3 9.5 9.3 9.6

9.2 9.6 8.9

8.4 9.9

9.5 9.8

9.1

Replicate Mean 1: 9.0 9.2 9.0 9.4 8.9 9.5

Number 3 8 7 11 7 10

Standard Dev.: 0.425 0.429 0.536 0.433 0.313 0.338

CV (%): 4.7 4.7 6.0 4.6 3.5 3.6

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 256

Appendix 16 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 44 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

500 9.5 9.7 8.5 9.3 8.8 9.5

9.9 9.4 9.3 9.4 9.1 8.9

8.6 9.4 9.8 9.5 9.4 9.7

9.2 9.0 9.1 9.4 8.9 8.4

9.9 9.3 9.7 9.1 8.5

9.1 9.7 8.7 9.2

9.7 8.0 9.0

9.5

9.2

9.5

Replicate Mean 1: 9.3 9.4 9.2 9.5 8.8 9.0

Number 4 10 5 6 7 7

Standard Dev.: 0.545 0.283 0.486 0.157 0.455 0.483

CV (%): 5.9 3.0 5.3 1.7 5.2 5.4

1250 9.1 8.6 9.6 9.3 8.3 9.2

9.2 9.7 8.5 9.0 9.4 9.4

9.3 8.5 8.3 9.7 9.0 8.2

8.6 9.5 9.3 8.6 9.2 9.3

8.9 9.5 8.6 8.9 6.9 9.2

8.8 10.0 9.3 9.9 9.1 9.2

9.9 9.8 8.9 8.9 9.2 9.7

9.2 9.3 9.2

9.4

8.7

9.5

Replicate Mean 1: 9.1 9.3 8.9 9.2 8.7 9.1

Number 8 11 7 7 7 8

Standard Dev.: 0.389 0.510 0.469 0.479 0.896 0.415

CV (%): 4.3 5.5 5.3 5.2 10.3 4.5

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 257

Appendix 17 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 44 Mysids

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 1.12 1.48 1.22 1.37 1.06 1.28

1.09 1.50 1.12 1.06 1.09 1.03

1.17 1.61 1.30 1.30 1.22 1.46

1.26 1.18 0.99 1.51 1.16 1.41

1.31 1.12 1.14 1.15 1.07 1.21

1.09 0.96 1.12 1.92 0.98

1.08 0.96 1.06 1.45

1.04 0.84 1.57

0.94 1.67

1.10 1.35

1.35

Replicate Mean 1: 1.12 1.26 1.10 1.43 1.10 1.28

Number 10 7 8 11 6 5

Standard Dev.: 0.106 0.269 0.140 0.239 0.084 0.171

CV (%): 9.4 21.4 12.8 16.8 7.6 13.4

32 1.03 1.30 1.20 1.22 1.32 1.80

1.26 1.25 1.20 1.61 1.25 1.67

1.08 1.50 1.30 1.37 1.20 1.51

1.17 1.54 1.40 1.37 1.68

1.17 1.22 1.32 1.11 1.70

1.28 1.08 1.31 1.33 1.64

1.47 1.72 1.63 1.26 1.70

1.43 1.26 1.31

1.13 0.95

1.06 1.20

1.23 1.44

1.47 1.44

Replicate Mean 1: 1.21 1.38 1.23 1.33 1.27 1.67

Number 7 8 3 12 12 7

Standard Dev.: 0.146 0.206 0.058 0.174 0.139 0.087

CV (%): 12.1 14.9 4.7 13.1 11.0 5.2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 258

Appendix 17 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 44 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

80 1.30 1.50 1.21 1.39 1.22 1.77

1.11 1.40 1.03 1.77 1.26 1.26

1.35 1.41 1.27 1.53 1.53 1.65

1.14 1.65 1.18 1.23 1.37 1.62

1.38 1.48 1.08 1.50 1.25

1.26 1.60 1.24 1.79 1.54

1.50 1.11 1.80 1.59

1.35

Replicate Mean 1: 1.29 1.51 1.18 1.57 1.35 1.53

Number 7 6 8 7 4 7

Standard Dev.: 0.136 0.101 0.106 0.222 0.139 0.198

CV (%): 10.6 6.7 9.0 14.1 10.3 13.0

200 1.36 1.20 1.13 1.74 1.38 1.82

1.05 1.52 0.85 1.86 1.36 1.94

1.18 1.40 1.43 2.11 1.33 1.82

1.45 1.44 1.25 1.29 2.00

1.58 1.14 1.21 1.18 1.55

1.48 1.40 1.90 1.44 1.79

1.72 1.28 1.58 1.47 1.65

1.08 1.82 1.08

1.07 1.89

1.48 1.76

1.30

Replicate Mean 1: 1.20 1.43 1.24 1.57 1.35 1.73

Number 3 8 7 11 7 10

Standard Dev.: 0.156 0.205 0.215 0.337 0.097 0.263

CV (%): 13.0 14.3 17.4 21.4 7.2 15.2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 259

Appendix 17 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 44 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

500 1.30 1.66 1.04 1.17 1.18 1.35

1.37 1.66 1.38 1.70 1.45 1.41

1.12 1.27 1.48 1.48 1.40 1.52

1.36 1.59 1.32 1.26 1.14 1.17

1.88 1.40 1.45 1.33 1.05

1.45 1.77 1.05 1.61

1.45 0.97 1.49

1.64

1.24

1.34

Replicate Mean 1: 1.29 1.52 1.32 1.47 1.22 1.37

Number 4 10 5 6 7 7

Standard Dev.: 0.116 0.203 0.169 0.236 0.181 0.200

CV (%): 9.0 13.4 12.7 16.0 14.9 14.5

1250 1.11 1.58 1.38 1.47 0.93 1.08

1.28 1.73 0.94 1.98 1.30 1.55

1.49 1.23 0.98 1.53 1.19 1.11

1.13 1.56 1.40 1.13 1.23 1.55

1.15 1.60 1.07 1.53 0.78 1.48

1.27 1.59 1.17 1.76 1.28 1.58

1.06 1.53 1.40 1.53 1.39 1.58

1.22 1.35 1.54

1.34

1.39

1.76

Replicate Mean 1: 1.21 1.51 1.19 1.56 1.16 1.43

Number 8 11 7 7 7 8

Standard Dev.: 0.136 0.167 0.202 0.262 0.220 0.212

CV (%): 11.2 11.1 17.0 16.8 19.0 14.8

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 260

Appendix 18 Replicate Mean Body Lengths for Male and Female F1 Generation 

Day 35 Mysids

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 8.6 9.5 9.0 9.5 -- 9.0

9.5 9.0

9.5

Replicate Mean 1: 8.6 9.5 9.3 9.3 -- 9.0

Number 1 1 2 3 1

Standard Dev.: 2 2 0.354 0.289 2

CV (%): 2 2 3.8 3.1 2

32 8.4 9.4 8.8 9.3 8.7 7.6

8.4 9.2 8.8 9.1 9.0 8.7

8.8 9.3 9.0 8.9 9.1 8.8

8.6 9.6 8.1

8.9

Replicate Mean 1: 8.5 9.1 8.9 9.2 8.9 8.3

Number 3 5 3 4 3 4

Standard Dev.: 0.231 0.327 0.115 0.299 0.208 0.560

CV (%): 2.7 3.6 1.3 3.2 2.3 6.7

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 261

Appendix 18 Replicate Mean Body Lengths for Male and Female F1 Generation 

Day 35 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

80 8.2 8.7 9.6 8.9 9.5 9.5

8.1 9.5 9.3 8.8 9.5

8.3 9.5 9.3 8.7 9.5

9.2 8.8 9.1

8.5 8.4

8.0

Replicate Mean 1: 8.5 9.2 9.1 8.7 9.5 9.5

Number 4 3 5 6 3 1

Standard Dev.: 0.507 0.462 0.442 0.394 0.000 2

CV (%): 6.0 5.0 4.9 4.6 0.0 2

200 9.0 10.0 8.1 8.6 9.3 9.7

10.5 10.0 8.7 9.1

10.0 9.5 8.8 8.7

9.5

10.5

Replicate Mean 1: 9.8 9.9 8.5 8.8 9.3 9.7

Number 3 5 3 3 1 1

Standard Dev.: 0.764 0.418 0.379 0.265 2 2

CV (%): 7.8 4.2 4.4 3.0 2 2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 262

Appendix 18 Replicate Mean Body Lengths for Male and Female F1 Generation 

Day 35 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

500 -- 10.5 9.5 9.5 -- --

9.5

9.0

Replicate Mean 1: -- 10.5 9.5 9.3 -- --

Number 1 1 3

Standard Dev.: 2 2 0.289

CV (%): 2 2 3.1

1250 -- 9.0 8.4 -- -- --

9.8

Replicate Mean 1: -- 9.4 8.4 -- -- --

Number 2 1

Standard Dev.: 0.566 2

CV (%): 6.0 2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 263

Appendix 19 Replicate Mean Dry Weights for Male and Female F1 Generation 

Day 35 Mysids

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 1.10 1.15 0.89 1.34 -- 0.93

1.14 1.48

1.56

Replicate Mean 1: 1.10 1.15 1.01 1.46 -- 0.93

Number 1 1 2 3 1

Standard Dev.: 2 2 0.177 0.111 2

CV (%): 2 2 17.4 7.6 2

32 1.13 1.26 1.24 1.35 1.10 0.76

1.04 1.67 0.89 1.10 0.96 1.07

1.45 1.60 1.17 1.12 1.24 1.42

1.21 1.15 1.07

1.47

Replicate Mean 1: 1.21 1.44 1.10 1.18 1.10 1.08

Number 3 5 3 4 3 4

Standard Dev.: 0.215 0.203 0.185 0.115 0.140 0.270

CV (%): 17.9 14.1 16.8 9.8 12.7 25.0

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 264

Appendix 19 Replicate Mean Dry Weights for Male and Female F1 Generation 

Day 35 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

80 1.37 0.99 1.52 1.33 1.02 1.66

0.70 1.38 1.52 1.32 1.30

1.05 1.42 1.22 1.28 1.33

0.91 0.95 1.41

0.96 0.96

0.86

Replicate Mean 1: 1.01 1.26 1.23 1.19 1.22 1.66

Number 4 3 5 6 3 1

Standard Dev.: 0.281 0.238 0.283 0.226 0.171 2

CV (%): 27.9 18.8 22.9 18.9 14.1 2

200 1.22 1.38 1.21 1.52 0.91 1.38

1.18 1.40 1.53 1.30

1.25 1.52 1.20 0.38

1.15

1.58

Replicate Mean 1: 1.22 1.41 1.31 1.07 0.91 1.38

Number 3 5 3 3 1 1

Standard Dev.: 0.035 0.165 0.188 0.605 2 2

CV (%): 2.9 11.8 14.3 56.7 2 2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 265

Appendix 19 Replicate Mean Dry Weights for Male and Female F1 Generation 

Day 35 Mysids (Continued)

Nominal Test 

Concentration

(ng/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

500 -- 1.42 1.19 1.20 -- --

1.33

1.02

Replicate Mean 1: -- 1.42 1.19 1.18 -- --

Number 1 1 3

Standard Dev.: 2 2 0.156

CV (%): 2 2 13.2

1250 -- 1.22 0.83 -- -- --

1.58

Replicate Mean 1: -- 1.40 0.83 -- -- --

Number 2 1

Standard Dev.: 0.255 2

CV (%): 18.2 2

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly. 2 Standard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 266

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

Negative A 1 14 14.0 22 25.7 33 33.8 11 8.2

Control 2 14 0.0 25 3.14 29 5.15 4 5.88

3 14 0.0 30 12.2 35 15.2 5 72.0

4 14 24 43 19

5 14 24 29 5

6 14 -- -- --

7 14 29 34 5

8 14 N/A N/A N/A

9 3 N/A N/A N/A

B 1 14 14.8 24 25.9 31 34.2 7 10.0

2 14 2.12 30 3.34 -- 7.50 -- 6.48

3 14 14.4 21 12.9 26 21.9 5 64.8

4 14 24 44 20

5 14 25 30 5

6 14 30 -- --

7 14 27 40 13

8 3 N/A N/A N/A

9 20 N/A N/A N/A

C 1 14 14.9 21 27.0 25 31.7 4 4.7

2 14 2.67 -- 5.18 -- 5.09 -- 1.37

3 14 17.9 34 19.2 37 16.1 3 29.3

4 14 21 26 5

5 14 30 34 4

6 14 27 32 5

7 14 29 36 7

8 14 N/A N/A N/A

9 22 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 267

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

32 A 1 14 14.0 28 31.3 38 38.8 10 9.4

2 14 0.00 32 5.20 39 0.45 7 2.30

3 14 0.0 41 16.6 -- 1.2 -- 24.5

4 14 31 39 8

5 14 26 39 13

6 14 -- -- --

7 14 30 39 9

8 14 N/A N/A N/A

9 14 N/A N/A N/A

B 1 14 14.0 -- 29.0 -- 34.0 -- 7.3

2 14 0.00 22 6.39 33 5.03 11 2.87

3 14 0.0 30 22.0 -- 14.8 -- 39.6

4 14 37 -- --

5 14 24 29 5

6 14 36 41 5

7 14 25 33 8

8 3 N/A N/A N/A

9 14 N/A N/A N/A

C 1 14 14.0 17 28.6 22 32.8 5 6.8

2 14 0.00 44 8.98 -- 7.41 -- 4.71

3 14 0.0 22 31.4 32 22.6 10 68.9

4 14 28 43 15

5 14 25 29 4

6 14 28 32 4

7 14 36 39 3

8 14 N/A N/A N/A

9 3 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 268

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

80 A 1 14 14.0 27 30.4 33 31.3 6 5.5

2 14 0.00 38 7.61 -- 3.50 -- 0.58

3 14 0.0 43 25.0 -- 11.2 -- 10.5

4 14 25 30 5

5 14 21 27 6

6 14 29 -- --

7 14 30 35 5

8 14 N/A N/A N/A

9 14 N/A N/A N/A

B 1 14 14.0 23 25.1 28 30.6 5 5.4

2 14 0.00 20 3.98 -- 3.21 -- 2.70

3 14 0.0 25 15.9 28 10.5 3 50.0

4 14 31 35 4

5 14 24 29 5

6 14 23 33 10

7 14 30 -- --

8 14 N/A N/A N/A

9 14 N/A N/A N/A

C 1 14 14.0 -- 24.5 -- 30.0 -- 5.0

2 14 0.00 18 4.46 -- 3.46 -- 0.00

3 14 0.0 29 18.2 -- 11.5 -- 0.0

4 14 24 29 5

5 14 24 29 5

6 14 30 35 5

7 14 22 27 5

8 3 N/A N/A N/A

9 14 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 269

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

200 A 1 14 14.0 21 25.9 26 29.7 5 7.3

2 14 0.00 22 4.53 34 4.04 12 4.04

3 14 0.0 34 17.5 -- 13.6 -- 55.1

4 14 29 -- --

5 14 24 29 5

6 14 24 -- --

7 14 27 -- --

8 14 N/A N/A N/A

9 14 N/A N/A N/A

B 1 14 14.4 31 29.4 34 37.6 3 8.1

2 14 1.06 25 2.76 35 3.60 10 4.34

3 14 7.4 30 9.4 35 9.6 5 53.3

4 14 29 44 15

5 14 28 40 12

6 14 29 36 7

7 14 34 39 5

8 3 N/A N/A N/A

9 17 N/A N/A N/A

C 1 14 14.0 16 23.1 21 25.7 5 5.8

2 14 0.00 18 9.79 23 5.43 5 2.04

3 14 0.0 21 42.4 31 21.1 10 35.0

4 14 18 23 5

5 14 43 -- --

6 14 17 22 5

7 14 29 34 5

8 14 N/A N/A N/A

9 14 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 270

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

500 A 1 14 14.0 22 24.4 -- 36.3 -- 10.3

2 14 0.00 -- 5.41 -- 8.33 -- 1.53

3 14 0.0 -- 22.2 -- 22.9 -- 14.8

4 14 29 39 10

5 14 18 27 9

6 14 31 43 12

7 14 22 -- --

8 14 N/A N/A N/A

9 14 N/A N/A N/A

B 1 14 14.0 -- 21.2 -- 29.8 -- 10.5

2 14 0.00 23 4.36 33 4.72 10 4.20

3 14 0.0 29 20.6 -- 15.8 -- 40.0

4 14 18 23 5

5 14 18 30 12

6 14 18 33 15

7 14 21 -- --

8 3 N/A N/A N/A

9 3 N/A N/A N/A

C 1 14 15.0 29 27.1 38 34.3 9 6.3

2 14 3.00 20 6.67 -- 3.51 -- 2.31

3 14 20.0 19 24.6 -- 10.2 -- 36.5

4 14 29 34 5

5 14 26 31 5

6 14 28 -- --

7 14 39 -- --

8 14 N/A N/A N/A

9 23 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be used 

to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 271

Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

1250 A 1 14 14.0 -- 25.0 -- 32.3 -- 7.3

2 14 0.00 -- 6.08 -- 1.16 -- 6.66

3 14 0.0 -- 24.3 -- 3.6 -- 90.8

4 14 -- -- --

5 14 29 33 4

6 14 18 33 15

7 14 28 31 3

8 14 N/A N/A N/A

9 14 N/A N/A N/A

B 1 14 14.0 -- 21.0 -- 31.0 -- 5.0

2 14 0.00 15 5.57 -- 3 -- 3

3 14 0.0 -- 26.5 -- 3 -- 3

4 14 -- -- --

5 14 26 31 5

6 14 22 -- --

7 14 -- -- --

8 14 N/A N/A N/A

9 14 N/A N/A N/A

C 1 14 14.0 -- 24.0 -- 28.7 -- 7.7

2 14 0.00 -- 6.48 -- 4.51 -- 6.43

3 14 0.0 24 27.0 29 15.7 5 83.9

4 14 21 24 3

5 14 -- -- --

6 14 18 33 15

7 14 33 -- --

8 14 N/A N/A N/A

9 14 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 Standard deviation and CV were not determined as only one chamber was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 272

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F1

Generation

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

Negative A 1 14 14.0 -- 4 -- 4 -- 4

Control 2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

B 1 14 14.0 17.0 17.0 -- 23.0 -- 6.0

2 14 0.00 17.0 0.00 23 4 6 4

3 3 0.0 3 0.0 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 N/A N/A N/A

C 1 3 14.0 3 4 3 4 3 4

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were not enough organisms to initiate the brood chamber. 4 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 273

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F1

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

32 A 1 14 14.0 -- 15.0 -- 4 -- 4

2 14 0.00 15 4 -- 4 -- 4

3 14 0.0 -- 4 -- 4 -- 4

4 14 -- -- --

5 14 -- -- --

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 N/A N/A N/A

B 1 13 13.0 19 26.75 29 31.00 10 9.50

2 13 0.00 24 6.85 33 2.83 9 0.71

3 13 0.0 35 25.61 -- 9.12 -- 7.44

4 13 29 -- --

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

C 1 14 14.00 -- 24.00 -- 29.00 -- 5.00

2 14 0.00 -- 7.07 -- 7.07 -- 0.00

3 14 0.00 29 29.46 34 24.38 5 0.00

4 14 19 24 5

5 14 -- -- --

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were not enough organisms to initiate the brood chamber. 4 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 274

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F1

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

80 A 1 14 14.0 29 26.7 -- 4 -- 4

2 14 0.00 18 7.77 -- 4 -- 4

3 14 0.0 33 29.1 -- 4 -- 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 14 N/A N/A N/A

9 3 3 3 3

B 1 14 14.0 28 27.0 -- 33.0 -- 6.0

2 14 0.00 27 0.71 -- 4 -- 4

3 14 0.0 -- 2.62 -- 4 -- 4

4 14 26 -- --

5 14 27 -- --

6 14 27 33 6

7 14 -- -- --

8 14 N/A N/A N/A

9 3 3 3 3

C 1 14 14.0 -- 17.0 -- 4 -- 4

2 14 0.00 -- 4 -- 4 -- 4

3 14 0.0 17 4 -- 4 -- 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were not enough organisms to initiate the brood chamber. 4 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 275

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F1

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

200 A 1 14 14.0 26 22.0 35 33.33 9 9.33

2 14 0.00 16 4.24 -- 2.89 -- 2.52

3 14 0.0 23 19.3 35 8.66 12 26.96

4 14 23 30 7

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 N/A N/A N/A

B 1 14 14.0 -- 16.0 -- 21.0 -- 5.00

2 14 0.00 16 4 21 4 5 4

3 14 0.0 -- 4 -- 4 -- 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 14 N/A N/A N/A

9 3 3 3 3

C 1 14 14.0 22 22.0 -- 4 -- 4

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were not enough organisms to initiate the brood chamber. 4 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 276

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F1

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

500 A 1 14 14.0 16 16.0 26 26.0 10 10.0

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

B 1 14 14.0 35 35.0 -- 4 -- 4

2 3 0.00 3 4 3 4 3 4

3 3 0.0 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 3 3 3

C 1 3 14.0 -- 4 -- 4 -- 4

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 N/A N/A N/A 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were not enough organisms to initiate the brood chamber. 4 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 277

Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F1

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

Replicate

Mean

SD

CV(%)2

1250 A 1 3 14.00 3 4 3 4 3 4

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 14 N/A N/A N/A

B 1 3 14.00 3 4 3 4 3 4

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 14 N/A N/A N/A

9 3 3 3 3

C 1 3 4 3 4 3 4 3 4

2 3 4 3 4 3 4 3 4

3 3 4 3 4 3 4 3 4

4 3 3 3 3

5 3 3 3 3

6 3 3 3 3

7 3 3 3 3

8 3 3 3 3

9 3 3 3 3

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids. 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 There were not enough organisms to initiate the brood chamber. 4 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 278

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

Negative A 1 6 4.7 2 4.8 9 74

Control 2 2 3.0 5 2.4 12 10.6

3 4 64.5 8 49.7 18 6.7

4 2 2 4 63.2

5 4 6 14

6 04 04 0

7 10 6 17

B 1 3 4.7 1 2.9 4 55

2 10 3.6 0 5.0 10 7.9

3 1 76.2 1 175.5 4 6.6

4 1 3 4 84.6

5 8 14 22

6 7 0 7

7 3 1 4

C 1 5 2.7 9 4.8 31 86

2 04 1.6 04 3.7 0 12.3

3 4 61.2 2 76.8 6 10.9

4 3 4 9 89.0

5 1 10 14

6 2 2 22

7 1 2 4 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 279

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

32 A 1 6 3.0 1 2.7 19 46

2 1 2.4 2 2.5 3 6.6

3 1 78.9 0 93.9 1 6.5

4 2 4 6 98.2

5 2 7 9

6 04 04 0

7 6 2 8

B 1 04 4.0 04 2.3 0 57

2 1 3.3 1 3.0 5 8.1

3 10 82.2 0 129.0 10 7.5

4 2 0 2 92.0

5 4 3 16

6 2 2 4

7 5 8 20

C 1 1 2.4 4 5.6 24 112

2 4 1.4 0 4.6 4 16.0

3 3 57.5 7 82.8 20 12.1

4 1 2 3 75.9

5 4 14 37

6 1 8 9

7 3 4 15 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 280

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

80 A 1 4 3.6 2 1.4 22 67

2 4 1.5 0 1.6 4 9.6

3 2 42.3 0 113.3 2 7.1

4 1 4 13 74.1

5 5 3 14

6 5 0 5

7 4 1 7

B 1 2 2.9 6 3.1 12 77

2 1 2.1 0 3.1 1 11.0

3 4 74.0 8 98.0 18 9.3

4 6 1 11 84.8

5 5 4 27

6 1 3 7

7 1 0 1

C 1 04 3.2 04 1.7 0 40

2 1 1.9 0 1.6 1 5.7

3 6 61.3 0 98.0 6 5.1

4 4 2 9 89.1

5 4 3 15

6 1 4 5

7 3 1 4 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 281

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

200 A 1 2 3.3 1 1.4 11 45

2 8 2.7 1 2.9 9 6.4

3 1 81.9 0 205.5 1 6.5

4 1 0 1 100.8

5 6 8 18

6 3 0 3

7 2 0 2

B 1 1 2.0 1 2.3 9 52

2 2 1.0 3 1.0 10 7.4

3 2 50.0 1 41.6 8 2.1

4 2 3 5 27.9

5 4 2 6

6 1 3 9

7 2 3 5

C 1 1 2.0 3 3.4 11 79

2 3 1.5 5 2.4 16 11.3

3 5 76.4 5 71.2 13 7.4

4 2 3 21 65.3

5 1 0 1

6 1 7 15

7 1 1 2 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 282

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

500 A 1 3 2.8 0 4.4 3 39

2 04 2.0 04 5.6 0 5.6

3 04 73.2 04 127.2 0 6.5

4 3 13 16 116.7

5 1 2 6

6 6 7 13

7 1 0 1

B 1 04 2.7 04 4.2 0 66

2 8 2.7 3 4.2 17 9.4

3 2 99.7 0 100 2 8.9

4 1 5 6 94.4

5 1 6 18

6 2 11 21

7 2 0 2

C 1 1 1.7 3 1.9 4 31

2 1 1.3 0 3.3 1 4.4

3 1 73.1 0 179.7 1 6.2

4 4 1 5 140.3

5 3 9 18

6 1 0 1

7 1 0 1 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 283

Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

1250 A 1 04 4.0 04 2.7 0 22

2 04 4.4 04 2.1 0 3.1

3 04 109.0 04 78.1 0 4.3

4 04 04 0 136.8

5 2 5 8

6 1 2 4

7 9 1 10

B 1 04 1.0 04 0.3 0 4

2 1 0.0 0 0.6 1 0.6

3 04 0.0 04 173.2 0 0.8

4 04 04 0 137.7

5 1 1 2

6 1 0 1

7 04 04 0

C 1 04 2.3 04 1.0 0 22

2 04 1.3 04 0.8 0 3.1

3 1 55.9 1 81.6 2 3.8

4 2 1 6 121.1

5 04 04 0

6 2 2 10

7 4 0 4 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 284

Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F1 

Generation

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

Negative A 1 06 5 06 5 0 0.0

Control 2 4 5 4 5 4 5

3 4 5 4 5 4 5

4 4 4 4

5 4 4 4

6 4 4 4

7 4 4 4

B 1 3 3.5 0 3.5 3 22

2 4 0.71 7 4.95 19 11.0

3 4 20.2 4 141.4 4 11.31

4 4 4 4 102.9

5 4 4 4

6 4 4 4

7 4 4 4

C 1 4 5 4 5 4 5

2 4 5 4 5 4 5

3 4 5 4 5 4 5

4 4 4 4

5 4 4 4

6 4 4 4

7 4 4 4

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 The F1 generation did not produce enough living organisms to initiate the Brood Chamber. 5 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group. 6 Represents a female that never produced a brood throughout the F1 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
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Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F1 

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

32 A 1 06 1.0 06 0.0 0 1

2 1 5 0 5 1 0.2

3 06 5 06 5 0 0.45

4 06 06 0 223.6

5 06 06 0

6 4 4 4

7 4 4 4

B 1 1 1.5 2 0.8 3 9

2 1 1.00 1 0.96 2 2.3

3 1 66.7 0 127.7 1 0.96

4 3 0 3 42.6

5 4 4 4

6 4 4 4

7 4 4 4

C 1 06 4 06 3.5 0 19

2 06 4.24 06 0.71 0 3.8

3 7 106.1 3 20.2 10 5.22

4 1 4 9 137.2

5 06 06 0

6 4 4 4

7 4 4 4

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 The F1 generation did not produce enough living organisms to initiate the Brood Chamber. 5 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group. 6 Represents a female that never produced a brood throughout the F1 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
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Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F1 

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

80 A 1 5 5.7 0 0.0 5 17

2 2 4.04 0 0.00 2 5.7

3 10 71.3 0 5 10 4.04

4 4 4 4 71.3

5 4 4 4

6 4 4 4

7 4 4 4

B 1 1 4.2 0 0.6 1 24

2 2 3.56 0 1.34 2 3.4

3 06 84.9 06 223.6 0 3.74

4 10 0 10 108.9

5 5 0 5

6 3 3 6

7 06 06 0

C 1 06 8.0 06 0.0 0 8

2 06 5 06 5 0 2.7

3 8 5 0 5 8 4.62

4 4 4 4 173.2

5 4 4 4

6 4 4 4

7 4 4 4

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 The F1 generation did not produce enough living organisms to initiate the Brood Chamber. 5 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group. 6 Represents a female that never produced a brood throughout the F1 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
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Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F1 

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

200 A 1 8 2.8 2 2.0 10 19

2 1 3.50 0 2.16 1 4.8

3 1 127.3 1 108.0 2 4.11

4 1 5 6 86.6

5 4 4 4

6 4 4 4

7 4 4 4

B 1 06 1.0 06 1.0 0 4

2 1 5 1 5 4 1.3

3 06 5 06 5 0 2.31

4 4 4 4 173.2

5 4 4 4

6 4 4 4

7 4 4 4

C 1 2 2.0 0 0.0 2 2

2 4 5 4 5 4 2.0

3 4 5 4 5 4 5

4 4 4 4 5

5 4 4 4

6 4 4 4

7 4 4 4

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 The F1 generation did not produce enough living organisms to initiate the Brood Chamber. 5 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group. 6 Represents a female that never produced a brood throughout the F1 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
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Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F1 

Generation (Continued)

Nominal Test 

Concentration

(ng/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total Number 

of Offspring 

Produced3

Replicate Total 

Produced

Mean

SD

CV%2

500 A 1 2 2.0 2 2.0 4 4

2 4 5 4 5 4 4.0

3 4 5 4 5 4 5

4 4 4 4 5

5 4 4 4

6 4 4 4

7 4 4 4

B 1 3 3.0 0 0.0 3 3

2 4 5 4 5 4 3.0

3 4 5 4 5 4 5

4 4 4 4 5

5 4 4 4

6 4 4 4

7 4 4 4

C 1 4 5 4 5 4 5

2 4 5 4 5 4 5

3 4 5 4 5 4 5

4 4 4 4

5 4 4 4

6 4 4 4

7 4 4 4

1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2000. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. 4 The F1 generation did not produce enough living organisms to initiate the Brood Chamber. 5 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatment group. 6 Represents a female that never produced a brood throughout the F1 generation reproductive phase of the study. The number of offspring produced in the first and second 

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
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Appendix 24 Replicate Mean Percent of Males Sex Ratio and Percent of 

Females Producing Offspring F0 Generation

Nominal 

Concentration

(ng/L) Replicate

Percent of 

Males

Sex Ratio

Group

Mean

SD

CV(%)

Percent of 

Females 

Producing 

Offspring

Group

Mean

SD

CV(%)

Negative Control A 53.1 50.3 85.7 90.5

B 40.6 8.61 100.0 8.25

C 57.1 17.1 85.7 9.12

32 A 54.6 59.9 85.7 90.5

B 51.5 11.9 85.7 8.25

C 73.5 19.9 100.0 9.12

80 A 51.6 43.9 100.0 95.2

B 45.5 8.61 100.0 8.2

C 34.6 19.6 85.7 8.66

200 A 50.0 43.6 100.0 100.0

B 41.4 5.68 100.0 0.0

C 39.3 13.0 100.0 0.0

500 A 48.4 52.2 71.4 85.7

B 52.9 3.46 85.7 14.3

C 55.2 6.63 100.0 16.7

1250 A 35.3 44.1 42.9 47.6

B 48.4 7.59 42.9 8.25

C 48.5 17.2 57.1 17.3
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Appendix 25 Replicate Mean Percent of Males Sex Ratio and Percent of Females 

Producing Offspring F1 Generation

Nominal 

Concentration

(ng/L) Replicate

Percent of 

Males

Sex Ratio

Group

Mean

SD

CV(%)

Percent of 

Females 

Producing 

Offspring

Group

Mean

SD

CV(%)

Negative Control A 50.0 30.0 0.0 50.0

B 40.0 26.5 100.0 70.7

C 0.0 88.3 --1 141.4

32 A 35.7 40.5 20.0 53.3

B 42.9 4.12 100.0 41.6

C 42.9 10.2 40.0 78.1

80 A 61.5 62.3 100.0 68.3

B 45.5 17.3 71.4 33.4

C 80.0 27.8 33.3 49.0

200 A 37.5 45.8 100.0 77.8

B 50.0 7.22 33.3 38.5

C 50.0 15.8 100.0 49.5

500 A 0.0 12.5 100.0 100.0

B 25.0 17.7 100.0 0.0

C --1 141.6 --1 0.0

1250 A 0.0 50.0 --1 --1

B 100 70.7

C --1 141.4

1 The F1 generation did not produce enough living organisms to initiate the Replicate
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STUDY TRIAL 2 – 185A-102-2A
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SUMMARY

Two-Generation Life-Cycle Toxicity Test with Mysids (Americamysis bahia) 

Inter-Laboratory Comparison

SPONSOR: U.S. Environmental Protection Agency 

WILDLIFE INTERNATIONAL, LTD

STUDY NUMBERS: 185A-102-2A

RTI

PROJECT NUMBER: 0210114.030

EPA CONTRACT

NUMBER: EP-W-06-026

TEST SUBSTANCE: 4-tert-octylphenol, Lot No. 08118jhv, CAS No. 140-66-9, 

reported to have a purity of 98.0% and was received from Battelle 

Marine Sciences Laboratory on November 10, 2011.

DEFINITIVE

TEST DATES: 4 August 2012 to October 18, 2012

TEST ORGANISM: The test was initiated with Americamysis bahia (newly released, 

≤ 24 hours old, F0 generation) that were grown to maturity. Their 

offspring (F1 generation) were collected, which in turn were 

grown to maturity, and their offspring (F2 generation) were 

collected.

Origin: Wildlife International, Ltd culture

DILUTION WATER: Natural seawater collected at Indian River Inlet, Delaware and 

diluted with laboratory well water to a salinity of approximately 

20 ‰.

TEST CONDITIONS: Test duration: 74 days

Temperature range: 25.0 – 26.8oC

Salinity range: 19 - 21‰

Dissolved oxygen range: 6.7 – 7.2 mg/L

pH range: 7.7 – 8.1

Light intensity range: 143 – 387 lux

Photoperiod: Illumination of 14 hours light: 

10 hours dark
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NOMINAL TEST

CONCENTRATIONS: Dilution water (negative) control (0.0), 0.26, 0.64, 1.6, 4.0 and 

10 μg 4-tert-octylphenol/L

ENDPOINTS:

Parental Generation (F0)

• Survival

• Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14 and termination)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14 and termination)

• Time to maturation, appearance of marsupial pouch

• Time to first brood release, number of days after test initiation to release of young 

in the first brood

• Time to second brood release, number of days after test initiation to release of 

young in the second brood

• Interbrood duration, time between release of first brood and release of second 

brood

• Number of young per female in each of the first two broods

• Total number of young in each replicate population

• Sex ratio

• Percentage of females that are reproductively active

First Filial Generation (F1)

• Survival

• Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14 and termination)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14 and termination)

• Time to maturation, appearance of marsupial pouch

• Time to first brood release, number of days after test initiation to release of young 

in the first brood

• Time to second brood release, number of days after test initiation to release of 

young in the second brood

• Interbrood duration, time between release of first brood and release of second 

brood

• Number of young per female in each of the first two broods

• Total number of young in each replicate population

• Sex ratio

• Percentage of females that are reproductively active
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RESULTS: The mean number of offspring in the second brood per reproducing 

female, Day 7 total mysid lengths and Day 14 female dry weights in 

the F0 generation were the most sensitive endpoints in the mysid twogeneration exposure to 4-tert-octylphenol. Reproductive effects were 

apparent in all test concentrations with insufficient production of live 

young to continue an F1 generation in all treatment groups. However, 

the negative control F1 produced as expected. Based on these

reproductive and growth endpoints, the NOEC and LOEC were

determined to be <0.26 and 0.26 μg/L, respectively. The MATC could 

not be calculated since a NOEC could not be determined for the study.
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9.0 INTRODUCTION

The Environmental Protection Agency’s EDSP has completed validation of a battery of Tier 1 

assays to identify chemicals that have the potential to interact with the endocrine system, and is 

pursuing validation of longer term Tier 2 assays to identify adverse endocrine effects and provide 

dose response information needed for risk assessment. One of the longer term Tier 2 assays 

being developed is the Mysid Two-generation Test with Americamysis bahia. This test contrasts 

with existing mysid life cycle methods (that represent a single generation life cycle) in that an 

additional generation is included to allow evaluation of potential trans-generational effects. The 

inclusion of the invertebrate assay in Tier 2 is important because invertebrates make up the 

majority of species and the endocrine systems of invertebrates are not always directly analogous 

to those of vertebrates. Additionally, a method for testing crustaceans for effects of EDCs is 

relevant to assessing the adverse consequences of chemicals indicated to be endocrine active in 

Tier 1 assays.

As part of the validation of the two generation test method, the value-added of this method must 

be demonstrated over the existing mysid life cycle test method. There is a theoretical basis that 

the two-generation test is more comprehensive for endocrine disrupting chemicals (EDCs) than 

the life cycle test, but the single generation test is less logistically complex and in practical 

aspects and general sensitivity may be sufficient. The value-added of the two-generation test 

over the existing life cycle test must be empirically demonstrated before the two-generation test 

can be accepted. An inter-laboratory evaluation is needed to support a value-added comparison. 

In addition, a copepod development and reproduction test method with Amphiascus tenuiremis 

has been under development within the Organization for Economic Cooperation and 

Development (OECD). This method offers some logistical advantages in time and resources, but 

it is uncertain to what extent this may be at the expense of sensitivity. The OECD validation 

effort was led by Dr. G. Thomas Chandler (Dean, Arnold School of Public Health, University of 

South Carolina, Columbia, SC 29208) who developed this test method, a related ASTM method, 

and provided training to the OECD participants. An inter-laboratory exercise where both 

methods are compared for their practical transferability and quantitative reproducibility is needed 
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to support the ultimate selection of the most appropriate EDSP Tier 2 invertebrate test. Two 

chemicals (lindane and 4-tert-Octylphenol) will be tested in three laboratories for both the mysid 

two-generation test method and the copepod development and reproduction test method for a 

total of 12 experiments.

The main objective of this Task Order was to obtain data on the inter-laboratory reproducibility 

and comparability of the mysid two-generation test method and the copepod development and 

reproduction test method in support of the validation of the mysid two-generation test method 

and copepod development and reproduction test method being considered for the Endocrine 

Disruptor Screening Program (EDSP).

All of the work supporting these studies was conducted in accordance with EPA Good 

Laboratory Practice (GLP) regulations. RTI International was responsible for overall Task Order 

management, ensuring the work of all six independent laboratories was of high quality and 

delivered on schedule. The RTI Team for mysid work included Smithers Viscient, LLC (Lead 

Laboratory, SV), Wildlife International, Ltd. (WLI), and a Laboratory at the University of North 

Texas (UNT). The RTI team for the copepod work included Brixham Environmental Laboratory 

(Lead Laboratory, Brixham), Centre for Fisheries, Environment & Aquaculture (Cefas 

Weymouth) and Block Environmental Services, Inc. (BES). The lead laboratories’ roles included 

responsibility for coordinating the inter-laboratory efforts and ensuring that participants adhere 

to the standardized guidelines/protocols.

10.0 DESCRIPTION OF THE METHOD

10.1 QAPP/Protocol

This study was conducted according to the procedures outlined in the Quality Assurance Project 

Plan (QAPP) for Task Order 30 (QAPP). The study protocol (Protocol) describes a 

two-generation test with an estuarine mysid that considers reproductive fitness in parents and 

offspring as an integrated measure of toxicant exposure. It also enables measurement of a suite 

of other endpoints that allow for diagnostic and definitive evaluation of endocrine disrupting 
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chemicals (EDCs) or other types of reproductive toxicants (Verslycke et al., 2004). The 

two-generation test described in the guideline is an extension of existing standard practice for 

conducting a mysid life-cycle test (ASTM, 1999; McKenney, 1986, 1998; and Nimmo et al., 

1977, 1978).

10.2 Test Organism

The saltwater mysid (Americamysis bahia) was selected as the test species for this study. The 

saltwater mysid is representative of an important group of marine invertebrates and was selected 

for use in the test based upon past use in the laboratory. Mysids used in the test were obtained as 

juveniles (<24 hours old) from cultures maintained by Wildlife International, Ltd., Easton, 

Maryland. The brood stock was originally obtained from two different commercial suppliers: 

Reed Mariculture, Inc. located in Pasadena, California and Marinco Bioassay Laboratory located 

in Sarasota, Florida, and is refreshed periodically. The last time brood stock mysids were added 

to the cultures prior to this study was July 18, 2012. 

Adult mysids in the cultures were held in the laboratory for at least 14 days before juveniles were 

collected for testing. The culture was maintained in a recirculating saltwater system using water 

from the same source as used during the test. During the 2-week period immediately preceding 

the test, water temperatures in the cultures ranged from 25.4 to 26.2oC, measured with a 

liquid-in-glass thermometer. The pH of the water ranged from 8.1 to 8.3, measured with a Fisher 

Scientific Accumet Model 4 Star Plus pH meter. Dissolved oxygen ranged from 6.3 to 7.1 mg/L 

(≥88% of saturation), measured with a Yellow Springs Instruments Model 3 Star Plus dissolved 

oxygen meter. Salinity of the filtered saltwater was 20 parts per thousand (‰), measured with a 

VitalSine refractometer. The area in which the mysids were cultured received a regulated 

photoperiod of 14 hours of light and 10 hours of darkness and had light intensity range of 221 to 

465 lux. 
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10.3 Feeding

During culturing, mysids were typically fed three times daily. The test mysids from both generations (F0

and F1) for all control and treatment groups were fed three times daily from Day 0 to Day 7 and four 

times daily from Day 8 to termination with brine shrimp (Artemia sp.) nauplii ≤ 24 – 48 hours old (posthydration) supplied by INVE Aquaculture, Salt Lake City, Utah (CSP Numbers:12-58, 12-68 and 12-

84). For each generation, at least one feeding each day was with brine shrimp enriched with A1 

DHA Selco, a supplemental substance high in saturated fatty acids, supplied by INVE (Thailand, 

Ltd.), Amphoe Wachirabarami, Phichit, Thailand (CSP Number 12-69). Densities (nauplii/mL) 

of concentrated collections of nauplii (a slurry of nauplii and dilution water) from Artemia

hatched daily from hydrated cysts were determined by replicate counts of aliquots pipetted onto 

filter paper. Based on these densities, the volume of nauplii fed to mysids was adjusted daily in 

accordance with the following regime: rations for reproduction/growth chambers (containing 

20 mysids each) were approximately 1800 nauplii/chamber on days 0 to 3, 2250 nauplii/chamber 

on days 4 to 6, 2700 nauplii/chamber on days 7 to 9, 3150 nauplii/chamber on days 10 to 12, and 

3600 nauplii/chamber on days 12 through the end of the study, while brood chambers 

(containing individually paired mysids) were fed approximately 900 nauplii/chamber throughout 

the test. All treatments and controls were fed using the same regime throughout the test. Mysid 

food was also daily supplemented with Skeletonema costatum, a saltwater alga, cultured by 

Wildlife International, Ltd. Excess brine shrimp and organic debris in the growth/reproductive 

chambers and brood chambers were removed periodically when observed. Periodic analyses of 

representative samples of the food were conducted using U.S. EPA standard methods (U.S. EPA, 

1997) by Lancaster Laboratories, Lancaster, PA 17605-2425 to ensure the absence of potential 

toxicants, including pesticides, PCBs and selected toxic metals, at concentrations which may be 

harmful to mysids (Appendix 28). Specifications for acceptable levels of contaminants in mysid 

diets have not been established. However, there are no known levels of contaminants reasonably 

expected to be present in the diet that are considered to interfere with the purpose or conduct of 

the test.
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10.4 Handling

Mysids were handled as gently as possible to avoid unnecessary stress. Great care was exercised 

to avoid injury to juvenile mysids during counting. All mysids were transferred below the water 

surface using a smooth-edged wide-bore pipette.

10.5 Dilution Water

The water used for culturing and testing was natural seawater collected at Indian River Inlet, 

Delaware. The freshly-collected seawater was passed through a sand filter to remove particles 

greater than approximately 25 μm, and pumped into a 37,800-L storage tank. The filtered 

saltwater then was diluted to a salinity of approximately 20‰ with freshwater from a well on the 

Wildlife International, Ltd. site and was aerated with spray nozzles. Prior to use, the 20‰ water 

was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) 

sterilizer.

Salinity and pH measurements taken during the four-week period immediately preceding the test 

are presented in Appendix 26. The results of periodic analyses performed to measure the 

concentrations of selected organic and inorganic constituents in the saltwater are presented in 

Appendix 27.

During the four-week period immediately preceding the test, the pH was 7.9 (range 7.9 – 7.9) 

and salinity was 20 ‰ (range of 20 – 20 ‰). Salinity and pH were measured using a VitalSine 

refractometer and a Thermo Orion Model 525Aplus pH Meter, respectively.

10.6 Water Quality Measurements

10.6.1 Temperature

The test vessels were indiscriminately positioned by treatment group in an environmental 

chamber designed to maintain the desired test temperature throughout the test period. The target 
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test temperature during the study was 26 ± 1°C. Temperature was measured in each test vessel at 

the beginning of the F0 generation, and in alternating replicates daily thereafter in both the F0 and 

F1 generations, using a hand-held liquid-in-glass thermometer. Temperature also was monitored 

continuously in one negative control test vessel using a Fulscope ER/C Recorder, which was 

verified prior to test initiation, and verified or calibrated approximately weekly during the test 

with a hand-held liquid-in-glass thermometer.

10.6.2 Salinity

The salinity of the test solutions was maintained at 20 ± 2‰ as described in the dilution water 

section above. Salinity was measured in each test vessel at the beginning test, and in alternating 

replicates daily thereafter. Salinity was measured using a VitalSine refractometer. 

10.6.3 Lighting

Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that 

emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a 

photoperiod of 14 hours of light and 10 hours of darkness. A 120-minute transition period of 

low light intensity was provided when lights went on and off to avoid sudden changes in 

lighting. Light intensity was measured at the water surface of each test vessel once during each 

generation using a SPER Scientific Model 840006C light meter.

10.6.4 Dissolved Oxygen

Total dissolved oxygen was measured to be at or near 100% of saturation at the start of the test 

for the F0 generation and was not allowed to drop below 60% saturation (4.3 mg/L at 26oC and 

20‰ salinity) throughout the test. The dilution water was aerated prior to the addition of 

organisms to the test system and continually through the test to maintain dissolved oxygen levels 

above 60% saturation. Dissolved oxygen was measured in each test vessel at the beginning of 

the test for the F0 generation, and in alternating replicates daily thereafter for both generations 
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during the test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus 

dissolved oxygen meter.

10.6.5 pH

Measurements of pH were made in each test vessel at the beginning of the test for the F0

generation, and in alternating replicates daily thereafter for both generations during the test. 

Measurements of pH were made using a Thermo Orion Model 525Aplus meter. 

10.7 Assay System

10.7.1 Test Vessels

The test vessels were 38-L glass aquaria measuring 50 x 25 x 30 cm equipped with self-starting 

siphon systems to ensure that test solution flowed through the vessels. The total solution volume 

maintained in each vessel was approximately 21 L. The aquaria were indiscriminately 

distributed by concentration within the environmental chamber, and labeled to identify the 

project number, treatment/control group, and the replicate designation. The depth of the water in 

a representative test vessel was 14.3 -14.4 cm. 

10.7.2 Retention Chambers (Reproduction/Growth Chambers)

Mysid juveniles were held in retention chambers within test chambers to facilitate observations 

and eliminate loss through outflow water. These retention chambers were constructed of 15-cm 

glass Petri dishes to which an approximately 15-cm high cylinder of nylon mesh screen (210-μm 

mesh) had been attached with aquarium grade (without fungicides) silicone adhesive. The depth 

of the water in a representative test retention compartment was 13.9 cm.

For the individual mysid reproduction or growth chambers, the following code was used to 

identify retention chambers and was marked on each Reproduction (R) or Growth chamber (G): 
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Treatment Level – Replicate Vessel – Chamber Type (R or G) – Generation (F0 or F1) – date 

chamber was initiated.

10.7.3 Brood Chambers

Upon maturing, gravid females were paired with males and isolated in brood chambers 

constructed of 10-cm glass Petri dishes to which an approximately 15-cm high cylinder of nylon 

mesh screen (210-μm mesh) was attached. The depth of the water in a representative test brood 

chamber was 14.1 cm.

For the individual mysid brood chambers, the following code was used to identify brood 

chambers and was marked on each Brood Chamber (BC): Treatment Level – Replicate – Brood 

Chamber (BC) – Brood Chamber Number (1-9) – Generation (F0 or F1) – date brood chamber 

was initiated.

10.8 Replication and Number of Test Organisms

For each mysid, the time to maturation was defined as the day of the study in which the mysid 

could be sexually identified as being male or female. For this study, both the F0 and F1 mysids 

were all first examined for reproductive characteristics starting on Day 14. In both generations, 

sex could be assigned to all of the mysids in each treatment group on Day 14. For each mysid, 

the time to first brood release was defined as the first day of the study in which the mysid 

released young. For each mysid, the time to second brood release was defined as the day of the 

study in which the mysid released its second brood. The typical gestational interval for mysids is 

between 5 to 9 days. Therefore, any mysids released within four days of the first brood were still 

considered part of that first brood. Any young released 5 days or more out from the first brood 

release were considered part of the second brood. For each mysid, the time of interbrood 

duration is defined as the time between release of the first brood and the release of the second 

brood.
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10.8.1 Exposed First Generation Mysids (F0)

Three replicate vessels per treatment were used in the study. The exposure was initiated with 

20 newly released mysid juveniles (< 24 hours old) assigned to reproduction chambers within 

each replicate tank. Within the three replicate vessels, one additional growth chamber was also 

initiated with 20 newly released mysid juveniles (< 24 hours old) to be sub-sampled weekly for 

growth measurements. Upon reaching maturity (Day 14 in this study), one male and one gravid 

female were indiscriminately assigned to separate brood chambers within each replicate (with a 

maximum of seven male/female pairs possible per replicate). Individuals in the reproduction 

chambers not paired in brood chambers were maintained and observed for survival and sex 

determination within the retention chambers until they were paired or until the test was 

terminated (one week after the mean day of release of the second brood). Unused mysids from 

the growth retention chambers were used to complete male:female pairings in the event that sex 

ratio in the reproduction chamber was skewed, preventing obtaining seven pairs. The F0

exposure continued until Day 46, which was at least seven days after the median day of release 

of the second brood (Day 33) in the negative control group. 

10.8.2 Exposed Second Generation Mysids (F1)

The F1 generation exposure was initiated with up to 15 newly released mysid juveniles 

(< 24 hours old) per day produced by the F0 generation. F1 generation mysids were assigned to 

reproduction chambers within the same treatment group, for each of the F0 exposure conditions 

(e.g., juveniles from Replicate A of the F0 generation vessel were assigned to Replicate A of the 

F1 generation vessel). Mixing of young released on the same day and across replicate chambers 

from the same treatment or control group, was conducted to minimize reproduction between 

siblings. An additional F1 generation growth chamber was initiated with up to 15 newly released 

mysid juveniles (when possible) within each replicate treatment level to be sub-sampled weekly 

for growth measurements. Priority was given to filling the reproduction retention chambers first, 

followed by the growth chambers. If possible, F1 offspring were indiscriminately placed into 

chambers designated as reproduction or growth. The ages of offspring in a given chamber were 

tracked to ensure growth measurements were collected on the appropriate day (e.g. Day 7 and 
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Day 14). Upon reaching maturity (Day 14 in this study), one male and one gravid female were 

indiscriminately assigned to separate F1 generation brood chambers within each replicate (with a 

maximum of 7 m/f pairs possible per replicate). Individuals in the F1 generation reproduction 

chambers not paired in brood chambers were maintained and observed for survival and sex 

determination within the F1 generation retention chambers until they were paired or until the test 

was terminated. Unused mysids from the growth retention chambers were used to complete 

male:female pairings in the event that sex ratio in the reproduction chamber was skewed, 

preventing obtaining seven pairs. The F1 exposure continued until chamber Day 35 (i.e., the 

organisms in each reproductive chamber reached an age of 35 days).

10.9 Diluter

The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter 

used to deliver each concentration of the test substance and a negative control (dilution water) to 

test vessels. Fluid metering pumps (Fluid Metering, Inc., Syosset, New York) were used to 

deliver volumes of a single test substance stock solution (see Stock Preparation below) to mixing 

chambers indiscriminately assigned to each treatment. The stock solution was diluted with well 

water in the mixing vessels in order to obtain the desired test concentrations prior to delivery to 

the test vessels. The flow of dilution water into each mixing chamber was controlled using 

rotameters, and was adjusted to provide each test vessel with at least 5 volume additions of test 

water per day (approximately 83 mL/min solution exchange for each test vessel). After mixing, 

the flow from each mixing chamber was split to deliver test water to three replicate test vessels.

The pumps used to deliver stock solutions to the mixing chambers were calibrated prior to the 

test and verified or recalibrated at least weekly during the test. The rotameters used to control 

the flow of dilution water to the mixing chambers were calibrated prior to the test and verified or 

recalibrated approximately weekly during the test. The proportion of the test water that was split 

into each replicate test vessel was checked prior to the test and approximately weekly during the 

test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the three 

replicates. Delivery of test solutions to the F0 and F1 generation test vessels was initiated 8 and 
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20 days, respectively, prior to the introduction of the test organisms to the test water in order to 

achieve equilibrium of the test substance. The general operation of the exposure system was 

checked visually at least once on the first and last days of the test and at least two times per day 

during the test, except on Day 28 of the F0 generation when the checklist was only done once.

10.10 Test Substance

The test substance, 4-tert-octylphenol, was received from Battelle Marine Sciences Laboratory, 

Sequim, Washington. The following information was provided:

Name: 4-tert-octylphenol

Synonyms (per MSDS): 4-(1,1,3,3-Tetramethylbutyl)phenol

Lot No.: 08118jhv

CAS No.: 140-66-9

Purity: 98.0%

Description: Solid

Date Received: November 10, 2011

Expiration Date: November 2015

4-tert-octylphenol was assigned Wildlife International, Ltd. identification number 10305 upon 

receipt and was stored under ambient conditions.

10.11 Test Substance Concentrations

Test substance concentrations for the study were selected by the EPA to be dilution water 

(negative) control (0), 0.26, 0.64, 1.6, 4.0 and 10 μg 4-tert-octylphenol/L. 

10.12 Saturation Column Preparation

Glass saturator columns were prepared for the delivery of 4-tert-octylphenol. Each glass column 

(1” ID x 24” L) was packed with approximately 90 grams of 8 micron glass wool. The glass 

wool in the column was then rinsed with approximately 500 mL of acetone to remove 

contaminants. The stock suspension was prepared in acetone to achieve the nominal 

concentration of approximately 150 mg/mL by adding 5.0 grams of 4-tert-octylphenol to 
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approximately 30 mL of acetone by stirring to mix until the test material was dissolved into 

solution. The beaker containing the solution was then poured and rinsed well using acetone onto 

the pre-washed glass wool in the column. The solution was then pulled down through the 

column using the vacuum to ensure even coating of the glass wool, making sure not to soak the 

glass wool at the end of the column. The column was vacuum dried overnight to remove the 

acetone. Once the acetone was removed, the column was wrapped in aluminium foil and stored 

refrigerated until use. Columns were stable for approximately 30 days under these storage 

conditions. One column was set up in the test system at a time. Columns were replaced weekly 

with a new, previously prepared column.

10.13 Stock Preparation

A single stock solution was prepared by applying the dilution water to the saturator column at a 

rate of 20 mL/min. The column delivered the test stock solution based at an approximate 

concentration of 11 mg/L, based on analysis of stock prior to experimental start (Table 23). The 

stock solution appeared clear and colorless. The stock solution from the column was delivered to 

a reservoir where it was further diluted using dilution water to a nominal concentration of 

500 μg/L. This diluted stock solution was held under ambient conditions, was protected from 

light, and was mixed continuously using a top-down electric mixer. The stock was then pumped 

from the reservoir into the diluter mixing chambers assigned to the treatment groups at target 

rates of 0.13 to 5.0 mL/min, and was mixed with filtered saltwater in the mixing chambers, 

delivered at target rates of 245 to 250 mL/min, to achieve the nominal test concentrations of 

0.26, 0.64, 1.6, 4.0 and 10 μg/L, respectively. The test solutions in the mixing chambers and test 

vessels were covered with Plexiglas to prevent volatilization. The negative control received 

dilution water only. Both the F0 and F1 generation stock solutions were delivered from the same 

diluted stock solution in the reservoir tank.
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10.14 Analytical Method and Verification

10.14.1 Analytical Sampling

Samples were collected from one test vessel of each treatment and control group five days prior 

to the start of the test after conditioning the diluter for approximately three days. Water samples 

were collected from alternating replicate test vessels at test initiation, at approximately weekly 

intervals during the study and at test termination to measure concentrations of the test substance. 

An additional water sample was collected from the negative control group on Day 21 in the F0

generation due to a high recovery noticed in the negative control solution. The stock solution 

being delivered to the reservoir from the column and the solution from the reservoir itself were 

also sampled prior to test initiation. All samples were collected from mid-depth in the test 

vessel, placed in glass separatory funnels outside of the test system room, and processed 

immediately for analysis. 

10.14.2 Analytical Determinations

The method used for the analysis of 4-tert-octylphenol samples in saltwater was developed by 

Wildlife International, Ltd. Samples were extracted with toluene and the extracts were analyzed 

by gas chromatography (GC) with mass selective detector (MS). 

Concentrations of 4-tert-octylphenol in the samples were determined using an Agilent Model 

5890 Gas Chromatograph equipped with an Agilent Model 5971A Mass Selective Detector 

operated in SIM mode. Chromatographic separations were achieved using a ZB-5 column 

(0.25 mm x 30 m, 0.25 μm film thickness). A method flowchart is provided in Appendix 29.1

and typical instrumental parameters for 4-tert-octylphenol are summarized in Appendix 29.2.

Five calibration standards of 4-tert-octylphenol, ranging in concentration from 2.50 to 20.0 μg/L, 

were prepared in toluene using a stock solution of 4-tert-octylphenol test substance in methanol 

(Appendix 29.3). Calibration standards were analyzed with each sample set. Linear regression 

equations were generated using the peak area responses versus the respective concentrations of the 
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calibration standards. The concentration of 4-tert-octylphenol in the samples was determined by 

substituting the peak area responses of the samples into the applicable linear regression equation. 

An example of the calculations for a representative sample is included in Appendix 29.4.

The limit of quantitation (LOQ) for the analysis of 4-tert-octylphenol in saltwater study samples 

was set at 0.125 μg/L, calculated as the product of the lowest calibration standard analyzed 

(2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). Nine matrix blank 

samples were analyzed for the initial trial to determine possible interferences (Appendix 29.5). 

Three matrix blank samples were analyzed for the F-1 generation trial to determine possible 

interferences (Appendix 29.6). 

Saltwater samples were fortified at 0.250, 2.00 and 10.0 μg/L using a stock solution of 4-tertoctylphenol test substance prepared in methanol and were analyzed concurrently with the 

samples. The measured concentrations for the matrix fortification samples for the initial trial 

ranged from 94.4 to 133% of nominal concentrations (Appendix 29.5). The measured 

concentrations for the matrix fortification samples for the F-1 generation ranged from 106 to 

126% of nominal concentrations (Appendix 29.6). 

A representative calibration curve is presented in Appendix 29.7. Representative chromatograms 

of low and high-level calibration standards are presented in Appendices 29.8 and 29.9, 

respectively. A representative chromatogram of a matrix blank sample is presented in 

Appendix 29.10, and a representative chromatogram of a matrix fortification sample is presented 

in Appendix 29.11. A representative chromatogram of a test sample is presented in Appendix 

29.12.

11.0 EXPOSURE INITIATION AND CONDUCT

The test substance was introduced into the test vessels approximately 8 days prior to test 

initiation in order to allow the test solutions to reach an equilibrium concentration of test 
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substance in the exposure system. Exposure for the F0 mysid began when all of the <24 hour old 

individuals were indiscriminately transferred into the test vessels from the stock cultures. 

11.1 Exposed First Generation Mysids (F0)

11.1.1 F0 Exposure Initiation

At exposure initiation, 20 <24-hour-old individuals were impartially transferred into 

reproduction and growth chambers. For each treatment level, triplicate exposure tanks included 

one growth and one retention chamber in each tank (Figure 4). Mysids were distributed among 

growth and reproduction chambers in the following manner: a pool of > 630 mysids (< 24 hours 

old) were removed from the culture tanks. Mysids (< 24 hours old) were distributed, one to four 

at a time, to each of the unlabeled intermediate beakers (two per replicate) containing dilution 

water until each beaker contained four mysids. This process was then repeated five more times 

until each intermediate beaker contained 20 mysids. Following distribution, the mysids in each 

intermediate beaker were then transferred to a labeled retention chamber. The exposure was 

initiated when each growth and reproduction chamber was added to the respective exposure tank.

In addition on Day 0, 45 mysids were collected from the population used to initiate the exposure. 

These mysids were separated into groups of five, euthanized in ice-cold de-ionized water, blotted 

dry and measured for total length to the nearest 0.1 mm. Following length measurements, each 

group was placed on pre-weighed aluminum tins and dried in an oven at approximately 100 oC 

for approximately 24 hours. Dried mysid samples were kept in a desiccator for at least 30 

minutes (to allow for cooling of the sample) prior to weighing to the nearest 0.01 mg on an 

analytical balance.

11.1.2 F0 Growth Chambers

At two time intervals (Day 7 and Day 14), mysids were sampled from each growth chamber. On 

Day 7, six mysids were removed from the growth chamber, separated into three groups of two 

mysids, euthanized in ice-cold reverse-osmosis water, blotted dry and measured for total length 
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to the nearest 0.1 mm. Following length measurements, each group was transferred to 

pre-weighed aluminum tins and dried in an oven at approximately 100 oC for approximately 

24 hours. Dried mysid samples were kept in a desiccator for at least 30 minutes prior to 

weighing to the nearest 0.01 mg on an analytical balance.

On Day 14, the remaining mysids (those not sampled at Day 7 and those not used to complete 

reproduction pairings) were removed, euthanized in ice-cold reverse-osmosis water, blotted dry 

and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 

100 oC for approximately 24 hours. Mysids on Day 14 were identified as male, female or 

immature organisms. Dried mysid samples were kept in a desiccator for at least 30 minutes prior 

to weighing to the nearest 0.01 mg on an analytical balance.

In addition, total length and dried body weight were measured at termination from mature 

mysids housed initially in reproductive chambers then transferred to brood chambers.

11.1.3 F0 Reproductive Chambers

When developing embryos were discerned within the majority of females in the reproduction 

chambers (Day 14 in this study), an ovigerous female was paired with one male and the pair was 

isolated in a separate brood chamber within that replicate chamber for that specific exposure 

condition (Figure 4). Mysids that were not paired were transferred to a separate brood chamber 

within the respective replicate test vessel. Remaining male mysids (not used in the initial pairing 

of mysids) were held in a separate brood chamber and used to replace male mysids that died in 

brood chambers during the reproduction phase of the exposure. Unused mysids from the growth 

retention chambers were used to complete the male:female pairings in the event that the sex ratio 

in the reproduction chamber was skewed, preventing obtaining seven pairs. The F0 exposure 

terminated at least seven days after the median day of release of the second brood (F0”) in the 

negative control treatment group. 
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Upon termination, each surviving F0 mysid was removed from the reproduction chambers, 

euthanized in ice-cold reverse-osmosis water, blotted dry, sexually identified as male or female 

and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 

100 oC for approximately 24 hours. Dried mysid samples were kept in a desiccator for at least 

30 minutes prior to weighing to the nearest 0.01 mg on an analytical balance.

Offspring of the F0 mysids (< 24 hours old) were collected and transferred to nylon screen 

chambers in a separate test system at the same test substance concentrations to which they were 

exposed during the F0 generation. 

11.1.4 Feeding

The mysids were fed daily newly hatched (24 or 48 hour old) brine shrimp (Artemia sp.) nauplii 

at the recommended densities described in section 10.3.

11.2 Exposed Second Generation Mysids (F1)

11.2.1 F1 Exposure Initiation

Groups of <24-hour-old offspring (F1) from either the first (F1’) or second brood (F1”) of the F0

mysids were isolated in reproduction chambers (as previously described), and placed in the same 

treatment group for the respective exposure conditions from which they originated. Mixing of 

young released on the same day and across replicate chambers from the same treatment or 

control group was conducted to minimize reproduction between siblings. In addition, groups of 

F1 mysids released on the same day (i.e. replicate Day 0) were indiscriminately assigned to 

growth chambers within the exposure vessels of the same exposure treatment from which they 

originated, if available. Priority was given to filling the reproduction retention chambers first, 

and then the growth chambers.
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In addition, four groups of five Day 0 mysids, when available, were collected from the negative 

control group from the F1 generation. These mysids were separated into groups of five, 

euthanized in ice-cold de-ionized water, blotted dry and measured for total length to the nearest 

0.1 mm. Following length measurements, each group was placed on pre-weighed aluminum tins 

and dried in an oven at approximately 100 oC for approximately 24 hours. Dried mysid samples 

were kept in a desiccator for at least 30 minutes (to allow for cooling of the sample) prior to 

weighing to the nearest 0.01 mg on an analytical balance.

11.2.2 F1 Growth Chambers

At two time intervals (Day 7 and Day 14), mysids were sampled from each growth chamber, 

when available. On Day 7, up to six mysids were removed from the growth chamber, separated 

into three groups of two mysids, euthanized in ice-cold reverse-osmosis water, blotted dry and 

measured for total length to the nearest 0.1 mm. Following length measurements, each group 

was transferred to pre-weighed aluminium tins and dried in an oven at approximately 100 oC for 

approximately 24 hours. 

On Day 14, the remaining mysids (those not sampled at Day 7 or used to complete reproduction 

pairings) when available, were removed, euthanized in ice-cold reverse-osmosis water, blotted 

dry and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminium tin and dried in an oven at 100 oC for 24 

hours. Mysids on Day 14 were identified as male, female or immature organisms. Dried mysid 

samples were kept in a desiccator for at least 30 minutes prior to weighing to the nearest 0.01 mg 

on an analytical balance.

In addition, total length and dried body weight were measured at termination from mature 

mysids housed initially in reproductive chambers then transferred to brood chambers.
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11.2.3 F1 Reproductive Chambers

When developing embryos were discerned within the majority of individual females in the 

reproduction chambers (Day 14 of the F1 generation exposure in this study), an ovigerous female 

was paired with one male and the pair was isolated in a separate brood chamber within that 

replicate chamber for that specific exposure condition. Mysids that were not paired were 

transferred to a separate brood chamber within the respective replicate test vessel. Remaining 

male mysids (not used in the initial pairing of mysids) were held in a separate brood chamber 

and used to replace male mysids that died in brood chambers during the reproduction phase of 

the exposure. Unused mysids from the growth retention chambers were used to complete the 

male:female pairings in the event that the sex ratio in the reproduction chamber was skewed, 

preventing obtaining seven pairs. The F1 exposure was terminated 35 days following initiation 

of the final F1 replicate (Day 42 of F1 exposure).

Upon termination, each surviving F1 mysid was removed from the reproduction chambers, 

euthanized in ice-cold reverse-osmosis water, blotted dry, sexually identified as male or female 

and measured for total length to the nearest 0.1 mm. Following length measurements, each 

mysid was transferred to a pre-weighed aluminum tin and dried in an oven at approximately 

100 oC for approximately 24 hours or 60 °C for approximately 72 hours. Dried mysid samples 

were kept in a desiccator for at least 30 minutes prior to weighing to the nearest 0.01 mg on an 

analytical balance.

11.2.4 Feeding

The mysids were fed newly hatched (24 or 48 hour old) brine shrimp (Artemia sp.) nauplii at the 

recommended densities described in section 10.3.
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11.3 Chamber Cleaning

Dead brine shrimp and other debris in the retention chambers and brood chambers were removed 

daily. Based on daily observations of water flow through the screens of these containers, mysids 

were transferred to clean retention chambers or brood chambers twice a week to prevent water 

flow restriction.

11.4 Conditions of Exposure 

Offspring of the F1 mysids were enumerated daily until Day 35 of the last surviving replicate, at 

which time the test was terminated. The study length, measured from the F0 initiation to the F1

termination, was 74 days.

12.0 OBSERVATION AND MEASUREMENTS

12.1 Biological Observations

At the start of the test and daily thereafter, observations of stress, abnormal behavior (including 

discoloration, lethargy, immobilization, erratic swimming, and inability to maintain position in 

the water column), differences in food consumption, and mortality were made. Dead mysids 

were recorded and removed from the test vessels at these intervals. Mysids which were missing 

were considered dead. The criterion for death was absence of mobility and failure to respond to 

gentle prodding. Survival was based on the number of mysids initially present in each test 

vessel. Daily assessment of survival was necessary to provide a basis for expression and 

interpretation of reproductive output, that is, number of offspring/female/day. 

The primary observations made during the test included:

• Survival (F0, F1) 

• Growth, weight to the nearest 0.01 mg and length to the nearest 0.1 mm (Days 0, 

7, 14 and termination for F0 and F1, when possible)

• Time to first brood release, number of days after exposure initiation to release of 

young in the first brood (F0, F1, when possible)
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• Time to second brood release, number of days after exposure initiation to release 

of young in the second brood (F0, F1, when possible)

• Interbrood duration, time between release of first brood and release of second 

brood (F0, F1, when possible)

• Number of young per female in each of the first two broods (F0, F1, when 

possible)

• Total number of young in the first two broods in each replicate population (F0, F1, 

when possible)

• Sex ratio (F0, F1)

• Percentage of females that are reproductively active (F0, F1)

13.0 STATISTICAL METHODS

13.1 Endpoints

The endpoints used for determination of significant effects by statistical evaluation are outlined 

below: 

• Survival, (F0, F1)

• Growth - length and dry weight, (Days 0, 7, 14 and termination for F0 and F1)

• Time to maturation, (F0, F1)

• Time to first brood release, (F0, F1)

• Time to second brood release, (F0, F1)

• Interbrood duration, (F0, F1)

• Number of young per female in each of the first two broods, (F0, F1)

• Total number of young in the first two broods in each replicate population (F0, F1)

• Sex ratio, (F0, F1)

• Percentage of females that are reproductively active, (F0, F1)

13.2 Statistical Methods

Survival data, sex ratio and reproducing females were considered to be discrete-variable data, 

while the remaining reproduction endpoints and growth data were considered 

continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s 

Exact tests to identify treatment groups that showed a statistically significant difference from the 

negative control (p ≤ 0.05). All continuous-variable data were evaluated for normality using the 

Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (p = 0.01). The data for 

all parameters passed the assumptions of normality and homogeneity of variance. Those 
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treatment means that were significantly different from the negative control means were identified 

using Dunnett’s test (p ≤ 0.05). Survival and percent of reproducing females were tested using 

Fisher’s Exact test (p ≤ 0.05). Sex ratio was tested using Chi-Square test (p ≤ 0.05).

Statistically significant treatment effects were used to define the Lowest-Observed-EffectConcentration (LOEC; the lowest test concentration with statistically significant differences from 

controls) and the no-Observed-Effect-Concentration (NOEC; and the highest test concentration 

that shows no statistically significant difference from the control). All statistical analysis was 

conducted using SAS software and a personal computer.

14.0 RESULTS 

Measurements of temperature, dissolved oxygen, pH, light intensity and salinity in the test 

chambers are summarized for F0 in Table 20 and for F1 in Table 21. Water temperatures were 

within the 26 ± 1°C range established for the test. Dissolved oxygen concentrations were at or 

near 100% of air saturation at test initiation and remained ≥ 93% of saturation (6.7 mg/L) 

throughout the exposure period for both the F0 and F1 generations tested. Measurements of pH 

ranged from 7.7 – 8.1 and salinity ranged from 19 to 21‰ during the test. Light intensity at test 

initiation was 222 lux for the F0 generation and 152 lux for the F1 generation, measured at the 

surface of the water of one representative test chamber. Also, on Day 0 of the study, light 

intensity measured at the surface of each test vessel ranged from 223 to 387 lux for the F0

generation and 143 to 235 lux for the F1 generation.

14.1 In-life Analytical Results

14.1.1 F0 Generation

Test solutions were premixed in mixing chambers and then delivered into tanks. Samples used 

for analytical measurements to determine mean measured concentrations were only collected 

from tanks. The appearance of test solutions in the mixing chambers and test chambers appeared 
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clear and colorless during the test, with no evidence of precipitation observed in any control or 

treatment solution. 

The 4-tert-octylphenol concentrations measured in pretest diluter verification samples ranged 

from approximately 69 to 119% of nominal (Table 22). The 4-tert-octylphenol concentration 

measured in the stock solution from the column prior to test initiation was 9.9 mg/L, which was 

89.6% of nominal (Table 23). The 4-tert-octylphenol concentrations measured in the test 

solution samples collected during the in-life phase of the study ranged from approximately 58 to 

128% of nominal in the four highest test concentrations and from 105 to 166% of nominal in the 

lowest test concentration, which was near (i.e. only twice the LOQ) the limit of the analytical 

method (Table 24). When measured concentrations of the samples collected during the test were 

averaged, the mean measured test concentrations for this study were 0.33, 0.69, 1.4, 3.2 and 

7.4 μg/L, representing 127, 108, 88, 80 and 74% of nominal concentrations, respectively 

(Figure 5). The results of the F0 generation were based on the nominal concentrations.

14.1.2 F1 Generation

Test solutions were premixed in mixing chambers and then delivered into tanks. Samples used 

for analytical measurements to determine mean measured concentrations were only collected 

from tanks. The appearance of test solutions in the mixing chambers and test chambers appeared 

clear and colorless during the test, with no evidence of precipitation observed in any control or 

treatment solution. 

The 4-tert-octylphenol concentrations measured in pretest diluter verification samples ranged 

from approximately 75.1 to 168% of nominal (Table 25). The 4-tert-octylphenol concentrations 

measured in the test solution samples collected during the in-life phase of the study ranged from 

approximately 79 to 118% of nominal in the four highest test concentrations and 163% of 

nominal in the lowest test concentration, which was near (i.e. only twice the LOQ) the limit of 

the analytical method (Table 26). When measured concentrations of the samples collected 

during the test were averaged, the mean measured test concentrations for this study 0.42, 0.71, 
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1.4, 3.3 and 8.2 μg/L, representing 162, 111, 88, 83 and 82% of nominal concentrations, 

respectively (Figure 6). The results of the F1 generation were based on the nominal 

concentrations.

14.2 Biological Results

Table 27 through Table 32 present endpoint summaries of the total means, standard deviations 

(SD) and coefficients of variability (CV) for the data collected during the study. Data from 

individual animals and replicates are summarized in Appendix 30 to Appendix 41. The results of 

the F0 generation are discussed before the F1 generation. The F1 generation results are limited to 

the negative control, since there were dramatic and apparent affects in reproduction in all 

4-tert-octylphenol test concentrations resulting in little or no reproduction. 

14.3 Survival

14.3.1 Survival from Day 0 to Day 7

A summary of survival of F0 juvenile mysids from test initiation to the Day 7 growth 

sub-sampling interval is located in Table 27. Survival during the first 7 days of exposure in the 

negative control (untreated filtered saltwater) and in the 0.26, 0.64, 1.6, 4.0 and 10 μg/L 

treatment groups was 100, 99.2, 100, 99.2, 100 and 97.5%, respectively. There were no apparent 

effects on survival during the first seven days of exposure and Fisher’s Exact test indicated there 

were no statistically significant decreases in any treatment group when compared to the negative 

control (p > 0.05). 

14.3.2 Survival from Day 8 to Day 14

A summary of survival of F0 juvenile mysids from Day 8 to the Day 14 growth sub-sampling 

interval is located in Table 27. Survival in the negative control and in the 0.26, 0.64, 1.6, 4.0 and 

10 μg/L treatment groups was 95.8, 96.7, 91.0, 97.5, 98.0 and 96.8%, respectively. There were 

no apparent effects on survival from days 8 to 14 and Fisher’s Exact test indicated there were no 
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statistically significant decreases in survival in any of the treatment groups when compared to the 

negative control (p > 0.05).

14.3.3 Survival from Day 15 to Test Termination of F0

A summary of survival of F0 adult mysids after pairing (from Day 15 to test termination on 

Day 46) is presented in Table 27. Survival in the negative control and in the 0.26, 0.64, 1.6, 4.0 

and 10 μg/L treatment groups (excluding mysids that died as a result of impingement) was 77.2, 

85.6, 66.2, 79.8, 76.2 and 65.1%, respectively. There were no apparent effects on survival from 

Days 15 to termination of the of F0 generation and Fisher’s Exact test indicated there were no 

statistically significant decreases in survival in any treatment group when compared to the 

negative control (p > 0.05). Female adult survival was higher than male adult survival in all 

treatment and control groups. However, there also were no apparent differences in the survival 

within the same gender group from Day 15 to termination based on the daily survival data by 

gender presented in Appendix 30.

14.4 Growth

Mysid size was measured at four time points (Days 0, 7, 14 and 46) during the F0 generation. 

Mysid dry weight and mysid total body length were measured on Days 0, 7 and 14 from the 

additional growth chamber that was subsampled for growth measurements and on all surviving 

mysids at the F0 generation test termination.

Summaries of the total lengths and dry weights of mysids throughout the study are presented in 

Table 28 to Table 31. Individual animals and their replicate mean measurements of total lengths 

and dry weights are provided in Appendix 31 to Appendix 37. Results for each of the four time 

points are presented below.
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14.4.1 Day 0 (less than 24 Hours old) – Dry Weight and Total Length from F0 Generation

A representative sample of Day 0 mysids (n = 45) was collected from the same pool of 

organisms used to initiate the test. The mean total lengths and dry weights of Day 0 mysids from 

the F0 generation are presented in Table 28, with measurements for individual test replicates 

presented in Appendix 31. The mean total length and dry weight of 45 Day 0 mysids for the F0

generation were 1.92 mm and 0.045 mg, respectively. 

14.4.2 Day 7 - Dry Weight and Total Length from F0 Generation

Summaries of the Day 7 total lengths and dry weights of surviving juvenile mysids in the 

treatment and control groups for the F0 generation are presented in Table 29, with replicate mean 

measurements of total lengths and dry weights presented in Appendix 32 to Appendix 33, 

respectively. 

Sex could not be determined for Day 7 mysids so the data represent all juveniles. The mean total 

length of the Day 7 mysids in the negative control, 0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment 

groups from the F0 generation was 5.55, 5.08, 5.09, 5.23, 4.91 and 4.99 mm, respectively, while 

the corresponding mean dry weights were 0.298, 0.254, 0.263, 0.300, 0.267 and 0.259 mg, 

respectively. There were no apparent effects or statistically significant differences in Day 7 weight 

measurements. However, there were apparent effects on the length of Day 7 mysids that were 

significantly less than controls in all of the treatment groups except the 1.6 μg/L treatment group 

(Dunnett’s test; p ≤ 0.05).

14.4.3 Day 14 – Male and Female Dry Weight and Total Length from F0 Generation

Summaries of the Day 14 total lengths and dry weights of surviving male and female mysids in 

the treatment and control groups for the F0 generation are presented in Table 30, with replicate 

mean measurements of total lengths and dry weights presented in Appendix 34 to Appendix 35. 

There were no immature mysids present in the Day 14 growth measurements.
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14.4.3.1 Day 14 – Male Dry Weight and Total Length from F0 Generation

The mean total length of the Day 14 male mysids in the F0 generation in the negative control, 0.26, 

0.64, 1.6, 4.0 and 10 μg/L treatment groups was 5.80, 6.24, 6.26, 6.26, 6.24 and 6.29 mm, 

respectively, while the corresponding mean dry weight of males was 0.606, 0.573, 0.605, 0.598, 

0.554 and 0.542 mg, respectively. There were no apparent effects or statistically significant 

decreases in mean total length or dry weight for the Day 14 male mysids from the F0 generation in 

any of the treatment groups (Table 30). The length differences observed on Day 7 were no longer 

apparent in male mysids by Day 14. 

14.4.3.2 Day 14 – Female Dry Weight and Total Length from F0 Generation

The mean total length of the Day 14 female mysids in the F0 generation in the negative control, 

0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups was 5.87, 5.95, 6.21, 6.16, 6.24 and 6.07 mm, 

respectively, while the corresponding mean dry weights of females in the 0.26, 0.64, 1.6, 4.0 and 

10 μg/L treatment groups was 0.706, 0.618, 0.687, 0.648, 0.642 and 0.580 mg, respectively. 

There were no apparent effects or statistically significant decreases in mean total length for the 

Day 14 female mysids from the F0 generation in any of the treatment groups. The length 

differences observed on Day 7 were no longer apparent in female mysids by Day 14. However, 

there were apparent decreases in mean dry weight for the Day 14 female mysids in comparison to 

the negative control group in all of the treatment groups, except the 0.64 μg/L treatment group, 

(p ≤ 0.05), which were statistically significant and appeared to be treatment related (Table 30).

14.4.4 Test Termination – Dry Weight and Total Length from F0 (Day 46) Generation

Summaries of total lengths and dry weights of surviving male and female mysids in the treatment 

and control groups at test termination for the F0 generation (Day 46) are presented in Table 31, 

with replicate mean measurements of total lengths and dry weights presented in Appendix 36 to 

Appendix 37. 
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14.4.4.1 Males at Test Termination from F0 (Day 46) Generation

The mean total length of the Day 46 male mysids in the F0 generation in the negative control, 0.26, 

0.64, 1.6, 4.0 and 10 μg/L treatment groups at test termination was 8.58, 8.63, 8.54, 8.72, 8.95 

and 8.56 mm, respectively, while the corresponding mean dry weights were 1.27, 1.26, 1.24, 1.21, 

1.29 and 1.22 mg, respectively. There were no apparent effects or statistically significant

decreases in mean total length or dry weight in males from the F0 generation in any of the 

treatment groups at test termination (Day 46). Any differences in length or weight seen on Days 7 

or 14 were no longer apparent at test termination.

14.4.4.2 Females at Test Termination from F0 (Day 46) Generation

The mean total length of the Day 46 female mysids in the F0 generation in the negative control,

0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups were 9.05, 9.08, 9.06, 9.07, 8.94 and 9.18 mm, 

respectively, while the corresponding mean dry weights were 1.57, 1.58, 1.52, 1.53, 1.44 and 

1.48 mg, respectively. There were no apparent effects or statistically significant decreases in mean 

total length or dry weight in females for the Day 46 mysids from the F0 generation in any of the 

treatment groups. Any differences in length or weight seen on Days 7 or 14 were no longer 

apparent at test termination.

14.5 Reproduction

Mysids in the F0 generation were evaluated for several reproductive endpoints, including time to 

maturation, time to first brood and second brood, interbrood duration, number of offspring per 

reproducing female in the first and second broods, total offspring produced, sex ratio and the 

percent of females that produced offspring. 

Summaries of the reproductive endpoints for the mysids throughout the study are presented in 

Table 32. Individual animals and their replicate means for each endpoint are provided in 

Appendix 38 to Appendix 40.
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14.5.1 Time to Maturation, First Brood, Second Brood and Interbrood Duration from F0

Generation

There were no significant differences in mean number of days to maturation, days to first brood, 

days to second brood, or mean interbrood duration for any treatment group in the F0 generation 

(Table 32). Summaries of the mean time to maturation, number of days to first and second 

brood, and interbrood duration in the negative control and treatment groups for the F0 generation 

are presented in Table 32. The replicate mean time to maturation, number of days to first and 

second brood, and interbrood duration are presented in Appendix 38. 

14.5.2 Number of Offspring in the First Brood from F0 Generation

Summaries of the mean number of offspring in the first brood per reproductive female in the 

negative control and treatment groups for the F0 generation are presented in Table 32. The 

replicate mean number of offspring in the first brood is presented in Appendix 39. 

The mean number of offspring in the first brood for the F0 generation in the negative control and 

the 0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups was 3.7, 3.4, 3.8, 3.1, 2.6 and 4.0 young per 

reproducing female, respectively. There were no apparent effects or statistically significant 

decreases in mean number of offspring in the first brood for the mysids from the F0 generation in 

any of the treatment groups.

14.5.3 Number of Offspring in the Second Brood from F0 Generation

Summaries of the mean number of offspring in the second brood per reproductive female in the 

negative control and treatment groups for the F0 generation are presented in Table 32. The 

replicate mean number of offspring in the second brood is presented in Appendix 39.

The mean number of offspring in the second brood for the F0 generation in the negative control 

and the 0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups was 5.4, 2.1, 1.9, 1.5, 0.7 and 

2.1 young per reproductive female, respectively. There were apparent effects and statistically 
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significant decreases in the number of offspring in the second brood per reproductive female in all 

of the treatment groups (p ≤ 0.05), which appeared to be treatment related. 

14.5.4 Total Number of Offspring from F0 Generation

Summaries of the mean total number of offspring produced in the study in the negative control 

and treatment groups for the F0 generation are presented in Table 32. The replicate mean total 

number of offspring produced in the study for the F0 generation is presented in Appendix 39.

The mean total number of offspring produced in the study for the F0 generation in the negative 

control and the 0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups was 79.7, 51.3, 44.3, 22.7, 16.7 

and 36.0, respectively. There were apparent effects and statistically significant decreases in the 

total number of offspring produced in the 1.6 and 4.0 μg/L treatment groups (p ≤ 0.05), which 

appeared to be treatment related. Also, while not statistically significant, there was an apparent 

decrease in the total number of offspring produced in the 10 μg/L treatment group that appeared 

to be treatment related. 

14.5.5 Sex Ratio (Male/Female) from F0 Generation

Summaries of the mean percent of males present (used to determine the sex ratio in the study) in 

the negative control and treatment groups for the F0 generation are presented in Table 32. The 

replicate mean percent male sex ratio in the study for the F0 generation is presented in 

Appendix 40.

The mean percent of males present in the study for the F0 generation in the negative control and 

the 0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups was 50.2, 46.1, 55.5, 46.9, 50.9 and 47.9%, 

respectively. There were no apparent effects or statistically significant decreases in the mean 

percent male sex ratio in the study for the mysids from the F0 generation in any of the treatment 

groups. 
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14.5.6 Percent of Reproducing Females from F0 Generation

Summaries of the mean percent of reproducing females in the study in the negative control and 

treatment groups for the F0 generation are presented in Table 32. The replicate mean percent of 

reproducing females in the study for the F0 generation is presented in Appendix 40.

The mean percent of reproducing females in the study for the F0 generation in the negative 

control and the 0.26, 0.64, 1.6, 4.0 and 10 μg/L treatment groups was 76.2, 85.7, 76.2, 71.4, 66.7 

and 71.4%, respectively. There were no apparent effects or statistically significant decreases in 

the percent of females producing young in any of the treatment groups when compared to the 

negative control.

14.6 Summary of Survival, Growth Measurements and Reproduction Endpoints for 

the F1 Generation

There appeared to be treatment related effects on reproduction in all treatment groups that greatly 

reduced the number of viable young that could be used to initiate the F1 generation. The mysids 

in the F0 generation negative control produced enough live young to initiate all three F1 

generation replicate reproductive retention chambers, as well as providing enough organisms to 

capture Day 0, 7, 14 and termination growth measurements, for the F1 generation negative 

control group. A small number of young were produced only in the 0.64 and 10 μg/L treatment 

groups, 4 and 10 live young respectively. However, this was an inadequate number of young to 

provide meaningful results for these treatment groups. Therefore, only the negative control was 

continued to evaluate F1 generation mysids. Summaries of data collected from the negative 

control mysids in the F1 generation are presented in Appendix 41.

14.6.1 Summary of Survival for the F1 Generation

A summary of survival of F1 negative control juvenile mysids from replicate chamber initiation 

to the Day 7 growth sub-sampling interval, Day 8 to pairing on Day 14 and Day 15 until each 

replicate chamber’s termination on replicate Day 35 is presented in Appendix 41.1, and was 
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83.5, 98.8 and 75.2%, respectively. The termination survival data presented excluded mysids 

that died as a cause of impingement. Day 15 to termination daily survival data by gender are 

presented in Appendix 41.5.

14.6.2 Summary of Growth Measurements for the F1 Generation

Summaries of the mean dry weight and total body length measurements in the negative control 

for the F1 generation mysids are presented in Appendix 41.2 to Appendix 41.3. Day 0 and 7 

mean dry weight measurements of F1 negative control mysids were 0.044 and 0.33 mg, 

respectively, while the corresponding mean total body length measurements were 1.89 and 

5.4 mm. By Days 14 and 35, male mysids in the F1 negative control had mean dry weights of 

0.85 and 1.35 mg, while corresponding total body length measurements were 8.13 and 8.10 mm. 

Female mysid dry weight measurements on Days 14 and 35 were 0.80 and 1.58 mg, while 

corresponding total body length measurements were 7.62 and 8.21 mm. 

14.6.3 Summary of Reproduction for the F1 Generation

Summaries of all reproductive endpoints evaluated in the negative control for the F1 generation 

mysids are presented in Appendix 41.4. As with the F0 generation, all mysids were sexually 

identified and therefore mature on Replicate Day 14 for each negative control replicate. 

The mean time to first and second brood, as well as the interbrood duration for the negative 

control, was 20.1, 25.5 and 6.5 days, respectively. The mean number of offspring in the first and 

second brood per female, as well as the mean total number of offspring produced in the negative 

control group, was 2.6, 5.2 and 8.7, respectively. While the amount of young produced were 

lower than what was observed in the F0 generation, it is important to note that the F1 generation 

was conducted for 35 replicate days versus the F0 generation of 46 days. The shorter number of 

days in the F1 generation also may help explain why 61.7% of females produced young in the F1

generation versus the 76.2% in the F0 generation. The lower percent of females producing young 

in the F1 generation was skewed by three female mysids that were dead five days into the adult 

reproductive phase (Appendix 41.5). The mean percent of males present in the negative control 
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F1 generation was 65.6%, about 15% higher than the F0 generation. Overall, reproduction was 

good in the negative control replicates in the F1 generation.

15.0 PERFORMANCE CRITERIA

The following criteria were for judging test data acceptability:

15.1 Water quality characteristics should remain within the tolerance limits 

illustrated in Tables 1 and 2 of the Protocol.

The water quality characteristics remained within the tolerance limits defined in the protocol 

throughout the study.

15.2 There should be more than 70% survival of control animals in all test phases 

over the duration of the chemical exposure.

Survival exceeded 70% of control animals in all test phases over the duration of the chemical 

exposure.

15.3 At least 75% of the females in the F0 and F1 controls must produce young. 

More than 75% of females in the F0 generations produced young in the control. In the F1

generation, 61.7% of females produced young. The lower percent of females producing young 

in the F1 generation was skewed by three female mysids that were dead five days into the adult 

reproductive phase.

15.4 The average number of young produced per control female in the first two 

broods is at least eight. 

The average combined total number of young in the first two broods was greater than eight per 

control female in the F0 and F1 generations.
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16.0 CONCLUSIONS

Saltwater mysids (Americamysis bahia) were exposed to 4-tert-octylphenol at nominal 

concentrations of 0.26 to 10 μg/L for the F0 and F1 generations. The test was conducted under 

flow-through conditions for 46 and 35 days for the F0 and F1 generations, respectively, and the 

mysids were evaluated for survival, growth and reproductive endpoints. The most sensitive 

biological endpoints were reproduction and growth in the F0 generation. There was a treatment 

related and statistically significant decrease in mean number of offspring in the second brood per 

reproductive female in all of the treatment groups tested, in the Day 7 total lengths for the 

0.26, 0.64, 4.0 and 10 μg/L treatment groups, and in the Day 14 female dry weights for the 

0.26, 1.6, 4.0 and 10 μg/L treatment groups. Although not statistically significant, there was an 

apparent decrease in the total number of offspring produced in all treatment groups in the F0

generation. Reproductive effects were apparent in all test concentrations with insufficient 

production of live young to continue an F1 generation in all treatment groups. However, the 

negative control F1 produced as expected. Consequently, the NOEC, based on reproduction and 

growth for the F0 generation, was <0.26 μg/L. The LOEC was 0.26 μg/L and the MATC could 

not be calculated.
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REFERENCES AND LITERATURE

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Reitsema, L.A. and J.M. Neff. 1980. A recirculating seawater system for the laboratory culture 

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on Testing and Assessment No. 50. 

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Table 20. Means and Ranges of Water Quality Measurements Taken During 

the F0 Generation Exposure to 4-tert-octylphenol 

Nominal

Concentration

(μg/L)

Mean ± SD and Range of Measured Parameters

Temperature1

(°C)

DO2

(mg/L) pH

Salinity

(‰)

Negative Control 26.2 ± 0.29 7.1 ± 0.11 7.9 ± 0.06 20 ± 0.14

(25.5 – 26.7) (6.7 – 7.2) (7.8 – 8.0) (20 – 21)

0.26 26.0 ± 0.32 7.1 ± 0.12 7.9 ± 0.05 20 ± 0.14

(25.3 – 26.7) (6.7 – 7.2) (7.8 – 8.0) (20 – 21)

0.64 25.9 ± 0.32 7.2 ± 0.09 7.9 ± 0.06 20 ± 0.14

(25.3 – 26.7) (6.9 – 7.2) (7.8 – 8.0) (20 – 21)

1.6 25.8 ± 0.32 7.1 ± 0.10 7.9 ± 0.05 20 ± 0.14

(25.2 – 26.4) (6.9 – 7.2) (7.7 – 8.0) (20 – 21)

4.0 25.8 ± 0.33 7.1 ± 0.10 7.9 ± 0.05 20 ± 0.14

(25.2 – 26.4) (6.8 – 7.2) (7.8 – 8.0) (20 – 21)

10 25.8 ± 0.36 7.1 ± 0.10 7.9 ± 0.06 20 ± 0.14

(25.0 – 26.4) (6.8 – 7.2) (7.7 – 8.0) (20 – 21)

1 Temperature monitored continuously during the test ranged from approximately 25 to 27oC, measured to the nearest 1oC. 2 A dissolved oxygen concentration of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 333

Table 21. Means and Ranges of Water Quality Measurements Taken During 

the F1 Generation Exposure to 4-tert-octylphenol 

Nominal

Concentration

(μg/L)

Mean ± SD and Range of Measured Parameters

Temperature1

(°C)

DO2

(mg/L) pH

Salinity

(‰)

Negative Control 26.0 ± 0.38 7.1 ± 0.13 7.9 ± 0.11 20 ± 0.33

(25.1 – 26.8) (6.8 – 7.2) (7.7 – 8.1) (19 – 21)

0.64 26.2 ± 0.41 7.1 ± 0.11 7.8 ± 0.08 20 ± 0.33

(25.3 – 26.6) (6.9 – 7.2) (7.7 – 7.9) (20 – 21)

10 26.2 ± 0.35 7.2 ± 0.00 7.9 ± 0.06 20 ± 0.00

(25.9 – 26.6) (7.2 – 7.2) (7.8 – 7.9) (20 – 20)

1 Temperature monitored continuously during the test ranged from approximately 25 to 27oC, measured to the nearest 1oC. 2 A dissolved oxygen concentration of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 334

Table 22. Measured Concentrations of 4-tert-octylphenol in Pretest Solution 

Samples for the F0 Generation 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-2-)

Sampling

Time

(Day)

Measured 

Concentration

(μg/L) 1

Percent

of

Nominal2

0.0

(Negative Control) PT-37 -5 < LOQ --

0.26 PT-38 -5 0.310 119

0.64 PT-39 -5 0.509 79.6

1.6 PT-40 -5 1.24 77.4

4.0 PT-41 -5 2.82 70.4

10 PT-42 -5 6.87 68.7 1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 335

Table 23. Measured Concentrations of 4-tert-octylphenol for the F0

Generation in Stock Solution Samples 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-2-)

Measured 

Concentration

(μg/L) 1

Percent

of

Nominal2

11000 S-1 9858 89.6

1 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 336

Table 24. Measured Concentrations of 4-tert-octylphenol in Saltwater Samples 

for the F0 Generation

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-2A-)

Sampling

Time

(Days)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Percent of

Nominal

0.0

(Negative Control)

1

7

13

19

25

26

32

38

44

0

6

13

20

21

27

34

41

46

< LOQ

< LOQ

< LOQ

0.523

< LOQ

< LOQ

0.203

< LOQ

< LOQ

--

--

--

--

--

--

--

--

--

-- --

0.26 2

8

14

20

27

33

39

45

0

6

13

20

27

34

41

46

0.290

0.432

0.275

0.343

0.273

0.350

0.345

0.339

111

166

106

132

105

135

133

131

0.33 127

0.64 3

9

15

21

28

34

40

46

0

6

13

20

27

34

41

46

0.587

0.755

0.566

0.755

0.609

0.719

0.735

0.819

91.6

118

88.4

118

95.1

112

115

128

0.69 108

1.6 4

10

16

22

29

35

41

47

0

6

13

20

27

34

41

46

1.22

1.20

1.17

1.45

1.22

1.44

1.57

1.59

76.4

75.2

73.2

90.9

76.4

89.7

97.9

99.1

1.4 88

1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 337

Table 24. Measured Concentrations of 4-tert-octylphenol in Saltwater 

Samples for the F0 Generation (Continued)

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-2A-)

Sampling

Time

(Days)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Percent of

Nominal

4.0 5

11

17

23

30

36

42

48

0

6

13

20

27

34

41

46

2.69

3.13

4.38

3.57

2.72

3.14

3.05

3.22

67.2

78.2

109

89.4

68.0

78.4

76.4

80.6

3.2 80

10 6

12

18

24

31

37

43

49

0

6

13

20

27

34

41

46

5.80

7.77

7.32

9.66

5.98

7.38

8.25

7.24

58.0

77.7

73.2

96.6

59.8

73.8

82.5

72.4

7.4 74

1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 338

Table 25. Measured Concentrations of 4-tert-octylphenol in Pretest Solution 

Samples for the F1 Generation 

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-2A-)

Sampling

Time

(Day)

Measured 

Concentration

(μg/L) 1

Percent

of

Nominal2

0.0

(Negative Control) F1-PT-7 -19 < LOQ --

0.26 F1-PT-2 -20 0.436 168

0.64 F1-PT-3 -20 0.622 97.2

1.6 F1-PT-4 -20 1.54 96.1

4.0 F1-PT-5 -20 3.01 75.1

10 F1-PT-6 -20 7.78 77.5 1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (2.50 μg a.i./L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 339

Table 26. Measured Concentrations of 4-tert-octylphenol in Saltwater Samples 

for the F1 Generation

Nominal Test

Concentration

(μg/L)

Sample

Number

(185A-102-2A-)

Sampling

Time

(Days)

Measured

Concentration

(μg/L)1

Percent

of

Nominal2

Mean

Measured

Concentration

(μg/L)

Mean

Percent of

Nominal

0.0

(Negative Control)

F1-1

F1-7

F1-13

F1-BU1

F1-BU2

F1-BU3

F1-BU4

0

7

14

21

28

35

42

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

--

--

--

--

--

--

--

-- --

0.26 F1-2

F1-8

0

7

0.423

0.423

163

163

0.42 162

0.64 F1-3

F1-9

F1-14

0

7

14

0.683

0.699

0.758

107

109

118

0.71 111

1.6 F1-4

F1-10

0

7

1.35

1.44

84.6

89.7

1.4 88

4.0 F1-5

F1-11

0

7

3.26

3.32

81.4

83.0

3.3 83

10 F1-6

F1-12

F1-15

0

7

14

7.99

7.94

8.59

79.9

79.4

85.9

8.2 82

1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest calibration 

standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 340

Table 27. Summary of F0 Generation Survival of Saltwater Mysids Exposed to 

4-tert-octylphenol

Nominal

Concentration

(μg/L)

Mean Percent Juvenile

Survival to

Lengths

On Day 7 ± SD

(CV%)1,2

Mean Percent Juvenile

Survival to

Pairing

On Day 14 ± SD

(CV%)1,2

Mean Percent Adult

Survival Day 15 to

Test Termination

On Day 46 ± SD

(CV%)1,2

Negative Control

100 ± 0.0

(0.0)

n = 120

95.8 ± 6.65

(6.93) 

n = 97

77.2 ± 12.95

(16.76) 

n = 32

0.26

99.2 ± 2.04

(2.06)

n = 119

96.7 ± 5.80

(5.99) 

n = 98

85.6 ± 7.33

(8.56) 

n = 39

0.64

100 ± 0.0

(0.0) 

n = 120

91.0 ± 10.6

(11.6) 

n = 92

66.2 ± 3.84

(5.80) 

n = 28

1.6

99.2 ± 2.04

(2.06)

n = 119

97.5 ± 4.18

(4.29) 

n = 98

79.8 ± 7.72

(9.68) 

n = 36

4.0

100 ± 0.0

(0.0) 

n = 120

98.0 ± 3.21

(3.27) 

n = 100

76.2 ± 6.59

(8.66) 

n = 33

10

97.5 ± 2.74

(2.81)

n = 117

96.8 ± 5.78

(5.97) 

n = 96

65.1 ± 14.35

(22.05) 

n = 26

1 For all treatment and control groups, n = number of surviving organisms. 2 There were no statistically significant decreases in survival in comparison to the negative control using Fisher’s Exact test 

(p > 0.05).
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 341

Table 28. Summary of Day 0 Growth Measurements of Culture Saltwater 

Mysids

Mean Total Body Length (mm) ± SD

(CV%) 1

Mean Dry Weight (mg) ± SD

(CV%) 1

1.92 ± 0.111

(5.78)

n = 45

0.045 ± 0.010

(22.8)

n = 49

1 Mean, standard deviation, and coefficient of variation was calculated using Excel 2010. Manual calculations may differ 

slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 342

Table 29. Summary of F0 Generation Day 7 Growth Measurements of 

Saltwater Mysids Exposed to 4-tert-octylphenol

Nominal

Concentration

(μg/L)

Growth Parameters on Day 71,2

Mean Total Length (mm) ± SD

(CV%)

Mean Dry Weight (mg) ± SD

(CV%)

Negative Control 5.55 ± 0.209

(3.76)

0.298 ± 0.016

(5.50)

0.26 5.08 ± 0.104*

(2.05)

0.254 ± 0.014

(5.58)

0.64 5.09 ± 0.284*

(5.57)

0.259 ± 0.045

(17.3)

1.6 5.23 ± 0.087

(1.65)

0.300 ± 0.008

(2.55)

4.0 4.91 ± 0.251*

(5.11)

0.267 ± 0.042

(15.8)

10 4.99 ± 0.186*

(3.73)

0.259 ± 0.014

(5.56)

* Statistically significant decrease in growth in comparison to the negative control using Dunnett’s test (p ≤ 0.05). 1 For all treatment and control groups tested, statistics were based on replicate means. Therefore, n = 3 for all treatment and 

control groups presented. 2 Coefficient of Variation was calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 343

Table 30. Summary of F0 Generation Day 14 Growth Measurements of 

Saltwater Mysids Exposed to 4-tert-octylphenol

Nominal

Concentration

(μg/L)

Growth Parameters on Day 14

Mean Total Length (mm) ± SD

(CV%) 1,2

Mean Dry Weight (mg) ± SD

(CV%) 1,2

Males Females Males Females

Negative Control 5.80 ± 0.160

(2.76)

5.87 ± 0.048

(0.818)

0.606 ± 0.048

(7.94)

0.706 ± 0.021

(2.97)

0.26 6.24 ± 0.042

(0.668)

5.95 ± 0.047

(0.794)

0.573 ± 0.034

(5.87)

 0.618 ± 0.019*

(3.13)

0.64 6.26 ± 0.014

(0.228)

6.21 ± 0.038

(0.611)

0.605 ± 0.043

(7.19)

0.687 ± 0.031

(4.58)

1.6 6.26 ± 0.159

(2.54)

6.16 ± 0.157

(2.55)

0.598 ± 0.013

(2.09)

 0.648 ± 0.030*

(4.65)

4.0 6.24 ± 0.176

(2.83)

6.24 ± 0.078

(1.25)

0.554 ± 0.046

(8.29)

 0.642 ± 0.042*

(6.53)

10 6.29 ± 0.069

(1.10)

6.07 ± 0.171

(2.81)

0.542 ± 0.035

(6.51)

 0.580 ± 0.024*

(4.11)

* Statistically significant decrease in growth in comparison to the negative control using Dunnett’s test (p ≤ 0.05). 1 For all treatment and control groups tested, statistics were all based on replicate means. Therefore, n = 3 for all treatment 

and control groups presented. 2 Coefficient of Variation was calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 344

Table 31. Summary of F0 Generation Day 46 Growth Measurements of 

Saltwater Mysids Exposed to 4-tert-octylphenol

Nominal

Concentration

(μg/L)

Growth Parameters on Day 46 1

Mean Total Length (mm) ± SD

(CV%) 2,3

Mean Dry Weight (mg) ± SD

(CV%) 2,3

Males Females Males Females

Negative Control 8.58 ± 0.169

(1.96)

9.05 ± 0.178

(1.96)

1.27 ± 0.042

(3.30)

1.57 ± 0.138

(8.82)

0.26 8.63 ± 0.149

(1.72)

9.08 ± 0.023

(0.254)

1.26 ± 0.113

(8.92)

1.58 ± 0.095

(6.03)

0.64 8.54 ± 0.360

(4.22)

9.06 ± 0.299

(3.30)

1.24 ± 0.181

(14.6)

1.52 ± 0.107

(7.05)

1.6 8.72 ± 0.237

(2.72)

9.07 ± 0.091

(0.999)

1.21 ± 0.065

(5.39)

1.53 ± 0.023

(1.52)

4.0 8.95 ± 0.070

(0.777)

8.94 ± 0.293

(3.27)

1.29 ± 0.126

(9.74)

1.44 ± 0.043

(3.01)

10 8.56 ± 0.513

(5.99)

9.18 ± 0.086

(0.937)

1.22 ± 0.135

(11.1)

1.48 ± 0.013

(0.861)

1 There were no statistically significant decreases in growth in comparison to the negative control using Dunnett’s test 

(p > 0.05). 2 For all treatment and control groups tested, statistics were all based on replicate means. Therefore, n = 3 for all treatment 

and control groups presented. 3 Coefficient of Variation was calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 345

Table 32. Summary of F0 Generation Reproduction of Saltwater Mysids Exposed to 4-tert-octylphenol

Nominal

Conc.

(μg/L)

Mean Time 

to 

Maturation

Days ± SD

(CV%)1

Mean 

Time to 1st

Brood

Days ± SD

(CV%)1

Mean 

Time to 2nd

Brood

Days ± SD

(CV%)1

Mean

Interbrood 

Duration

Days ± SD

(CV%)1

Mean Number 

of Offspring in 

1st Brood Per 

Female ± SD

(CV%)1

Mean Number 

of Offspring in 

2nd Brood Per 

Female ± SD

(CV%)1

Mean

Total 

Number of

Offspring ± 

SD

(CV%)1

Mean

Percent of 

Males Sex

Ratio ± SD

(CV%) 1

Mean

Percent of

Females

Producing 

Offspring ± SD

(CV%) 1,2

Negative 

Control

14.0 ± 0.000

(0.00)

26.4 ± 2.03

(7.71)

34.7 ± 3.76

(10.83)

9.6 ± 2.40

(25.1)

4.1 ± 0.79

(19.3)

5.0 ± 0.74

(14.6)

79.7 ± 54.17

(68.0)

50.2 ± 10.37

(20.6)

n = 97

76.2 ± 8.25

(10.8) 

n = 16

0.26 14.0 ± 0.000

(0.00)

26.8 ± 0.252

(0.94)

36.3 ± 7.54

(20.76)

9.4 ± 0.52

(5.53)

3.4 ± 1.11

(32.2)

2.0 ± 1.32*

(66.0)

51.3 ± 34.24

(66.7)

46.1 ± 7.48

(16.2)

n = 98

85.7 ± 14.29

(16.7) 

n = 18

0.64 14.0 ± 0.000

(0.00)

28.7 ± 2.86

(9.94)

31.6 ± 9.45

(29.96)

7.2 ± 2.89

(40.0)

3.8 ± 0.95

(25.2)

1.9 ± 1.30*

(68.4)

44.3 ± 18.18

(41.0)

55.5 ± 0.86

(1.55)

n = 92

76.2 ± 8.25

(10.8) 

n = 16

1.6 14.0 ± 0.000

(0.00)

30.4 ± 5.11

(16.65)

40.0 ± 5.57

(13.92)

8.7 ± 4.16

(48.0)

3.1 ± 0.98

(32.0)

1.5 ± 0.58*

(39.4)

22.7 ± 4.93*

(21.8)

46.9 ± 5.62

(12.0)

n = 98

71.4 ± 14.29

(20.0) 

n = 15

4.0 14.0 ± 0.000

(0.00)

30.8 ± 5.98

(19.41)

35.0 ± 7.94

(22.68)

6.7 ± 2.89

(43.3)

2.6 ± 1.29

(50.2)

0.7 ± 0.70*

(100)

16.7 ± 4.04*

(24.2)

50.9 ± 9.59

(18.9)

n = 100

66.7 ± 8.25

(12.4) 

n = 14

10 14.0 ± 0.000

(0.00)

25.9 ± 1.55

(5.98)

35.2 ± 2.47

(7.01)

8.2 ± 4.65

(56.9)

4.0 ± 1.82

(45.2)

2.1 ± 0.38*

(17.7)

36.0 ± 7.94 3

(22.0)

47.9 ± 0.90

(1.88)

n = 96

71.4 ± 14.29

(20.0) 

n = 15

* Statistically significant decrease in reproduction in comparison to the negative control using Dunnett’s test (p ≤ 0.05). 1 Unless otherwise noted, n = 3, the number of replicates, for all treatment and control groups. For male sex ratio n = the total number of organisms that were able to be sexually 

identified throughout the test. For percent females producing offspring, n = the number of females that produced offspring. 2 There were no statistically significant decreases in reproduction in comparison to the negative control using Fisher’s Exact test (p > 0.05). 3 While not statistically significant from the negative control, the decrease in total number of offspring in the 10 μg/L treatment group appeared to be a treatment related affect.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 346

Figure 4. Mysid Two-Generation Test Method Schematic

Source: Wildlife International, Ltd. Protocol No.: 185/021511/MYS-2GeN/SUB185; Page 44.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 347

Figure 5. F0 Generation Analytical Recoveries Over Time
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 348

Figure 6. F1 Generation Analytical Recoveries Over Time
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 349

Appendix 26. Dilution Water Chemistry Parameters Measured During the 4-

Week Period Immediately Preceding the Test

Parameter Mean Range

Salinity (‰) 20

(N = 4)

20 – 20

pH 7.9

(N = 4)

7.9 – 7.9
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 350

Appendix 27. Analyses of Pesticides, Organics and Metals in Wildlife 

International, Ltd. Saltwater

Pesticides and Organics 1

Component

Measured Concentration

(μg/L) Component

Measured Concentration

(μg/L)

Aldrin < 0.0097 Gamma BHC – 4-tertoctylphenol < 0.0097

Alpha BHC < 0.0097 Gamma Chlordane < 0.019

Alpha Chlordane < 0.0097 Guthion (Azinphos-methyl) < 5.8

Beta BHC < 0.0097 HCB < 0.0097

Bolstar < 1.9 Heptachlor < 0.0097

Chlordane < 0.49 Heptachlor Epoxide < 0.0097

Coumaphos < 2.9 Kepone < 0.19

Delta BHC < 0.0097 Malathion < 2.9

Demeton-O < 1.9 Merphos < 1.9

Demeton-S < 2.9 Methoxychlor < 0.097

Diazinon < 5.8 Methyl Parathion < 1.9

Dichlorvos < 2.9 Mevinphos < 4.8

Dieldrin < 0.019 Mirex < 0.24

Disulfoton < 1.9 Naled < 5.8

Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.019

Endosulfan I < 0.0097 o,p-DDE < 0.73

Endosulfan II < 0.019 o,p-DDT < 0.019

Endosulfan Sulfate < 0.019 p,p-DDD < 0.019

Endrin < 0.019 p,p-DDE < 0.019

Endrin Aldehyde < 0.097 p,p-DDT < 0.019

Endrin Ketone < 0.019 Phorate < 1.9

EPN < 1.9 Ronnel < 1.9

Ethion < 1.9 Stirophos < 1.9

Ethoprop < 2.9 Telodrin < 0.0097

Ethyl Parathion < 1.9 Tokuthion < 1.9

Famphur < 2.9 Toxaphene < 2.9

Fensulfothion < 8.7 Trichloronate < 1.9

Fenthion < 1.9 Trithion < 1.9

1 Analyses performed by Lancaster Laboratories on samples collected on December 28, 2011.

.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 351

Appendix 27. Analyses of Pesticides, Organics and Metals in Wildlife 

International, Ltd. Saltwater (Continued)

Metals 1

Component

Measured Concentration

(mg/L) Component

Measured Concentration

(mg/L)

Aluminum < 0.200 Magnesium 722

Antimony < 0.0200 Manganese < 0.0050

Arsenic < 0.0200 Mercury < 0.00020

Barium 0.0071 Nickel < 0.0100

Beryllium < 0.0050 Nitrate Nitrogen < 0.50

Bromide 42.0 Nitrite Nitrogen < 10.0

Cadmium < 0.0050 Potassium 235

Calcium 240 Selenium < 0.0200

Chloride 15,600 Silver < 0.0050

Chromium < 0.0150 Sodium 5870

Cobalt < 0.0050 Sulfate 1680

Copper < 0.0100 Thallium < 0.0300

Fluoride < 10.0 Vanadium < 0.0050

Iron < 0.200 Zinc < 0.0200

Lead < 0.0150

1 Analyses performed by Lancaster Laboratories on samples collected on December 28, 2011.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 352

Appendix 28. Analyses of Pesticides, Organics and Metals in Brine (Artemia) Egg 

Sample

Pesticides and Organics 1

Component

Measured Concentration

(μg/kg) Component

Measured Concentration

(μg/kg)

Aldrin < 0.93 Heptachlor Epoxide < 0.93

Alpha BHC < 0.93 Kepone < 7.8

Alpha Chlordane < 0.93 Malathion < 75

Beta BHC < 2.1 Merphos < 75

Bolstar < 75 Methoxychlor < 9.3

Chlordane < 19 Methyl Parathion < 75

Coumaphos < 75 Mevinphos < 75

Delta BHC < 0.93 Mirex < 1.9

Demeton-O < 75 Naled < 75

Demeton-S < 75 o,p-DDD < 1.9

Diazinon < 75 o,p-DDE < 1.9

Dichlorvos < 75 o,p-DDT < 1.9

Dieldrin < 1.9 p,p-DDD < 1.9

Disulfoton < 84 p,p-DDE < 1.9

Dursban (Chlorpyrifos) < 75 p,p-DDT < 1.9

Endosulfan I < 0.93 PCB-1016 < 19

Endosulfan II < 1.9 PCB-1221 < 19

Endosulfan Sulfate < 1.9 PCB-1232 < 19

Endrin < 1.9 PCB-1242 < 19

Endrin Aldehyde < 1.9 PCB-1248 < 19

Endrin Ketone < 2.0 PCB-1254 < 19

EPN < 75 PCB-1260 < 19

Ethion < 75 PCB-1262 < 19

Ethoprop < 75 PCB-1268 < 19

Ethyl Parathion < 75 TOTAL PCBs < 19

Famphur < 75 Phorate < 75

Fensulfothion < 220 Ronnel < 75

Fenthion < 75 Stirophos < 75

Gamma BHC – 4-tertoctylphenol < 0.93 Telodrin < 1.3

Gamma Chlordane < 0.93 Tokuthion < 75

Guthion (Azinphos-methyl) < 75 Toxaphene < 37

HCB < 0.93 Trichloronate < 75

Heptachlor < 0.93 Trithion < 75

1 Analyses performed by Lancaster Laboratories on samples collected on December 22, 2011.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 353

Appendix 28. Analyses of Pesticides, Organics and Metals in Brine (Artemia) Egg 

Sample (Continued)

Metals 1

Component

Measured Concentration

(mg/kg) Component

Measured Concentration

(mg/kg)

Aluminum < 21.5 Magnesium 2180

Antimony < 2.15 Manganese 7.07

Arsenic 15.8 Mercury < 0.537

Barium < 0.538 Nickel < 1.08

Beryllium < 0.538 Nitrate by IC (solid) 2.8

Bromide by IC (solid) < 5.6 Nitrite by IC (solid) < 1.1

Cadmium < 0.538 Potassium 7370

Calcium 503 Selenium 2.24

Chloride by IC (solid) 2010 Silver < 0.538

Chromium < 1.62 Sodium 2180

Cobalt < 0.538 Sulfate by IC (solid) 358

Copper 7.07 Thallium < 3.23

Fluoride by IC (solid) < 11.2 Vanadium < 0.538

Iron 574 Zinc 83.1

Lead < 1.62

1 Analyses performed by Lancaster Laboratories on samples collected on December 22, 2011.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 354

Appendix 29. The Analysis of 4-Tert-Octylphenol in Saltwater
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 355

Appendix 29.1 Method Flowchart for the Analysis of 4-TertOctylphenol in Saltwater

METHOD FLOWCHART FOR THE ANALYSIS OF 4-TERT-OCTYLPHENOL

IN SALTWATER

Rinse separatory funnels flasks with toluene.

↓

Prepare recovery samples by fortifying saltwater (contained in a separatory funnel) with the 

appropriate 4-tert-octyphenol stock solution. Unfortified saltwater will

serve as the matrix blank.

↓

Volumetrically pipet exposure samples into separatory funnels.

↓

Using a volumetric pipet, add the requisite volume of toluene to each sample. Stopper and 

shake each sample (with venting) for approximately one minute. Allow the organic 

and aqueous layers to separate. Drain the aqueous (lower) layer to waste.

↓

Drain the toluene layer through a small column of anhydrous sodium sulfate.

↓

Transfer each extract to an autosampler vial and submit samples for analysis by GC/MS.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 356

Appendix 29.2 Typical Gas Chromatograph (GC) Operational 

Parameters

INSTRUMENT: Agilent Model 5890 Gas Chromatograph (GC) 

DETECTOR: Agilent Model 5971A Mass Selective Detector operated in 

SIM mode

ANALYTICAL COLUMN: ZB-5 (0.25 mm x 30 m, 0.25 μm film thickness)

INJECTOR TEMPERATURE: 250°C

OVEN: Initial temperature: 100°C

Initial hold time: 1.00 minute

Ramp: 20.0°C/minute

Final temperature: 280°C

Final hold time: 0.00 minutes

Run time: 10.00 minutes

Ion monitoring: 135 m/z

DETECTOR TEMPERATURE: 280°C

INJECTION VOLUME: 2.00 μL (Splitless)

CARRIER GAS: Helium

HEAD PRESSURE: 12 psi

4-TERT-OCTYLPHENOL PEAK 

RETENTION TIME: Approximately 7.2 minutes
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 357

Appendix 29.3 Analytical Stocks and Standards Preparation

A stock solution of 4-tert-octylphenol test substance was prepared by weighing 0.1000 g of the 

test substance on an analytical balance. The test substance was transferred to a 100-mL class-A 

volumetric flask brought to volume using methanol. This primary stock solution contained 

1.00 mg/mL of 4-tert-octylphenol and was diluted in methanol to prepare 0.100, 0.0100 and 

0.00100 mg/mL stock solutions. The 0.00100 mg/mL stock solution was used to fortify the 

quality control samples. Calibration standards were prepared in toluene using the 0.0100 mg/mL 

stock solution. The following shows the dilution scheme for the set of calibration standards:

Stock

Concentration

(mg/mL)

Aliquot

(μL)

Final

Volume

(mL)

Standard

Concentration

(μg/L)

0.0100

0.0100

0.0100

0.0100

0.0100

25.0

50.0

100

150

200

100

100

100

100

100

2.50

5.00

10.0

15.0

20.0
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 358

Appendix 29.4 Example Calculations for a Representative Test 

Sample

The analytical result and percent recovery for sample number 185A-102-2A-3, with a 

nominal concentration of 0.64 μg/L, were calculated using the following equations:

Concentration of 4-tert-octylphenol in sample (μg/L) = 

Peak Area - Y-intercept

Slope X Dilution 

Factor 

Percent of nominal concentration = measured concentration in sample (μg/L)

nominal concentration in sample (μg/L) X 100 

Peak Area = 20486

Y-intercept = -2777.52355

Slope = 2379.83269

Dilution factor = 0.0600

Concentration of 4-tert-octylphenol in sample (μg/L) = 

20486 + 2777.52355

2379.83269 X 0.0600

Concentration of 4-tert-octylphenol in sample (μg/L) = 0.587

Percent of nominal concentration = 0.587 μg/L

0.64 μg/L X 100

Percent of nominal concentration = 91.6%
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 359

Appendix 29.5 Quality Control Samples of 4-Tert-Octylphenol in

Saltwater for the F0 Generation

Sample Number

(185A-102-2A-) Sample

Type

Concentration of 4-Tert-Octylphenol

Percent

Recovery2

Fortified

(μg/L)

Measured

(μg/L)1

Pre Test

PT-MAB-7

PT-MAS-19

PT-MAS-20

PT-MAS-21

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.250

2.00

10.0

< LOQ

0.270

2.00

10.2

--

108

100

102

Exposure

MAB-1

MAB-2

MAB-3

MAB-4

MAB-5

MAB-6

MAB-7

MAB-8

MAB-9

MAS-1

MAS-2

MAS-3

MAS-4

MAS-5

MAS-6

MAS-7

MAS-8

MAS-9

MAS-10

MAS-11

MAS-12

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.250

2.00

10.0

0.250

2.00

10.0

0.250

2.00

10.0

0.250

2.00

10.0

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

0.288

2.04

9.71

0.254

2.09

10.2

0.258

1.93

9.44

0.290

2.18

10.6

--

--

--

--

--

--

--

--

--

115

102

97.1

102

105

102

103

96.4

94.4

116

109

106 1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 V in full precision mode. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 360

Appendix 29.5 Quality Control Samples of 4-Tert-Octylphenol in 

Saltwater for the F0 Generation (Continued)

Sample 

Number

(185A-102-2A-)

Sample

Type

Concentration of 4-Tert Octylphenol

Percent

Recovery2

Fortified

(μg/L)

Measured

(μg/L)1

MAS-14

MAS-15

MAS-16

MAS-17

MAS-18

MAS-19

MAS-20

MAS-21

MAS-22

MAS-23

MAS-24

MAS-25

MAS-26

MAS-27

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

2.00

10.0

0.250

2.00

10.0

0.250

2.00

10.0

0.250

2.00

10.0

0.250

2.00

10.0

2.67

12.5

0.283

2.20

11.1

0.293

2.32

11.3

0.309

2.34

11.3

0.300

2.20

10.7

133

125

113

110

111

117

116

113

124

117

113

120

110

107

Mean3 =

Standard Deviation =

CV =

111

9.27

 8.35% 1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 V in full precision mode. Manual calculations may differ slightly. 3 Based on exposure samples only.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 361

Appendix 29.6 Quality Control Samples of 4-Tert-Octylphenol in 

Saltwater for the F1 Generation

Sample

Number

(185A-102-2A-)

Sample

Type

Concentration of 4-Tert Octylphenol

Percent

Recovery2

Fortified

(μg/L)

Measured

(μg/L)1

Pre Test

F1-PT-MAB-1

F1-PT-MAS-1

F1-PT-MAS-2

F1-PT-MAS-3

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.250

2.00

10.0

< LOQ

0.253

1.91

9.36

--

101

95.7

93.6

Exposure

F1-MAB-1

F1-MAB-2

F1-MAB-3

F1-MAS-1

F1-MAS-2

F1-MAS-3

F1-MAS-4

F1-MAS-5

F1-MAS-6

F1-MAB-7

F1-MAB-8

F1-MAB-9

Matrix Blank

Matrix Blank

Matrix Blank

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

Matrix Fortification

0.0

0.0

0.0

0.250

2.00

10.0

0.250

2.00

10.0

0.250

2.00

10.0

< LOQ

< LOQ

< LOQ

0.308

2.19

10.7

0.316

2.12

10.7

0.297

2.24

10.6

--

--

--

123

109

107

126

106

107

119

112

106

Mean3 =

Standard Deviation =

CV =

113

7.84

 6.94% 1 The limit of quantitation (LOQ) was 0.125 μg/L, calculated as the product of the concentration of the lowest 

calibration standard (2.50 μg/L) and the dilution factor of the matrix blank samples (0.0500). 2 Results were generated using Excel 2000 V in full precision mode. Manual calculations may differ slightly. 3 Based on exposure samples only.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 362

Appendix 29.7 Representative Calibration Curve for 4-TertOctylphenol 

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0.0 5.0 10.0 15.0 20.0 25.0

Concentration (ug/L)

Peak Area m/z 135

Slope = 2379.83269; Y-Intercept = -2777.52355; R2 = 0.99390
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 363

Appendix 29.8 Representative Chromatogram of a Low-Level 4-

Tert-Octylphenol Calibration Standard 

Nominal concentration: 2.50 μg/L. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 364

Appendix 29.9 Representative Chromatogram of a High-Level 4-

Tert-Octylphenol Calibration Standard 

Nominal concentration: 20.0 μg/L. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 365

Appendix 29.10 Representative Chromatogram of a Matrix Blank

Sample number 185A-102-2A-MAB-1. The arrow indicates the approximate retention time of 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 366

Appendix 29.11 Representative Chromatogram of a Matrix 

Fortification

Sample number 185A-102-2A-MAS-1; 0.250 μg/L, nominal concentration. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 367

Appendix 29.12 Representative Chromatogram of a Test Sample

Sample number 185A-102-2A-3; 0.64 μg/L, nominal concentration. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 368

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

Day of

Test

Negative Control – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 1 6 0 11 0 12 97

16 0 6 1 10 1 11 90

17 1 5 0 11 2 0 11 90

18 0 5 0 11 0 11 90

19 0 5 0 11 1 10 87

20 0 5 0 11 0 10 87

21 0 5 0 11 1 9 83

22 0 5 0 11 0 9 83

23 0 5 0 11 0 9 83

24 0 5 2 9 0 9 77

25 0 5 1 8 0 9 73

26 0 5 0 8 0 9 73

27 0 5 0 8 0 9 73

28 0 5 0 8 0 9 73

29 0 5 1 7 0 9 70

30 0 5 0 7 0 9 70

31 0 5 0 7 0 9 70

32 0 5 0 7 0 9 70

33 0 5 0 7 0 9 70

34 0 5 0 7 0 9 70

35 0 5 0 7 0 9 70

36 0 5 0 7 0 9 70

37 0 5 0 7 1 8 67

38 0 5 0 7 0 8 67

39 0 5 1 6 0 8 63

40 0 5 0 6 0 8 63

41 0 5 0 6 0 8 63

42 0 5 0 6 1 7 60

43 0 5 0 6 0 7 60

44 0 5 1 4 3 0 7 53

45 0 5 0 4 0 7 53

46 0 5 0 4 0 7 53 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 The mysid that was observed as missing and assumed dead on Day 16 was discovered on Day 17. 3 One male mysid was determined to be female on Day 44.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 369

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

Negative Control – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 10 0 9 0 7 100

16 0 10 0 9 0 7 100

17 0 10 0 9 1 6 96

18 0 10 0 9 0 6 96

19 0 10 0 9 0 6 96

20 0 10 0 9 0 6 96

21 0 10 0 9 0 6 96

22 0 10 0 9 0 6 96

23 0 10 0 9 0 6 96

24 0 10 0 9 0 6 96

25 0 10 0 9 0 6 96

26 0 10 0 9 1 5 92

27 0 10 0 9 0 5 92

28 0 10 0 9 0 5 92

29 0 10 0 9 0 5 92

30 0 10 0 9 0 5 92

31 0 10 1 8 0 5 88

32 1 9 0 8 0 5 85

33 0 9 0 8 0 5 85

34 0 9 0 8 0 5 85

35 0 9 1 7 0 5 81

36 1 8 1 6 0 5 73

37 0 8 0 6 0 5 73

38 0 8 0 6 0 5 73

39 0 8 0 6 0 5 73

40 0 8 1 5 0 5 69

41 0 8 0 5 0 5 69

42 0 8 0 5 0 5 69

43 0 8 0 5 0 5 69

44 1 7 0 6 2 1 4 65

45 0 7 0 6 0 4 65

46 1 6 0 6 0 4 62 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 One male mysid was determined to be female on Day 44.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 370

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

0.26 μg/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 1 8 0 7 0 9 96

16 0 8 0 7 0 9 96

17 0 8 0 7 0 9 96

18 0 8 0 7 0 9 96

19 0 8 0 7 1 8 92

20 0 8 0 7 0 8 92

21 0 8 0 7 0 8 92

22 0 8 0 7 0 8 92

23 0 8 0 7 1 7 88

24 0 8 0 7 0 7 88

25 0 8 0 7 0 7 88

26 0 8 0 7 0 7 88

27 0 8 0 7 0 7 88

28 0 8 0 7 0 7 88

29 0 8 0 7 0 7 88

30 0 8 0 7 1 6 84

31 0 8 2 5 0 6 76

32 0 8 0 5 0 6 76

33 0 8 0 5 0 6 76

34 0 8 0 5 0 6 76

35 0 8 0 5 0 6 76

36 1 7 1 4 0 6 68

37 0 7 0 4 0 6 68

38 0 7 0 4 0 6 68

39 0 7 0 4 0 6 68

40 0 7 0 4 1 5 64

41 0 7 0 4 0 5 64

42 0 7 0 4 0 5 64

43 0 7 0 4 0 5 64

44 0 7 0 4 0 5 64

45 0 7 0 4 1 4 60

46 0 7 0 4 0 4 60

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 371

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

0.26 μg/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 11 0 11 0 11 100

16 0 11 0 11 0 11 100

17 0 11 0 11 0 11 100

18 0 11 0 11 0 11 100

19 0 11 0 11 0 11 100

20 0 11 0 11 0 11 100

21 1 10 1 10 0 11 94

22 0 10 0 10 0 11 94

23 0 10 0 10 0 11 94

24 0 10 0 10 0 11 94

25 0 10 0 10 0 11 94

26 0 10 0 10 0 11 94

27 0 10 0 10 0 11 94

28 0 10 0 10 0 11 94

29 0 10 0 10 0 11 94

30 0 10 0 10 0 11 94

31 0 10 0 10 0 11 94

32 0 10 0 10 0 11 94

33 0 10 1 9 0 11 91

34 0 10 0 9 0 11 91

35 0 10 0 9 0 11 91

36 0 10 0 9 1 10 88

37 0 10 0 9 0 10 88

38 0 10 0 9 0 10 88

39 1 9 0 9 0 10 85

40 0 9 0 9 0 10 85

41 0 9 0 9 0 10 85

42 1 8 0 9 1 9 79

43 0 8 0 9 0 9 79

44 0 8 0 9 0 9 79

45 0 8 0 9 1 8 76

46 1 7 0 9 0 8 73

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 372

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

0.64 μg/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 11 0 9 1 8 97

16 0 11 0 9 0 8 97

17 0 11 1 8 0 8 93

18 0 11 0 8 0 8 93

19 1 10 0 8 0 8 90

20 0 10 0 8 0 8 90

21 0 10 0 8 0 8 90

22 0 10 0 8 0 8 90

23 0 10 0 8 0 8 90

24 0 10 0 8 1 7 86

25 0 10 0 8 1 6 83

26 0 10 1 7 1 5 76

27 0 10 0 7 0 5 76

28 0 10 0 7 0 5 76

29 0 10 1 6 0 5 72

30 0 10 1 5 0 5 69

31 0 10 0 5 0 5 69

32 0 10 1 4 0 5 66

33 0 10 0 4 0 5 66

34 2 8 0 4 0 5 59

35 0 8 0 4 0 5 59

36 1 7 0 4 0 5 55

37 0 7 0 4 0 5 55

38 0 7 0 4 1 4 52

39 0 7 0 4 0 4 52

40 1 6 0 4 0 4 48

41 0 6 0 4 0 4 48

42 0 6 0 4 0 4 48

43 1 5 0 4 0 4 45

44 0 5 1 3 0 4 41

45 0 5 0 3 0 4 41

46 0 5 0 3 1 3 38

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 373

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

0.64 μg/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 8 2 8 0 7 92

16 0 8 0 8 0 7 92

17 0 8 0 8 0 7 92

18 1 7 0 8 0 7 88

19 0 7 0 8 0 7 88

20 0 7 0 8 0 7 88

21 0 7 0 8 0 7 88

22 0 7 0 8 0 7 88

23 0 7 0 8 0 7 88

24 0 7 0 8 1 6 84

25 0 7 0 8 1 5 80

26 0 7 0 8 0 5 80

27 0 7 0 8 0 5 80

28 0 7 0 8 0 5 80

29 0 7 0 8 0 5 80

30 0 7 0 8 0 5 80

31 0 7 0 8 0 5 80

32 0 7 0 8 0 5 80

33 0 7 0 8 0 5 80

34 0 7 1 7 0 5 76

35 0 7 0 7 0 5 76

36 0 7 0 7 0 5 76

37 0 7 0 7 0 5 76

38 0 7 0 7 0 5 76

39 0 7 0 7 0 5 76

40 0 7 0 7 0 5 76

41 0 7 0 7 0 5 76

42 0 7 0 7 0 5 76

43 0 7 0 7 0 5 76

44 0 7 1 6 1 4 68

45 0 7 0 6 0 4 68

46 0 7 0 6 0 4 68

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 374

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

1.6 μg/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 7 0 9 0 9 100

16 0 7 0 9 1 8 96

17 0 7 0 9 1 7 92

18 0 7 0 9 0 7 92

19 0 7 0 9 0 7 92

20 0 7 0 9 0 7 92

21 0 7 0 9 0 7 92

22 0 7 0 9 0 7 92

23 0 7 0 9 0 7 92

24 0 7 0 9 0 7 92

25 0 7 1 8 0 7 88

26 0 7 0 8 0 7 88

27 0 7 0 8 0 7 88

28 0 7 0 8 1 6 84

29 0 7 1 7 0 6 80

30 0 7 0 7 1 5 76

31 0 7 0 7 0 5 76

32 0 7 1 6 0 5 72

33 1 6 0 6 0 5 68

34 0 6 0 6 0 5 68

35 0 6 1 5 0 5 64

36 0 6 0 5 0 5 64

37 0 6 0 5 0 5 64

38 0 6 0 5 1 4 60

39 1 5 0 5 0 4 56

40 0 5 0 5 0 4 56

41 0 5 0 5 0 4 56

42 0 5 0 5 0 4 56

43 1 4 0 5 0 4 52

44 0 4 0 5 1 3 48

45 0 4 0 5 0 3 48

46 0 4 0 5 0 3 48

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 375

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

1.6 μg/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 12 0 11 0 10 100

16 0 12 0 11 0 10 100

17 0 12 0 11 0 10 100

18 0 12 0 11 0 10 100

19 0 12 0 11 0 10 100

20 0 12 0 11 0 10 100

21 0 12 0 11 0 10 100

22 0 12 0 11 0 10 100

23 0 12 0 11 0 10 100

24 0 12 0 11 0 10 100

25 0 12 0 11 0 10 100

26 0 12 0 11 0 10 100

27 0 12 0 11 0 10 100

28 1 11 0 11 1 9 94

29 0 11 0 11 0 9 94

30 0 11 0 11 0 9 94

31 0 11 0 11 0 9 94

32 0 11 1 10 0 9 91

33 0 11 0 10 0 9 91

34 0 11 0 10 1 8 88

35 1 10 0 10 0 8 85

36 0 10 0 10 0 8 85

37 0 10 0 10 0 8 85

38 0 10 0 10 1 7 82

39 0 10 0 10 0 7 82

40 0 10 1 9 0 7 79

41 0 10 0 9 0 7 79

42 0 10 0 9 0 7 79

43 0 10 0 9 0 7 79

44 1 9 0 9 0 7 76

45 0 9 0 9 0 7 76

46 0 9 1 8 0 7 73

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 376

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

4.0 μg/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 1 9 0 10 0 8 96

16 0 9 0 10 1 7 93

17 0 9 0 10 0 7 93

18 0 9 0 10 0 7 93

19 0 9 0 10 0 7 93

20 0 9 0 10 0 7 93

21 0 9 0 10 0 7 93

22 0 9 1 9 0 7 89

23 0 9 0 9 0 7 89

24 0 9 0 9 0 7 89

25 0 9 0 9 0 7 89

26 0 9 0 9 0 7 89

27 0 9 1 8 0 7 86

28 0 9 0 8 0 7 86

29 0 9 0 8 0 7 86

30 0 9 0 8 0 7 86

31 1 8 1 7 0 7 79

32 0 8 1 6 0 7 75

33 0 8 0 6 0 7 75

34 0 8 0 6 0 7 75

35 0 8 1 5 0 7 71

36 0 8 0 5 0 7 71

37 0 8 0 5 1 6 68

38 1 7 0 5 0 6 64

39 2 5 0 5 0 6 57

40 0 5 0 5 2 4 50

41 1 4 1 4 0 4 43

42 0 4 0 4 0 4 43

43 1 3 0 4 0 4 39

44 1 2 0 4 0 4 36

45 0 2 0 4 0 4 36

46 0 2 1 3 0 4 32

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 377

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

4.0 μg/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 10 0 10 0 11 100

16 1 9 0 10 0 11 97

17 0 9 0 10 0 11 97

18 0 9 0 10 0 11 97

19 0 9 0 10 0 11 97

20 0 9 0 10 0 11 97

21 0 9 0 10 0 11 97

22 0 9 0 10 0 11 97

23 0 9 0 10 0 11 97

24 0 9 0 10 0 11 97

25 0 9 0 10 0 11 97

26 0 9 0 10 0 11 97

27 1 8 0 10 1 10 90

28 0 8 1 9 0 10 87

29 0 8 0 9 0 10 87

30 0 8 0 9 0 10 87

31 0 8 1 8 0 10 84

32 0 8 0 8 0 10 84

33 0 8 1 7 0 10 81

34 0 8 0 8 2 0 10 84

35 0 8 0 8 0 10 84

36 0 8 0 8 0 10 84

37 0 8 0 8 0 10 84

38 0 8 0 8 0 10 84

39 1 7 0 8 0 10 81

40 0 7 0 8 0 10 81

41 0 7 1 7 0 10 77

42 0 7 0 7 0 10 77

43 0 7 0 7 0 10 77

44 0 7 0 7 0 10 77

45 0 7 0 7 0 10 77

46 0 7 0 7 0 10 77 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study. 2 There was an error in the count of mysids on Day 33 that was discovered on Day 34.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 378

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

10 μg/L – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 8 1 8 0 9 96

16 1 7 0 8 0 9 92

17 0 7 0 8 0 9 92

18 0 7 0 8 1 8 88

19 0 7 0 8 0 8 88

20 0 7 0 8 0 8 88

21 0 7 0 8 0 8 88

22 0 7 0 8 0 8 88

23 1 6 0 8 0 8 85

24 0 6 0 8 0 8 85

25 0 6 0 8 0 8 85

26 0 6 0 8 1 7 81

27 0 6 1 7 1 6 73

28 0 6 0 7 0 6 73

29 0 6 0 7 0 6 73

30 1 5 1 6 0 6 65

31 0 5 0 6 0 6 65

32 0 5 0 6 0 6 65

33 0 5 1 5 1 5 58

34 0 5 0 5 0 5 58

35 0 5 0 5 0 5 58

36 0 5 3 2 0 5 46

37 0 5 0 2 0 5 46

38 0 5 0 2 0 5 46

39 0 5 0 2 0 5 46

40 1 4 0 2 0 5 42

41 0 4 0 2 0 5 42

42 0 4 0 2 0 5 42

43 0 4 0 2 0 5 42

44 1 3 0 2 1 4 35

45 0 3 0 2 0 4 35

46 0 3 0 2 0 4 35

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 379

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4-Tert-Octylphenol - F0 Generation

(Continued)

Day of

Test

10 μg/L – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 11 0 10 0 10 100

16 0 11 0 10 0 10 100

17 0 11 1 9 0 10 97

18 0 11 1 8 0 10 94

19 0 11 0 8 0 10 94

20 0 11 0 8 0 10 94

21 0 11 0 8 0 10 94

22 0 11 0 8 0 10 94

23 0 11 0 8 0 10 94

24 0 11 0 8 0 10 94

25 0 11 0 8 0 10 94

26 0 11 0 8 1 9 90

27 0 11 0 8 0 9 90

28 0 11 0 8 1 8 87

29 0 11 0 8 0 8 87

30 1 10 0 8 0 8 84

31 0 10 0 8 0 8 84

32 0 10 1 7 1 7 77

33 0 10 1 6 2 5 68

34 1 9 0 6 0 5 65

35 0 9 0 6 0 5 65

36 0 9 0 6 0 5 65

37 0 9 0 6 0 5 65

38 0 9 0 6 0 5 65

39 0 9 0 6 0 5 65

40 0 9 0 6 0 5 65

41 0 9 0 6 0 5 65

42 0 9 1 5 0 5 61

43 0 9 0 5 0 5 61

44 0 9 0 5 1 4 58

45 1 8 0 5 0 4 55

46 0 8 0 5 0 4 55 1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 380

Appendix 31 Replicate Mean Growth Measurements for the F0 Generation 

Day 0 Culture Mysids

Pan# Mysid#

Total Body 

Length (mm)

Dry Weight of 

Mysids Per Pan

(mg)

Avg. Dry 

Weight Per 

Mysid (mg) 

1

1-1 1.90

0.23

1-2 1.90

1-3 1.90 0.046

1-4 2.00

1-5 1.90

2

2-1 1.80

0.18

2-2 1.80

2-3 1.70 0.036

2-4 1.90

2-5 1.80

3

3-1 1.80

0.19

3-2 1.90

3-3 1.80 0.038

3-4 1.80

3-5 1.90

4

4-1 1.90

0.31

4-2 2.00

4-3 1.90 0.062

4-4 2.10

4-5 1.90

5

5-1 2.20

0.21

5-2 1.90

5-3 2.10 0.042

5-4 2.10

5-5 1.90
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 381

Appendix 31 Replicate Mean Growth Measurements for the F0 Generation Day 

0 Culture Mysids (Continued)

Pan# Mysid# Total Body 

Length (mm)

Dry Weight of 

Mysids Per Pan

(mg)

Avg. Dry 

Weight Per 

Mysid (mg) 

6

6-1 1.90

0.22

6-2 2.00

6-3 1.90 0.044

6-4 2.10

6-5 1.90

7

7-1 2.00

0.27

7-2 1.70

7-3 1.90 0.054

7-4 1.80

7-5 1.90

8

8-1 1.90

0.28

8-2 1.90

8-3 2.10 0.056

8-4 1.90

8-5 1.80

9

9-1 1.90

0.15

9-2 2.00

9-3 1.80 0.030

9-4 1.90

9-5 2.10

Average 1.92 0.23 0.045

N 45 9 --

S.D. 0.111 0.052 0.010

CV 5.78 22.8 22.8
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 382

Appendix 32 Replicate Mean Total Body Lengths for the F0 Generation Day 7 

Mysids

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Negative Control 5.80 5.40 5.90

5.80 5.80 5.00

5.70 5.70 6.10

5.60 5.30 5.20

6.00 5.80 4.90

5.60 5.40 4.90

Replicate Mean 1: 5.75 5.57 5.33

Number 6 6 6

Standard Dev.: 0.152 0.225 0.532

CV (%): 2.6 4.0 10.0

0.26 5.00 5.00 5.00

4.90 5.00 4.60

5.40 5.30 5.10

4.80 5.10 5.40

4.90 5.20 5.40

5.00 5.60 4.80

Replicate Mean 1: 5.00 5.20 5.05

Number 6 6 6

Standard Dev.: 0.210 0.228 0.321

CV (%): 4.2 4.4 6.4

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 383

Appendix 32 Replicate Mean Total Body Lengths for the F0 Generation Day 7 

Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

0.64 5.00 5.30 4.60

5.10 5.00 5.80

5.20 5.10 3.90

5.10 5.60 4.50

5.00 5.50 4.90

5.80 5.20

Replicate Mean 1: 5.08 5.38 4.82

Number 5 6 6

Standard Dev.: 0.084 0.306 0.649

CV (%): 1.6 5.7 13.5

1.6 5.00 5.00 5.70

5.00 5.20 4.80

5.30 5.60 5.10

5.50 5.40 4.80

5.40 5.50 5.20

5.50 5.00 5.20

Replicate Mean 1: 5.28 5.28 5.13

Number 6 6 6

Standard Dev.: 0.232 0.256 0.333

CV (%): 4.4 4.9 6.5

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 384

Appendix 32 Replicate Mean Total Body Lengths for the F0 Generation Day 7 

Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

4.0 4.90 4.90 4.90

5.00 5.00 5.00

5.20 5.10 3.30

4.50 5.60 5.20

5.30 5.20 4.60

4.70 5.10 4.90

Replicate Mean 1: 4.93 5.15 4.65

Number 6 6 6

Standard Dev.: 0.301 0.243 0.689

CV (%): 6.1 4.7 14.8

10 5.20 4.70 5.00

4.60 5.40 5.00

3.80 4.20 5.50

5.40 4.90 5.50

4.90 4.80 5.00

5.20 5.50 5.20

Replicate Mean 1: 4.85 4.92 5.20

Number 6 6 6

Standard Dev.: 0.586 0.479 0.245

CV (%): 12.1 9.7 4.7

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 385

Appendix 33 Replicate Mean Dry Weights for the F0 Generation Day 7 Mysids

Nominal Test 

Concentration

(μg/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

Negative Control

Pan 1 0.25 0.29 0.32

Pan 2 0.31 0.30 0.27

Pan 3 0.28 0.32 0.35

Replicate Mean 2: 0.28 0.30 0.31

Number 3 3 3

Standard Dev.: 0.030 0.015 0.043

CV (%): 10.7 5.0 13.8

0.26

Pan 1 0.24 0.26 0.26

Pan 2 0.26 0.30 0.26

Pan 3 0.22 0.25 0.25

Replicate Mean 2: 0.24 0.27 0.25

Number 3 3 3

Standard Dev.: 0.020 0.028 0.003

CV (%): 8.3 10.3 1.1

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two. 2 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 386

Appendix 33 Replicate Mean Dry Weights for the F0 Generation Day 7 Mysids

(Continued)

Nominal Test 

Concentration

(μg/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

0.64

Pan 1 0.28 0.24 0.26

Pan 2 0.24 0.31 0.17

Pan 3 0.34 3 0.34 0.20

Replicate Mean 2: 0.27 0.30 0.21

Number 3 3 3

Standard Dev.: 0.053 0.049 0.048

CV (%): 19.2 16.7 23.1

1.6

Pan 1 0.25 0.26 0.32

Pan 2 0.33 0.32 0.26

Pan 3 0.30 0.35 0.33

Replicate Mean 2: 0.29 0.31 0.30

Number 3 3 3

Standard Dev.: 0.040 0.043 0.041

CV (%): 13.7 14.1 13.5

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two. 2 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly. 3 Due to biologist oversight, only five mysids were measured in the 0.64 μg/L treatment group Replicate A. Pan 3 only 

contained one mysid, so this value was not divided by two.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 387

Appendix 33 Replicate Mean Dry Weights for the F0 Generation Day 7 Mysids 

(Continued)

Nominal Test 

Concentration

(μg/L)

Dry Weight (mg) 1

Replicate A Replicate B Replicate C

4.0

Pan 1 0.29 0.29 0.27

Pan 2 0.24 0.33 0.17

Pan 3 0.29 0.31 0.23

Replicate Mean 2: 0.27 0.31 0.22

Number 3 3 3

Standard Dev.: 0.029 0.020 0.050

CV (%): 10.6 6.6 22.6

10

Pan 1 0.30 0.21 0.24

Pan 2 0.20 0.24 0.29

Pan 3 0.32 0.29 0.26

Replicate Mean 2: 0.27 0.24 0.26

Number 3 3 3

Standard Dev.: 0.067 0.043 0.023

CV (%): 24.7 17.7 8.6

1 Dry weight measurements were performed on weigh pans containing two mysids. The values for individual mysids 

presented in this appendix reflect the dry weight of the two mysids measured divided by two. 2 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 388

Appendix 34 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 14 Mysids

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 6.3 6.6 5.9 6.0 5.6 6.0

6.4 5.7 5.7 6.2 5.7 5.8

5.9 5.8 5.5 5.6 5.8 6.0

5.6 5.8 5.7 5.8 5.8 5.9

5.7 5.7 5.7 5.5 5.5 6.0

5.8 5.9 5.7

6.0 5.8 6.1

5.9 5.6 5.8

5.7 5.8

Replicate Mean 1: 5.98 5.89 5.73 5.82 5.68 5.91

Number 5 9 9 5 5 8

Standard Dev.: 0.356 0.285 0.132 0.286 0.130 0.136

CV (%): 5.96 4.84 2.31 4.92 2.30 2.29

0.26 6.3 6.1 6.5 6.0 6.1 5.6

6.1 6.5 6.4 6.7 6.6 5.7

6.3 5.9 6.0 5.9 6.3 5.9

6.1 6.2 6.1 5.4 6.1 6.2

6.0 6.4 6.3 6.0

5.9 6.2 6.3 6.1

4.5 6.2

6.1 5.9

6.1 6.3

5.9 6.4

Replicate Mean 1: 6.20 5.92 6.24 6.00 6.28 5.92

Number 4 10 10 4 6 6

Standard Dev.: 0.116 0.531 0.196 0.535 0.185 0.232

CV (%): 1.86 8.97 3.13 8.92 2.92 3.92

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 389

Appendix 34 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 14 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

0.64 6.4 6.1 6.2 5.6 6.3 5.6

6.3 6.4 6.1 6.1 6.0 6.4

6.4 6.0 6.0 6.7 6.2 6.5

5.8 6.2 6.4 6.3 6.7 6.0

6.3 6.0 6.6 6.4 6.5 6.7

6.6 6.3 6.0 5.8

6.1 6.1

6.2

6.6

Replicate Mean 1: 6.27 6.17 6.24 6.22 6.25 6.24

Number 7 6 9 5 6 5

Standard Dev.: 0.256 0.163 0.235 0.409 0.327 0.439

CV (%): 4.09 2.65 3.77 6.57 5.23 7.04

1.6 6.0 6.1 6.4 6.7 6.5 5.9

6.2 5.6 6.5 6.1 6.0 6.2

6.4 6.2 6.5 4.8 6.3 6.1

5.8 6.6 6.2 6.5 6.6 5.6

6.1 6.8 6.2 6.7 6.5 6.4

6.0 6.4 6.2 6.1 6.0

6.5 6.7 6.2 5.8

6.4

Replicate Mean 1: 6.08 6.31 6.39 6.16 6.31 6.00

Number 6 7 8 5 7 7

Standard Dev.: 0.204 0.393 0.181 0.799 0.227 0.265

CV (%): 3.36 6.23 2.83 13.0 3.59 4.41

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 390

Appendix 34 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 14 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

4.0 6.4 6.9 6.1 6.3 6.4 6.5

6.2 6.6 6.0 6.1 6.4 5.8

6.2 5.7 5.1 6.2 6.3 6.2

6.7 6.0 6.5 6.1 6.6

6.5 5.8 5.8 6.7 6.2

6.1 5.9 6.7 6.1 6.4

6.3 6.7 6.6

6.3 5.8

6.2

5.0

6.5

Replicate Mean 1: 6.35 6.19 6.04 6.20 6.33 6.33

Number 6 8 11 3 6 7

Standard Dev.: 0.226 0.416 0.584 0.100 0.225 0.287

CV (%): 3.56 6.72 9.67 1.61 3.55 4.54

10 6.3 6.5 5.9 5.6 6.3 6.8

5.9 6.2 6.4 5.8 6.3 6.2

6.4 5.8 6.7 5.5 5.9 5.4

6.3 6.1 6.2 6.2 6.3 6.0

6.2 6.7 6.7 5.8 6.7 6.0

6.4 6.0 6.3 6.4 6.0 6.1

6.1 6.5 6.3

Replicate Mean 1: 6.23 6.22 6.37 5.88 6.29 6.11

Number 7 6 6 6 7 7

Standard Dev.: 0.180 0.331 0.308 0.349 0.273 0.418

CV (%): 2.89 5.33 4.83 5.93 4.35 6.84

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 391

Appendix 35 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 14 Mysids

Nominal Test 

Concentration

(μg/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 0.55 0.85 0.69 0.53 0.57 0.62

0.78 0.70 0.59 0.75 0.59 0.84

0.75 0.66 0.61 0.59 0.73 0.68

0.64 0.80 0.60 0.81 0.44 0.71

0.54 0.75 0.60 0.73 0.45 0.63

0.64 0.62 0.78

0.67 0.65 0.73

0.82 0.53 0.74

0.59 0.59

Replicate Mean 1: 0.652 0.720 0.609 0.682 0.556 0.716

Number 5 9 9 5 5 8

Standard Dev.: 0.111 0.089 0.044 0.117 0.119 0.074

CV (%): 17.0 12.4 7.23 17.2 21.3 10.3

0.26 0.54 0.65 0.56 0.72 0.65 0.54

0.59 0.69 0.65 0.67 0.58 0.55

0.50 0.48 0.63 0.68 0.49 0.62

0.58 0.73 0.52 0.49 0.59 0.78

0.69 0.68 0.52 0.57

0.72 0.57 0.50 0.61

0.35 0.55

0.60 0.61

0.60 0.68

0.52 0.67

Replicate Mean 1: 0.553 0.603 0.612 0.640 0.555 0.612

Number 4 10 10 4 6 6

Standard Dev.: 0.041 0.122 0.059 0.102 0.062 0.088

CV (%): 7.44 20.2 9.61 16.0 11.2 14.5

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 392

Appendix 35 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 14 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

0.64 0.61 0.69 0.66 0.59 0.53 0.47

0.67 0.81 0.65 0.72 0.43 0.83

0.62 0.65 0.50 0.76 0.62 0.82

0.59 0.73 0.63 0.55 0.66 0.56

0.68 0.65 0.73 0.68 0.66 0.72

0.82 0.80 0.62 0.43

0.45 0.66

0.54

0.64

Replicate Mean 1: 0.634 0.722 0.626 0.660 0.555 0.680

Number 7 6 9 5 6 5

Standard Dev.: 0.111 0.071 0.068 0.088 0.108 0.160

CV (%): 17.6 9.85 10.9 13.3 19.4 23.5

1.6 0.63 0.55 0.58 0.71 0.67 0.62

0.63 0.43 0.67 0.52 0.50 0.72

0.55 0.66 0.67 0.36 0.60 0.68

0.54 0.75 0.72 0.63 0.61 0.51

0.60 0.88 0.60 0.85 0.54 0.74

0.56 0.60 0.56 0.72 0.66

0.83 0.55 0.55 0.68

0.53

Replicate Mean 1: 0.585 0.671 0.610 0.614 0.599 0.659

Number 6 7 8 5 7 7

Standard Dev.: 0.040 0.160 0.068 0.186 0.077 0.076

CV (%): 6.90 23.8 11.2 30.3 12.8 11.6

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 393

Appendix 35 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 14 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

4.0 0.57 0.92 0.58 0.68 0.55 0.61

0.62 0.85 0.56 0.60 0.53 0.55

0.59 0.52 0.35 0.63 0.62 0.53

0.69 0.71 0.63 0.47 0.68

0.63 0.50 0.50 0.54 0.54

0.53 0.57 0.59 0.53 0.64

0.66 0.64 0.67

0.76 0.52

0.50

0.24

0.57

Replicate Mean 1: 0.605 0.686 0.516 0.637 0.540 0.603

Number 6 8 11 3 6 7

Standard Dev.: 0.055 0.153 0.121 0.040 0.048 0.063

CV (%): 9.10 22.3 23.5 6.35 8.92 10.5

10 0.41 0.60 0.50 0.41 0.48 0.68

0.55 0.56 0.66 0.55 0.54 0.45

0.53 0.56 0.66 0.44 0.35 0.41

0.52 0.64 0.51 0.69 0.45 0.70

0.51 0.65 0.56 0.53 0.63 0.65

0.60 0.46 0.60 0.72 0.56 0.59

0.49 0.68 0.75

Replicate Mean 1: 0.516 0.578 0.582 0.557 0.527 0.604

Number 7 6 6 6 7 7

Standard Dev.: 0.058 0.069 0.071 0.127 0.112 0.129

CV (%): 11.3 12.0 12.1 22.8 21.2 21.4

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 394

Appendix 36 Replicate Mean Body Lengths for Male and Female F0

Generation Day 46 Mysids

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 8.17 8.99 8.73 8.76 8.58 9.37

8.30 8.99 8.57 8.65 8.19 9.04

8.79 9.51 8.26 9.15 8.76 9.11

8.82 9.04 8.50 8.67 9.46 8.87

8.21 9.42 9.38 8.44

9.27 8.53 8.75

9.24

Replicate Mean 1: 8.46 9.20 8.52 8.86 8.77 9.10

Number 5 6 4 6 7 4

Standard Dev.: 0.320 0.229 0.195 0.333 0.443 0.208

CV (%): 3.79 2.49 2.29 3.76 5.05 2.28

0.26 8.68 9.26 8.19 9.36 8.92 8.85

8.49 8.79 8.21 8.81 9.27 8.55

8.59 9.39 8.79 9.47 8.15 9.18

8.96 8.92 8.67 9.39 8.42 8.83

8.72 9.65 9.29 9.11

9.11 8.94 8.49 9.50

8.67 8.68 8.84 9.71

9.10 8.67

9.06

Replicate Mean 1: 8.75 9.09 8.47 9.09 8.69 9.05

Number 7 7 4 9 4 8

Standard Dev.: 0.216 0.352 0.310 0.326 0.501 0.404

CV (%): 2.47 3.87 3.66 3.58 5.77 4.46

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 395

Appendix 36 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 46 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

0.64 8.88 9.21 7.98 8.68 8.79 9.19

8.66 8.91 8.19 8.99 9.04 8.93

8.41 8.44 8.25 8.99 8.70 9.86

8.74 9.01 8.80 9.63

8.44 8.89 9.21

8.42 8.98

9.01

Replicate Mean 1: 8.63 8.84 8.14 8.94 8.84 9.40

Number 5 7 3 6 3 4

Standard Dev.: 0.200 0.300 0.142 0.183 0.176 0.420

CV (%): 2.32 3.39 1.74 2.04 1.99 4.47

1.6 8.32 8.85 8.63 9.17 9.72 9.12

8.15 8.80 9.09 9.27 7.70 9.34

8.52 9.03 8.32 9.36 8.65 9.26

9.01 9.01 9.46 9.36 8.41

9.16 9.36 9.17 9.40

9.01 8.43 8.85

8.44 8.93 9.16

9.32 9.58

9.18

Replicate Mean 1: 8.50 8.98 8.97 9.16 8.69 9.08

Number 4 9 5 8 3 7

Standard Dev.: 0.372 0.258 0.486 0.349 1.011 0.345

CV (%): 4.38 2.88 5.42 3.81 11.6 3.80

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 396

Appendix 36 Replicate Mean Body Lengths for Male and Female F0 Generation 

Day 46 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Total Body Lengths (mm)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

4.0 9.62 8.60 9.10 8.66 9.00 8.32

8.45 8.43 9.13 9.11 8.81 9.40

8.80 8.52 9.27 8.94 9.17

8.95 9.27 8.90 9.31

8.86 9.36 9.16

8.26 9.30 9.24

8.36 9.21 9.06

8.50

8.96

9.24

Replicate Mean 1: 9.04 8.61 8.92 9.17 8.91 9.04

Number 2 7 3 7 4 10

Standard Dev.: 0.827 0.268 0.344 0.237 0.080 0.354

CV (%): 9.16 3.12 3.86 2.59 0.895 3.92

10 8.58 9.18 9.01 9.00 7.91 9.08

9.15 8.85 8.47 8.96 7.22 9.00

9.14 9.08 8.90 8.27 9.16

9.80 9.88 8.52 9.79

9.41 8.70

8.89

9.34

9.04

Replicate Mean 1: 8.96 9.20 8.74 9.09 7.98 9.26

Number 3 8 2 5 4 4

Standard Dev.: 0.326 0.312 0.382 0.458 0.565 0.361

CV (%): 3.64 3.39 4.37 5.03 7.08 3.90

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 397

Appendix 37 Replicate Mean Dry Weights for Male and Female F0

Generation Day 46 Mysids

Nominal Test 

Concentration

(μg/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

Negative Control 1.06 1.65 1.40 1.56 1.24 1.37

1.18 1.61 1.40 1.47 1.05 1.80

1.35 1.41 1.15 1.81 1.31 1.83

1.39 1.58 1.28 1.44 1.55 1.91

1.14 1.32 1.56 1.10

1.45 1.01 1.12

1.53

Replicate Mean 1: 1.22 1.50 1.31 1.48 1.27 1.73

Number 5 6 4 6 7 4

Standard Dev.: 0.141 0.130 0.119 0.262 0.203 0.243

CV (%): 11.5 8.62 9.12 17.8 16.0 14.1

0.26 1.38 1.86 1.00 1.52 1.51 1.51

1.34 1.55 1.08 1.27 1.25 1.45

1.44 1.63 1.55 1.52 0.94 1.95

1.43 1.68 1.12 1.75 1.15 1.39

1.33 1.73 1.50 1.90

1.52 1.89 1.53 1.57

1.32 1.31 1.49 1.68

1.28 1.26

1.42

Replicate Mean 1: 1.39 1.66 1.19 1.48 1.21 1.59

Number 7 7 4 9 4 8

Standard Dev.: 0.073 0.197 0.247 0.144 0.237 0.242

CV (%): 5.24 11.9 20.8 9.78 19.5 15.2

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 398

Appendix 37 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 46 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

0.64 1.26 1.77 0.81 1.35 1.40 1.40

1.16 1.35 1.16 1.78 1.42 1.39

1.29 1.07 1.15 1.69 1.36 1.97

1.38 1.54 1.37 1.68

1.34 1.31 1.45

1.25 1.60

1.51

Replicate Mean 1: 1.29 1.40 1.04 1.54 1.39 1.61

Number 5 7 3 6 3 4

Standard Dev.: 0.084 0.228 0.199 0.177 0.031 0.275

CV (%): 6.54 16.3 19.2 11.5 2.19 17.1

1.6 1.07 1.81 1.00 1.62 1.48 1.76

0.87 1.62 1.33 1.92 0.96 1.68

1.30 1.50 1.08 1.46 1.19 1.39

1.32 1.84 1.38 1.47 1.19

1.76 1.56 1.59 1.72

1.33 1.19 1.42

1.31 1.14 1.51

1.31 1.74

1.56

Replicate Mean 1: 1.14 1.56 1.27 1.52 1.21 1.52

Number 4 9 5 8 3 7

Standard Dev.: 0.213 0.214 0.228 0.263 0.261 0.208

CV (%): 18.7 13.7 18.0 17.3 21.5 13.6

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 399

Appendix 37 Replicate Mean Dry Weights for Male and Female F0 Generation 

Day 46 Mysids (Continued)

Nominal Test 

Concentration

(μg/L)

Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Females

4.0 1.37 1.24 1.54 1.12 1.14 1.21

1.42 1.57 1.29 1.24 1.32 1.46

1.37 1.17 1.66 0.99 1.44

1.86 1.76 1.16 1.60

1.25 1.69 1.42

1.23 1.19 1.85

1.21 1.64 1.65

1.01

1.35

1.57

Replicate Mean 1: 1.39 1.39 1.33 1.47 1.15 1.46

Number 2 7 3 7 4 10

Standard Dev.: 0.035 0.243 0.189 0.274 0.135 0.235

CV (%): 2.53 17.5 14.2 18.6 11.7 16.2

10 1.25 1.41 1.41 1.39 1.11 1.25

1.25 1.61 1.23 1.52 0.79 1.43

1.31 1.51 1.36 1.10 1.52

1.55 1.64 1.26 1.72

1.82 1.42

1.25

1.29

 --2

Replicate Mean 1: 1.27 1.49 1.32 1.47 1.07 1.48

Number 3 7 2 5 4 4

Standard Dev.: 0.035 0.196 0.127 0.114 0.197 0.195

CV (%): 2.73 13.2 9.64 7.80 18.5 13.2

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly. 2 This measurement was suspect, and was therefore excluded from statistical analysis.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 400

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

(Days)

Replicate

Mean

SD

CV(%)2

Negative A 1 14 14.0 -- 28.4 -- 38.8 -- 11.8

Control 2 14 0.00 32 4.34 44 5.56 12 5.25

3 14 0.00 34 15.27 -- 14.35 -- 44.70

4 14 24 43 19

5 14 26 33 7

6 14 26 35 9

7 14 -- -- --

8 -

3 -- -- --

9 14 -- -- --

B 1 14 14.0 -- 26.4 -- 34.0 -- 10.0

2 14 0.00 -- 7.20 -- 3.00 -- 4.58

3 14 0.00 35 27.26 -- 8.82 -- 45.83

4 14 23 34 11

5 14 17 31 14

6 14 32 37 5

7 14 25 -- --

8 14 -- -- --

9 14 -- -- --

C 1 14 14.0 23 24.3 32 31.3 9 7.0

2 14 0.00 -- 6.15 -- 6.83 -- 1.67

3 14 0.00 28 25.29 33 21.80 5 23.90

4 14 18 24 6

5 14 25 31 6

6 14 18 25 7

7 14 34 43 9

8 14 -- -- --

9 -

3 -- -- -- 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids.

2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 401

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

(Days)

Replicate

Mean

SD

CV(%)2

0.26 A 1 14 14.0 32 26.5 37 32.8 5 9.0

2 14 0.00 16 6.72 32 2.99 16 5.23

3 14 0.00 22 25.34 32 9.12 10 58.09

4 14 33 -- --

5 14 25 30 5

6 14 31 -- --

7 14 -- -- --

8 14 -- -- --

9 14 -- -- --

B 1 14 14.0 -- 26.8 -- 45.0 -- 10.0

2 14 0.00 27 8.26 -- -

4 -- -4

3 14 0.00 15 30.81 -- -

4 -- -4

4 14 23 -- --

5 14 35 45 10

6 14 -- -- --

7 14 34 -- --

8 -

3 -- -- --

9 14 -- -- --

C 1 14 14.0 33 27.0 -- 31.3 -- 9.3

2 14 0.00 25 6.51 34 3.78 9 3.10

3 14 0.00 34 24.10 -- 12.08 -- 33.47

4 14 19 31 12

5 14 21 26 5

6 14 23 34 11

7 14 34 -- --

8 14 -- -- --

9 14 -- -- -- 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids.

2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed. 4 There was only one value and so neither standard deviation nor CV could be calculated.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 402

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

(Days)

Replicate

Mean

SD

CV(%)2

0.64 A 1 14 14.0 34 31.8 39 39.3 5 10.5

2 14 0.00 23 7.81 40 0.96 17 6.40

3 14 0.00 -- 24.53 -- 2.44 -- 60.98

4 14 45 -- --

5 14 25 40 15

6 14 33 38 5

7 14 31 -- --

8 14 -- -- --

9 14 -- -- --

B 1 14 14.0 33 28.2 38 34.4 5 6.2

2 14 0.00 22 6.98 27 6.58 5 3.27

3 14 0.00 38 24.75 42 19.13 4 52.76

4 14 25 37 12

5 14 -- -- --

6 14 23 28 5

7 14 -- -- --

8 14 -- -- --

9 14 -- -- --

C 1 14 14.0 16 26.2 21 21.0 5 5.0

2 14 0.00 -- 7.46 -- -

4 -- -4

3 14 0.00 33 28.49 -- -

4 -- -4

4 14 23 -- --

5 14 -- -- --

6 14 25 -- --

7 14 34 -- --

8 14 -- -- --

9 -

3 -- -- -- 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids.

2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed. 4 There was only one value and so neither standard deviation nor CV could be calculated.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 403

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

(Days)

Replicate

Mean

SD

CV(%)2

1.6 A 1 14 14.0 23 24.7 -- 34.0 -- 12.0

2 14 0.00 18 8.26 -- 7.21 -- 6.08

3 14 0.00 24 33.50 40 21.21 16 50.69

4 14 -- -- --

5 14 21 36 15

6 14 41 -- --

7 14 21 26 5

8 -

3 -- -- --

9 14 -- -- --

B 1 14 14.0 -- 34.5 -- 41.0 -- 10.0

2 14 0.00 -- 4.65 -- 2.83 -- 5.66

3 14 0.00 -- 13.49 -- 6.90 -- 56.57

4 14 33 39 6

5 14 36 -- --

6 14 40 -- --

7 14 29 43 14

8 14 -- -- --

9 14 -- -- --

C 1 14 14.0 33 32.0 -- 45.0 -- 4.0

2 14 0.00 -- 6.71 -- -

4 -- -4

3 14 0.00 -- 20.96 -- -

4 -- -4

4 14 35 -- --

5 14 24 -- --

6 14 41 45 4

7 14 27 -- --

8 14 -- -- --

9 14 -- -- -- 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids.

2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed. 4 There was only one value and so neither standard deviation nor CV could be calculated.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 404

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

(Days)

Replicate

Mean

SD

CV(%)2

4.0 A 1 14 14.0 39 36.8 -- 38.0 -- 10.0

2 14 0.00 -- 5.91 -- -

4 -- -4

3 14 0.00 -- 16.08 -- -

4 -- -4

4 14 28 38 10

5 14 -- -- --

6 14 41 -- --

7 14 39 -- --

8 14 -- -- --

9 14 -- -- --

B 1 14 14.0 21 24.8 -- 26.0 -- 5.0

2 14 0.00 21 6.94 26 -

4 5 -4

3 14 0.00 24 27.99 -- -

4 -- -4

4 14 -- -- --

5 14 -- -- --

6 14 37 -- --

7 14 21 -- --

8 14 -- -- --

9 14 -- -- --

C 1 14 14.0 35 30.8 -- 41.0 -- 5.0

2 14 0.00 21 6.91 -- -

4 -- -4

3 14 0.00 36 22.42 -- -

4 -- -4

4 14 36 41 5

5 14 -- -- --

6 14 -- -- --

7 14 26 -- --

8 14 -- -- --

9 14 -- -- -- 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be used 

to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids.

2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed. 4 There was only one value and so neither standard deviation nor CV could be calculated.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 405

Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Time to 

Maturation

(Day)

Replicate

Mean

SD

CV(%)2

Time to 

1st Brood

(Day)

Replicate

Mean

SD

CV(%)2

Time to 2nd

Brood

(Day)

Replicate

Mean

SD

CV(%)2

Interbrood 

Duration

(Days)

Replicate

Mean

SD

CV(%)2

10 A 1 14 14.0 34 26.2 -- 33.5 -- 6.0

2 14 0.00 30 5.04 35 2.12 5 1.41

3 14 0.00 23 19.25 -- 6.33 -- 23.57

4 14 25 -- --

5 14 25 32 7

6 14 20 -- --

7 14 -- -- --

8 14 -- -- --

9 14 -- -- --

B 1 14 14.0 21 24.2 -- 38.0 -- 13.5

2 14 0.00 -- 6.46 -- 8.49 -- 2.12

3 14 0.00 17 26.68 32 22.33 15 15.71

4 14 30 -- --

5 14 21 -- --

6 14 32 44 12

7 14 -- -- --

8 14 -- -- --

9 14 -- -- --

C 1 14 14.0 -- 27.3 -- 34.0 -- 5.0

2 14 0.00 -- 4.72 -- 7.07 -- 0.00

3 14 0.00 -- 17.31 -- 20.80 -- 0.00

4 14 27 -- --

5 14 34 39 5

6 14 24 29 5

7 14 24 -- --

8 14 -- -- --

9 14 -- -- -- 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). Brood chamber 8 contained any extra male mysids to be 

used to replace male mortality in the paired chambers. Brood chamber 9 contained extra females and/or immature mysids.

2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 There were no extra organisms; therefore, the extra brood chamber was not needed. 4 There was only one value and so neither standard deviation nor CV could be calculated.

-- = The female in the compartment did not release a first and/or second brood. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 406

Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total 

Number of 

Offspring 

Produced3

Replicate Total Produced

Per Number of Females 

at Pairing Mean

SD

CV%2

Negative A 1 -

4 3.8 -4 4.2 0 44

Control 2 5 2.17 3 3.03 8

3 6 57.05 0 72.22 6 6.3

4 1 8 9 4.79

5 5 4 13 76.14

6 2 6 8

7 -

4 -4 0

B 1 -

4 5.0 -4 5.6 0 53

2 -

4 3.03 -4 6.23 0

3 4 60.66 0 111.23 4 7.6

4 3 14 17 7.39

5 2 4 6 97.61

6 8 10 18

7 8 0 8

C 1 3 3.5 7 5.3 39 142

2 -

4 2.59 -4 2.50 0

3 2 73.96 6 46.94 12 20.3

4 1 1 45 16.94

5 5 6 23 83.50

6 2 4 6

7 8 8 16 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second 

brood. This total was then based on the total number of females present at pairing in the study (7 for each replicate) 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and 

second brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 407

Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total 

Number of 

Offspring 

Produced3

Replicate Total Produced

Per Number of Females 

at Pairing Mean

SD

CV%2

0.26 A 1 5 3.3 8 3.2 13 56

2 1 2.42 7 3.54 8

3 2 72.66 1 111.95 7 8.0

4 4 0 4 7.96

5 7 3 23 99.48

6 1 0 1

7 -

4 -4 0

B 1 -

4 2.4 -4 0.6 0 15

2 3 1.34 0 1.34 3

3 1 55.90 0 223.61 1 2.1

4 1 0 1 2.27

5 3 3 6 105.83

6 -

4 -4 0

7 4 0 4

C 1 6 4.6 0 2.3 6 83

2 8 2.82 6 2.43 32

3 4 61.69 0 106.31 4 11.9

4 1 4 15 9.51

5 3 4 9 80.22

6 2 2 9

7 8 0 8 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second 

brood. This total was then based on the total number of females present at pairing in the study (7 for each replicate) 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and 

second brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 408

Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total 

Number of 

Offspring 

Produced3

Replicate Total Produced

Per Number of Females at 

Pairing Mean

SD

CV%2

0.64 A 1 8 4.7 2 2.7 23 59

2 1 2.80 9 3.44 10

3 -

4 60.10 -4 129.18 0 8.4

4 4 0 4 7.63

5 8 4 12 90.58

6 4 1 7

7 3 0 3

B 1 5 3.8 4 2.6 13 50

2 2 1.64 1 1.34 3

3 6 43.24 2 51.60 8 7.1

4 3 4 7 7.01

5 -

4 -4 0 98.14

6 3 2 19

7 -

4 -4 0

C 1 3 2.8 2 0.4 13 24

2 -

4 2.05 -4 0.89 0

3 1 73.19 0 223.61 1 3.4

4 1 0 1 4.72

5 -

4 -4 0 137.69

6 3 0 3

7 6 0 6 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. This total 

was then based on the total number of females present at pairing in the study (7 for each replicate) 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second brood 

was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 409

Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total 

Number of 

Offspring 

Produced3

Replicate Total Produced

Per Number of Females 

at Pairing Mean

SD

CV%2

1.6 A 1 1 2.5 0 1.8 1 26

2 1 2.07 0 2.23 1

3 6 82.95 2 121.56 8 3.7

4 -

4 -4 0 3.82

5 2 4 6 102.77

6 1 0 1

7 4 5 9

B 1 -

4 2.5 -4 1.8 0 17

2 -

4 1.29 -4 2.87 0

3 -

4 51.64 -4 164.13 0 2.4

4 2 6 8 3.05

5 4 0 4 125.47

6 1 0 1

7 3 1 4

C 1 9 4.2 0 0.8 9 25

2 -

4 3.27 -4 1.79 0

3 -

4 77.88 -4 223.61 0 3.6

4 6 0 6 3.69

5 1 0 1 103.33

6 3 4 7

7 2 0 2 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. This total was 

then based on the total number of females present at pairing in the study (7 for each replicate) 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second brood was 

based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 410

Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total 

Number of 

Offspring 

Produced3

Replicate Total Produced

Per Number of Females at 

Pairing Mean

SD

CV%2

4.0 A 1 1 1.5 0 1.5 1 16

2 -

4 0.58 -4 3.00 0

3 -

4 38.49 -4 200.00 0 2.3

4 2 6 12 4.35

5 -

4 -4 0 190.22

6 2 0 2

7 1 0 1

B 1 3 2.2 0 0.4 3 13

2 2 0.45 2 0.89 4

3 2 20.33 0 223.61 2 1.9

4 -

4 -4 0 1.46

5 -

4 -4 0 78.82

6 2 0 2

7 2 0 2

C 1 11 4.0 0 0.2 11 21

2 1 4.12 0 0.45 1

3 3 103.08 0 223.61 3 3.0

4 4 1 5 3.96

5 -

4 -4 0 131.94

6 -

4 -4 0

7 1 0 1 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. This total was 

then based on the total number of females present at pairing in the study (7 for each replicate) 4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second brood was 

based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 411

Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F0

Generation (Continued)

Nominal Test 

Concentration

(μg/L) Replicate

Brood

Chamber1

Number of 

Offspring in 1st

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Number of 

Offspring in 2nd

Brood per 

Reproducing 

Female

Replicate

Mean

SD

CV%2

Total 

Number of 

Offspring 

Produced3

Replicate Total Produced

Per Number of Females at 

Pairing Mean

SD

CV%2

10 A 1 3 3.7 0 1.7 3 39

2 5 1.97 8 3.20 20

3 2 53.63 0 192.25 2 5.6

4 5 0 5 6.90

5 6 2 8 123.86

6 1 0 1

7 -

4 -4 0

B 1 5 2.4 0 2.4 5 27

2 -

4 1.67 -4 4.34 0

3 2 69.72 2 180.66 7 3.9

4 3 0 3 4.10

5 1 0 1 106.29

6 1 10 11

7 -

4 -4 0

C 1 -

4 6.0 -4 2.3 0 42

2 -

4 6.63 -4 2.63 0

3 -

4 110.55 -4 116.89 0 6.0

4 1 0 1 8.00

5 15 4 19 133.33

6 1 5 15

7 7 0 7 1 Each compartment contained 1 male/female pair, with a maximum of seven pairs per replicate test chamber (1-7). 2 Replicate means, standard deviation and CV were calculated using Excel 2010. Manual calculations may differ slightly. 3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods following the release of the second brood. This total was

then based on the total number of females present at pairing in the study (7 for each replicate) 

4 Represents a female that never produced a brood throughout the F0 generation reproductive phase of the study. The number of offspring produced in the first and second brood was 

based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 412

Appendix 40 Replicate Mean Percent of Males Sex Ratio and Percent of 

Females Producing Offspring F0 Generation

Nominal

Concentration

(μg/L) Replicate

Percent of 

Males

Sex Ratio

Percent of 

Females 

Producing 

Offspring

Negative Control A 38.7 71.4

B 58.8 71.4

C 53.1 85.7

0.26 A 38.2 85.7

B 53.1 71.4

C 46.9 100.0

0.64 A 56.3 85.7

B 54.5 71.4

C 55.6 71.4

1.6 A 40.6 85.7

B 51.5 57.1

C 48.5 71.4

4.0 A 47.1 57.1

B 61.8 71.4

C 43.8 71.4

10 A 46.9 85.7

B 48.4 71.4

C 48.5 57.1
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 413

Appendix 41 F1 Summary Tables
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 414

Appendix 41.1 Summary of F1 Generation Survival of Saltwater 

Mysids Exposed to 4-tert-octylphenol

Nominal

Concentration

(μg/L)

Mean Percent Juvenile

Survival to

Lengths

On Day 7 ± SD

(CV%)1

Mean Percent Juvenile

Survival to

Pairing

On Day 14 ± SD

(CV%)1

Mean Percent Adult

Survival Day 15 to

Test Termination

On Day 35 ± SD

(CV%)1

Negative Control 83.5 ± 18.4

(22.0)

n = 65

98.8 ± 2.1

(2.1)

n = 52

75.2 ± 10.92

(14.52)

n = 20

1 For survival, n = number of surviving organisms.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 415

Appendix 41.2 Summary of Day 0 and 7 F1 Generation Negative 

Control Growth Measurements of Culture 

Saltwater Mysids

Mean Total Body Length (mm) ± 

SD

(CV%) 1

Mean Dry Weight (mg) ± SD

(CV%) 1

Day 0 1.89 ± 0.13

(6.81)

n = 19

0.044 ± 0.011

(24.72)

n = 4

Day 7 5.4 ± 0.49

(9.17)

n = 3

0.33 ± 0.05

(13.7)

n = 3

1 Mean, standard deviation, and coefficient of variation was calculated using Excel 2010. Manual calculations may differ 

slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 416

Appendix 41.3 Summary of F1 Generation Day 14 and 35 Growth 

Measurements in the Negative Control of Saltwater 

Mysids Exposed to 4-tert-octylphenol

Growth Parameters

Mean Total Length (mm) ± SD

(CV%) 1

Mean Dry Weight (mg) ± SD

(CV%) 1

Males Females Males Females

Day 14

7.89 ± 0.70

(8.83)

n = 2

7.85 ± 0.024

(0.30)

n = 2

0.79 ± 0.12

(15.5)

n = 2

0.86 ± 0.054

(6.32)

n = 2

Day 35

8.10 ± 0.98

(12.13)

n = 3

8.21 ± 1.14

(13.86)

n = 3

1.35 ± 0.22

(16.59)

n = 3

1.58 ± 0.29

(18.65)

n = 3

1 Mean, standard deviation, and coefficient of variation was calculated using Excel 2010. Manual calculations may differ 

slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 417

Appendix 41.4 Summary of F1 Generation Reproduction of Saltwater Mysids Exposed to 4-tertoctylphenol

Nominal

Conc.

(μg/L)

Mean Time 

to 

Maturation

Days ± SD

(CV%)

Mean 

Time to 1st

Brood

Days ± SD

(CV%)

Mean 

Time to 2nd

Brood

Days ± SD

(CV%)

Mean

Interbrood 

Duration

Days ± SD

(CV%)

Mean Number 

of Offspring in 

1st Brood Per 

Female ± SD

(CV%)

Mean Number 

of Offspring in 

2nd Brood Per 

Female ± SD

(CV%)

Mean

Total 

Number of

Offspring ± 

SD

(CV%)

Mean

Percent of 

Males Sex

Ratio ± SD

(CV%) 1

Mean

Percent of

Females

Producing 

Offspring ± SD

(CV%) 1

Negative 

Control

14.0 ± 0.000

(0.00)

n = 3

20.1 ± 2.83

(14.10)

n = 3

24.8 ± 4.25

(17.12)

n = 3

6.0 ± 1.73

(28.87)

n = 3

2.6 ± 1.3

(49.4)

n = 3

2.9 ± 0.20

(6.66)

n = 3

16.0 ± 3.61

(22.53)

n = 3

60.6 ± 18.75

(30.92)

n = 3

61.7 ± 12.58

(20.4)

n = 3 1 Mean, standard deviation, and coefficient of variation was calculated using Excel 2010. Manual calculations may differ slightly.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 418

Appendix 41.5 Daily Percent Survival of Saltwater Mysids Exposed to 4-tert-octylphenol – F1

Generation

Day of

Test

Negative Control – Males

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. A (%) live No. Dead No. Alive No. Dead No. Alive

 151 0 9 0 5 0 10 100

16 0 9 1 4 0 10 96

17 0 9 0 4 1 9 92

18 0 9 0 4 0 9 92

19 0 9 0 4 0 9 92

20 0 9 0 4 0 9 92

21 0 9 0 4 0 9 92

22 0 9 0 4 0 9 92

23 0 9 0 4 0 9 92

24 0 9 0 4 0 9 92

25 0 9 0 4 0 9 92

26 0 9 0 4 0 9 92

27 0 9 0 4 1 8 88

28 0 9 0 4 0 8 88

29 1 8 0 4 0 8 83

30 1 7 0 4 1 7 75

31 0 7 0 4 2 5 67

32 0 7 1 3 0 5 63

33 0 7 0 3 2 3 54

34 0 7 0 3 0 3 54

35 0 7 0 3 0 3 54

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026

Wildlife International, Ltd. Study No. 185A-102 Page 419

Appendix 41.5 Daily Percent Survival of Saltwater Mysids Exposed to 4-tert-octylphenol – F1

Generation (Continued)

Day of

Test

Negative Control – Females

Replicate A Replicate B Replicate C Percent Survival

No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive (%)

 151 0 4 0 7 0 2 100

16 0 4 1 6 0 2 92

17 0 4 0 6 0 2 92

18 0 4 0 6 0 2 92

19 0 4 0 6 0 2 92

20 2 2 0 6 0 2 77

21 0 2 0 6 0 2 77

22 0 2 0 6 0 2 77

23 0 2 0 6 0 2 77

24 0 2 0 6 0 2 77

25 0 2 1 5 0 2 69

26 0 2 0 5 0 2 69

27 1 1 0 5 0 2 62

28 0 1 0 5 0 2 62

29 0 1 0 5 0 2 62

30 0 1 0 5 0 2 62

31 0 1 1 4 0 2 54

32 0 1 0 4 0 2 54

33 0 1 0 4 0 2 54

34 0 1 0 4 0 2 54

35 0 1 0 4 0 2 54

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.