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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

Decem ber 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 Internation al, Ltd. Project No. 185A

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RTI Project No. 0210114.030

EPA Contract No. EP

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Page 1 of 419

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## GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR: U.S. Environmental Protection Agency

TITL E: 

### Two

- Generation Life

- Cycle Toxicity Test with Mysids ( Americamysis bahia )

Inter

- Laboratory Comparison

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 185A

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RTI PROJECT NUMBER: 0210114.030

EPA CONTRACT NUMBER: EP

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STUDY COMPLETION: Decembe r 2 1 , 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 M AFF (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 perfo rmed 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 determin ed 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

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## QUALITY ASSURANCE STATEMENT

The study con duct, 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 Assuran ce

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 Repo rted to

Management

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

Trial 1 (185A

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- 1)

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

May 16, 2011 Assignment of 

### F 0 

### Pairs to May 16, 2011 May 25, 2011

Brood Chambers

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

F 1 

### Growth Chambers

August 25

- 31 and Analytical Data and September 9, 2011 September 2 8 , 2011

September 1, 2011 Draft Report

April 2 to 19, 2012 Biological Data and April 19, 2012 May 21, 201 2

Draft Report

Trial 2 (185A

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

Trial 2 (185A

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

September 21, 2012 F 1 

### 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 Decemb er 20, 2012 December 2 1 , 2012



### WILDLIFE INTERNATIONAL, LTD

________________________________________________ ________________________ Linda R. Mitchell Date

Director of Regulatory and Ecotox Operations

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## KEY STUDY PERSONNEL

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

and r eporting 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

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## TABLE OF CONTENTS

Page

GOOD LABORATORY PRAC TICE COMPLIANCE STAT EMENT

................................ ... 2

QUALITY ASSURANCE ST ATEMENT

................................ ................................ ............... 3

KEY STUDY PERSONNEL

................................ ................................ ................................ .... 4

TABLE OF CONTENTS

................................ ................................ ................................ .......... 5

PROTOCOL

................................ ................................ ................................ ........................... 15

AMENDMENTS AND DEVIA TIONS

................................ ................................ ................. 60

QAPP

................................ ................................ ................................ ................................ ...... 85

STUDY TRIAL 1

– 185A

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- 1

................................ ................................ .......................... 121

SUMMARY

................................ ................................ ................................ .......................... 122

1.0 INTRODUCTION

................................ ................................ ................................ ....... 125

2.0 DESCRIPTION OF THE M ETHOD

................................ ................................ .......... 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)

.................. 1 32

2.7.3 Brood Chambers

................................ ................................ ............... 132

2.8 Replication and Number of Test Organisms

................................ ................. 132

2.8.1 Exposed First Generation Mysids (F 0 

### )

................................ .............. 133

2.8.2 Exposed Second Generation Mysids (F 1 

### )

................................ ......... 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 (F 0 

### )

................................ .......................... 139

3.1.1 F 0 

### Exposure Initiation

................................ ................................ ........ 139

3.1.2 F 0 

### Growth Chambers

................................ ................................ ......... 140

3.1.3 F 0 

### Reproductive Chambers

................................ ................................ 140

3.1.4 Feeding

................................ ................................ .............................. 141

3.2 Exposed Second Generation Mysids (F 1 

### )

................................ ..................... 141

3.2.1 Initiation of F 1 

### Exposure

................................ ................................ ... 141

3.2.2 F 1 

### Growth Chambers

................................ ................................ ......... 142

3.2 .3 F 1 

### Reproductive Chambers

................................ ................................ 142

3.2.4 Feeding

................................ ................................ .............................. 143

3.3 Chamber Cleaning

................................ ................................ ........................ 143

3.4 Conditions of Exposure

................................ ................................ ................. 144

4.0 OBSERVATION AND MEAS UREMENTS

................................ ............................. 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 F 0 

### Generation

................................ ................................ .................... 146

6.1.2 F 1 

### Generation

................................ ................................ .................... 147

6.2 Biological Results

................................ ................................ ......................... 148

6.3 Survi val

................................ ................................ ................................ ......... 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

F 0 

### and F 1 

### Generations

................................ ................................ ....... 150

6.4.2 Day 7

- Dry Weight and Total Length from F 0 

### and F 1 

### Generations

. 150

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6.4.3 Day 14

– Male and Female Dry Weight and Total Length from F 0



### and F 1 

### Generations

................................ ................................ ............ 151

6.4.3.1 Day 14

– Male Dry Weight and Total Length from F 0 

### and

F 1 

### Generations

................................ ................................ .. 152

6.4.3.2 Day 14

– Female Dry Weight and Total Length from F 0



### and F 1 

### Generations

................................ ........................... 152

6.4.4 Test Termination

– Dry Weight and Total Length from F 0 

### (Day 44)

and F 1 

### (Day 35) Generations

................................ ............................. 153

6.4.4.1 Males at Test Termination from F 0 

### (Day 44) and F 1 

### (Day

35) Generations

................................ ................................ 153

6.4 .4.2 Females at Test Termination from F 0 

### (Day 44) and F 1 

### (Day 35) Generations

................................ ....................... 154

6.5 Reproduction

................................ ................................ ................................ . 154

6.5.1 Time to Maturation from F 0 

### and F 1 

### Generations

.............................. 155

6.5.2 Number of Offspring in the First Brood from F 0 

### and F 1 

### Generations 155

6.5.3 Number of Offsp ring in the Second Brood from F 0 

### and F 1



### Generations

................................ ................................ ....................... 156

6.5.4 Total Number of Offspring from F 0 

### and F 1 

### Generations

.................. 156

6.5.5 Sex Ratio (Male/Female) from F 0 

### and F 1 

### Generations

..................... 157

6.5.6 Percent of Reproducing Females from F 0 

### and F 1 

### Generations

......... 158

7.0 PERF ORMANCE 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 a nd 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 LITER ATURE

................................ ................................ .................. 161

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

F 0 

### Generation Exposure to Lindane

................................ .............................. 164

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

F 1 

### Generation Exposure to Lindane

................................ .............................. 165

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

the F 0 

### Generation

................................ ................................ .......................... 166

Table 4. Measured Concentrations of Lindane for the F 0 

### Gener ation in Stock

Solution Samples

................................ ................................ .......................... 167

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Table 5. Measured Concentrations of Lindane in Saltwater Samples for the F 0



### Generation

................................ ................................ ................................ ..... 168

T able 6. Measured Concentrations of Lindane in Pretest Diluter Verification

Samples for the F 1 

### Generation

................................ ................................ ...... 170

Table 7. Measured Concentration of Lindane in F 1 

### Generation Stock Solution

Samples

................................ ................................ ................................ ......... 171

Table 8. Measured Concentrations of Lindane Saltwater Samples for the F 1



### Generation

................................ ................................ ................................ ..... 172

Table 9. Summary of F 0 

### Generation Su rvival of Saltwater Mysids Exposed to

Lindane

................................ ................................ ................................ ......... 174

Table 10. Summary of F 1 

### 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 F 0 

### Generation Day 7 Growth Measurements of Saltwater

Mysids Exposed to Lindane

................................ ................................ .......... 177

Table 13. Summary of F 1 

### Generation Day 7 Growth Measurements of Saltwater

Mysids Exposed to Lindane

................................ ................................ .......... 178

Table 14. Summary of F 0 

### Generation Day 14 Growth Measurements of Saltwater

Mysids Exposed to Lindane

................................ ................................ .......... 179

Table 15. Summary of F 1 

### Generation Day 14 Growth Measurements of Saltwater

Mysids Exposed to Lindane

................................ ................................ .......... 180

Table 16. Summary of F 0 

### Generation Day 44 Growth Measurements of Saltwater

Mysids Exposed to Lindane

................................ ................................ .......... 181

Table 17. Summary of F 1 

### Gener ation Day 35 Growth Measurements of Saltwater

Mysids Exposed to Lindane

................................ ................................ .......... 182

Table 18. Summary of F 0 

### Generation Reproduction of Saltwater Mysids Exposed

to Lindane

................................ ................................ ................................ ..... 183

Table 19. Summary of F 1 

### Generation Reproduction of Saltwater Mysids Exposed

to Lindane

................................ ................................ ................................ ..... 184

Figure 1. Mysid Two

- Generation Test Method Schematic

................................ .......... 185

Figure 2. F 0 

### Analytical Recoveries Over Time

................................ ............................ 186

Figure 3. F 1 

### 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 a nd Metals in Wildlife International,

Ltd. Saltwater

................................ ................................ ................................ 18 9

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

- F 0



### Generation

................................ ................................ ................................ ..... 208

Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1



### Generation

................................ ................................ ................................ ..... 220

Appendix 7 Replicate Mean Growth Measurements for the F 0 

### Gener ation Day 0

Culture Mysids

................................ ................................ .............................. 232

Appendix 8 Replicate Mean Total Body Lengths for the F 0 

### Generation Day 7

Mysids

................................ ................................ ................................ ........... 234

Appendix 9 Re plicate Mean Dry Weights for the F 0 

### Generation Day 7 Mysids

............. 237

Appendix 10 Replicate Mean Total Body Lengths for the F 1 

### Generation Day 7

Mysids

................................ ................................ ................................ ........... 240

Appendix 11 Replicate Mean Dry Weights for the F 1 

### Generation Day 7 Mysids

............. 243

Appendix 12 Replicate Mean Body Lengths for Male and Female F 0 

### Generation

Day 14 Mysids

................................ ................................ .............................. 246

Appendix 13 Replicate Mean Dry Weights for Male and Female F 0 

### Generation Day

14 Mysids

................................ ................................ ................................ ...... 249

Appendix 14 Replicate Mean Body Length s for Male and Female F 1 

### Generation

Day 14 Mysids

................................ ................................ .............................. 252

Appendix 15 Replicate Mean Dry Weights for Male and Female F 1 

### Generation Day

14 Mysids

................................ ................................ ................................ ...... 253

Appendix 16 Replicate Mean Body Lengths for Male and Female F 0 

### Generation

Day 44 Mysids

................................ ................................ .............................. 254

Appendix 17 Replicate Mean Dry Weights for Male and Female F 0 

### Generation Day

44 Mys ids

................................ ................................ ................................ ...... 257

Appendix 18 Replicate Mean Body Lengths for Male and Female F 1 

### Generation

Day 35 Mysids

................................ ................................ .............................. 260

Appendix 19 Replicate Mean D ry Weights for Male and Female F 1 

### Generation Day

35 Mysids

................................ ................................ ................................ ...... 263

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

Interbrood Duration F 0 

### Generation

................................ ............................... 266

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

Interbrood Duration F 1 

### Generation

................................ ............................... 272

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

and Total Produced F 0 

### Generation

................................ ................................ 278

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

and Total Produced F 1 

### Gene ration

................................ ................................ 284

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

Producing Offspring F 0 

### Generation

................................ .............................. 289

Ap pendix 25 Replicate Mean Percent of Males Sex Ratio and Percent of Females

Producing Offspring F 1 

### Generation

................................ .............................. 290

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STUDY TRIAL 2

– 185A

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- 2A

................................ ................................ ....................... 291

SUMMARY

................................ ................................ ................................ .......................... 292

9.0 INTRODUCTION

................................ ................................ ................................ ....... 295

10.0 DESCRIPTION OF THE M ETHOD

................................ ................................ .......... 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 Bro od Chambers

................................ ................................ ............... 302

10.8 Replication and Number of Test Organisms

................................ ................. 302

10.8.1 Exposed First Generation Mysids (F 0 

### )

................................ .............. 303

10.8.2 Exposed Second Generation Mysids (F 1 

### )

................................ ......... 303

10.9 Diluter

................................ ................................ ................................ ........... 304

10.10 Test S ubstance

................................ ................................ .............................. 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 Samplin g

................................ ................................ .. 307

10.14.2 Analytical Determinations

................................ ......................... 307

11.0 EXPOSURE INITIATION AND CONDUCT

................................ ........................... 308

11.1 Exposed First Generation Mysids (F 0 

### )

................................ .......................... 309

11.1.1 F 0 

### Exposure Initiation

................................ ................................ ........ 309

11.1.2 F 0 

### Growth Chambers

................................ ................................ ......... 309

11.1.3 F 0 

### Reproductive Chambers

................................ ................................ 310

11.1.4 Feeding

................................ ................................ .............................. 311

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11.2 E xposed Second Generation Mysids (F 1 

### )

................................ ..................... 311

11.2.1 F 1 

### Exposure Initiation

................................ ................................ ........ 311

11.2.2 F 1 

### Growth Chambers

................................ ................................ ......... 312

11.2.3 F 1 

### Reproductive Chambers

................................ ................................ 313

11.2.4 Feeding

................................ ................................ .............................. 313

11.3 Chamber Cleaning

................................ ................................ ........................ 314

11.4 Conditions of Exposure

................................ ................................ ................. 314

12.0 OBSERVATION AND MEAS UREMENTS

................................ ............................. 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 F 0 

### Generation

................................ ................................ .................... 316

14.1.2 F 1 

### 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 F 0



### .............................. 319

14.4 Growth

................................ ................................ ................................ .......... 319

14.4.1 Day 0 (less than 24 Hours old)

– Dry Weight and Total Length

from F 0 

### Generation

................................ ................................ ........... 320

14.4.2 Day 7

- Dry Weight and Total Length from F 0 

### Generation

.............. 320

14.4.3 Day 14

– Male and Female Dry Weight and Total Length from

F 0 

### Generation

................................ ................................ .................... 320

14.4.3.1 Day 14

– Male Dry Weight and Total Length from F 0



### Generation

................................ ................................ ........ 321

14.4.3.2 Day 14

– Female Dry Weight and Total Length from

F 0 

### Generation

................................ ................................ ... 321

14.4.4 Test Termination

– Dr y Weight and Total Length from F 0 

### (Day

46) Generation

................................ ................................ .................. 321

14.4.4.1 Males at Test Termination from F 0 

### (Day 46)

Generation

................................ ................................ ........ 322

14. 4.4.2 Females at Test Termination from F 0 

### (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 F 0 

### Generation

................................ ............................ 323

14.5.2 Number of Offspring in the First Brood from F 0 

### Generation

........... 323

14.5.3 Number of Offspring in the Second Brood from F 0 

### Generation

....... 323

14.5.4 Total Number of Offspring from F 0 

### Generation

............................... 324

1 4.5.5 Sex Ratio (Male/Female) from F 0 

### Generation

................................ .. 324

14.5.6 Percent of Reproducing Females from F 0 

### Generation

...................... 325

14.6 Summary o f Survival, Growth Measurements and Reproduction

Endpoints for the F 1 

### Generation

................................ ................................ ... 325

14.6.1 Summary of Survival for the F 1 

### Generation

................................ ..... 325

14.6.2 Summary of Growth Measurements for the F 1 

### Generation

.............. 326

14.6.3 Summary of Reproduction for the F 1 

### 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 fema les in the F 0 

### and F 1 

### 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 LITER ATURE

................................ ................................ .................. 329

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

F 0 

### Generation Exposure to 4

- tert

- octylphenol

................................ .............. 332

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

F 1 

### Generation Exposure to 4

- tert

- octyl phenol

................................ .............. 333

Table 22. Measured Concentrations of 4

- tert

- octylphenol in Pretest Solution

Samples for the F 0 

### Generation

................................ ................................ ...... 334

Table 2 3. Measured Concentrations of 4

- tert

- octylphenol for the F 0 

### Generation

in Stock Solution Samples

................................ ................................ ............ 335

Table 24. Measured Concentrations of 4

- tert

- octylphenol in Saltwater Samples

for the F 0 

### Gen eration

................................ ................................ ..................... 336

Table 25. Measured Concentrations of 4

- tert

- octylphenol in Pretest Solution

Samples for the F 1 

### Generation

................................ ................................ ...... 338

Table 26. Measured Concentrations of 4

- tert

- octylphenol in Saltwater Samples

for the F 1 

### Generation

................................ ................................ ..................... 339

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Table 27. Summary of F 0 

### 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 F 0 

### Generation Day 7 Growt h Measurements of Saltwater

Mysids Exposed to 4

- tert

- octylphenol

................................ .......................... 342

Table 30. Summary of F 0 

### Generation Day 14 Growth Measurements of Saltwater

Mysids Exposed to 4

- tert

- octylphenol

................................ .......................... 343

Table 31. Summary of F 0 

### Generation Day 46 Growth Measurements of Saltwater

Mysids Exposed to 4

- tert

- octylphenol

................................ .......................... 344

Table 32. Summary o f F 0 

### Generation Reproduction of Saltwater Mysids Exposed

to 4

- tert

- octylphenol

................................ ................................ ...................... 345

Figure 4. Mysid Two

- Generation Test Method Schematic

................................ .......... 346

Figure 5. F 0 

### Generation Analytical Recoveries Over Time

................................ ......... 347

Figure 6. F 1 

### 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 I nternational,

Ltd. Saltwater

................................ ................................ ................................ 350

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

Sample

................................ ................................ ................................ ........... 352

Appe ndix 29. The Analysis of 4

- Tert

- Octylphenol in Saltwater

................................ ......... 354

Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

Octylphenol

- F 0 

### Generation

................................ ................................ ......... 368

Appendix 31 Replicate Mean Growth Measurements for the F 0 

### Generation Day 0

Culture Mysids

................................ ................................ .............................. 380

Appendix 32 Replicate Mean Total Body Lengths for the F 0 

### Gen eration Day 7

Mysids

................................ ................................ ................................ ........... 382

Appendix 33 Replicate Mean Dry Weights for the F 0 

### Generation Day 7 Mysids

............. 385

Appendix 34 Replicate Mea n Body Lengths for Male and Female F 0 

### Generation

Day 14 Mysids

................................ ................................ .............................. 388

Appendix 35 Replicate Mean Dry Weights for Male and Female F 0 

### Generation Day

14 Mysids

................................ ................................ ................................ ...... 391

Appendix 36 Replicate Mean Body Lengths for Male and Female F 0 

### Generation

Day 46 Mysids

................................ ................................ .............................. 394

Appendix 37 Replicate Mean Dry Weights for Male and Female F 0 

### Generati on Day

46 Mysids

................................ ................................ ................................ ...... 397

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

Interbrood Duration F 0 

### Generation

................................ ............................... 400

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Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods

and Total Produced F 0 

### Generation

................................ ................................ 406

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

Producing Offspring F 0 

### Generation

................................ .............................. 412

Appendix 41 F 1 

### Summary Tables

................................ ................................ ....................... 413

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## PROTOCOL

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## AMENDMENTS AND DEVIATI ONS

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## QAPP

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# STUDY TRIAL 1

– 185A

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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

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TEST SUBSTANCE: Lindane , Lot No. 14419EB , CAS No. 58

- 89

- 9 , reported to have a

purity of 99.6% and was received from Battell e 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, F 0 

### generation) that were grown to maturity. Their

offspring (F 1 

### generation) were collected, which in turn were

grown to maturity, and their offspring (F 2 

### generation) were

collected.

Origin: Wildlife International, Lt d 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

Salin ity 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) con trol (0.0), 32, 80, 200, 500 and

1250 ng Lindane/L

ENDPOINTS:

Parental Generation (F 0 

### )

• 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 maturati on, 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 (F 1 

### )

• 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, num ber 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 seco nd

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 s econd

brood per reproducing female, mean total number of offspring during

the study and the mean percent of reproducing females, in the F 0 

### generation was the most sensitive endpoint in the mysid two

generation exposure to Lindane. Based on reproduction, t he 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 tr ans

- 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. Additionall y, 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 i s 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

Developmen t (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 q uantitative 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 two

generation test method and t he 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 d evelopment 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 suppor ting 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 L aboratory

(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 ens uring that participants

adhere to the standardized guidelines/protocols.

RTI arrange d 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 include d 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 t he authorization of

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

An archive inventory was maintained and storage capability was provide d for the expedient

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

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



## 2.0 DESCRIPTION OF THE METHOD

2.1 QAPP/Protocol

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

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 an d

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 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 ).



## 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 b rood stock was originally obtained from two different commercial suppliers:

C hesapeake 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 mys ids 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 p eriod immediately preceding

the test, water temperatures in the cultures ranged from 23.0 to 27.5oC, measured with a liquid

in

- 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 me ter. 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 V italSine 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 (F 0 

### and F 1 

### ) for all control and

treatm ent 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 s ubstance 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 hyd rated 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 (co ntaining 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 gro wth/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 handle d 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 smooth

edged pipette.



## 2.5 Dilution Water

The water used for culturing and testing was natur al 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 org anic 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 co nductance 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

chambe r 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 F 0 

### generation, and in alternating replicates d aily thereafter in both the F 0 

### and

F 1 

### 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 a nd 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. F luorescent 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 F 0



### 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

F 0 

### 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 F 0



### 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 flow ed 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/con trol 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 o bservations

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 fun gicides) 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 e ach Reproduction (R) or Growth chamber (G):

Treatment Level

– Replicate Vessel

– Chamber Type (R or G)

– Generation (F 0 

### or F 1 

### )

– 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 broo d 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 (F 0 

### or F 1 

### )

– date brood chamber

was initiated.



## 2.8 Replic ation 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 F 0 

### and F 1 

### mysids

were all first examined for re productive 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 F 0 

### 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 bro od 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 wa s defined as the time between release of the first brood and the release of the second

brood.

2.8.1 Exposed First Generation Mysids (F 0 

### )

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 reproducti on 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 gr owth 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 F 0



### exposure continued until Day 44, which was one week after the median day of rel ease of the

second brood (Day 37) in the negative control group.

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2.8.2 Exposed Second Generation Mysids (F 1 

### )

The F 1 

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

(< 24 hours old) per day produced by the F 0 

### generation. F 1 

### gene ration mysids were assigned to

reproduction chambers within the same treatment group, for each of the F 0 

### exposure conditions

(e.g. juveniles from Replicate A of the F 0 

### generation vessel were assigned to Replicate A of the

F 1 

### 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 F 1 

### 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, F 1 

### offspring was indiscriminat ely 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 F 1 

### generation brood chambers within each replicate (with a

maximum of 7 m/f pairs possible per replicate). Individuals in the F 1 

### generation reproduction

chambers not paired in br ood chambers were maintained and observed for survival and sex

determination within the F 1 

### 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 F 1 

### 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, I nc., 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 chamb er 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

s plit 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 prio r 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 F 0 

### and F 1 

### 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 a nd at

least two times per day during the test, except on Day 23 of the F 0 

### 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 followi ng 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) cont rol (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 F 0 

### and F 1 

### generations combined. A primary stock solutio n 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 hour s. 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 a pproximately 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 achiev e the desired test concentrations for both the F 0 

### and F 1 

### 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 ves sel 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 F 0 

### and F 1 

### generations as well as on Day 2 of the F 1 

### 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 (H 3 

### PO 4 

### ) 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 Mo del 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 Ap pendix 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 μ g a.i./L, were

prepared in hexane using a stock solu tion 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 o f 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 i ncluded in Appendix 4.4

.

A stock analysis for the F 0 

### generation was conducted at nominal concentrations of 2.1, 5.1, 13,

32 and 80 μ g/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 F 1 

### 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 st udy samples was set at

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

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 F 0 

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

possible interferences in the F 1 

### generation ( Appendix 4.6 ).

Acidified saltwater samples were fortified at 0.0320, 0.400 and 1.25 μ g 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 F 0 

### generation

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

concentrations for the matrix fortification samples for the F 1 

### generation ranged from 70.6 to

141% of nominal concent rations ( Appendix 4.6 )

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

of low and high

- level calibration standards are p resented 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 p resented 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 appro ximately 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 F 0 

### 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 (F 0 

## )

3.1.1 F 0 

### Exposure Initiation

At exposure initiation, 20 < 24

- hour

- old individuals were impartially transferred into

reproduction and growth chambers. For each tre atment 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 fou r 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 m ysid 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 F 0 

### Growth Chambers

At two time intervals (Day 7 and Day 14), mysids were sampled from ea ch 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 measu rements, 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

– 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

10 0 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 F 0 

### Reproductive Chambers

When developing embryos were discerned within the majority of femal es 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 F 0



### exposure terminated at least seven days after the median day of release of the second brood (F 0 

### ”)

in the negative control treatment group.

Upon termination, each surviving F 0 

### mysid was removed from the reproduction chamber s,

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 a n 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 F 0 

### 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 F 0 

### generation.

3.1.4 Feeding

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

recommended densities described in section 2.3.



## 3.2 Exposed Second Generation Mysids (F 1 

## )

3.2.1 Initiation of F 1 

### Exposure

Groups of < 24

- hour old offspring (F 1 

### ) from either the first (F 1 

### ’) or second brood (F 1 

### ”) of the F 0



### mysids were isolated in reprod uction 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 F 1 

### 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. P riority was given to

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

There were not enough of F 1 

### mysids available to conduct the Day 0 mysid growth measurements

for the F 1 

### generation.

3.2.2 F 1 

### Growth Chambers

At two time interv als (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 n ot 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 mysi d 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 t o 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 F 1 

### Reproductive Chambers

When de veloping 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 ch amber 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 reprod uction chamber was skewed preventing obtaining seven pairs. The F 1



### exposure terminated on Reproductive Chamber Day 35 or 36.

Upon termination, each surviving F 1 

### mysid was removed from the reproduction chambers,

euthanized in ice

- cold reverse

- osmosis wat er, 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 approximat ely 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, mysid s

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 F 1 

### 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 F 0



### initiation to the F 1 

### 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 asses sment 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 (F 0 

### , F 1 

### )

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

7 and 14 for F 0 

### and F 1, 

### when possible)

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

young in the first brood (F 0 

### , F 1, 

### when possible)

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

of young in the second brood (F 0 

### , F 1, 

### when possible)

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

brood (F 0 

### , F 1, 

### when possible)

• Number of young per fem ale in each of the first two broods (F 0 

### , F 1, 

### when

possible)

• Total number of young in the first two broods in each replicate population (F 0 

### , F 1, 

### when possible)

• Sex ratio (F 0 

### , F 1 

### )

• Percentage of females that are reproductively active (F 0 

### , F 1 

### )

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## 5.0 STATISTICAL MET HODS

5.1 Endpoints

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

below:

• Survival, (F 0 

### , F 1 

### )

• Growth

- length and dry weight, (Days 0, 7 and 14 for F 0 

### and F 1 

### )

• Time to maturation, (F 0 

### , F 1 

### )

• Time to first brood release, (F 0 

### , F 1 

### )

• Time to second brood release, (F 0 

### , F 1 

### )

• Interbrood duration, (F 0 

### , F 1 

### )

• Number of young per female in each of the first two broods, (F 0 

### , F 1 

### )

• Total number of young in the first two broods in each replicate population (F 0 

### , F 1 

### )

• Sex ratio, (F 0 

### , F 1 

### )

• Percentage of females that are reproductively active, (F 0 

### , F 1 

### )



## 5.2 Statistical Methods

Survival data, sex ratio and females that were reproductively active w ere 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 conti nuous

- 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 F 1 

### generation time to maturation, F 1 

### generation time to second brood

release, and F 1 

### generation interbrood duration. The data was not conducive to transformation to

correct the condition. Therefore, the Nonparametric Wilcoxon rank

- sum test was used fo r

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 negat ive 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 conce ntration with statistically

significant differences from controls) and the N o

- Observed

- Effect

- Concentration (NOEC; and

the highest test concentration that show ed no statistically significant difference from the control).

All statistical analys e s w ere cond ucted 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 F 0 

### in Table 1 and for F 1 

### 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 F 1



### generation when the temperature fell to 24.1 ° C in replicate B in the 32 ng/L treatment group.

Dissolved oxygen conce ntrations 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 F 0 

### and F 1 

### 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 F 0 

### generation and 326 lux for the F 1



### 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 eac h test vessel ranged from 190

to 308 lux for the F 0 

### generation and 238 to 367 lux for the F 1 

### generation.



## 6.1 In

- life Analytical Results

6.1.1 F 0 

### Generation

Test solutions were premixed in mixing chambers and then delivered into tanks. Samples used

for analytical m easurements 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 t est initiation ranged from approximately 75.3 to 87.4% of nominal ( Table 4 ).

Lindane concentration s in the test solution samples collected during the test ranged from

approximately 68 to 102% in the four highest test concentrati ons and from 93 to 137% of

nominal in the lowest test concentration

, which was near the limit of the anal ytical method

( Table 5 ). When measured concentrations of the samples collected during the test were

averaged, the mean measu red 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 F 0 

### generation were based on nominal concentrat ions.

6.1.2 F 1 

### 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

pres ented in Table 8 . Lindane concentrations ranged from approximately 6 7 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 m ethod. 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, respec tive ly ( Figure 3 ). The resu lts of the F 1 

### generation also were based on the

nominal concentrations.



## 6.2 Biological Results

Table 9 through Table 19 present endpoint summaries of the to tal 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 F 0 

### juvenile mysids from test initiation to the Day 7 growth sub

sampling 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 F 1 

### juvenile mysids from test initiation to the Day 7 growth sub

sampling 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 wh en compared to the negative control ( p > 0.05)

.

6.3.2 Survival from Day 8 to Day 14

A summary of survival of F 0 

### juvenile mysids from Day 8 to the Day 14 growth sub

- sampling

interval is located in Table 9 . Survival in the negative co ntrol 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 comp ared 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 F 1 

### juvenile mysids from test D ay 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

Ex act 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 F 0 

### 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. F isher’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 Appen dix 5

.

A summary of survival of F 1 

### 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 wa s 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

F 1 

### generation. Fisher’s Exact test indicated there were no statistically significant decreases i n

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 4 4) during the F 0 

### generation and

three time points (Days 7, 14 and 35) for the F 1 

### 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 subsample d for growth measurements. This was done for the F 0



### generation and the F 1 

### generation when possible. There were not always enough mysids available

in the F 1 

### to fill the reproductive retention chambers and the retention chamber used to monitor

growth. Pri ority 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.

Summar ies 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 F 0 

### and F 1



### 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 F 0



### 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 F 0 

### 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 F 1 

### generation.

6.4.2 Day 7

- Dry Weight and Total Length from F 0 

### and F 1 

### Generations

Summaries of the Day 7 total lengths and dry weights of surviving juvenile mysids in the

treatment and contro l groups for the F 0 

### and F 1 

### 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 F 0 

### 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 decr eases in mean total

length or dry weight for the Day 7 mysids from the F 0 

### generation in any of the treatment groups

( p > 0.05) ( Table 12 )

.

Results for the F 1 

### generation were similar to the F 0 

### generation. The mean total length of the

Day 7 mysids in the F 1 

### 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 , respec tively. Dunnett’s test indicated

there were no statistically significant decreases in mean total length or dry weight for the Day 7

mysids from the F 1 

### generation in any of the treatment groups ( p > 0.05) ( Table 13 )

. Because of

low production of offspring in the two highest treatment level s in the F 0 

### generation, there were

not enough F 1 

### mysids to set aside mysids for growth determinations.

6.4.3 Day 14

– Male and Female Dry Weight and Total Length from F 0 

### and F 1



### 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 F 0 

### and F 1 

### generations are presented in Table 14 and

Table 15 , with replicate m ean 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 F 0 

### and F 1 

### Generations

The mean total length of the Day 14 male mysids in the F 0 

### 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 weight s of males w ere 0.403, 0.498, 0.474, 0.45 7,

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 F 0 

### generation in any

of the treatment groups ( p > 0.05) ( Table 14 ).

The mean total length of the Day 14 male mysids in the F 1 

### 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 F 0 

### generation

. F 0 

### control mysids did not

produce enough live mysids to initiate as many F 1 

### 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 F 0 

### generation, there were not enough F 1 

### mysids to set aside mysids for

growth determinations ( Table 15 ).

6.4.3.2 Day 14

– Female Dry Weight and Total Length f rom F 0 

### and F 1 

### Generations

The mean total length of the Day 14 female mysids in the F 0 

### 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 me an 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

weigh t for the Day 14 mysids from the F 0 

### generation in any of the treatment groups ( p > 0.05)

( Table 14 ).

The mean total length of the Day 14 female mysids in the F 1 

### 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 F 0



### genera tion, there were not enough F 1 

### mysids to set aside mysids for growth determinations.

Male and female mysids from the F 1 

### generation on Day 14 were, overall, larger than the F 0



### generation in both total length and dry weight ( Table 15 ).

6.4.4 Test Termination

– Dry Weight and Total Length from F 0 

### (Day 44) and F 1 

### (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 F 0 

### (Day 44) and F 1 

### (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 F 0 

### (Day 44) and F 1 

### (Day 35) Generations

The mean total length of the Day 44 male mysids in the F 0 

### generation in the negative control, 32,

80, 200, 500 and 1250 ng/L treatment groups at test terminati on 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 l ength or dry weight in males from the F 0 

### 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 F 1 

### generation in the nega tive 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 F 1



### 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 F 0 

### and F 1



### generations, even though there was a 12

- day differen ce in age at termination of the two

generations.

6.4.4.2 Females at Test Termination from F 0 

### (Day 44) and F 1 

### (Day 35) Generations

The mean total length of the Day 44 female mysids in the F 0 

### generation in the negative control, 32,

80, 200, 500 and 1250 ng/L treatm ent 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 F 0 

### 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 F 1 

### 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 t here were no statistically significant decreases in

mean total length or dry weight in males from the F 1 

### generation in any of the treatment groups at

test termination (Day 35) ( p > 0.05) presented in Table 17 . In general, at tes t termination female

mysids in the F 0 

### generation were slightly heavier than females from the F 1 

### generation, but they

were also 12

- days older than the F 1 

### females.



## 6.5 Reproduction

Mysids in both generations were evaluated for several reproductive endpoints, in cluding 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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Sum maries 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 F 0 

### and F 1 

### Generations

There were no significant differences in mean number of days to maturation, days to 1 st 

### brood,

days to second brood, or mean interbrood d uration for any treatment group in the F 0 

### and F 1



### 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 F 0 

### and F 1 

### 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 F 1 

### generation, therefore this level was excluded

from statistical analysis of the F 1 

### generation reproduction data.

6.5.2 Number of Offspring i n the First Brood from F 0 

### and F 1 

### Generations

Summaries of the mean number of offspring in the first brood per reproductive female in the

negative control and treatment groups for the F 0 

### and F 1 

### 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 b rood for the F 0 

### 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

dec reases in mean number of offspring in the first brood for the mysids from the F 0 

### generation in

any of the treatment groups ( p > 0.05) ( Table 18 )

.

The mean number of offspring for the F 1 

### generation in the negative control and t he 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 indic ated there were no statistically significant

decreases in mean number of offspring in the first brood for the mysids from the F 1 

### generation in

any of the treatment groups ( p > 0.05) ( Table 19 ).

6.5.3 Number of Offspring in the Secon d Brood from F 0 

### and F 1 

### Generations

Summaries of the mean number of offspring in the second brood per reproductive female in the

negative control and treatment groups for the F 0 

### and F 1 

### 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 F 0 

### 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 decre ases 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 F 1 

### 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 F 1 

### generation in any of the

treatment groups ( p > 0.05) ( Table 19 )

.

6.5.4 Total Number of Offspring from F 0 

### and F 1 

### Generations

Summaries of the mean total number of offspring produced in the study in the negative control

and treatment groups for the F 0 

### and F 1 

### generations are pre sented 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 F 0 

### 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. Altho ugh 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 F 1 

### 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 o ffspring produced in the study for the mysids from the F 1



### 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 F 0 

### and F 1 

### Generations

Summaries of the mean percent of males present (used to determine the sex ratio in the study) in

the negative c ontrol and treatment groups for the F 0 

### and F 1 

### 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 F 0 

### 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 F 0 

### generation in any of the treatment

groups ( p > 0.05) ( Table 18 )

.

The mean percent of males present for the F 1 

### 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 F 1 

### generation to generate reliable

percentages of males to perform statistical analyses ( Table 19 )

.

6.5.6 Percent of Reproducing Females from F 0 

### and F 1 

### Generations

Summaries of the mean percent of reproducing females in the st udy in the negative control and

treatment groups for the F 0 

### and F 1 

### 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 F 0 

### 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 F 1 

### 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 F 1 

### 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

illu strated 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 F 1 

### generation

when the temperature fe ll 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 durat ion 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 F 0 

### generation produced young. For the F 1 

### 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 ei ght per

control female in the F 0 

### generation. While the F 1 

### 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 Li ndane at nominal concentrations of

32 to 1250 ng/L for the F 0 

### and F 1 

### generations. The test was conducted under flow

- through

conditions for 44 and 35 days for the F 0 

### and F 1 

### generations, respectively, and the mysids were

evaluated for survival, growth and r eproductive endpoints. The most sensitive biological

endpoints were reproduction variables in the F 0 

### 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 F 0 

### generation. Consequently, the NOEC, based on

reproduction for the F 0 

### generation, was 500 ng/L. The LOEC was 1250 ng/ L and the MATC was

calc ulated to be 791 ng/L.

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## REFERENCES AND LITERATURE

ASTM. 2008. Committee E

- 47 on Biological Effects and Environmental Fate. Standard Guide

for Conducting Life

- Cycle Toxicity Tests with Mysids. ASTM Designation E 1191

- 039

(re

- approved 2008). American Soci ety for Testing and Materials, 100 Barr Harbor Drive,

West Conshohocken, PA.

Hutchinson, T.H., Shillabeer, N, Winter, M.J., Pickford, D.B. 2006. Acute and Chronic Effects

of Carrier Solvents in Aquatic Organisms: A Critical Review. Review. Aquatic

Tox icology, 76: 69

- 92.

Lemke, A.E., W.A. Brungs and B.J. Halligan. 1978. Manual for construction and operation of

toxicity

- testing proportional diluters. EPA 600/3

- 78

- 072. U.S. Environmental Protection

Agency, Washington, D.C.

McKenney CL, Jr. 1986 Infl uence of the organophosphate insecticide fenthion on Mysidopsis

bahia exposed during a complete life cycle. I. Survival, reproduction, and age

- specific

growth. Dis quat Org 1:131

- 139.

McKenney CL, Jr. 1994 Resistance patterns to salinity and temperatu re in an estuarine mysid

( Mysidopsis bahia ) in relation to its life cycle. Comp Biochem Physiol 109A:199

- 208.

M cKenney CL, Jr. 1996 The combined effects of salinity and temperature on various aspects of

the reproductive biology of t he estuarine mysid, Mysidopsis bahia

. Invertebr Reprod Dev

29(1): 9

- 18.

McKenney CL, Jr. and Celestial DM. 1995. Interactions among salinity, temperature, and age

on growth of the estuarine mysid Mysidopsis bahia reared in the labo ratory through a

complete life cycle. I. Body mass and age

- specific growth rate. J Crust Biol 15:169

-

178.

McKenney CL, Jr. 1998. Physiological dysfunction in estuarine mysids and larval decapods

with chronic pesticide exposure. In: Microscale Testing in Aquatic Toxicology:

Advances, Techniques, and Practice , PG Wells, K Lee, and C Blaise, Eds., CRC Press,

Boca Raton, FL, Chapter 32, pp. 465

- 476.

Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicity studies.

Water Resear ch 1: 21

- 29.

Nimmo DR, Bahner LH, Rigby RA, Sheppard JM and Wilson AJ, Jr. 1977. Mysidopsis bahia :

an estuarine species suitable for life

- cycle toxicity tests to determine the effects of a

pollutant. In: Aquatic Toxicology and Hazard Evaluation, ASTM ST P 634 . FL Mayer and

JL Hamelink, Eds., American Society for Testing and Materials, Philadelphia, PA, pp.

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 P rocedures 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

Monito ring. 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 Crustace a: 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 Two

Generation 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 R odenticide 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

receivin g 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 co mpared with a zero dose control. Biometrics 27: 103

- 117.

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- 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.

20 04. Mysid crustaceans as potential test organisms for the evaluation of environmental

endocrine disruption: a review. Environ Tox Chem 23 (5): IN PRESS.

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

the F 0 

## Generation Exposure to Lindane

Nominal

Concentration

(ng/L) Mean ± SD and Range of Measured Parameters

Temperature 1

(°C) DO 2

(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

– 2 1)

1 Temperature monitored continuously during the test ranged from approximately 25 to 27 o C, measured to the nearest 1 o C.

2 A dissolved oxygen concentration of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰

.

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

the F 1 

## Generation Exposure to Lindane

Nominal

Concentration

(ng/L) Mean ± SD and Range of Measured Parameters

Temperature 1

(°C) DO 2

(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)

12 50 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 27 o C, measured to the nearest 1 o C.

2 A dissolved oxygen concentrati on of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰

.

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

for the F 0 

## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- ) Sampling

T ime

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2

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 μ g/L, calculated as the product of the concentration of the lowest

calibration standard (0.500 μ g 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.

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## Table 4. Measured Concentrations of Li ndane for the F 0 

## Generation in Stock

Solution Samples

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- ) Sampling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 1

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.

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## Table 5. Measured Concentrations of Lindane in Saltwater Samples for the

F 0 

## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- ) Sampling

Time

(Days) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Concentration

( μ g/L) Mean

Percent of

Nominal

0.0

(Negative Control) 1

7

13

19

25

31

37

43 0

7

14

15

22

2 9

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 μ g/L, calculated as the product of the concentration of the lowes t calibration

standard (0.500 μ g 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.

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## Table 5. Measured Concentrations of Lindane in Saltwater Samples for the F 0



## Generation (Continued)

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- ) Sampling

Time

(Days) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Conc entration

( μ g/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 μ g/L, calculated as the product of the concentration of the lowest calibration

standard (0.500 μ g 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 usin g Excel 2000 in full precision mode. Manual calculations may differ slightly.

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## Table 6. Measured Concentrations of Lindane in Pretest Diluter Verification

Samples for the F 1 

## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- F1

- ) Sa mpling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2

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 limi t of quantitation (LOQ) was 0.0151 μ g/L, calculated as the product of the concentration of the lowest calibration

standard (0.500 μ g a.i./L) and the dilution factor of the matrix blank samples (0.0300), divided by test substance purity

(99.6%).

2 Results w ere generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.

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## Table 7. Measured Concentration of Lindane in F 1 

## Generation Stock

Solution Samples

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- ) Sampling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 1

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.

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## Table 8. Measured Concentrations of Lindane Saltwater Samples for the F 1



## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- F1

- ) Sampling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Concentration

( μ g/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

0.04 79

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 μ g/L, calculated as the product of the concentration of the lowest calibration

standard (0.500 μ g 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.

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## Table 8. Measured Concentratio ns of Lindane Saltwater Samples for the F 1



## Generatio n (Continued)

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 1

- F1

- ) Sampling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Concentration

( μ g/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 μ g/L, calculated as the product of the concentration of the lowest

calibration standard (0.500 μ g a.i./L) and the dilution factor of the matrix blank samples (0.0300), div ided by test

substance purity (99.6%).

2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.

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## Table 9. Summary of F 0 

## 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 survi val 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 wa s slight, was within control acceptability and was not dose

- responsive.

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## Table 10. Summary of F 1 

## 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 Me an 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 s urvival in comparison to the negative control using Fisher’s Exact test

( p > 0.05).

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## 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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## Table 12. Summary of F 0 

## 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 an d

control groups presented.

2 There were no statistically significant decreas es in growth in comparison to the negative control using Dunnett’s test

( p > 0.05).

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## Table 13. Summary of F 1 

## 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

20 0 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

F 0 adults.

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## Table 14. Summary of F 0 

## 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).

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## Table 15. Summary of F 1 

## Generation Day 14 Growth Mea surements 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 F 0

adults.

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## Table 16. Summary of F 0 

## 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 Contro l 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)

20 0 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 treatmen t

and control groups presented.

2 There were no statistically significant decreases in growth in comparison to the negative cont rol using Dunnett’s test

( p > 0.05).

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## Table 17. Summary of F 1 

## 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. Th erefore, 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 r eplicates were available for the treatment group.

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## Table 18. Summary of F 0 

## Generation Reproduction of Saltwater Mysids Exposed to Lindane

Nominal

Conc.

(ng/L) Mean Time

to

Maturation

Days ± SD

(CV%) 1 Mean

Time to 1 st

Brood

Days ± SD

(CV%) 1 Mean

Time to 2 nd

Brood

Days ± SD

(CV%) 1 Mean

Interbrood

Duration

Days ± SD

(CV%) 1 Mean Number

of Offspring in

1 st Brood Per

Female ± SD

(CV%) 1 Mean Number

of Offspring in

2 nd 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

(1 2.2) 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 we re able to be sexually

identified throughout the test. For percent females producing offspring, n = the number of females that pr oduced 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 t est ( p ≤ 0.05).

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## Table 19. Summary of F 1 

## Generation Reproduction of Saltwater Mysids Exposed to Lindane

Nominal

Conc.

(ng/L) Mean

Time to

Maturation

Days ± SD

(CV%) 1,2 Mean

Time to 1 st

Brood

Days ± SD

(CV%) 1,2 Mean

Time to 2 nd

Brood

Days ± SD

(CV%) 1,2 Mean

Interbrood

Duration

Days ± SD

(CV%) 1,2 Mean Number

of Offspring in

1 st Brood Per

Female ± SD

(CV%) 1,2 Mean Number

of Offspring in

2 nd 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 23.0 3

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 replicat es 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 of fspri ng.

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 a s only one replicate was available for the treatment group

4 No offspring were produced by the F 1 adults in the 1250 ng/L treatment group.

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## Figure 1. Mysid Two

- Generation Test Method Schematic

Source: Wildlife International, Ltd. Protocol No.: 185/02 1511/MYS

- 2GeN/SUB185; Page 44.

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## Figure 2. F 0 

## Analytical Recoveries Over Time

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## Figure 3. F 1 

## Analytical Recoveries Over Time

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## Appendix 1. Dilution Water Chemistry Parameters Measured During the 4

-

Wee k 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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## Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife

International, Ltd. Saltwater

Pesticides and Organics 1

C omponent 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 < 0.01 0

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

D ichlorvos < 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 < 0.0 20

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 Tox aphene < 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.

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

Inte rnational, 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 Ni ckel < 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

Co pper < 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.

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## Appendix 3. Analyses of Pesticides, Organics and Me tals 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 Lancas ter Laboratories on samples collected on December 29, 2010.

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## 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

C alcium 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.

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## Appendix 4. The Analysis of Lindane in Saltwater

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## Appendix 4.1 Method Flowchart for the Analysis of Lindane in

Saltwater

METHOD FLOWCHART FOR THE ANALYSIS OF LI NDANE 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 appropria te 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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## Appendix 4.2 Typical Gas Chromatograph (GC) Operational

Parameter s

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 μ m 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 μ L

CARRIER GAS: Helium

HEAD PRESSURE: 4 psi

MAKE

- UP GAS: Argon / Methane, 60 psi.

A PPROXIMATE LINDANE PEAK

RETENTION TIME: 8.9 minutes

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## 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 analy tical 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 0.00 0100 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 f or the set of calibration standards:

Stock

Concentration

(mg a.i./mL) Aliquot

( μ L) Final

Volume

(mL) Standard

Concentration

( μ g 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

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## Append ix 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 μ g/L, were calculated using the following equations:

Concentration of Lindane in sample ( μ g/L) = Peak Area

- Y

- intercept

Slope X Dilution Factor ÷ Purity

Percent of nominal concentration = measured concentration in sample ( μ g/L )

nominal concentration in sample ( μ g/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 ( μ g/L) = 9287.33301 + 701.0670

5429.6864 X 0.0400 ÷ 0.996

Concentration of Lindane in sample ( μ g/L) = 0.0739

Percent of nominal concentr ation = 0.0739 μ g/L

0.080 μ g/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.

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## Appendix 4.5 Quality Control Samples of Lindane in Saltwater for the

F 0 

## Generation

Sample Number

(185A

- 102

- 1

- ) Sample

Type Concentration of Lindane

Percent

Recovery 2 Fortified

( μ g a.i./L) Measured

( μ g 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 Fortific ation

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 μ g a.i./L, calculated as the product of the concentration of the lowest

calibration standard (0.500 μ g 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.

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## Appendix 4.5. Quality Control Samples of Lindane in Saltwater for the

F 0 

## Generation (C ontinued)

Sample Number

(185A

- 102

- 1

- ) Sample

Type Concentration of Lindane

Percent

Recovery 2 Fortified

( μ g a.i./L) Measured

( μ g 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

Ma trix 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

0.032 0

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

Mean 3

=

Standard Deviation =

CV = 83.7

4.25

5.08%

1 The li mit of quantitation (LOQ) was 0.0150 μ g a.i./L, calculated as the product of the concentration of the lowest

calibration standard (0.500 μ g a.i./L) and the dilution factor of the matrix blank samples (0.0300).

2 Results were generated using Excel 2000 in t he full precision mode. Manual calculations may differ slightly.

3 Based on exposure samples only.

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## Appendix 4.6. Quality Control Samples of Lindane in Saltwater for the

F 1 

## Generation

Sample Number

(185A

- 102

- 1

- ) Sample

Type Concentration of Lindane

Percent

Recovery 2 Fortified

( μ g a.i./L) Measured

( μ g 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 Blan k

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

8 5.5

78.5

78.2

1 The limit of quantitation (LOQ) was 0.0150 μ g a.i./L, calculated as the product of the concentration of the lowest

calibration standard (0.500 μ g a.i./L) and the dilution factor of the matrix blank samples (0.0300).

2 Results were generate d using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

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## Appendix 4.6. Quality Control Samples of Lindane in Saltwater for the

F 1 

## Generation (Continued)

Sample Number

(185A

- 102

- 1

- ) Sample

Type Concentration of Lindan e

Percent

Recovery 2 Fortified

( μ g a.i./L) Measured

( μ g 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 Fortificati on

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 μ g a.i./L, calculated as the product of the concentration of the lowest

calibration standard (0.500 μ g 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. Man ual calculations may differ slightly.

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## 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; R 2 

### = 0.99779

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## Appendix 4.8. Representative Chromatogram of a Low

- Level Lind ane Calibration Standard

Nominal concentration: 0.500 μ g a.i./L.

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## Appendix 4.9. Representative Chromatogram of a High

- Level Lindane

Calibration Standard

Nominal concentration: 5.00 μ g a.i./L. The arrow indicates the approximate retention tim e of Lindane.

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## 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.

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## Appendix 4.11. Representative Chromatogram of a Matrix Fortification

S ample number 185A

- 102

- 1

- MAS

- 1; 0.0320 μ g a.i./L, nominal concentration. The arrow indicates the

approximate retention time of Lindane.

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## Appendix 4.12. Representative Chromatogram of a Test Sample

Sample number 185A

- 102

- 1

- 4; 0.20 μ g/L, nominal conc entration. The arrow indicates the approximate

retention time of Lindane.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 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 th e study.

2 One male mysid was identified as female on Day 32.

3 One female mysid was identified as male on Day 38.

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## A ppendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 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 7 2 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 ot herwise 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 fem ale. 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.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## Generation (Con tinued)

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

15 1 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 th e study.

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## Appendix 5 Daily P ercent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 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 o rganisms used to initiate the adult phase of the study.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 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 in dicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## Generation (Continued)

Day of

Test 80 ng/L

– Females

Repl icate A Replicate B Replicate C Percent Survival

(%) No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive

15 1 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

4 3 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 th e study.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 0 9 0 7 0 8 100

16 0 9 0 7 0 8 100

17 0 9 0 7 0 8 100

1 8 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 th e study.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 0 9 0 10 (2 I) 2 0 11 1 00

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 th e 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).

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## Generati on (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

15 1 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 th e study.

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## Appendix 5 Daily Percent Survival of Saltwater Mysi ds Exposed to Lindane

- F 0 

## 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

15 1 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 8 2 100

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 als o 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 ).

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 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.

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## Appendix 5 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

- F 0 

## 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

15 1 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 th e study.

2 I = Mysid was not sexually identified (immature) during the Day 14 p airing 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).

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to L indane

– F 1 

## 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

15 1 0 1 0 2 0 0 100

16 0 1 0 2 0 0 100

17 0 1 0 2 0 0 1 00

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.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## 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

15 1 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 th e study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## Generatio n (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

15 1 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 th e study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## 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

15 1 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, t he number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## Generation (Continued)

Day of

Test 80 ng/L

– Males

Replicate A Rep licate B Replicate C Percent Survival

(%) No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive

15 1 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 th e study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exp osed to Lindane

– F 1 

## 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

15 1 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.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## 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

15 1 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 7 0

1 Unless otherwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of th e study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## 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

15 1 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 1 00

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 th e study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## 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

15 1 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 10 0

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.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## Generation (Continued)

Day of

Test 500 ng/L

– Females

Replicate A Re plicate B Replicate C Percent Survival

(%) No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive

15 1 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 th e study.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## 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

15 1 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 10 0

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.

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## Appendix 6 Daily Percent Survival of Saltwater Mysids Exposed to Lindane

– F 1 

## Generation (Continued)

Day of

Test 1250 ng/L

– Females

Replicate A Replicate B Replicate C Percent Sur vival

(%) No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive

15 1 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 th e study.

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## Appendix 7 Replicate Mean Growth Measurements for the F 0 

## Generation

Da y 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

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## Appendix 7 Replicate Mean Growth Measurements for the F 0 

## 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

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## Appendix 8 Replicate Mean Total Body Lengths for the F 0 

## 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.

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## Appendix 8 Replicate Mean Total Body Lengths for the F 0 

## Generation Day 7 Mysids (Continued)

Nominal Test

Concentration

(ng/L) Total Body Len gths (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 De v.: 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.

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## Appendix 8 Replicate Mean Total Body Lengths for the F 0 

## 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.

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## Appendix 9 Replicate Mean Dry Weights for the F 0 

## 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

Numb er 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 my sids measured divided by two.

2 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.

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## Appendix 9 Replicate Mean Dry Weights for the F 0 

## 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 0.03 2

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 usin g Excel 2000. Manual calculations may differ slightly.

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## Appendix 9 Replicate Mean Dry Weights for the F 0 

## 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.

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## Appendix 10 Replicate Mean Total Body Lengths for the F 1 

## 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

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## Appendix 10 Replicate Mean Total Body Lengths for the F 1 

## 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 calculat ions may differ slightly.

-- = No mysid(s) available for measurement

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## Appendix 10 Replicate Mean Total Body Lengths for the F 1 

## Generation Day 7 Mysids (Continued)

Nominal Test

Concentration

(ng/L) Total Body Lengths (mm)

Replicate A Replicate B Repl icate 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

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## Appendix 11 Replicate Mean Dry Weights for the F 1 

## Generation Day 7 Mysids

Nominal Test

Concentration

(ng/L) Dr y 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 my sid).

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

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## Appendix 1 1 R eplicate 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

-- -- --

Replic ate 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 weig ht 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 avail able for the replicate.

-- = No mysid(s) available for measurement

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## 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

-- -- --

P an 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

pres ented 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

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## Appendix 1 2 Replicate Mean Body Lengths for Male and Female F 0 

## Generation Day 14 Mysids

Nominal Test

Concentration

(ng/L) Total Body Lengths (m m)

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

Immature 2 Immature 2

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 1 0.0 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 calcula tions 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.

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## Appendix 1 2 Replicate Mean Body Len gths for Male and Female F 0 

## 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 Replic ate means were calculated using Excel 2000. Manual calculations may differ slightly.

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## Appendix 12 Replicate Mean Body Lengths for Male and Female F 0 

## Generation Day 14 Mysids (Continued)

Nominal Test

Concentration

(ng/L) Total Body Lengths (mm)

Replica te 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 Immature 3

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 dete rmined 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.

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## App endix 1 3 Replicate Mean Dry Weights for Male and Female F 0 

## 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

Immature 2 Immature 2

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 0.6 5 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 Immatur e 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.

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## Appendix 1 3 Replicate Mean Dry Weights for Male and Female F 0 

## Generati on

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.

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## Appendix 13 Replicate Mean Dry Weights for Male and Female F 0 

## 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 Im mature 3

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 calcula ted 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, incl uded in the replicate

mean, standard deviation or CV calculations or in the statistical analyses.

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## Appendix 1 4 Replicate Mean Body Lengths for Male and Female F 1 

## Generation

Day 14 Mysids

Nominal Test

Concentration

(ng/L) Total Body Lengths (mm)

Replic ate 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

Re plicate 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

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## Appendix 1 5 Replicate Mean Dry Weights for Male and Female F 1 

## Generation

Day 14 Mysids

Nomina l 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

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## Appendix 1 6 Replicate Mean Body Lengths for Male and Female F 0 

## 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.

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## Appendix 1 6 Replicate Mean Body Lengths for Male and F emale F 0 

## 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

St andard 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.

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## Appendix 16 Replicate Mean Body Lengths for Male and Female F 0 

## Generation

Day 44 Mysids (Continued)

Nominal Test

Concentration

(ng/L) Total B ody 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 calc ulations may differ slightly.

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## Appendix 1 7 Replicate Mean Dry Weights for Male and Female F 0 

## Generation

Day 44 Mysids

Nominal Test

Concentration

(ng/L) Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Males Females Males Female s

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.

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## Appendix 1 7 Replicate Mean Dry Weights for Male and Female F 0 

## Generation

Day 44 Mysids (Continued)

Nominal Test

Concentration

(ng/L) Dry Weights (mg)

Replica te 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.

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## Appendix 17 Replicate Mean Dry Weights for Male and Female F 0 

## 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 1.3 3 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 1 6.8 19.0 14.8

1 Replicate means were calculated using Excel 2000. Manual calculations may differ slightly.

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## Appendix 1 8 Replicate Mean Body Lengths for Male and Female F 1 

## 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

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## Appendix 1 8 Replicate Mean Body Lengths for Male and Female F 1 

## 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 D ev.: 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 S tandard deviation and CV were not determined as only one value was available for the replicate.

-- = No mysid(s) available for measurement

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## Appendix 18 Replicate Mean Body Lengths for Male and Female F 1 

## 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

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## Appendix 1 9 Replicate Mean Dry Weights for Male and Female F 1 

## Generation

Day 35 Mysids

Nominal Test

Concentra tion

(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

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## Appendix 1 9 Replicate Mean Dry Weights for Male and Female F 1 

## Generation

Day 35 Mysids (Continued)

Nominal Test

Concentration

(ng/L) Dry Weights (mg)

Replicate A Replicate B Replicate C

Males Females Male s 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

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## Appendix 19 Repl icate Mean Dry Weights for Male and Female F 1 

## 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

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## Appendi x 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

C V(%) 2 Time to 2 nd

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 1 4 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 mysi ds 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 Ther e 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.

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## Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Durati on F 0



## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 contain ed 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 f emale in the compartment did not release a first and/or second brood.

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## Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 3.9 8

-- 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 p er 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 wer e 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.

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## Appendix 20 Rep licate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 4 0 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.

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## Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Gen eration (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 contai ned 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.

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## Appendix 20 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 1 5 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 pe r 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 broo d.

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## Appendix 2 1 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 1



## Generation

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Repli cate

Mean

SD

CV(%) 2 Time to 2 nd

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 mean s, 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 on ly one value was available for the treatment group.

-- = The female in the compartment did not release a first and/or second brood.

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## Appendix 2 1 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 1



## Generation (Continue d)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 3 1.00 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 0.0 0

-- 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.

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## Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/ L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 m ale 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 treatm ent group.

-- = The female in the compartment did not release a first and/or second brood.

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## Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

S D

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 c hamber 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, stand ard 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.

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## Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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. M anual 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 treatm ent group.

-- = The fema le in the compartment did not release a first and/or second brood.

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## Appendix 21 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Tim e to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 compartmen t 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 im mature 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 determine d 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.

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## Appendix 2 2 Replicate Mean Number of Offspring in the First and Second Broods and Total P roduced F 0



## Generation

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of O ffspring

Produced 3 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 0 4 0 4 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 0 4 1.6 0 4 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 F 0 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 repli cate mean, SD and CV.

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## Appendix 2 2 Replicate Mean Number of O ffspring in the First and Second Broods and Total Produced F 0



## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 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 0 4 0 4 0

7 6 2 8

B 1 0 4 4.0 0 4 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. Manua l calculations may differ slightly.

3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods foll owing the release of the second brood.

4 Represents a female that never produced a brood thro ughout the F 0 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 repli cate mean, SD a nd CV.

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## Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 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 0 4 3.2 0 4 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 w ere 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 foll owing the release of the second brood.

4 Represents a female that never produced a brood throughout the F 0 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 c alculations of the replicate mean, SD and CV.

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## Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 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 Replica te 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 foll owing the release of the second brood.

4 Represents a female that never produced a brood throughout the F 0 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 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Broo d

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 Replicate Total

Produced

Mean

SD

CV% 2

50 0 A 1 3 2.8 0 4.4 3 39

2 0 4 2.0 0 4 5.6 0 5.6

3 0 4 73.2 0 4 127.2 0 6.5

4 3 13 16 116.7

5 1 2 6

6 6 7 13

7 1 0 1

B 1 0 4 2.7 0 4 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 pa irs 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, incl uding any subsequent broods following the release of the second brood.

4 Represents a female that never produced a brood throughout the F 0 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 an d CV.

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## Appendix 22 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 Re plicate Total

Produced

Mean

SD

CV% 2

1250 A 1 0 4 4.0 0 4 2.7 0 22

2 0 4 4.4 0 4 2.1 0 3.1

3 0 4 109.0 0 4 78.1 0 4.3

4 0 4 0 4 0 136.8

5 2 5 8

6 1 2 4

7 9 1 10

B 1 0 4 1.0 0 4 0.3 0 4

2 1 0.0 0 0.6 1 0 .6

3 0 4 0.0 0 4 173.2 0 0.8

4 0 4 0 4 0 137.7

5 1 1 2

6 1 0 1

7 0 4 0 4 0

C 1 0 4 2.3 0 4 1.0 0 22

2 0 4 1.3 0 4 0.8 0 3.1

3 1 55.9 1 81.6 2 3.8

4 2 1 6 121.1

5 0 4 0 4 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 F 0 generation reproductive phase of the stud y. 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 repli cate mean, SD and CV.

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## Appendix 2 3 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 1



## Generation

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Fema le Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 Replicate Total

Produced

Mean

SD

CV% 2

Negative A 1 0 6 5 0 6 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 Eac h 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 off spring produced includes all offspring produced during the study, including any subsequent broods following the release of th e second brood.

4 The F 1 generation did not produce enough living organisms to initiate the Brood Chamber.

5 Mean, standard deviat ion 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 F 1 generation reproductive phase of the study. The number of offspring prod uced in the first and second

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the repli cate mean, SD and CV.

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## Appendix 2 3 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 1



## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 Replicate Total

Produced

Mean

SD

CV% 2

32 A 1 0 6 1.0 0 6 0.0 0 1

2 1 5 0 5 1 0.2

3 0 6 5 0 6 5 0 0.45

4 0 6 0 6 0 223.6

5 0 6 0 6 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 0 6 4 0 6 3.5 0 19

2 0 6 4.24 0 6 0.71 0 3.8

3 7 106.1 3 20.2 10 5.22

4 1 4 9 137.2

5 0 6 0 6 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 foll owing the release of the second brood.

4 The F 1 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 treatm ent group.

6 Represents a female that never produced a brood throughout the F 1 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 repli cate mean, SD and CV.

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## Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 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 0 6 84.9 0 6 223.6 0 3.74

4 10 0 10 108.9

5 5 0 5

6 3 3 6

7 0 6 0 6 0

C 1 0 6 8.0 0 6 0.0 0 8

2 0 6 5 0 6 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 di ffer slightly.

3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods foll owing the release of the second brood.

4 The F 1 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 treatm ent group.

6 Represents a female that never produced a brood throughout the F 1 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 repli cate mean, SD and CV.

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## Appendix 23 Replicate Mean Number of O ffspring in the First and Second Broods and Total Produced F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 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 0 6 1.0 0 6 1.0 0 4

2 1 5 1 5 4 1.3

3 0 6 5 0 6 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 slight ly.

3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods foll owing the release of the second brood.

4 The F 1 generation did not produce enough living organisms to initiate the Brood Cham ber.

5 Mean, standard deviation and/or CV were not determined as there were no values or only one value was available for the treatm ent group.

6 Represents a female that never produced a brood throughout the F 1 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 repli cate mean, SD and CV.

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## Appendix 23 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 1 

## Generation (Continued)

Nominal Test

Concentration

(ng/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reprodu cing

Female Replicate

Mean

SD

CV% 2 Total Number

of Offspring

Produced 3 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 a ll offspring produced during the study, including any subsequent broods following the release of the second brood.

4 The F 1 generation did not produce enough living organisms to initiate the Brood Chamber.

5 Mean, standard deviation and/or CV were not det ermined 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 F 1 generation reproductive phase of the study. The number of offspring produced in the first and seco nd

brood was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the repli cate mean, SD and CV.

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## Appendix 2 4 Replicate Mean Percent of Males Sex Ratio and Percent of

Females Producing Offspring F 0 

## Gene ration

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 2 5 Replicate Mean Percent of Males Sex Ratio and Percent of Females

Producing Offspring F 1 

## 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 F 1 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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## SUM MARY

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

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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, F 0 

### generation) that were grown to maturity. Their

offspring (F 1 

### generation) were collected, which in turn were

grown to maturity, and their offspring (F 2 

### 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 h ours 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 (F 0 

### )

• 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 bro od 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 i n each replicate population

• Sex ratio

• Percentage of females that are reproductively active

First Filial Generation (F 1 

### )

• 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

you ng 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 a re reproductively active

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RESULTS: The mean number of offspring in the secon d brood per reproducing

female, Day 7 total mysid lengths and Day 14 female dry weights in

the F 0 

### generation were the most sensitive endpoints in the mysid two

generation exposur e 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 th ese

reproductive and growth endpoint s , 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 h as 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 respon se 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 cy cle) 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 d isrupting 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 tes t 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 vali dation

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

- laborat ory 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

- Octylphe nol) 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

- laborator y 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 b eing 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

manage ment, 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 Univer sity 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 labor atories’ 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 procedur es 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 con siders 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 o ther 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 b rood 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 prio r 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 sourc e 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 Fi sher

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 (F 0



### and F 1 

### ) 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 (post

hydration) 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 subs tance 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/grow th 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

fo od 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. Pe riodic 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 pest icides, 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 avo id 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,

Delawa re. 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 take n 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

refractomete r 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 throughou t 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 F 0 

### generation, and in alternating replicates daily thereafter in both the F 0 

### and

F 1 

### generations, using a ha nd

- 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 te st

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

r eplicates daily thereafter. Salinity was measured using a VitalSine refractometer.

10.6.3 Lighting

Ambient laboratory light was used to illuminate the test systems. F luorescent 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 F 0 

### generation a nd 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 lev els

above 60% saturation. Dissolved oxygen was measured in each test vessel at the beginning of

the test for the F 0 

### generation, and in alternating replicates daily thereafter for both generations

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during the test. Dissolved oxygen was measured using a The rmo 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 F 0



### 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 replicat e 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 los s 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 chamb er (G):

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Treatment Level

– Replicate Vessel

– Chamber Type (R or G)

– Generation (F 0 

### or F 1 

### )

– 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 d ishes 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 identi fy brood

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

– Replicate

– Brood

Chamber (BC)

– Brood Chamber Number (1

- 9)

– Generation (F 0 

### or F 1 

### )

– 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 F 0 

### and F 1 

### mysids

were all first examined for reproductive characteristics starting on Day 14. I n 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 sec ond 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

consider ed 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 r elease of the second

brood.

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10.8.1 Exposed First Generation Mysids (F 0 

### )

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

eac h 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 chamber s were used to complete male:female pairings in the event that sex

ratio in the reproduction chamber was skewed, preventing obtaining seven pairs. The F 0



### 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 (F 1 

### )

The F 1 

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

(< 24 hours old) per day produced by the F 0 

### generation. F 1 

### generation mysi ds were assigned to

reproduction chambers within the same treatment group, for each of the F 0 

### exposure conditions

(e.g., juveniles from Replicate A of the F 0 

### generation vessel were assigned to Replicate A of the

F 1 

### generation vessel). Mixing of young rele ased on the same day and across replicate chambers

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

siblings. An additional F 1 

### generation growth chamber was initiated with up to 15 newly released

mysid juveniles (wh en 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, F 1 

### 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 F 1 

### generation brood chambers within each replicate (with a

maximum of 7 m/f pairs possible per replicate). Individuals in the F 1 

### generation reproduction

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

determination within the F 1 

### 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:fema le pairings in the event that sex ratio in the reproduction chamber was skewed,

preventing obtaining seven pairs. The F 1 

### exposure continued until chamber Day 35 (i.e., the

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



## 10.9 Diluter

The tox icity 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

wat er 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 F 0 

### and F 1 

### 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 F 0 

### generation when the checklist was only done once.



## 10.10 Test Subst ance

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.: 0 8118jhv

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 ambi ent 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 satu rator 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 remo ve

contaminants. The s tock 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 dissol ved 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 wo ol, 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 stab le 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 ) . Th e

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. Th is diluted stock solution was held under ambient co nditions, 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 trea tment groups at target

rates of 0.13 to 5.0 mL/min, and was m ixed with filtered saltw ater 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 w ere covered with Plexiglas to prevent volatilization . The negative control received

dilution water only. Both the F 0 

### and F 1 

### generation stock solutions were delivered from the same

diluted stock solution in the reservoir tank.

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## 10.14 Analytical Method and Veri fication

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 alternati ng 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 F 0



### 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 wer e 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 saltw ater 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 usin g an Agilent Model

5890 Gas Chromat ograph 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 pro vided i n 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 μ g/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 genera ted using the peak area responses versus the respective concentrations o f 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 re gression 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 μ g/ L, calculated as the product of the lowest calibration standard analyzed

(2.50 μ g/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 μ g/L usin g a stock solution of 4

- tert

octylphenol 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 9 4 .4 to 133% of nominal concen trations ( Appendix 29.5 ). The measured

concentrations for the matrix fortification samples for the F

- 1 generation ranged from 106 to

12 6 % of nominal concentrations ( Appendix 29.6 ).

A representati ve calibration curve is presented i n Appendix 29.7 . Representative chromatograms

of low and high

- level calibration standards are p resented in Appendices 29.8 and 29.9

,

res pectively. 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 F 0 

### mysid began when all of the <24 hour old

individuals were indiscriminately transferred into the test vessels from the stock cultur es.



## 11.1 Exposed First Generation Mysids (F 0 

## )

11.1.1 F 0 

### 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 gro wth 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 intermed iate 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 exposur e 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 F 0 

### 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, separa ted 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 alumi num 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 neares t 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 F 0 

### Reproductive Chambers

When developing emb ryos 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 expos ure

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 sep arate 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 F 0 

### exposure

terminated at least seven days after the median day of release of the second brood (F 0 

### ”) in the

negative control treatment group.

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Upon termination, each surviving F 0 

### 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 trans ferred 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.

Offspri ng of the F 0 

### 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 F 0 

### 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 (F 1 

## )

11.2.1 F 1 

### Exposure Initiation

Groups of <24

- hour

- old offspring (F 1 

### ) from either the first (F 1 

### ’) or secon d brood (F 1 

### ”) of the F 0



### 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

F 1 

### 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 avail able, were collected from the negative

control group from the F 1 

### 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 measu rements, 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

weighi ng to the nearest 0.01 mg on an analytical balance.

11.2.2 F 1 

### 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 app roximately 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 mysi d

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 F 1 

### Reproductive Chambers

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

reproduction chambers (Day 14 of the F 1 

### 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 tes t 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 ret ention 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 F 1 

### exposure was terminated 35 days following initiation

of the final F 1 

### replicate (Day 42 of F 1 

### exposure).

Upon termination, each surviving F 1 

### 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 a t 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 Cleanin g

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 F 1 

### 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 F 0 

### initiatio n to the F 1



### 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 i nability 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 f or 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 (F 0 

### , F 1 

### )

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

7, 14 and termination for F 0 

### and F 1, 

### when possible)

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

young in the first brood (F 0 

### , F 1, 

### when possible)

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

of young in the second brood (F 0 

### , F 1, 

### when possible)

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

brood (F 0 

### , F 1, 

### when possible)

• Number of young per female in each of the first two broods (F 0 

### , F 1, 

### when

possible)

• Total num ber of young in the first two broods in each replicate population (F 0 

### , F 1, 

### when possible)

• Sex ratio (F 0 

### , F 1 

### )

• Percentage of females that are reproductively active (F 0 

### , F 1 

### )



## 13.0 STATISTICAL METHODS

13.1 Endpoints

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

below:

• Survival, (F 0 

### , F 1 

### )

• Growth

- length and dry weight, (Days 0, 7, 14 and termination for F 0 

### and F 1 

### )

• Time to maturation, (F 0 

### , F 1 

### )

• Time to first brood release, (F 0 

### , F 1 

### )

• Time to second brood release, (F 0 

### , F 1 

### )

• Interbrood duration, (F 0 

### , F 1 

### )

• Number of young per female in each of the first two broods, (F 0 

### , F 1 

### )

• Total number of young in the first two broods in each replicate population (F 0 

### , F 1 

### )

• Sex ratio, (F 0 

### , F 1 

### )

• Percentage of females that are reproductively active

, (F 0 

### , F 1 

### )



## 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 te st 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 i dentified

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

- Ob served

- 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 shows no statistically significant difference from the control). All statistical analysis was

conducted using SAS software a nd a personal computer.



## 14.0 RESULTS

Measurements of temperature, dissolved oxygen, pH, light intensity and salinity in the test

chambers are summarized for F 0 

### in Table 20 and for F 1 

### 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 ≥ 9 3% of saturation (6.7 mg/L)

throughout the exposure period for both the F 0 

### and F 1 

### 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 F 0 

### g eneration and 152 lux for the F 1 

### 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 F 0



### generation and 143 to 235 lux for the F 1 

### generation.



## 14.1 In

- life Analytical Results

14.1.1 F 0 

### Generation

Test solutions were premixed in mixing chambers and then delivered into tanks. Samples used

for analytical measurements to determine mean measured concentrations were onl y 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 conc entrations 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 F 0 

### generation were based on the nominal concentrations.

14.1.2 F 1 

### 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

- octylphe nol 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 i n

- 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 concen trations,

respectively ( Figure 6 ). The results of the F 1 

### generation were based on the nominal

concentrations.



## 14.2 Biological Results

Table 27 through Table 32 present endpoint su mmaries 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 F 0 

### generation are discussed before the F 1 

### generation. The F 1 

### generation results are limited to

the negative control, since there were dramatic and apparent affects in reproduction in all

4

- tert

- octylphenol test conc entrations resulting in little or no reproduction.



## 14.3 Survival

14.3.1 Survival from Day 0 to Day 7

A summary of survival of F 0 

### juvenile mysids from test initiation to the Day 7 growth

sub

- sampling interval is located in Table 27 . Survi val 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 d uring 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 F 0 

### 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 F 0



### A summary of survival of F 0 

### 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, 0.6 4, 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 F 0 

### generation and F isher’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 grou ps. 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 F 0 

### 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 F 0 

### 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 me asurements 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 To tal Length from F 0 

### 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 F 0 

### 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 F 0



### generation were 1.92 mm and 0.045 mg, respectively.

14.4.2 D ay 7

- Dry Weight and Total Length from F 0 

### Generation

Summaries of the Day 7 total lengths and dry weights of surviving juvenile mysids in the

treatment and control groups for the F 0 

### generation are presented in Table 29 , with rep licate 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 F 0 

### 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 co ntrols 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 F 0 

### 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 F 0 

### 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 measure ments.

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14.4.3.1 Day 14

– Male Dry Weight and Total Length from F 0 

### Generation

The mean total length of the Day 14 male mysids in the F 0 

### 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 m m

,

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 ma le mysids from the F 0 

### 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 F 0 

### Genera tion

The mean total length of the Day 14 female mysids in the F 0 

### 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 f emales 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 F 0 

### 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 neg ative 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 F 0 

### (Day 46) Generation

Summaries of total lengths and dry weights of surviving m ale and female mysids in the treatment

and control groups at test termination for the F 0 

### 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 F 0 

### (Day 46) Generation

The mean total length of the Day 46 male mysids in the F 0 

### generation in the negative control, 0.26,

0.64, 1.6, 4.0 and 10 μg/L treat ment 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

d ecreases in mean total length or dry weight in males from the F 0 

### 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 F 0 

### (Day 46) Generation

The mean total length of the Day 46 female mysids in the F 0 

### 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 th e 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 F 0 

### 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 F 0 

### 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 Appendi x 40

.

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14.5.1 Time to Maturation, First Brood, Second Brood and Interbrood Duration from F 0



### 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 trea tment group in the F 0 

### 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 F 0 

### generation

are pre sented 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 F 0 

### Generation

Summaries of the mean number of offspring in the first brood per reproductive female in the

negative control and treatment groups for the F 0 

### generation are presented in Table 32 . The

replicate mean number of offsp ring in the first brood is presented in Appendix 39

.

The mean number of offspring in the first brood for the F 0 

### 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 F 0 

### generation in

any of the treatment groups.

14.5.3 Number of Offspring in the Second Brood from F 0 

### Generation

Summaries of the mean number of offspring in the second brood per reproductive female in the

negative control and treatment groups for the F 0 

### 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 F 0 

### generation in the negative control

and the 0.26, 0.64, 1.6, 4.0 and 10 μg/L tr eatment 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 treatm ent groups ( p ≤ 0.05) , which appeared to be treatment related.

14.5.4 Total Number of Offspring from F 0 

### Generation

Summaries of the mean total number of offspring produced in the study in the negative control

and treatment groups for the F 0 

### generation are pres ented in Table 32 . The replicate mean total

number of offspring produced in the study for the F 0 

### generation is presented in Appendix 39

.

The mean total number of offspring produced in the study for th e F 0 

### 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 F 0 

### 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 F 0 

### generation are presented i n Table 32 . The

replicate mean percent male sex ratio in the study for the F 0 

### generation is presented in

Appendix 40

.

The mean percent of males present in the study for the F 0 

### 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 mysi ds from the F 0 

### generation in any of the treatment

groups.

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14.5.6 Percent of Reproducing Females from F 0 

### Generation

Summaries of the mean percent of reproducing females in the study in the negative control and

treatment groups for the F 0 

### generation are presente d in Table 32 . The replicate mean percent of

reproducing females in the study for the F 0 

### generation is presented in Appendix 40

.

The mean percent of reproducing females in the study for the F 0 

### generat ion 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 youn g in any of the treatment groups when compared to the

negative control.



## 14.6 Summary of Survival, Growth Measurements and Reproduction Endpoints for

the F 1 

## Generation

There appeared to be treatment related effects on reproduction in all treatment groups that g reatly

reduced the number of viable young that could be used to initiate the F 1 

### generation. The mysids

in the F 0 

### generation negative control produced enough live young to initiate all three F 1



### generation replicate reproductive retention chambers, as well as providing enough organisms to

capture Day 0, 7, 14 and termination growth measurements, for the F 1 

### 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 F 1 

### generation mysids. Summaries of data collected from the negative

control mysids in the F 1 

### generation are presented in Appendix 41

.

14.6.1 Summary of Survival for the F 1 

### Generation

A summary of survival of F 1 

### negative control juvenile mysids from replicate chamber initiation

to the Day 7 growth sub

- sampling interv al, 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 exclude d 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 F 1 

### Generation

Summaries of the mean dry weight and total b ody length measurements in the negative control

for the F 1 

### generation mysids are presented in Appendix 41.2 to Appendix 41.3

. Day 0 and 7

mean dry weight measurements of F 1 

### 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 F 1 

### 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 F 1 

### Generation

Summaries of all r eproductive endpoints evaluated in the negative control for the F 1 

### generation

mysids are presented in Appendix 41.4 . As with the F 0 

### generation, all mysids were sexually

identified and therefore mature on Replicate Day 14 for eac h 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 wel l 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 F 0 

### generation, it is important to note that the F 1 

### generation

w as conducted for 35 replicate days versus the F 0 

### generation of 46 days. The shorter number of

days in the F 1 

### generation also may help explain why 61.7% of females produced young in the F 1



### generation versus the 76.2% in the F 0 

### generation. The lower percen t of females producing young

in the F 1 

### 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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F 1 

### generat ion was 65.6%, about 15% higher than the F 0 

### generation. Overall, reproduction was

good in the negative control replicates in the F 1 

### generation.



## 15.0 PERFORMANCE CRITERIA

The following criteria were for judging test data acceptability:



## 15.1 Water quality characteri stics 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 co ntrol 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 F 0 

## and F 1 

## controls must produce young.

More than 75% of females in the F 0 

### generations produced young in the control. In the F 1



### generation, 61.7% of females produced young. The lower percent of females producing young

in the F 1 

### 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 t he F 0 

### and F 1 

### 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 F 0 

### and F 1 

### generations. The test was conducted under

flow

- through conditions for 46 and 35 days for the F 0 

### and F 1 

### generations, respectively, and the

mysids were evaluated for survival, growth and reproductive endpoints. The most sensitive

biological endpoints were reproduction and growth in the F 0 

### 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 F 0



### 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 F 0 

### genera tion, was <0.26 μg/L. The LOEC was 0.26 μg/L and the MATC could

not be calculated.

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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 differ ences between treatment means when several dose levels

are compared with a zero dose control. Biometrics 27: 103

- 117.

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Williams, D.A. 1972. A comparison of several dose levels with a zero control. Biometrics 28:

519

- 531.

Verslycke TA, Fockedey N, McKenne y 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.

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## Table 20

. Means and Ranges of Water Qu ality Measurements Taken During

the F 0 

## Generation Exposure to 4

- tert

- octylphenol

Nominal

Concentration

(μg/L) Mean ± SD and Range of Measured Parameters

Temperature 1

(°C) DO 2

(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 27 o C, measured to the nearest 1 o C.

2 A dissolved oxygen concentration of 4.3 mg/L repr esents 60% saturation at 26oC in saltwater with a salinity of 20‰

.

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## Table 2 1

. Means and Ranges of Water Quality Measurements Taken During

the F 1 

## Generation Exposure to 4

- tert

- octylphenol

Nominal

Concentration

(μg/L) Mean ± SD and Range of Measured Param eters

Temperature 1

(°C) DO 2

(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 27 o C, measured to the nearest 1 o C.

2 A dissolved oxygen concentration of 4.3 mg/L represents 60% saturation at 26oC in saltwater with a salinity of 20‰

.

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## Table 22

. Measured Concentrations of 4

- tert

- octylphenol in Pretest Solution

Samples for the F 0 

## Generation

Nominal Test

Concentration

( μ g/ L) Sample

Number

(185A

- 102

- 2

- ) Sampling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2

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 μ g/L, calculated as the product of the concentration of the lowest

calibration standard (2.50 μ g/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.

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## Table 23

. Measured Concentrations of 4

- tert

- octylphenol for the F 0 

## Generation in Stock Solution Samples

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 2

- ) M easured

Concentration

( μ g/L) 1 Percent

of

Nominal 2

11000 S

- 1 9858 89.6

1 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.

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## Table 24

. Measured Concentrations of 4

- tert

- octylphenol in Saltwa ter Samples

for the F 0 

## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 2A

- ) Sampling

Time

(Days) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Concentration

( μ g/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

13 2

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 μ g/L, calculated as the product of the concentration of the lowest calibration

standard (2.50 μ g/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.

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## Table 24

. Measured Concentrations of 4

- tert

- octylphenol in Saltwater

Samples for the F 0 

## Generation (Continued)

Nominal Test

Concentration

( μ g/ L) Sample

Number

(185A

- 102

- 2A

- ) Sampling

Time

(Days) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Concentration

( μ g/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 μ g/L, calculated as the pr oduct of the concentration of the lowest calibration

standard (2.50 μ g/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.

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## Table 25

. Measured Concentrations of 4

- tert

- octylphenol in Pretest Solution

Samples for the F 1 

## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 2A

- ) Sampling

Time

(Day) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2

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 μ g/L, calculated as the product of the concentration of the lowest

calibration standard (2.50 μ g 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.

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## Table 26

. Measu red Concentrations of 4

- tert

- octylphenol in Saltwater Samples

for the F 1 

## Generation

Nominal Test

Concentration

( μ g/L) Sample

Number

(185A

- 102

- 2A

- ) Sampling

Time

(Days) Measured

Concentration

( μ g/L) 1 Percent

of

Nominal 2 Mean

Measured

Concentration

( μ g/ 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 μ g /L, calculated as the product of the concentration of the lowest calibration

standard (2.50 μ g/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 diffe r slightly.

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## Table 27

. Summary of F 0 

## 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 1 4 ± 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 co mparison to the negative control using Fisher’s Exact test

( p > 0.05).

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## 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 0.0 45 ± 0.010

(22.8)

n = 49

1 Mean, standard deviation, and coefficient of variation was calculated using Excel 2010 . Manual calculations may differ

slightly.

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## Table 29

. Summary of F 0 

## Generation Day 7 Growth Measurements of

Saltwater Mysids Exposed to 4

- t ert

- octylphenol

Nominal

Concentration

(μg/L) Growth Parameters on Day 7 1,2

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 an d

control groups presented.

2 C oefficient of V ariation was calculated using Excel 2010 . Manual calculations may differ slightly.

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## Table 30

. Summary of F 0 

## 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 treatmen t

and control groups presented.

2 C oefficient of V ariation was calculated using Excel 2010 . Manual calculations may di ffer slightly.

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## Table 31

. Summary of F 0 

## 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) ± S D

(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

(4.2 2) 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 ± 0.51 3

(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, sta tistics were all based on replicate means. Therefore, n = 3 for all treatment

and control groups presented.

3 C oefficient of V ariation was calculated using Excel 2010 . Manual calculations may differ slightly.

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## Table 32

. Summary of F 0 

## Generation Reprodu ction of Saltwater Mysids Exposed to 4

- tert

- octylphenol

Nominal

Conc.

(μg/L) Mean Time

to

Maturation

Days ± SD

(CV%) 1 Mean

Time to 1 st

Brood

Days ± SD

(CV%) 1 Mean

Time to 2 nd

Brood

Days ± SD

(CV%) 1 Mean

Interbrood

Duration

Days ± SD

(CV%) 1 Mean Number

of Offspring in

1 st Brood Per

Female ± SD

(CV%) 1 Mean Number

of Offspring in

2 nd 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 ± 7.5 4

(20.76) 9.4 ± 0.5 2

(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 ± 1.3 0*

(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 Dun nett’s test ( p ≤ 0.05).

1 Unless otherwise noted, n = 3, the number of replicate s , 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 female s 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.

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## Figure 4

. Mysid Two

- Generation Test Method Schematic

Source: Wildlife International, Ltd. Protocol No.: 1 85/021511/MYS

- 2GeN/SUB185; Page 44.

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## Figure 5

. F 0 

## Generation Analytical Recoveries Over Time

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## Figure 6

. F 1 

## Generation Analytical Recoveries Over Time

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## Appendix 2 6

. Dilution Water Chemistry Paramet ers 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

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## Appendix 2 7

. Analyses of Pesticides, Organics and Metals in Wildlife

International, Ltd. Saltwate r

Pesticides and Organics 1

Component Measured Concentration

(μg/L) Component Measured Concentration

(μg/L)

Aldrin < 0.0097 Gamma BHC

– 4

- tert

octylphenol < 0.0097

Alpha BHC < 0.0097 Gamma Chlordane < 0.019

Alpha Chlordane < 0.0097 Guthion (Azin phos

- 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 Methoxych lor < 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.



### .

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## Appendix 2 7

. An alyses 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

Ch romium < 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.

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## 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 P CB

- 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 T OTAL PCBs < 19

Famphur < 75 Phorate < 75

Fensulfothion < 220 Ronnel < 75

Fenthion < 75 Stirophos < 75

Gamma BHC

– 4

- tert

octylphenol < 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.

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## Appendix 28

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

Sample (Continued)

Metal s 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 (soli d) 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 Thall ium < 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.

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## Appendix 29

. The Analysis of 4

- Tert

- Octylphenol in Saltwater

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## Appe ndix 29 .1 Method Flowchart for the Analysis of 4

- Tert

Octylphenol 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 vent ing) 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.

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## 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 μ m 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 μ L (Splitless)

CARRIER GAS: Helium

HEAD PRESSURE: 12 psi

4

- TERT

- OCTYLPHENOL PEAK

RETENTION TIME: Approximately 7.2 minutes

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## Appendix 29 .3 Analytical Stocks and Stand ards 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 metha nol. 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. Calibra tion 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

( μ L) Final

Volume

(mL) Standard

Concentration

( μ g/L)

0.010 0

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

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## 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

nomi nal concentration of 0.64 μ g/L, were calculated using the following equations:

Concentration of 4

- tert

- octylphenol in sample ( μ g/L) = Peak Area

- Y

- intercept

Slope X Dilution

Factor

Percent of nominal concentration = measured concent ration in sample ( μ g/L )

nominal concentration in sample ( μ g/L ) X 100

Peak Area = 20486

Y

- intercept =

- 2777.52355

Slope = 2379.83269

Dilution factor = 0.0600

Concentration of 4

- tert

- octylphenol in sample ( μ g/L) = 20486 + 2777.52355

2379.83269 X 0.0600

Concentration of 4

- tert

- octylphenol in sample ( μ g/L) = 0.587

Percent of nominal concentration = 0.587 μ g/L

0.64 μ g/L X 100

Percent of nominal concentration = 91.6%

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## 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

Recovery 2 Fortified

( μ g/L) Measured

( μ g/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 Fortificat ion

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

< LO Q

< 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 μ g/L, calculated as the product of the concent ration of the lowest

calibration standard (2.50 μ g/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.

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## Appendix 29 .5 Quality Cont rol Samples of 4

- Tert

- Octylphenol in

Saltwater for the F 0 

## Generation (Continued)

Sample

Number

(185A

- 102

- 2A

- ) Sample

Type Concentration of 4

- Tert Octylphenol

Percent

Recovery 2 Fortified

( μ g/L) Measured

( μ g/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

2.2 0

10.7 133

125

113

110

111

117

116

113

124

117

113

120

110

107

Mean 3

=

Standard Deviation =

CV = 111

9.27

8.35%

1 The limit of quantitation (LOQ) was 0.125 μ g/L, calculated as the product of the concentration of the lowest

calibration standard (2 .50 μ g/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.

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## Appendix 29 .6 Quality Control Sample s of 4

- Tert

- Octylphenol in

Saltwater for the F1 Generation

Sample

Number

(185A

- 102

- 2A

- ) Sample

Type Concentration of 4

- Tert Octylphenol

Percent

Recovery 2 Fortified

( μ g/L) Measured

( μ g/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

0.2 50

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

Mean 3

=

Standard Deviation =

CV = 113

7.84

6.94%

1 The limit of quantitation (LOQ) w as 0.125 μ g/L, calculated as the product of the concentration of the lowest

calibration standard (2.50 μ g/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 calculati ons may differ slightly.

3 Based on exposure samples only.

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## Appendix 29 .7 Representative Calibration Curve for 4

- Tert

Octylphenol

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; R 2 

### = 0.99390

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## Appendix 29 .8 Representati ve Chromatogram of a Low

- Level 4

-

Tert

- Octylphenol Calibration Standard

Nominal concentration: 2.50 μ g/L.

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## Appendix 29 .9 Representative Chromatogram of a High

- Level 4

-

Tert

- Octylphenol Calibration Standard

Nominal concentration: 20.0 μ g/L.

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## A ppendi x 29 .10 Representative Chromatogram of a Matrix Blank

Sample number 185A

- 102

- 2A

- MAB

- 1. The arrow indicates the approximate retention time of 4

- tert

octylphenol.

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## Appendix 29 .11 Representative Chromatogram of a Matrix

Fortification

Sample nu mber 185A

- 102

- 2A

- MAS

- 1; 0.250 μ g/L, nominal concentration.

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## Appendix 29 .12 Representative Chromatogram of a Test Sample

Sample number 185A

- 102

- 2A

- 3; 0.64 μ g/L, nominal concentration.

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## Appendix 3 0 Daily Percent Survival of Saltwater Mysids Expos ed to 4

- Tert

- O ctylphenol

- F 0 

## 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

15 1 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.

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## Appendix 3 0 Daily Percent Survival of Saltwater Mysids E xposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 0 10 0 9 0 7 100

16 0 1 0 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 nu mber of organisms used to initiate the adult phase of the study.

2 One male mysid was determined to be female on Day 44.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 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 th e study.

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## Appendix 30 Daily Percent Survival of Salt water Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 0 11 0 11 0 11 10 0

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 oth erwise indicated, the number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 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

3 1 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 th e study.

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## Appendix 30 Daily Pe rcent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 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.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 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 th e study.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## Ge neration

(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

15 1 0 12 0 11 0 10 100

16 0 12 0 11 0 10 100

17 0 12 0 11 0 10 1 00

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 1 0 100

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 i s also the number of organisms used to initiate the adult phase of the study.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## Generation

(Continued)

Day of

Test 4.0 μg/L

– Males

Replicate A Replicate B Repli cate C Percent Survival

(%) No. Dead No. Alive No. Dead No. Alive No. Dead No. Alive

15 1 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 th e study.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylp henol

- F 0 

## 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

15 1 0 10 0 10 0 11 100

16 1 9 0 10 0 11 97

17 0 9 0 1 0 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 numbe r 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.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## Generation

(Conti nued)

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

15 1 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 th e study.

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## Appendix 30 Daily Percent Survival of Saltwater Mysids Exposed to 4

- Tert

- Octylphenol

- F 0 

## 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

15 1 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, t he number alive on Day 15 is also the number of organisms used to initiate the adult phase of the study.

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## Appendix 3 1 Replicate Mean Growth Measurements for the F 0 

## Generation

Day 0 Culture Mysids

Pan# Mysid# Total Body

Length (mm) Dry Weight of

Mysids P er 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

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## Appendix 3 1 Replicate Mean Growth Measurements for the F0 Generation Day

0 Culture My sids (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

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## Appen dix 3 2 Replicate Mean Total Body Lengths for the F 0 

## 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 calcul ations may differ slightly.

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## Appendix 3 2 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

5.0 0 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.

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## 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.

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## Appendix 3 3 Replicate Mean Dry Weights for the F 0 

## Generation Day 7 Mysids

Nominal Test

Concentration

(μg/L) Dry Weight (mg) 1

Replicate A Replicate B Repl icate 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 mea surements 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 calculat ions may differ slightly.

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## Appendix 3 3 Replicate Mean Dry Weights for the F0 Gen eration 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 0.3 2 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.

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## Appendix 33 Replicate Mean Dry Weights for the F0 Gen eration Day 7 Mysids

(Continued)

Nominal Test

Concentra tion

(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 w ere calculated using Excel 2010. Manual calculations may differ slightly.

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## Appendix 3 4 Replicate Mean Body Lengths for Male and Female F 0 

## Generation

Day 14 Mysids

Nominal Test

Concentration

(μg/L) Total Body Lengths (mm)

Replicate A Replicate B Repl icate 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 Me an 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 sligh tly.

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## Appendix 3 4 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 M ean 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.: 0.20 4 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.

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## 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 D ev.: 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.

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## Appendix 3 5 Replicate Mean Dry Weights for Male and Female F 0 

## 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 1 6.0 11.2 14.5

1 Replicate means were calculated using Excel 2010. Manual calculations may differ slightly.

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## Appendix 3 5 Replicate Mean Dry Weights for Male and Female F 0 

## 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.

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## Appendix 35 Replicate Mean Dry Weights for Male and Female F 0 

## 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.

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## Appendix 3 6 Replicate Mean Body Lengths for Male and Female F 0 

## Generation Day 46 Mysids

Nominal Test

Concentration

(μg/L) To tal 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.

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## Appendix 3 6 Replicate Mean Body Lengths for Male and Female F 0 

## 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. Ma nual calculations may differ slightly.

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## Appendix 36 Replicate Mean Body Lengths for Male and Female F 0 

## 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

Numb er 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.

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## Appendix 3 7 Replicate Mean Dry Weight s for Male and Female F 0 

## 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.

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## Appendix 3 7 Replicate Mean Dry Weights for Male and Female F 0 

## Generation

Da y 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.

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## Appendix 37 Replicate Mean Dry Weights for Male and Female F 0 

## Generation

Day 46 Mysids (Continued)

Nominal Test

Concentration

(μg/L) Dry Weig hts (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 calculatio ns may differ slightly.

2 This measurement was suspect, and was therefore excluded from statistical analysis.

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## Appendix 3 8 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation

Nominal Test

Concentration

(μg/ L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 m ale/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.

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## Appendix 3 8 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation (Continued )

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

S D

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 3 0.81

-- - 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). Broo d 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 relea se a first and/or second brood.

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## Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation (Continued )

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mea n

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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). Broo d 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 relea se a first and/or second brood.

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## Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation (Continued )

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mea n

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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. Ma nual 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 r elease a first and/or second brood.

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## Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation (Continued )

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 calculatio ns 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.

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## Appendix 38 Replicate Mean Time to Maturation, First Brood, Second Brood and Interbrood Duration F 0



## Generation (Continued )

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Time to

Maturation

(Day) Replicate

Mean

SD

CV(%) 2 Time to

1 st Brood

(Day) Replicate

Mean

SD

CV(%) 2 Time to 2 nd

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 3 2 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 releas e a first and/or second brood.

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## Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0



## Generation

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Fem ale Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total

Number of

Offspring

Produced 3 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 1 6.94

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 20 1 0. Manual calculations may differ slightly.

3 The total number of offspring produced includes all offspring produced during the study, in cluding 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 F 0 g eneration 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 th e replicate mean, SD and CV.

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## Append ix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0



## Generation (Continued)

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 N umber of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total

Number of

Offspring

Produced 3 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 Repl icate means, standard deviation and CV were calculated using Excel 20 1 0. Manual calculations may differ slightly.

3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods foll owing the rele ase 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 F 0 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 th e replicate mean, SD and CV.

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## Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0



## Generation (Continued)

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total

Number of

Offspring

Produced 3 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 foll owing the release of the second brood. This total

was the n 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 F 0 generation reproductive phase of the study. The number of offspring produced in the first an d second brood

was based on the number of reproducing females. Therefore, this number was excluded from the calculations of the replicate m ean, SD and CV.

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## Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0



## Generation (Continued)

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total

Number o f

Offspring

Produced 3 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 calculati ons may differ slightly.

3 The total number of offspring produced includes all offspring produced during the study, including any subsequent broods foll owing 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 F 0 generation reproductive phase of the study. The number of offspring produced in the first and second brood was

based on the number of repr oducing females. Therefore, this number was excluded from the calculations of the replicate mean, SD and CV.

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## Appendix 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0



## Generation (Continued)

Nominal Test

Concentr ation

(μg/L) Replicate Brood

Chamber 1 Number of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total

Number of

Offspring

Produced 3 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 numb er of offspring produced includes all offspring produced during the study, including any subsequent broods following the rele ase of the second brood. This total was

then based on the total number of females present at pairing in the study (7 for each repl icate)

4 Represents a female that never produced a brood throughout the F 0 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 39 Replicate Mean Number of Offspring in the First and Second Broods and Total Produced F 0



## Generation (Continued)

Nominal Test

Concentration

(μg/L) Replicate Brood

Chamber 1 Num ber of

Offspring in 1 st

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Number of

Offspring in 2 nd

Brood per

Reproducing

Female Replicate

Mean

SD

CV% 2 Total

Number of

Offspring

Produced 3 Replicate Total Produced

Per Number of Females at

Pairing Mean

S D

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 of fspring 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 F 0 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 th e replicate mean, SD and CV.

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## Appendix 4 0 Replicate Mean Percent of Males Sex Ratio and Percent of

Females Producing Offspring F 0 

## 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

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## Appendix 4 1 F 1 

## Summary Tables

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## Appendix 4 1 .1 Summary of F 1 

## Generation Survival of Saltwater

Mysids Exposed to 4

- tert

- octylphenol

Nominal

Concentration

(μg/L) Mean Percent Juvenile

Survival to

Len gths

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.

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## Appendix 4 1 .2 Summary of Day 0 and 7 F 1 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.

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## Appendix 4 1 .3 Summary of F 1 

## 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 Fema les 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, stan dard deviation, and coefficient of variation was calculated using Excel 2010 . Manual calculations may differ

slightly.

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## Appendix 4 1 .4 Summary of F 1 

## Generation Reproduction of Saltwater Mysids Exposed to 4

- tert

octylphenol

Nominal

Conc.

(μg/L) Mean Ti me

to

Maturation

Days ± SD

(CV%) Mean

Time to 1 st

Brood

Days ± SD

(CV%) Mean

Time to 2 nd

Brood

Days ± SD

(CV%) Mean

Interbrood

Duration

Days ± SD

(CV%) Mean Number

of Offspring in

1 st Brood Per

Female ± SD

(CV%) Mean Number

of Offspring in

2 nd 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 ca lculations may differ slightly.

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## Appendix 4 1 .5 Daily Percent Survival of Saltwater Mysids Exposed to 4

- tert

- octylphenol

– F 1



## 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

15 1 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 th e study.

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## Appendix 4 1 .5 Daily Percent Survival of Saltwater Mysids Exposed to 4

- tert

- octylphenol

– F 1



## 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

15 1 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 th e stu dy.