### Study Title

Lindane and 4-Tert-octylphenol - Two-Ge neration Life-Cycle Toxicity Test with Mysids ( Americamysis bahia )

Smithers Viscient Study Director

Michael R. Lee

Study Completion Date

Revised Draft Version

22 October 2012

Submitted To

RTI International

3040 Cornwallis Road

P.O. Box 12194 Research Triangle Park, North Carolina 27709

Performing Laboratory

Smithers Viscient

790 Main Street Wareham, Massachusetts 02571-1037

Laboratory Project ID

Smithers Viscient Study Nos. 13888.6109 (lindane) 13888.6111 (4-tert-octylphenol) RTI Project No. 0210114.030

EPA Contract No. EP-W-06026

Page 1 of 468

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 2



## STATEMENT OF NO DATA CONFIDENTIALITY CLAIM

No claim of confidentiality, on a ny basis whatsoever, is made for any information contained in

this document. I acknowledge that information not designated as within the scope of FIFRA sec.

10(d)(1)(A), (B), or (C) and which pertains to a register ed or previously registered pesticide is

not entitled to confidential treatment and may be released to the public, subject to the provisions

regarding disclosure to multinationa l entities under FIFRA 10(g).

Company:

Company Agent: Date:

(Typed Name)

(Title) (Signature)

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 3



## GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

The data and report prepared for "Lindane and 4-Tert-octylphe nol - Two-Generation LifeCycle Toxicity Test with Mysids ( Americamysis bahia )" were produced and compiled in

accordance with all pertinent U.S. EPA Good Laboratory Practice Regulations ( 40 CFR,

Part 160 ) with the following exceptions : routine dilution water and food contaminant screening

analyses for pesticides, PCBs and toxic meta ls were conducted using standard U.S. EPA

procedures by GeoLabs, Inc., Braintree, Massachus etts and are considered facility records under

Smithers Viscient’s Standard Operating Pro cedures. Since the an alyses were conducted

following standard validated methods, these ex ceptions had no impact on the study results.

SMITHERS VISCIENT

________________________________________________ ________________________ Michael R. Lee Date Study Director

RTI INTERNATIONAL

________________________________________________ ________________________ Study Sponsor Date

________________________________________________ ________________________
Submitter Date

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## QUALITY ASSURANCE STATEMENT

The study conduct, raw data and report for "Lindane and 4-Tert-octylphenol - TwoGeneration Life-Cycle Toxi city Test with Mysids ( Americamysis bahia )" were inspected by

the Quality Assurance Unit at Smithers Viscient to determine adherence with the Quality

Assurance Project Plan (QAPP), the study plan a nd laboratory standard operating procedures.

Dates of study inspections, in spection type, and dates reporte d to Study Director and to

Management are listed below.

Inspection Inspection Reported to

Date Type Study Director/Management

13888.6109

2/3/11 Protocol review 2/3/11 9/13/11 In-life, F 0 

### termination 9/13/11 10/4/11 In-life, GC sequence table 10/5/11

1/4/12 Method validation data & appendix 1/5/12 2/6 & 2/7/12 Data & draft report 2/9/12 7/3/12 Revised draft report 7/3/12 Final report

13888.6111

2/3/11 Protocol review 2/3/11

4/26/12 In-life, biological observations 4/26/12

TEST RESET

5/15/12 Appendix audit 5/16/12

5/30/12 In-life, biological observations 5/30/12

7/9/12 In-life, F 1 

### termination 7/9/12 9/7, 9/10, 9/11, 9/14 &

9/16/12 Data & draft report 9/16/12

Final report

SMITHERS VISCIENT

________________________________________________ ________________________ Kathleen F. Terrio Date Quality Assurance Auditor

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## KEY STUDY PERSONNEL

The following Smithers Viscient personnel were re sponsible for the conduct of the work and

reporting of the study results.

Michael R. Lee Study Director

Leah Gallant Assistant Biologist

Amanda Schwader Assistant Biologist

Joseph Marini Assistant Biologist

Ryan T. Higgins Assistant Biologist

Nicola A. D'Agostino Biology Technician II

Barbara E. Smith Assistant Chemist

Marjorie E. Dix Senior Research Chemist

Andrea M. Soares Technical Report Writer

Mark A. Cafarella Di rector, Ecotoxicology

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 6



## TABLE OF CONTENTS

Page

STATEMENT OF NO DATA CONFIDENTIALITY CLAIM ......................................................2

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..........................................3

QUALITY ASSURANCE STATEMENT ......................................................................................4

KEY STUDY PERSONNEL ........................................................................................................... 5

TABLE OF CONTENTS ............................................................................................................. ....6

SUMMARY, PART A ............................................................................................................... ....18

1.0 INTRODUCTION ................................................................................................................21

2.0 MATERIALS AND METHODS .........................................................................................22

2.1 QAPP/Study Plan ...................................................................................................22

2.2 Test Substance .......................................................................................................22

2.3 Test Organism ........................................................................................................23

2.4 Feeding ...................................................................................................................24

2.5 Dilution Water .......................................................................................................25

2.6 Test Substance Concentrations ..............................................................................26

2.7 Stock Preparation ...................................................................................................26

2.8 Exposure System ....................................................................................................26

2.8.1 Exposure Vessels .......................................................................................26

2.8.2 Retention Chambers ...................................................................................26

2.8.3 Brood Chambers ........................................................................................27

2.8.4 Diluter System ...........................................................................................27

2.9 Test Conditions ......................................................................................................28

2.10 Exposure Conduct ..................................................................................................28

2.10.1 Exposure Initiation .....................................................................................28

2.10.2 Biological Methods ....................................................................................29

2.10.2.1 Exposed Parental Generation (F 0 

### ) .............................................29

2.10.2.1.1 F 0 

### Exposure Initiation ...............................................................29

2.10.2.1.2 F 0 

### Growth Chambers .................................................................30

2.10.2.1.3 F 0 

### Reproduction Chambers .......................................................31

2.10.2.2 Exposed First Filial Generation (F 1 

### ) .........................................32

2.10.2.2.1 Initiation of F 1 

### Generation .........................................................32

2.10.2.2.2 F 1 

### Growth Chambers .................................................................32

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2.10.2.2.3 F 1 

### Reproduction Chambers .......................................................33

2.10.3 Analytical Measurements ...........................................................................34

2.10.4 Water Quality Measurements ....................................................................35

2.11 Biological Observations .........................................................................................36

3.0 STATISTICAL METHODS ................................................................................................37

3.1 Endpoints ...............................................................................................................37

3.2 Statistical Procedures .............................................................................................38

4.0 PERFORMANCE CRITERIA .............................................................................................39

5.0 RESULTS ....................................................................................................................... ......39

5.1 Water Quality Measurements ................................................................................39

5.2 Analytical Results ..................................................................................................39

5.3 Biological Results ..................................................................................................41

5.3.1 F 0 

### Generation .............................................................................................41

5.3.1.1 F 0 

### Survival .................................................................................41

5.3.1.2 F 0 

### Growth ..................................................................................41

5.3.1.2.1 Day 0 (Exposure Initiation) – F 0 

### Total Body Length and Dry Body Weight ......................................................................42

5.3.1.2.2 Day 7 - F 0 

### Total Body Length and Dry Body Weight ..............42

5.3.1.2.3 Day 14 - F 0 

### Total Body Length and Dry Body Weight ............42

5.3.1.2.4 Day 27 (F 0 

### Termination) - Total Body Length and Dry Body Weight .............................................................................43

5.3.1.3 F 0 

### Time to Maturation ...............................................................44

5.3.1.4 F 0 

### Time to First Brood Release .................................................44

5.3.1.5 F 0 

### Number of Offspri ng in the First Brood ...............................45

5.3.1.6 F 0 

### Time to Second Brood Release ............................................45

5.3.1.7 F 0 

### Number of Offspring in the Second Brood ..........................45

5.3.1.8 F 0 

### Interbrood Duration ..............................................................46

5.3.1.9 F 0 

### Total Number of Offspring in First Two Broods .................46

5.3.1.10 F 0 

### Sex Ratio (Male:Female) ......................................................46

5.3.1.11 F 0 

### Percent of Reproductively Active Females ..........................46

5.3.2 F 1 

### Generation .............................................................................................47

5.3.2.1 F 1 

### Survival .................................................................................47

5.3.2.2 F 1 

### Growth ..................................................................................47

5.3.2.2.1 Day 0 (Exposure Initiation) – F 1 

### Total Body Length and Dry Body Weight ......................................................................47

5.3.2.2.2 Day 7 - F 1 

### Total Body Length and Dry Body Weight ..............48

5.3.2.2.3 Day 14 - F 1 

### Total Body Length and Dry Body Weight ............48

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5.3.2.2.4 Day 27 (Exposure Termination) - Total Body Length and Dry Body Weight ......................................................................49

5.3.2.3 F 1 

### Time to Maturation ...............................................................50

5.3.2.4 F 1 

### Time to First Brood Release .................................................50

5.3.2.5 F 1 

### Number of Offspri ng in the First Brood ...............................51

5.3.2.6 F 1 

### Time to Second Brood Release ............................................51

5.3.2.7 F 1 

### Number of Offspring in the Second Brood ..........................51

5.3.2.8 F 1 

### Interbrood Duration ..............................................................51

5.3.2.9 F 1 

### Total Number of Offspring in First Two Broods .................52

5.3.2.10 F 1 

### Sex Ratio (Male:Female) ......................................................52

5.3.2.11 F 1 

### Percent of Reproductively Active Females ..........................52

6.0 CONCLUSIONS ..................................................................................................................5 3

PROTOCOL DEVIATIONS .........................................................................................................54

REFERENCES .................................................................................................................... ..........55

Table 1. Summary of water quality parameters measured in the exposure solutions during the mysid ( Americamysis bahia ) two-generation

test with lindane. ........................................................................................57

Table 2. Concentrations of lindane measured in the exposure solutions during the mysid ( Americamysis bahia ) two-generation test

. ...............................58

Table 3. Results of the quality control (QC) samples analyzed concurrently with test solution samples during the mysid ( Americamysis bahia ) two-generation test

. ....................................................................................59

Table 4. F 0 

### mysid survival and growth (length and dry weight) during the mysid ( Americamysis bahia ) two-generation test with lindane. ................60

Table 5. F 0 

### mysid time to maturation, time to first brood release, time to second brood release, offspring re leased per female and interbrood duration during the mysid ( Americamysis bahia ) two-generation test

with lindane

. ...............................................................................................62

Table 6. F 0 

### mysid total number of young in the first two broods, sex ratio and percentage of reproductively active females during the mysid ( Americamysis bahia ) two-generation test with lindane

. ...........................63

Table 7. F 1 

### mysid survival and growth (length and dry weight) during the mysid ( Americamysis bahia ) two-generation test with lindane. ................64

Table 8. F 1 

### mysid time to maturation, time to first brood release, time to second brood release, offspring re leased per female and interbrood duration during the mysid ( Americamysis bahia ) two-generation test

with lindane

. ...............................................................................................66

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Table 9. F 1 

### mysid total number of young in the first two broods, sex ratio and percentage of reproductively active females during the mysid ( Americamysis bahia ) two-generation test with lindane

. ...........................67

Table 10. Summary of F 0 

### and F 1 

### endpoints, No-Observed-Effect Concentration (NOEC) and Lowest -Observed-Effect Concentration (LOEC) values determined during the mysid ( Americamysis bahia ) two-generation test with lindane

. ...............................................................68

Figure 1. Schematic of biological methods during the mysid ( Americamysis

bahia ) two-generation test with lindane

. ...................................................69

Figure 2. Relationship between mean measured concentrations and the nominal treatment levels during the mysid ( Americamysis bahia ) two-generation test with lindane

. ...............................................................70

Figure 3. Mean percent survival of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........................................................71

Figure 4. Day 7 mean total body length of F 0 

### mysids ( Americamysis bahia ) during the two-generati on test with lindane

. ..............................................72

Figure 5. Day 7 mean dry body weight of F 0 

### mysids ( Americamysis bahia ) during the two-generati on test with lindane

. ..............................................73

Figure 6. Day 14 male and female mean total body length of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........74

Figure 7. Day 14 male and female mean dry body weight of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........75

Figure 8. Day 27 male and female mean total body length of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........76

Figure 9. Day 27 male and female mean dry body weight of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........77

Figure 10. Mean time to maturation of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane. .........................................................78

Figure 11. Mean time to first brood release of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with lindane. ..............................................79

Figure 12. F 0 

### Mean number of offspring/fema le produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane. ........................................................................................80

Figure 13. Mean time to second brood release of F 0 

### mysids ( Americamysis

bahia ) during the two-generati on test with lindane. ..................................81

Figure 14. F 0 

### Mean number of offspring/fe male produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane. ........................................................................................82

Figure 15. F 0 

### Mean interbrood duration for mysids ( Americamysis bahia ) during the two-generation test with lindane. ..............................................83

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Figure 16. F 0 

### Mean total number of offspring released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test

with lindane. ...............................................................................................84

Figure 17. F 0 

### Mean male:female sex ratio for mysids ( Americamysis bahia ) during the two-generation test with lindane. ..............................................85

Figure 18. F 0 

### Mean percentage of reproductively active females for mysids ( Americamysis bahia ) during the two-generation test with lindane. .........86

Figure 19. Mean percent survival of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........................................................87

Figure 20. Day 0 mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generati on test with lindane

. ..............................................88

Figure 21. Day 0 mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generati on test with lindane

. ..............................................89

Figure 22. Day 7 mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generati on test with lindane

. ..............................................90

Figure 23. Day 7 mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generati on test with lindane

. ..............................................91

Figure 24. Day 14 male and female mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........92

Figure 25. Day 14 male and female mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........93

Figure 26. Day 27 male and female mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........94

Figure 27. Day 27 male and female mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane

. .........95

Figure 28. Mean time to maturation of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane. .........................................................96

Figure 29. Mean time to first brood release of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with lindane. ..............................................97

Figure 30. F 1 

### Mean number of offspring/fema le produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane. ........................................................................................98

Figure 31. Mean time to second brood release of F 1 

### mysids ( Americamysis

bahia ) during the two-generati on test with lindane. ..................................99

Figure 32. F 1 

### Mean number of offspring/fe male produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane. ......................................................................................100

Figure 33. F 1 

### Mean interbrood duration for mysids ( Americamysis bahia ) during the two-generation test with lindane. ............................................101

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Figure 34. F 1 

### Mean total number of offspring released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test

with lindane. .............................................................................................102

Figure 35. F 1 

### Mean male:female sex ratio for mysids ( Americamysis bahia ) during the two-generation test with lindane. ............................................103

Figure 36. F 1 

### Mean percentage of reproductively active females for mysids ( Americamysis bahia ) during the two-generation test with lindane. .......104

APPENDIX 1 - QAPP ............................................................................................................. ....105

APPENDIX 2 - STUDY PLAN ...................................................................................................141

APPENDIX 3 - ANALYTICAL METHODOLOGY - LINDANE ............................................185

Table 3A. Analytical results for the recovery of lindane present in filtered seawater during the method validation study. ..........................................193

Figure 3A. Representative chromatogram of a 25.0 μg a.i./L calibration standard used for quantificati on during the method validation study

with lindane. .............................................................................................194

Figure 3B. Representative chromatogram of a 0.200 μg a.i./L recovery sample fortified with lindane during the method validation study. ......................195

Figure 3C. Representative chromatogram of a control sample during the method validation study with lindane. ..................................................................196

Figure 3D. A typical regression analysis for the calibration standards used to quantitate lindane in recovery sa mples during the method validation study. ........................................................................................................197

APPENDIX 4 - POST-PAIRING SU RVIVAL DATA - LINDANE ..........................................198

SUMMARY, PART B ............................................................................................................... ..235

7.0 INTRODUCTION ..............................................................................................................239

8.0 MATERIALS AND METHODS .......................................................................................240

8.1 QAPP/Study Plan .................................................................................................240

8.2 Test Substance .....................................................................................................240

8.3 Test Organism ......................................................................................................241

8.4 Feeding .................................................................................................................242

8.5 Dilution Water .....................................................................................................243

8.6 Test Substance Concentrations ............................................................................244

8.7 Stock Preparation .................................................................................................244

8.8 Exposure System ..................................................................................................244

8.8.1 Exposure Vessels .....................................................................................244

8.8.2 Retention Chambers .................................................................................245

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8.8.3 Brood Chambers ......................................................................................245

8.8.4 Diluter System .........................................................................................245

8.9 Test Conditions ....................................................................................................247

8.10 Exposure Conduct ................................................................................................247

8.10.1 Failed Exposures ......................................................................................247

8.10.2 Exposure Initiation ...................................................................................248

8.10.3 Biological Methods ..................................................................................248

8.10.3.1 Exposed Parental Generation (F 0 

### ) ...........................................248

8.10.3.1.1 F 0 

### Exposure Initiation .............................................................248

8.10.3.1.2 F 0 

### Growth Chambers ...............................................................249

8.10.3.1.3 F 0 

### Reproduction Chambers .....................................................250

8.10.3.2 Exposed First Filial Generation (F 1 

### ) .......................................251

8.10.3.2.1 Initiation of F 1 

### Generation .......................................................251

8.10.3.2.2 F 1 

### Growth Chambers ...............................................................252

8.10.3.2.3 F 1 

### Reproduction Chambers .....................................................252

8.10.4 Analytical Measurements .........................................................................253

8.10.5 Water Quality Measurements ..................................................................255

8.11 Biological Observations .......................................................................................255

9.0 STATISTICAL METHODS ..............................................................................................256

9.1 Endpoints .............................................................................................................256

9.2 Statistical Procedures ...........................................................................................257

10.0 PERFORMANCE CRITERIA ...........................................................................................258

11.0 RESULTS ....................................................................................................................... ....258

11.1 Water Quality Measurements ..............................................................................258

11.2 Analytical Results ................................................................................................259

11.3 Biological Results ................................................................................................260

11.3.1 F 0 

### Generation ...........................................................................................260

11.3.1.1 F 0 

### Survival ...............................................................................260

11.3.1.2 F 0 

### Growth ................................................................................260

11.3.1.2.1 Day 0 (Exposure Initiation) – F 0 

### Total Body Length and Dry Body Weight ....................................................................260

11.3.1.2.2 Day 7 - F 0 

### Total Body Length and Dry Body Weight ............261

11.3.1.2.3 Day 14 - F 0 

### Total Body Length and Dry Body Weight ..........261

11.3.1.2.4 Day 30 (F 0 

### Termination) - Total Body Length and Dry Body Weight ...........................................................................262

11.3.1.3 F 0 

### Time to Maturation .............................................................263

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11.3.1.4 F 0 

### Time to First Brood Release ...............................................263

11.3.1.5 F 0 

### Number of Offspri ng in the First Brood .............................263

11.3.1.6 F 0 

### Time to Second Brood Release ..........................................264

11.3.1.7 F 0 

### Number of Offspring in the Second Brood ........................264

11.3.1.8 F 0 

### Interbrood Duration ............................................................264

11.3.1.9 F 0 

### Total Number of Offspring in First Two Broods ...............265

11.3.1.10 F 0 

### Sex Ratio (Male:Female) ....................................................265

11.3.1.11 F 0 

### Percent of Reproductively Active Females ........................265

11.3.2 F 1 

### Generation ...........................................................................................266

11.3.2.1 F 1 

### Survival ...............................................................................266

11.3.2.2 F 1 

### Growth ................................................................................266

11.3.2.2.1 Day 0 (Exposure Initiation) – F 1 

### Total Body Length and Dry Body Weight ....................................................................266

11.3.2.2.2 Day 7 - F 1 

### Total Body Length and Dry Body Weight ............267

11.3.2.2.3 Day 14 - F 1 

### Total Body Length and Dry Body Weight ..........267

11.3.2.2.4 Day 27 (Exposure Termination) - Total Body Length and Dry Body Weight ....................................................................268

11.3.2.3 F 1 

### Time to Maturation .............................................................269

11.3.2.4 F 1 

### Time to First Brood Release ...............................................269

11.3.2.5 F 1 

### Number of Offspri ng in the First Brood .............................269

11.3.2.6 F 1 

### Time to Second Brood Release ..........................................270

11.3.2.7 F 1 

### Number of Offspring in the Second Brood ........................270

11.3.2.8 F 1 

### Interbrood Duration ............................................................270

11.3.2.9 F 1 

### Total Number of Offspring in First Two Broods ...............271

11.3.2.10 F 1 

### Sex Ratio (Male:Female) ....................................................271

11.3.2.11 F 1 

### Percent of Reproductively Active Females ........................271

12.0 CONCLUSIONS ................................................................................................................271

PROTOCOL DEVIATIONS .......................................................................................................273

REFERENCES .................................................................................................................... ........274

Table 11. Summary of water quality parameters measured in the exposure solutions during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol. .....................................................................276

Table 12. Concentrations of 4-tert-octyl phenol measured in the exposure solutions during the mysid ( Americamysis bahia ) two-generation

test

. ...........................................................................................................277

Table 13. Results of the quality control (QC) samples analyzed concurrently with test solution samples during the mysid ( Americamysis bahia ) two-generation test

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

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Table 14. F 0 

### mysid survival and growth (length and dry weight) during the mysid ( Americamysis bahia ) two-generation test with 4-tertoctylphenol. ..............................................................................................279

Table 15. F 0 

### mysid time to maturation, time to first brood release, time to second brood release, offspring re leased per female and interbrood duration during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol

. ............................................................................281

Table 16. F 0 

### mysid total number of young in the first two broods, sex ratio and percentage of reproductively active females during the mysid ( Americamysis bahia ) two-generation test wi th 4-tert-octylphenol

. ........282

Table 17. F 1 

### mysid survival and growth (length and dry weight) during the mysid ( Americamysis bahia ) two-generation test with 4-tertoctylphenol. ..............................................................................................283

Table 18. F 1 

### mysid time to maturation, time to first brood release, time to second brood release, offspring re leased per female and interbrood duration during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol

. ............................................................................285

Table 19. F 1 

### mysid total number of young in the first two broods, sex ratio and percentage of reproductively active females during the mysid ( Americamysis bahia ) two-generation test wi th 4-tert-octylphenol

. ........286

Table 20. Summary of F 0 

### and F 1 

### endpoints, No-Observed-Effect Concentration (NOEC) and Lowest -Observed-Effect Concentration (LOEC) values determined during the mysid ( Americamysis bahia ) two-generation test wi th 4-tert-octylphenol

. ............................................287

Figure 37. Schematic of biological methods during the mysid ( Americamysis

bahia ) two-generation test w ith 4-tert-octylphenol

. ................................288

Figure 38. Relationship between mean measured concentrations and the nominal treatment levels during the mysid ( Americamysis bahia ) two-generation test wi th 4-tert-octylphenol

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

Figure 39. Mean percent survival of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

Figure 40. Day 7 mean total body length of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

Figure 41. Day 7 mean dry body weight of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

Figure 42. Day 14 male and female mean total body length of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

. ..............................................................................................293

Figure 43. Day 14 male and female mean dry body weight of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

. ..............................................................................................294

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Figure 44. Day 30 male and female mean total body length of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

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

Figure 45. Day 30 male and female mean dry body weight of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

. ..............................................................................................296

Figure 46. Mean time to maturation of F 0 

### mysids ( Americamysis bahia ) during the two-generation test w ith 4-tert-octylphenol. ......................................297

Figure 47. Mean time to first brood release of F 0 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ..........................298

Figure 48. F 0 

### Mean number of offspring/fema le produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. .....................................................................299

Figure 49. Mean time to second brood release of F 0 

### mysids ( Americamysis

bahia ) during the two-generation test with 4-tert-octylphenol. ...............300

Figure 50. F 0 

### Mean number of offspring/fe male produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. .....................................................................301

Figure 51. F 0 

### Mean interbrood duration for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ..........................302

Figure 52. F 0 

### Mean total number of offspring released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ............................................................................303

Figure 53. F 0 

### Mean male:female sex ratio for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ..........................304

Figure 54. F 0 

### Mean percentage of reproductively active females for mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol. ..............................................................................................305

Figure 55. Mean percent survival of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

. ......................................306

Figure 56. Day 0 mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

Figure 57. Day 0 mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

Figure 58. Day 7 mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

Figure 59. Day 7 mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

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

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Figure 60. Day 14 male and female mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

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

Figure 61. Day 14 male and female mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

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

Figure 62. Day 27 male and female mean total body length of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

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

Figure 63. Day 27 male and female mean dry body weight of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol

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

Figure 64. Mean time to maturation of F 1 

### mysids ( Americamysis bahia ) during the two-generation test w ith 4-tert-octylphenol. ......................................315

Figure 65. Mean time to first brood release of F 1 

### mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ..........................316

Figure 66. F 1 

### Mean number of offspring/fema le produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. .....................................................................317

Figure 67. Mean time to second brood release of F 1 

### mysids ( Americamysis

bahia ) during the two-generation test with 4-tert-octylphenol. ...............318

Figure 68. F 1 

### Mean number of offspring/fe male produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. .....................................................................319

Figure 69. F 1 

### Mean interbrood duration for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ..........................320

Figure 70. F 1 

### Mean total number of offspring released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ............................................................................321

Figure 71. F 1 

### Mean male:female sex ratio for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol. ..........................322

Figure 72. F 1 

### Mean percentage of reproductively active females for mysids ( Americamysis bahia ) during the two-generation test with 4-tertoctylphenol. ..............................................................................................323

APPENDIX 5 - QAPP ............................................................................................................. ....324

APPENDIX 6 - STUDY PLAN ...................................................................................................360

APPENDIX 7 - ANALYTICAL METHODOLOGY - 4-TERT-OCTYLPHENOL ..................404

Table 7A. Analytical results for the recovery of 4-tert-octylphenol from 20 ppt filtered seawater during the method validation study. .............................413

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Figure 7A. Representative chromatogram of a 5.00 μg/L calibration standard and internal standard during the method validation study with 4-tertoctylphenol. ..............................................................................................414

Figure 7B. Representative chromatogram of a 1.00 μg/L recovery sample fortified with 4-tert-octylphenol and internal standard during the method validation study. ..........................................................................415

Figure 7C. Representative chromatogram of a control sample and internal standard during the method validatio n study with 4-tert-octylphenol. ....416

Figure 7D. A typical regression analysis for the calibration standards used to quantitate 4-tert-octylphenol in the recovery samples. ............................417

APPENDIX 8 - INDIVIDUAL F 1 

### DAY 0 LENGTH AND WE IGHT MEASUREMENTS .....418

APPENDIX 9 - POST-PAIRING SURVIVAL DATA - 4-TERT -OCTYLPHENOL ................425

APPENDIX 10 - BRINE SHRI MP NAUPLII ANALYSIS ........................................................462

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## SUMMARY, Part A

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

SPONSOR: RTI International

SMITHERS VISCIENT

STUDY PLAN TITLE: Two-Generation Life-Cycle T oxicity Test with Mysids ( Americamysis bahia ) Inter-Laboratory Comparison, Smithers

Viscient Protocol No.: 0 91310/OECD/Mysid, two-generation

SMITHERS VISCIENT

STUDY NUMBER: 13888.6109

RTI

PROJECT NUMBER: 0210114.030

EPA CONTRACT

NUMBER: EP-W-06-026

TEST SUBSTANCE: Lindane, Lot No. 14419eb, CAS No. 58-89-9, reported to have a purity of 99.6%, was received from Battelle Marine Science Laboratory on 19 January 2011.

DEFINITIVE

TEST DATES: 17 August to 4 October 2011

TEST ORGANISM: Americamysis bahia ( ≤ 22 hours old post-release) Origin: Smithers Viscient culture

DILUTION WATER: Dilute, filtered, natural seawater Salinity: 19 to 20‰ pH: 7.8 to 8.0

TEST CONDITIONS: Exposure duration: 48-days Temperature range: 23 to 27 oC Salinity range: 19 to 22 ‰ Dissolved oxygen range: 5.6 to 8.0 mg/L

78 to 110% of saturation pH range: 7.7 to 8.1 Photoperiod: 14 hours light: 10 hours dark Light intensity range: 47 to 99 footcandles

510 to 1100 lux

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NOMINAL TEST

CONCENTRATIONS: 32, 80, 200, 500 and 1250 ng/L

MEAN MEASURED

CONCENTRATIONS: 30, 100, 170, 380 and 1100 ng/L

ENDPOINTS: Parental Generation (F 0 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 7, 14, 30)

o Dry weight to the nearest 0.01 mg (days 7, 14, 30)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

First Filial Generation (F 1 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 27)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 27)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

ENDPOINTS AND

RESULTS: For the F 0 

### exposure, male and female mysid dry weight (day 14) were the most sensitive endpoints in the mysid two-generation exposure to lindane. F 1 

### generation survival, day 27 male dry weight and total body length were the most sensitive endpoints in the mysid two-generation exposure to lindane.

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F 0 Endpoints Measured Concentration (ng/L)

30 100 170 380 1100

Survival Day 7 Dry Weight Day 14 Male Dry Weight ↑ ↑ ↑ Day 14 Female Dry Weight ↑ ↑ ↑ Termination (Day 27) Male Dry Weight ↓ Termination (Day 27) Female Dry Weight Day 7 Length Day 14 Male Length Day 14 Female Length ↑ ↑ Termination (Day 27) Male Length Termination (Day 27) Female Length

Time to Maturation

Time to 1 s t Brood Release ↓

1 s t

Brood Offspring/Female ↑

Time to 2 nd Brood Release ↓

2 nd Brood Offspring/Female

Interbrood Duration Total Offspring/Female

Sex Ratio % Reproducing Females
↓ Endpoint determined to be sta tistically significantly reduced (p ≤ 0.05) compared to control ↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control

F 1 Endpoints Measured Concentration (ng/L)

30 100 170 380 1100

Survival Day 0 Dry Weight Day 7 Dry Weight Day 14 Male Dry Weight ↓ Day 14 Female Dry Weight Termination (Day 27) Male Dry Weight ↓ Termination (Day 27) Female Dry Weight Day 0 Length Day 7 Length Day 14 Male Length ↓ Day 14 Female Length Termination (Day 27) Male Length ↓ Termination (Day 27) Female Length ↑

Time to Maturation

Time to 1 s t Brood Release ↑ ↑ ↑

1 s t

Brood Offspring/Female ↓

Time to 2 nd Brood Release

2 nd Brood Offspring/Female ↓

Interbrood Duration Total Offspring/Female ↓ ↓

Sex Ratio % Reproducing Females
↓ Endpoint determined to be sta tistically significantly reduced (p ≤ 0.05) compared to control

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# 1.0 INTRODUCTION

The U.S. Environmental Protection Agency's En docrine Disruptor Screening Program (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 endoc rine effects and provide dose re sponse information needed for

risk assessment. One of the longer term Tier2 assays being developed is the mysid twogeneration test with Americamysis bahia . This test contrasts with existing mysid life-cycle

methods (that represent a single ge neration life-cycle) in that an additional generation is included

to allow evaluation of potential trans-generational effects. The inclusion of the invertebrate

assay in Tier 2 is important because invertebrate s make up the majority of animal species and the

endocrine systems of invertebrate s often are not directly analogous to those of vertebrates.

Additionally, a method for testi ng crustaceans for effects of endocrine disrupting chemicals

(EDCs) is relevant to assessing the adverse consequences of chem icals indicated to be endocrine

active in Tier 1 assays.

The purpose of work to be conducted under this assignment is to obtain data on the interlaboratory reproducibility and comparability of the mysid two-generation test method in support

of the validation of the mysi d two-generation test method being considered for the EDSP.

This study was conducted under the Quality Assurance Project Plan (QAPP, Appendix 1 ), the

Smithers Viscient study plan entitled "Study Plan on Two-Generation Life-Cycle Toxicity Test

with Mysids ( Americamysis bahia ) Inter-Laboratory Comparison," Smithers Viscient protocol

No. 091310/OECD/Mysid, two-generation ( Appendix 2 ), relevant program and facility standard

operating procedures (SOPs), and guidance documents.

The experimental phase of the study was c onducted from 17 August to 4 October 2011 at

Smithers Viscient (SMV), located in Wareham, Ma ssachusetts. The original raw data, protocol,

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 22

and the original final report produced during this study are stored at Smithers Viscient,

Wareham, Massachusetts.



# 2.0 MATERIALS AND METHODS

2.1 QAPP/Study Plan

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

Plan (QAPP) for Task Order 30 ( Appendix 1 ). The study plan ( Appendix 2 ) describes a twogeneration test with an estuarine mysid that considers reproductive fitness in parents and

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

of other endpoints that allow for diagnostic and definitive evaluation of EDCs or other types of

reproductive toxicants ( Verslycke et al., 2004 ). The mysid two-generati on test is a relatively

long-term (normally 60 days or longer) assay th at assesses early development, growth and

reproduction in two generations (F 0 

### and F 1 

### ). The two-generation test described in the guideline

is an extension of existing standard pract ice for conducting a mysid life-cycle test ( ASTM, 2008 ;

McKenney, 1986, 1996 ; and Nimmo et al., 1977, 1978 ).



## 2.2 Test Substance

The test substance, lindane, was received on 19 January 2011 from Battelle Marine Science

Laboratory, Sequim, Washington. The fo llowing information was provided:

Name: Lindane, 97% Synonyms: Lindane; 1 α ,2 α ,3 β ,4 α ,5 α ,6 β -Hexachlorocyclohexane; y-BHC

Product No.: 233390

Lot No.: 14419eb

CAS No.: 58-89-9 Purity: 99.6% Expiry Date: December 2012

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Upon receipt at Smithers Viscient, the test s ubstance (SMV No. 4841) was stored at room

temperature in the original contai ner in a dark ventilated cabinet. Concentrations were adjusted

for the purity of the test substance.



## 2.3 Test Organism

Juvenile mysids ( ≤ 22 hours old), Americamysis bahia (formerly Mysidopsis bahia ) were used to

initiate the life-cycle test. The adult mysids (SMV Lot Nos. 11A79, 11A83, 11A86, 11A93 and

11A110) used to produce juvenile mysids for te sting were obtained from SMV cultures. The

brood stock was originally obtained from Aqua tic BioSystems, Inc., a commercial supplier

located in Fort Collins, Colorado, and are refr eshed periodically. Culture organisms were

received on June 9 and July 15, 2011 and were maintained in-house for approximately 1 to

2 months prior to exposure initiation. Mysids were cultured using dilute, filtered, natural

seawater and six 80-L glass aquaria under clos ed-loop, recirculating conditions. Standard

aquarium undergravel filters were used to prov ide aeration, biological fi ltration and a current

conducive to feeding.

The seawater used for culturing was characterize d as having a salinity range of 19 to 22‰ and a

pH range of 7.5 to 7.7 during the 14-day period prior to exposure initi ation. Dissolved oxygen

concentration ranged from 6.4 to 6.9 mg/L. The area in which the mysids were cultured received

a regulated photoperiod of 16 hour s of light and 8 hours of da rkness. Culture solution

temperature was maintained at 24 to 26  C. Culture condition and test conditions are based on

previously determined optimal salinity-temperature conditions for survival ( McKenney, 1994 ),

growth ( McKinney and Celestial, 1995 ) and reproduction ( McKenney, 1996 ).

Juvenile mysids were obtained by isolating mature gravid females in isolation baskets

approximately 22 hours prior to exposure initia tion. Young produced by these organisms were

harvested from designated mysid breeding tanks usi ng a variation of the method described in the

Short-Term Methods for Estimating the Chronic T oxicity of Effluents and Receiving Waters to

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 24

Marine and Estuarine Organisms ( U.S. EPA, 2002 ). Based on these procedures, the post-release

age of the juveniles used to initiate this assay was ≤ 22 hours.



## 2.4 Feeding

During this exposure, mysids (both F 0 

### and F 1 

### ) were fed twice daily with brine shrimp ( Artemia

salina , SMV Lot No. 100209, supplied by INVE Aquacu lture, Salt Lake City, Utah) nauplii

≤ 48 hours old (post-hydration). At least one of these feedings was with brine shrimp nauplii

enriched with Selco ® 

### , a substance high in unsaturated fatty acids. Mysid survival, growth and

reproduction are improved with high levels of Ω -3 highly unsaturated fatty acids. Selco ® 

### is

added to newly hatched brine shrimp. The newly hatched Artemia become enriched by feeding

on the lipid-coated micro particles. Densities per mL of concentrat ed collections of nauplii from

Artemia cultures created from daily hydrated cysts were determined by triplicate counts of

aliquots pipetted onto filter paper. Based on these densities, the following pre-determined

volumes of nauplii were fed to mysids at each feeding: rations for retention/brood chambers

were approximately 1800 naupli i/retention chamber on days 0 to 3, 2250 on days 4 to 6, 2700 on

days 7 to 9, 3600 on days 10 to 12, and 4500 on days > 12, while brood chambers (containing

individually paired mysids) were fed approximately 900 nauplii/brood chamber throughout the

test. All treatments and controls received, as near as reasonably possible, the same ration of

food. Excess brine shrimp and organic debris in the test chambers were removed daily prior to

the addition of new nauplii.

Representative samples of the brine shrimp naup lii were analyzed periodically for the presence

of pesticides, PCBs, and toxic metals, by GeoLabs, Inc., Braintree, Massachusetts

( Appendix 10 ). None of these compounds were detected at concentrations that are considered

toxic in any of the food sample s analyzed in agreement with ASTM (2002) standard practice.

Based on these analyses and the performance of the control organisms, the food source was

considered to be of acceptable quality since a ll analyte concentrations were below levels of

concern and control organisms exceeded guideline criteria.

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## 2.5 Dilution Water

Dilute, filtered, natural seawater was used as dilution and control wa ter during this study.

Seawater was collected from the Cape Cod Cana l in Bourne, Massachusetts from about 1 to

4 meters offshore at a depth of approximately 0.5 meters. The seawater was transferred with a

pump (fiberglass reinforced thermoplastic housin g) and a polyvinyl chloride (PVC) pipe and was

then transported to the testing facility in a 3400-L fi berglass tank. At the testing facility, the

seawater was transferred to an 8500-L epoxy-coat ed concrete tank, wher e it was diluted to a

salinity of 20 ± 3‰ with laborat ory well water using an actuated valve controlled by a Signet

Controller. The diluted seawater was mixed thoroughly prior to characterization. Following

characterization, the water was then pumped in to holding tanks where it was recirculated for

48 hours prior to use. The seawater was pumped through a series of polypropylene core filters

(20-, 5- and 1-μm) to the laboratory under cons tant pressure through PV C pipe to a head tank,

where it was heated to test temperature and ae rated prior to delivery to the intermittent-flow

proportional diluter system. The seawater used fo r this exposure had a salinity range of 19 to

20‰ and a pH range of 7.8 to 8.0.

Representative samples of the dilution water were analyzed periodically for the presence of

pesticides, PCBs and toxic metals by GeoLabs, In c., Braintree, Massachusetts. None of these

compounds have been detected at concentrations that are considered toxic in any of the water

samples analyzed, in agreement with ASTM (2002) standard practice. In addition, representative

samples of the dilution water were analy zed monthly for total organic carbon (TOC)

concentration. The TOC concentration in the se awater source ranged from 0.99 to 1.2 mg/L for

August through October 2011. Based on these anal yses and the performance of the control

organisms, the dilution water source was consider ed to be of acceptable qu ality since all analyte

concentrations were below levels of con cern and control organisms exceeded guideline

performance criteria.

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## 2.6 Test Substance Concentrations

Nominal concentrations of 32, 80, 200, 500 and 1250 ng/L, and a control were selected by the

EPA for the definitive exposure.



## 2.7 Stock Preparation

A 50 μg/L diluter stock solution was prepared weekly throughout th e exposure by diluting

approximately 0.0050 g (0.0050 g as active ingredient ) of lindane in a 100-L graduated Nalgene ®



### carboy containing 100 liters of dilu te, natural seawater. The stoc k was mixed overnight using a

3⁄4 horsepower mixer with a stainless steel shaf t and propeller blades. Following mixing, the

resultant solution was observed to be clear and colorless.



## 2.8 Exposure System

2.8.1 Exposure Vessels

The exposure vessels were 36-L glass aquaria measuring 60 x 30 x 30 cm equipped with side

drain at 12 cm. The total solution volume mainta ined in each vessel was approximately 21 L.

The aquaria were randomly distribu ted within the water bath, and labeled to identify the nominal

test substance treatment concentration and the replicate designation.



## 2.8.2 Retention Chambers

Retention chambers were construc ted of 10-cm glass Petri dishes to which a 15-cm high cylinder

of nylon mesh screen (250-μm mesh) had been attached w ith aquarium grade (without

fungicides) silicone adhesive. There were two retention chambers in each exposure vessel prior

to reaching sexual maturity. One chamber, the reproduction chamber, was used to rear mysids

which were used for reproductive ev aluation; these mysids were held in these retention chambers

until sexual maturity was reached. The second cham ber, the growth chamber, was used to rear

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 27

mysids which were used for collecting growth metr ics; these mysids were held in the retention

chamber until Day 14.



## 2.8.3 Brood Chambers

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

constructed of 6-cm glass Petri dishes to wh ich a 15-cm high cylinder of nylon mesh screen

(250-μm mesh) was attached.



## 2.8.4 Diluter System

A 2-L intermittent-flow, proportional diluter (e.g., Mount and Brungs, 1967 ) was employed to

deliver five test substance concentrations and a negative control. An FMI pump was calibrated

to deliver 82.5 mL/cycle of the stock solution (50 μg/L) into the diluter's chemical mixing

chamber, which also received 3.3 L of diluti on water per cycle. The mixing chamber was

positioned over a magnetic stir plate which aided in mixing the stock solution with the dilution

water. The solution contained in the mixi ng chamber constituted the highest nominal

concentration (1250 ng/L). This solution was subsequently diluted (40%) to provide the

remaining nominal exposure concentrations (500, 200, 80 and 32 ng/L). A set of control vessels

was also established which contained the same dilution water and was maintained under the

same conditions as the treatment level vessels, but contained no li ndane. Three replicates were

maintained for all treatments and the control.

A flow-splitting chamber was used between the d iluter cells and the aquaria to promote mixing

of the test substance solution and diluent water. In each chamber, three standpipes with umbrella

siphons were employed to split the test solution equa lly between test aquaria. The volume of test

solution delivered to each of three replicate aqua ria was 670 mL with a fl ow splitting accuracy of

± 5%. Solution was delivered to the exposure ve ssels at a rate of approximately six exposure

vessel volume additions per 24 hours and an approximate 9-hour 90% replacement time.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 28

The calibration of the diluter system was checked and recorded prior to exposure initiation. If

there was any indication during th e test that the diluter calib ration had changed (e.g., diluter

malfunction or unexplained differences in water quality or measured concentrations in the

aquaria), calibration of the necessary dilute r components was checked. During the test, a

complete check of diluter function (e.g. diluter cy cle rate, toxicant stock flow, etc.) and a visual

inspection was performed at the begi nning and end of the work day.



## 2.9 Test Conditions

The test area was illuminated with fluorescent bulbs at an intensity range of 47 to 99 footcandles

(510 to 1100 lux) at the solutions' surface. Th e photoperiod was set to be maintained at 14 hours

light:10 hours dark. The photoperiod was gradua l (i.e., began and ended with 30 minutes of

reduced lighting, and 13 hours of fu ll light intensity in between th e reduced lighting periods).

Photoperiods were maintained by using an automatic timer. Light intensity was measured with a

VWR light meter once during the exposure. Two Process Technology heaters were set to

maintain the protocol specified test solution te mperature at 26 ± 1 oC inside each of the

temperature-controlled water baths. The salinity of the test solutions was set to maintain the

protocol specified at 20 ± 2‰ and monitored prio r to use to ensure these parameters were

maintained. The total dissolved oxygen was ma intained between 80 to 100% of saturation at

exposure initiation and > 60% saturation (4.3 mg /L at 26 oC and 20‰ sa linity) throughout the

test.



## 2.10 Exposure Conduct

2.10.1 Exposure Initiation

The exposure was not initiated unt il the diluter and test substa nce delivery device had been

properly functioning for at least 48 hours prior. This allowed the test solution to reach an

equilibrium concentration of test substance in the exposure system and ensured concentrations

were maintained at exposure in itiation. The exposure was initia ted when the retention chambers

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 29

(growth and reproduction) containing F 0 

### mysids were added to the respective test vessels (see

Section 2.10.2.1.1 ).



## 2.10.2 Biological Methods



## 2.10.2.1 Exposed Parental Generation (F 0 

## )

2.10.2.1.1 F 0 

## Exposure Initiation

Three replicate aquaria per treatment were us ed for each test concentration (0, 32, 80, 200, 500,

1250 ng/L). Each replicate tank housed one gr owth and one reproduction chamber (days 0

through 14) or seven brood chambe rs (days 14 through termination; Figure 1 ).

At exposure initiation, 20 juvenile mysids ( ≤ 22 hours old, post-release) were impartially

transferred into both the growth and reproduction chambers in the following manner: (1) a pool

of > 630 juvenile mysids were remove d from the cultures; (2) mysids ( ≤ 22 hours old, postrelease) were distributed, four at a time, to unl abeled intermediate beakers (two per replicate)

containing dilution water until each beaker contains four mysids. This process was repeated

five more times until each intermediate beaker contained 20 mysids; (3) following distribution,

mysids in each intermediate beaker were then tr ansferred to a labeled re tention chamber. Care

was exercised to avoid injury to juvenile mysids during counting. Mysids were transferred using

either a smooth-edge pipette or a piece of fine sc reen mesh. Mysids that were dropped or injured

during transfer were removed and replaced with a new individual from the same cohort. The

exposure was initiated when each labeled growth and reproduction basket was added to the

respective exposure vessel.

At exposure initiation, 45 additi onal mysids (from the cohort used for initiating the exposure),

were to be obtained from the culture for growth metrics. Due to an oversight, these metrics were

not performed at exposure initiation. Rather , these metrics were performed approximately

7 days following exposure initiation (see Protocol Deviations ). These 45 mysids were pooled

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 30

then divided into 9 groups of 5 mysids and were placed in ice-cold, dei onized water, blotted dry,

and digital images were taken of each group of mysids for total length measurements. Each

group was then placed on pre-weighed aluminum tins, and placed in an oven at approximately

100  C for approximately 24 hours. Following the dr ying period, mysid samples were kept in a

desiccator for a minimum of 30 minutes (to allow for cooling of the sample) and weighed to the

nearest 0.01 mg on an analytical balance.

Length measurements were obtained from digita l photographs using the following software and

hardware: Zeiss Stemi-2000 CS, a Zeiss AxioCa m ICc 3 and Zeiss AxioVision image analyzing

software.



## 2.10.2.1.2 F 0 

## Growth Chambers

Following 7 and 14 days of exposure, mysids were sampled from each growth chamber for

survival, length and dry weight determinations . On day 7, six mysids were removed from each

growth chamber, separated into three groups of two mysids, euthanized in ice-cold deionized

water, blotted dry and photographed for total le ngth measurements. Each group was then placed

on pre-weighed aluminum tins and placed in an oven at approximately 100  C for approximately

24 hours. Following the drying period, mysid sample s were kept in a desiccator for a minimum

of 30 minutes (to allow for cooling of the sa mple) and weighed to the nearest 0.01 mg on an

analytical balance.

On day 14, the remaining mysids were sampled from each growth chamber for survival, length

and dry weight determinations. Prior to th e day 14 termination, mysids from the growth

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

the reproduction chamber was skewed pr eventing obtaining seven pairs (see Section 2.10.2.1.3 ).

The remaining mysids were removed, identified as male, female or immature (following the

criteria outlined in U.S. EPA, 2002 ), euthanized in ice-cold deionized water, blotted dry and

photographed for total length measurements. E ach mysid was then placed on pre-weighed

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 31

aluminum tins and placed in an oven at approximately 100  C for approximately 24 hours.

Following the drying period, mysid samples were kept in a desiccator for a minimum of

30 minutes (to allow for cooling of the samp le) and weighed to the nearest 0.01 mg on an

analytical balance.



## 2.10.2.1.3 F 0 

## Reproduction Chambers

Mysids maintained within reproduction cham bers were observed daily for survival, sex

determination and reproductive output. Begi nning on day 10, developing embryos could be

discerned within individual females at most tr eatment levels. By day 12 all females observed

had developed marsupial pouches. At sexual ma turity (designated by th e appearance of the

marsupial pouch), one ovigerous female was paired with one male, and the pair was transferred

to a brood chamber housed in the replicate tank. For each exposure concentration, 3 replicate

tanks housed up to seven brood chambers each ( Figure 1 ). Mysids that were not paired remained

in reproduction chambers until three or fewer rema ined; if three or fewer remained, these mysids

were transferred to a separate brood chamber. Remaining male mysids (e.g., those not used in

the initial male/female pairings) were held and us ed to replace male mysids that died in brood

chambers during the reproductive phase of the ex posure. Prior to th e day 14 terminations,

mysids from growth chambers we re recruited to form brood chambe r pairs in the event that the

sex ratio in the reproduction chamber was skewed, preventing obtaining seven pairs.

During the reproductive phase until F 0 

### termination, offspring were enumerated daily. Offspring

from the F 0 

### mysids (used to establish the F 1 

### generation) were collected and transferred to growth

and reproduction chambers exposed to the same test substance concentration from which they

originated. The F 0 

### exposure was terminated six days afte r the median day of release of the

second brood release in th e control treatments (see Protocol Deviations ).

At exposure termination, F 0 

### mysids were removed, identified as male, female or immature,

euthanized in ice-cold deionized water, blotted dry and photographed for total length

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 32

measurements. Each mysid was then placed on i ndividual aluminum tins and placed in an oven

at approximately 100  C for approximately 24 hours. Following the drying period, mysid

samples were kept in a desiccator for a mini mum of 30 minutes (to allow for cooling of the

sample) and weighed to the nearest 0.01 mg on an analytical balance.



## 2.10.2.2 Exposed First Filial Generation (F 1 

## )

2.10.2.2.1 Initiation of F 1 

## Generation

Between exposure days 18 and 24, groups of 15 < 24-hour old offspring (F 1 

### ), from either the first

(F 1 

### ') or second (F 1 " 

### ) brood of the F 0 

### mysids, were isolated in gr owth and reproduction chambers

(as previously described), with one excepti on. In replicate A of the 1250 ng/L treatment

concentration, only 12 offspring were available fo r initiation of the growth chamber prior to F 0



### exposure termination. F 1 

### offspring were exposed to the same test substance concentrations as

their F 0 

### parents. Mixing of young from replicate e xposure tanks released on the same day across

replicate chambers was encouraged to mini mize reproduction between siblings. When F 1 

### mysids

were limited, priority was given to populating all replicate reproduction chambers first, then

growth chambers.

In addition, five day 0 F 1 

### mysids were collected from each F 0 

### exposure concentration, euthanized

in cold-water deionized water, blotted dry, and digital images were taken for total length

measurements (see Protocol Deviations ). Each group was then placed on pre-weighed aluminum

tins, and placed in an oven at approximately 100  C for approximately 24 hours. Following the

drying period, mysid samples were kept in a desiccator for a mi nimum of 30 minutes (to allow

for cooling of the sample) and weighed to th e nearest 0.01 mg on an analytical balance.



## 2.10.2.2.2 F 1 

## Growth Chambers

For each growth chamber, on their respective day 7 and day 14, mysids were sampled for length

and dry weight determinations. On day 7, six my sids were removed from the growth chamber,

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 33

separated into three groups of two mysids, euthanized in ice-cold deionized water, blotted dry

and photographed for total length measurements. Each group was then placed on pre-weighed

aluminum tins and placed in an oven at approximately 100  C for approximately 24 hours.

Following the drying period, mysid samples were kept in a desiccator for a minimum of

30 minutes (to allow for cooling of the samp le) and weighed to the nearest 0.01 mg on an

analytical balance.

On day 14, mysids remaining in the growth cham ber, (e.g., those not sampled on day 7 and those

not used to complete reproduc tive pairings) were removed, identified as male, female or

immature (following the criteria outlined in U.S. EPA, 2002 ), euthanized in ice-cold deionized

water, blotted dry and photographed for total le ngth measurements. Each mysid was then placed

on pre-weighed aluminum tins and placed in an oven at approximately 100  C for approximately

24 hours. Following the drying period, mysid sample s were kept in a desiccator for a minimum

of 30 minutes (to allow for cooling of the sa mple) and weighed to the nearest 0.01 mg on an

analytical balance.



## 2.10.2.2.3 F 1 

## Reproduction Chambers

Mysids maintained within reproduction cham bers were observed daily for survival, sex

determination and reproductive output. Betw een the respective days 9 to 13 for the F 1



### generation, developing embryos coul d be discerned within individua l females at most treatment

levels. When developed marsupial pouches could be discerned within an individual female, the

ovigerous female was paired with one male, a nd the pair was isolated in a separate brood

chamber housed within the replicate tank for that specific exposure conc entration. A maximum

of seven pairs were isolated in br ood chambers for each replicate tank ( Figure 1 ). Mysids that

were not paired remained in reproduction chambe rs until three or fewer remained; if three or

fewer remained, these mysids were transferred to a separate brood chamber. Remaining male

mysids (e.g., those not used in the initial male /female pairings) were held and used to replace

male mysids that died in brood chambers during th e reproductive phase of the exposure. Prior to

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 34

their respective day 14 terminations, mysids from growth chambers were recruited to form brood

chamber pairs in the event that the sex ratio in the reproduction chamber was skewed, preventing

obtaining seven pairs.

During the reproductive phase , offspring from the F 1 

### mysids were removed, enumerated, and

recorded from their respective brood chamber daily until exposu re termination. The exposure

was terminated seven days after the median day of release of the second brood in the F 1 

### control

tanks.

At exposure termination, F 1 

### mysids were removed, identified as male, female or immature,

euthanized in ice-cold deionized water, blotted dry and photographed for total length

measurements. Each mysid was then placed on i ndividual aluminum tins and placed in an oven

at approximately 100  C for approximately 24 hours. Following the drying period, mysid

samples were kept in a desiccator for a mini mum of 30 minutes (to allow for cooling of the

sample) and weighed to the nearest 0.01 mg on an analytical balance.



## 2.10.3 Analytical Measurements

At exposure initiation (day 0), water samples from each replicate of each concentration and

control were removed and analyzed for lindane . Following day 0, water samples were removed

weekly from alternating replic ates (e.g., replicate B sample d week 2, replicate C sampled

week 3, replicate A sampled week 4, etc.). At exposure termination (seven days after the median

day of release of the s econd brood release in the F 1 

### control vessels), each replicate vessel was

sampled. Since the initiation of each reproducti on chamber was not uniform across the treatment

and control replicates, each e xposure vessel was sampled on its respective termination day.

Water samples were removed from a point appr oximately midway between the water surface,

bottom, and sides of each test vessel and analyzed immediately. A sample of the stock solution

was analyzed at each sampling interval, with one exception. Since exposure termination

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 35

encompassed multiple days, the stock sample was not analyzed at each sampling interval. The

diluter stock was typically prepared about once per week, therefore the stock was sampled once

during the termination period.

Three quality control (QC) samples were prep ared at each sampling interval at nominal

concentrations of 20.0, 200, and 1250 ng/L. These c oncentrations were intended to approximate

the test concentration range. The QC samples were processed and analyzed with the exposure

solution samples and remained with the exposur e solution samples thr oughout the analytical

process. Results of the analyses of the QC samples were used to evaluate the precision and the

quality control maintained during the an alysis of exposure solution samples.

All exposure solutions and QC samples were an alyzed for lindane using gas chromatography

with micro electron capture de tection (GC/μECD) based on me thodology validated at Smithers

Viscient. The method validation was conducted prior to the initiation of th e definitive test and

established an average recovery of 98.1% ± 6.59% for lindane from filtered seawater

( Appendix 3 ). Defined limits for acceptance of quality control sample performance in

subsequent studies were set at 70 to 120%. Conditions and pr ocedures used throughout the

analysis of exposure solutions and QC samples duri ng this study were similar to those used in the

method validation.



## 2.10.4 Water Quality Measurements

Temperature, dissolved oxygen concentration, pH and salinity were measured daily in each

replicate of each treatment and the control solutions throughout the exposure period, with the

exception of replicate C of the c ontrol at exposure initiation (see Protocol Deviations ). Exposure

solution temperature was continuously monitore d in one replicate of the control vessel

(replicate B in the upper water bath ; replicate C in the lower wate r bath) by using a VWR digital

minimum/maximum thermometer. Salinity was measured with a Yellow Springs Instrument

(YSI) Model 30 conductivity/salin ity meter. The dissolved oxygen concentration and daily

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 36

temperature were measured using a YSI Model 55 0A dissolved oxygen meter/temperature probe.

The pH was measured with a YSI Model pH100 pH meter.



## 2.11 Biological Observations

Biological observations of the mysids were reco rded at exposure initia tion and daily thereafter

until termination of the F 1 

### generation (day 48). Dead orga nisms were recorded and removed

when observed and discarded at each observation interval. The cr iteria used for determining

death were the absence of mobility and failure to respond to gentle prodding. Survival was

based on the number of mysids init ially present in each test vessel.

The primary biological observations made during the exposure were:

Parental Generation (F 0 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 30)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 30)

 Time to maturation, appearance of marsupial pouch

 Time to first brood release, number of days after test initiatio n to release of young

in the first brood

 Time to second brood release, number of da ys after exposure initiation to release of young in the second brood

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active First Filial Generation (F 1 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 27)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 27)

 Time to maturation, appearance of marsupial pouch

 Time to first brood release, number of days after test initiatio n to release of young

in the first brood

 Time to second brood release, number of da ys after exposure initiation to release of young in the second brood

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 37

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active



# 3.0 Statistical Methods

3.1 Endpoints

Parental Generation (F 0 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 7, 14, 30)

o Dry weight to the nearest 0.01 mg (days 7, 14, 30)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

First Filial Generation (F 1 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 27)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 27)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 38

3.2 

## Statistical Procedures

All statistical conclusions were made at the 95% level of certa inty except in the cases of

Modified Levene's Test ( U.S. EPA, 2002 ), Bartlett's Test ( U.S. EPA, 2002 ) and Shapiro-Wilks’

Test ( U.S. EPA, 2002 ), in which the 99% level of cer tainty was applied. The following

procedures were used:

 Prior to statistical analysis, data repr esenting endpoints obtained as proportions (e.g., percentage of females that are repr oductively active, survival, etc.) were transformed using the arcsine square-root transformation.

 As a check on the assumption of homogene ity of variance implicit in parametric statistics, data for each endpoint were analyzed by using Bartlett's Test ( U.S. EPA, 2002 )

, 

### which can be used for equal and unequal replicates as long as the populations are normally distri buted. Since Bartlett’s Te st can be overly sensitive to non-normality, Modified Levene's Test ( U.S. EPA, 2002 ) was also utilized for homogeneity of variance.

 Shapiro-Wilks’ Test for normality ( U.S. EPA, 2002 ) was conducted and compared the observed sample distribu tion with a normal distribution. The assumption that observations are normally distributed must be validated before subsequent analyses, following parametric procedures, can be performed. If the data were not normally distributed, then a non-parametric procedure was used for subsequent analyses.

 All endpoints met the assumptions of normality and homoscedasticity, with one exception. The data for percentage of females that are reproductively active (F 0 

### , F 1 

### ) did not meet the assumptions of nor mality. Therefore these data were compared with the performance of the cont rol data using Fisher's Exact Test with Bonferroni-Holm's Adjustment ( U.S. EPA, 2002 ).

 Significant treatment effects for all rema ining endpoints were determined from either Dunnett's Multiple Comparison Test ( U.S. EPA, 2002 ) or Bonferroni's Adjusted t-Test ( U.S. EPA, 2002 ).

CETIS TM 

### version 1.8.1.1 ( Ives, 2009 ) was used to perform the statistical computations.

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# 4.0 PERFORMANCE CRITERIA

The following performance criteria were us ed for judging test data acceptability:

 Water quality characteristics should remain within the tolerance limits illustrated in Tables 1 and 2 of the study plan ( Appendix 2 ).

 There should be more than 70% survival of control animals in all test phases over the duration of the chemical exposure.

 At least 75% of the females in the F 0 

### or F 1 

### controls must produce young.

 The average number of young produced per co ntrol female in the first two broods combined must be at least eight.



# 5.0 RESULTS

5.1 Water Quality Measurements

Water quality parameters measured during the te st were unaffected by the concentrations of

lindane tested and remained within acceptable lim its for the survival, reproduction and growth of

mysids ( Table 1 ). Daily measurements established di ssolved oxygen concentrations that were

≥ 91% of saturation at exposure initiation and maintained at ≥ 78% of saturation throughout the

remainder of the test. These measurements also determined a pH range of 7.7 to 8.1; a

temperature range of 23 to 27 oC (see Protocol Deviations ); and a salinity range of 19 to 22‰.

Continuous temperature monitoring of the control established a temperature range of 23 to 27 oC

during the exposure period.



## 5.2 Analytical Results

The diluter system which prepared and delivered the test solutions to the exposure vessels

functioned properly during the pre-exposure peri od and throughout the definitive study. During

the in-life phase of the definitive study, no undissolved test substance (e.g., precipitate) was

observed in the diluter system (e.g., mixing cham ber and chemical cells) or exposure solutions

throughout the exposure period.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 40

During the in-life exposure, test solutions were sampled for conc entrations of lindane on day 0,

4, 5, 8, 11, 14, 22, 29, 35, 42, 45/46, 47 and 48. The results of these analyses are presented in

Table 2 and Table 3 . Throughout the exposure period, the meas ured recoveries at each exposure

concentration approximated the nominal doses and were generally consiste nt, with the following

exceptions:

 On day 0, the measured concentrations at th e lowest treatment level (32 ng/L nominal)

were inconsistent between repl icates. Therefore, all replic ates at this dose were resampled on day 4. However, the results from the day 4 sampling were not used due to an

incorrect setting on the instrument. Therefor e, the system was re-sampled on day 5 to

confirm the initial day 0 results. These measurements from day 5 confirmed that

concentrations for all replicates at this dos e were similar; the anomalous measurements

from day 0 were attributed to a sample proc essing error and therefore, not used in the

calculation of mean measured concentrations.

 On day 8, the measured concentration for th e highest treatment level tested (1250 ng/L

nominal) was lower than expected. Since a ll doses are diluted from this concentration

and all remaining exposure con centrations approximated nom inal, this measurement was

believed to be erroneous. On day 11, all repl icates from the high dose were re-sampled to

confirm exposure dose. The results from the day 11 sampling approximated nominal

concentrations; the recoveries from the da y 8 sampling were attributed to a sample

processing error and therefore, not used in the calculation of mean measured

concentrations.

Analysis of the test solutions throughout the e xposure period demonstrated that the expected

concentration-gradient was generally maintained during the exposure. Exposure solutions were

generally consistent throughout the exposure period and were maintained at a CV of ≤ 16%.

Mean measured concentrations of lindane range d from 76 to 130% of no minal. Based on mean

measured concentrations of lindane, the treatmen t levels tested (and measured) were defined as

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 41

30, 100, 170, 380 and 1100 ng/L. The relationship betw een the nominal treatm ent levels and the

mean measured concentrations established during the exposure period is illustrated in Figure 2

.

Thirty-three of the thir ty-six QC samples ranged from 78.7% to 118% ( Table 3 ) of the nominal

fortified concentrations (20.0, 200 and 1250 ng/L) a nd were consistent with the predetermined

recovery range (i.e., set at 70 to 120%, Appendix 3 ). Based on these results, it was determined

that the appropriate quality control was maintain ed during the analyses of the lindane exposure

solution samples. Three of the thirty-six QC samples had recoveries outside the predetermined

recovery range at 127, 130 and 145% recovery. QC samples can be out of range due to a number

of factors, some of which ar e spiking errors, handling errors and instrument errors.



## 5.3 Biological Results

5.3.1 F 0 

## Generation



## 5.3.1.1 F 0 

## Survival

Mean percent survival in the control for the F 0 

### generation averaged 82% ( Table 4 and Figure 3 ).

Mean percent survival among F 0 

### mysids exposed to the treatmen t levels tested averaged 79 to

87%. Dunnett’s Multiple Comparison Test dete rmined no significant differences in survival

among mysids exposed to any of the treatment le vels tested, compared to the control.



## 5.3.1.2 F 0 

## Growth

Growth measurements were determined at three time points during the F 0 

### exposure. Mysid dry

body weight and mysid total length were measured at day 0, 7, 14 and 27 (see Protocol

Deviations ).

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 42



## 5.3.1.2.1 Day 0 (Exposure Initiation) – F 0 

## Total Body Length and Dry Body

Weight

Day 0 mean total body length for the F 0 

### generation was 1.84 mm and mean dry body weight for

the F 0 

### generation was 0.035 mg ( Table 4 ).



## 5.3.1.2.2 Day 7 - F 0 

## Total Body Length and Dry Body Weight

Day 7 mean total body length in the control for the F 0 

### generation averaged 4.46 mm ( Table 4 and

Figure 4 ). Mean total body length among F 0 

### mysids exposed to the treatment levels tested

averaged 4.46 to 4.84 mm. Dunnett’s Multiple Comparison Test determined no significant

differences in total body length among mysids expos ed to any of the treatment levels tested,

compared to the control.

Day 7 mean dry body weight in the control for the F 0 

### generation averaged 0.30 mg ( Table 4 and

Figure 5 ). Mean dry body weight among F 0 

### mysids exposed to the treatment levels tested

averaged 0.28 to 0.36 mg. Dunnett’s Multiple Comparison Test determined no significant

differences in dry body weight am ong mysids exposed to any of the treatment levels tested,

compared to the control.



## 5.3.1.2.3 Day 14 - F 0 

## Total Body Length and Dry Body Weight

Day 14 mean male total body lengt h in the control for the F 0 

### generation averaged 6.25 mm

( Table 4 and Figure 6 ). Mean total body length among F 0 

### male mysids exposed to the treatment

levels tested averaged 6.51 to 6.85 mm. Dunne tt’s Multiple Comparison Test determined no

significant differences in male total body length among mysids expos ed to any of the treatment

levels tested, compared to the control.

Day 14 mean female total body length in the control for the F 0 

### generation averaged 6.34 mm

( Table 4 and Figure 6 ). Mean total body length among F 0 

### female mysids exposed to the 32, 80,

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 43

200, 500 and 1250 ng/L nominal treatment levels averaged 6.69, 6.73, 6.86, 7.08 and 6.70 mm,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in female total body length among mysids expos ed to the 200 and 500 ng/L nominal treatment

levels compared to the control.

Day 14 mean male dry body weight in the control for the F 0 

### generation averaged 0.58 mg

( Table 4 and Figure 7 ). Mean dry body weight among F 0 

### male mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 0.57, 0.64, 0.75, 0.89 and 0.86 mg,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in male dry body weight among mysids e xposed to the 200, 500 and 1250 ng/L nominal

treatment levels compared to the control.

Day 14 mean female dry body weight in the control for the F 0 

### generation averaged 0.70 mg

( Table 4 and Figure 7 ). Mean dry body weight among F 0 

### female mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 0.73, 0.80, 0.99, 1.11 and 1.05 mg,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in female dry body weight among mysids e xposed to the 200, 500 and 1250 ng/L nominal

treatment levels compared to the control.



## 5.3.1.2.4 Day 27 (F 0 

## Termination) - Total Body Length and Dry Body Weight

Day 27 mean male total body lengt h in the control for the F 0 

### generation averaged 7.32 mm

( Table 4 and Figure 8 ). Mean total body length among F 0 

### male mysids exposed to the treatment

levels tested averaged 6.96 to 7.35 mm. Dunne tt’s Multiple Comparison Test determined no

significant differences in male total body length among mysids expos ed to any of the treatment

levels tested, compared to the control.

Day 27 mean female total body length in the control for the F 0 

### generation averaged 7.51 mm

( Table 4 and Figure 8 ). Mean total body length among F 0 

### female mysids exposed to the

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 44

treatment levels tested averaged 7.60 to 7.86 mm. Dunnett’s Multiple Comparison Test

determined no significant differences in fema le total body length among mysids exposed to any

of the treatment levels teste d, compared to the control.

Day 27 mean male dry body weight in the control for the F 0 

### generation averaged 0.96 mg

( Table 4 and Figure 9 ). Mean dry body weight among F 0 

### male mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 0.97, 0.88, 0.84, 0.95 and 0.72 mg,

respectively. Bonferroni's Adjusted t-Te st determined a significant difference (p ≤ 0.05) in male

dry body weight among mysids exposed to the 12 50 ng/L nominal treatment level compared to

the control.

Day 27 mean female dry body weight in the control for the F 0 

### generation averaged 1.30 mg

( Table 4 and Figure 9 ). Mean dry body weight among F 0 

### female mysids exposed to the

treatment levels tested averaged 1.20 to 1.45 mg. Dunnett’s Multiple Comparison Test

determined no significant differences in fema le dry body weight among mysids exposed to any

of the treatment levels tested, compared to the control.



## 5.3.1.3 F 0 

## Time to Maturation

Mean time to maturation for the F 0 

### control mysids averaged 10.3 days ( Table 5 and Figure 10 ).

Mean time to maturation among mysids exposed to the treatment levels av eraged 9.0 to 9.7 days.

Dunnett’s Multiple Comparison Test determined no significant differences in time to maturation

among mysids exposed to any of the treatment le vels tested, compared to the control.



## 5.3.1.4 F 0 

## Time to First Brood Release

Mean time to first brood release for the F 0 

### control mysids averaged 15.8 days ( Table 5 and

Figure 11 ). Mean time to first brood release for the F 0 

### mysids exposed to the 32, 80, 200, 500

and 1250 ng/L nominal treatment levels averaged 16.0, 15.2, 15.3, 14.2 and 15.2 days,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 45

in time to first brood release among mysids expos ed to the 500 ng/L nominal treatment level

compared to the control.



## 5.3.1.5 F 0 

## Number of Offspring in the First Brood

The mean total number produced in the first brood from the F 0 

### control mysids averaged

5.5 offspring/female ( Table 5 and Figure 12 ). The mean total number produced in the first brood

from the F 0 

### mysids exposed to the 32, 80, 200, 500 and 1250 ng/L nominal treatment levels

averaged 6.6, 6.4, 6.4, 8.1 and 6.7 offspring/fe male, respectively. Dunnett's Multiple

Comparison Test determined a significant difference (p ≤ 0.05) in number of offspring in the first

brood among mysids exposed to the 500 ng/L tr eatment level compared to the control.



## 5.3.1.6 F 0 

## Time to Second Brood Release

Mean time to second brood release for the F 0 

### control mysids averaged 21.3 days ( Table 5 and

Figure 13 ). Mean time to second brood release for the F 0 

### mysids exposed to the 32, 80, 200, 500

and 1250 ng/L nominal treatment levels averaged 20.6, 20.2, 20.3, 19.3 and 21.0 days,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in time to second brood release among mysids e xposed to the 500 ng/L nominal treatment level

compared to the control.



## 5.3.1.7 F 0 

## Number of Offspring in the Second Brood

The mean total number of offspring produced in the second brood from the F 0 

### control mysids

averaged 8.8 offspring/female ( Table 5 and Figure 14 ). The mean total number produced in the

second brood from the F 0 

### mysids exposed to the treatment levels averaged 8.6 to

10.7 offspring/female. Dunnett’s Multiple Co mparison Test determined no significant

differences in total number of offspring produced in the second brood among mysids exposed to

any of the treatment levels teste d, compared to the control.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 46



## 5.3.1.8 F 0 

## Interbrood Duration

Mean F 0 

### interbrood duration for the cont rol mysids averaged 5.4 days ( Table 5 and Figure 15 ).

Mean interbrood duration for mysids exposed to the treatment levels tested averaged 4.5 to

6.4 days. Dunnett’s Multiple Comparison Test determined no significant differences in

interbrood duration among mysids exposed to any of the treatment levels tested, compared to the

control.



## 5.3.1.9 F 0 

## Total Number of Offspring in First Two Broods

The total mean number of offspring re leased in the first two broods for F 0 

### control mysids

averaged 14.4 offspring/female ( Table 6 and Figure 16 ). The total mean number released in the

first two broods for F 0 

### mysids exposed to the treatmen t levels tested averaged 15.0 to

18.8 offspring/female. Dunnett’s Multiple Co mparison Test determined no significant

differences in number of offspri ng released among mysids exposed to any of the treatment levels

tested, compared to the control.



## 5.3.1.10 F 0 

## Sex Ratio (Male:Female)

The F 0 

### male to female sex ratio in the control averaged 1.3 ( Table 6 and Figure 17 ). The F 0 

### male

to female sex ratio among mysids exposed to th e treatment levels tested averaged 0.70 to 1.4.

Dunnett’s Multiple Comparison Test determined no significant differences in sex ratio among

mysids exposed to any of the treatment le vels tested, compared to the control.



## 5.3.1.11 F 0 

## Percent of Reproducti vely Active Females

The mean percent of reproducing females in the F 0 

### control averaged 100% ( Table 6 and

Figure 18 ). Mean percent of reproducing F 0 

### females among mysids exposed to the treatment

levels tested ranged from 95 to 100%. Dunnett ’s Multiple Comparison Test determined no

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 47

significant differences in mean percent of reproducing F 0 

### females among mysids exposed to any

of the treatment levels tested, compared to the control.



## 5.3.2 F 1 

## Generation



## 5.3.2.1 F 1 

## Survival

Mean percent survival in the control for the F 1 

### generation averaged 85% ( Table 7 and Figure 19 ).

Mean percent survival among F 1 

### mysids exposed to the treatment levels tested ranged from 70 to

92%. Dunnett’s Multiple Comparison Test dete rmined no significant differences in survival

among mysids exposed to any of the treatment levels tested, compared to the control.



## 5.3.2.2 F 1 

## Growth

Growth measurements were determin ed at four time points during the F 1 

### exposure. Mysid dry

body weight and mysid total length were measured at the respective day 0, 7, 14 and 27 for the

F 1 

### generation.



## 5.3.2.2.1 Day 0 (Exposure Initiation) – F 1 

## Total Body Length and Dry Body Weight

Day 0 mean total body length in the control for the F 1 

### generation averaged 1.63 mm ( Table 7 and

Figure 20 ). Mean total body length among F 1 

### mysids exposed to the treatment levels tested

averaged 1.64 to 1.70 mm. Dunnett’s Multiple Comparison Test determined no significant

differences in total body length among mysids expos ed to any of the treatment levels tested,

compared to the control.

Day 0 mean dry body weight in the control for the F 1 

### generation averaged 0.029 mg ( Table 7 and

Figure 21 ). Mean dry body weight among F 1 

### mysids exposed to the treatment levels tested

averaged 0.025 to 0.033 mg. Dunnett’s Multiple Comparison Test determined no significant

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 48

differences in dry body weight am ong mysids exposed to any of the treatment levels tested,

compared to the control.



## 5.3.2.2.2 Day 7 - F 1 

## Total Body Length and Dry Body Weight

Day 7 mean total body length in the control for the F 1 

### generation averaged 4.72 mm ( Table 7 and

Figure 22 ). Mean total body length among F 1 

### mysids exposed to the treatment levels tested

averaged 4.33 to 4.86 mm. Dunnett’s Multiple Comparison Test determined no significant

differences in total body length among mysids expos ed to any of the treatment levels tested,

compared to the control.

Day 7 mean dry body weight in the control for the F 1 

### generation averaged 0.38 mg ( Table 7 and

Figure 23 ). Mean dry body weight among F 1 

### mysids exposed to the treatment levels tested

averaged 0.29 to 0.38 mg. Dunnett’s Multiple Comparison Test determined no significant

differences in dry body weight am ong mysids exposed to any of the treatment levels tested,

compared to the control.



## 5.3.2.2.3 Day 14 - F 1 

## Total Body Length and Dry Body Weight

Day 14 mean male total body lengt h in the control for the F 1 

### generation averaged 6.32 mm

( Table 7 and Figure 24 ). Mean total body length among F 1 

### male mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 6.33, 5.98, 5.90, 6.10 and 5.74 mm,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

among mysids exposed to the 1250 ng/L nominal treatment level compar ed to the control.

Day 14 mean female total body length in the control for the F 1 

### generation averaged 6.46 mm

( Table 7 and Figure 24 ). Mean total body length among F 1 

### female mysids exposed to the

treatment levels tested averaged 6.23 to 6.66 mm. Dunnett’s Multiple Comparison Test

determined no significant differences in fema le total body length among mysids exposed to any

of the treatment levels tested, compared to the control.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 49

Day 14 mean male dry body weight in the control for the F 1 

### generation averaged 0.72 mg

( Table 7 and Figure 25 ). Mean dry body weight among F 1 

### male mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 0.76, 0.52, 0.47, 0.61 and 0.45 mg,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in male dry body weight among mysids exposed to the 500 ng/L nominal treatment level

compared to the control.

Day 14 mean female dry body weight in the control for the F 1 

### generation averaged 0.93 mg

( Table 7 and Figure 25 ). Mean dry body weight among F 1 

### female mysids exposed to the

treatment levels tested ranged from 0.73 to 0.90 mg. Dunnett’s Multiple Comparison Test

determined no significant differences in fema le dry body weight among mysids exposed to any

of the treatment levels tested, compared to the control.



## 5.3.2.2.4 Day 27 (Exposure Termination) - Total Body Length and Dry Body

Weight

Day 27 mean male total body lengt h in the control for the F 1 

### generation averaged 7.30 mm

( Table 7 and Figure 26 ). Mean total body length among F 1 

### male mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 7.32, 7.07, 6.91, 7.40 and 6.74 mm,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in male total body length among mysids exposed to the 1250 ng/L nomin al treatment level

compared to the control.

Day 27 mean female total body length in the control for the F 1 

### generation averaged 7.52 mm

( Table 7 and Figure 26 ). Mean total body length among F 1 

### female mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 7.37, 7.39, 7.33, 7.90 and 7.55 mm,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in female total body length among mysids expos ed to the 500 ng/L nomin al treatment level

compared to the control.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 50

Day 27 mean male dry body weight in the control for the F 1 

### generation averaged 0.86 mg

( Table 7 and Figure 27 ). Mean dry body weight among F 1 

### male mysids exposed to the 32, 80,

200, 500 and 1250 ng/L nominal treatment levels averaged 0.90, 0.81, 0.78, 0.87 and 0.70 mg,

respectively. Dunnett's Multiple Comparison Te st determined a significant difference (p ≤ 0.05)

in male dry body weight among mysids exposed to the 1250 ng/L nomin al treatment level

compared to the control.

Day 27 mean female dry body weight in the control for the F 1 

### generation averaged 1.25 mg

( Table 7 and Figure 27 ). Mean dry body weight among F 1 

### female mysids exposed to the

treatment levels tested averaged 1.10 to 1.29 mg. Dunnett’s Multiple Comparison Test

determined no significant differences in fema le dry body weight among mysids exposed to any

of the treatment levels tested, compared to the control.



## 5.3.2.3 F 1 

## Time to Maturation

Mean time to maturation for the F 1 

### control mysids averaged 9.7 days ( Table 8 and Figure 28 ).

Mean time to maturation among mysids exposed to the treatment levels tested averaged 8.7 to

11 days. Dunnett’s Multiple Comparison Test de termined no significant differences in time to

maturation among mysids exposed to any of the treatment levels test ed, compared to the control.



## 5.3.2.4 F 1 

## Time to First Brood Release

Mean time to first brood release for the F 1 

### control mysids averaged 14.9 days ( Table 8 and

Figure 29 ). Mean time to first brood release for the F 1 

### mysids exposed to the 32, 80, 200, 500

and 1250 ng/L nominal treatment levels averaged 16.6, 16.7, 16.7, 15.7, 16.0 days, respectively.

Dunnett's Multiple Comparison Test determined a significant difference (p ≤ 0.05) in time to first

brood release among mysids exposed to the 32, 80 and 200 ng/L nominal treatment levels

compared to the control.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 51



## 5.3.2.5 F 1 

## Number of Offspring in the First Brood

The mean total number produced in the first brood from the F 1 

### control mysids averaged

6.6 offspring/female ( Table 8 and Figure 30 ). The mean total number produced in the first brood

from the F 1 

### mysids exposed to the 32, 80, 200, 500 and 1250 ng/L nominal treatment levels

averaged 6.1, 4.8, 3.9, 4.8 and 5.3 offspring/fe male, respectively. Dunnett's Multiple

Comparison Test determined a significant difference (p ≤ 0.05) in number of offspring in the first

brood among mysids exposed to the 200 ng/L nominal treatment level compar ed to the control.



## 5.3.2.6 F 1 

## Time to Second Brood Release

Mean time to second brood release for the F 1 

### control mysids averaged 20.0 days ( Table 8 and

Figure 31 ). Mean time to second brood release for the F 1 

### mysids exposed to the treatment levels

tested averaged 20.9 to 21.8 days. Dunnett’s Multiple Comparison Test determined no

significant differences in time to second brood release among mysids exposed to any of the

treatment levels tested, compared to the control.



## 5.3.2.7 F 1 

## Number of Offspring in the Second Brood

The mean total number produced in the second brood from the F 1 

### control mysids averaged

9.6 offspring/female ( Table 8 and Figure 32 ). The mean total number produced in the second

brood from the F 1 

### mysids exposed to the 32, 80, 200, 500 and 1250 ng/L nominal treatment

levels averaged 9.1, 7.4, 5.3, 10.6 and 9.3 offspring/female, respectively. Dunnett's Multiple

Comparison Test determined a significant difference (p ≤ 0.05) in number of offspring in the first

brood among mysids exposed to the 200 ng/L nominal treatment level compar ed to the control.



## 5.3.2.8 F 1 

## Interbrood Duration

Mean F 1 

### interbrood duration for the cont rol mysids averaged 5.2 days ( Table 8 and Figure 33 ).

Mean interbrood duration for mysids exposed to the treatment levels tested averaged 4.8 to

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 52

5.6 days. Dunnett’s Multiple Comparison Test determined no significant differences in

interbrood duration among mysids exposed to any of the treatment levels tested, compared to the

control.



## 5.3.2.9 F 1 

## Total Number of Offspring in First Two Broods

The total mean number released in the first two broods for F 1 

### control mysids averaged

16.0 offspring/female ( Table 9 and Figure 34 ). The total mean number released in the first two

broods for F 1 

### mysids exposed to the 32, 50, 200, 500 and 1250 ng/L nominal treatment levels

averaged 14.9, 12.2, 9.2, 15.4 and 14.3 offspring/female, respectively. Dunnett's Multiple

Comparison Test determined a significant difference (p ≤ 0.05) in number offspring among

mysids exposed to the 80 and 200 ng/L nominal treatment levels compared to the control.



## 5.3.2.10 F 1 

## Sex Ratio (Male:Female)

The F 1 

### male to female sex ratio in the control averaged 1.3 ( Table 9 and Figure 35 ). The F 1 

### male

to female sex ratio among mysids exposed to th e treatment levels tested averaged 0.87 to 1.7.

Dunnett’s Multiple Comparison Test determined no significant differences in sex ratio among

mysids exposed to any of the treatment le vels tested, compared to the control.



## 5.3.2.11 F 1 

## Percent of Reproducti vely Active Females

The mean percent of reproducing females in the F 1 

### control averaged 100% ( Table 9 and

Figure 36 ). Mean percent of reproducing F 1 

### females among mysids exposed to the treatment

levels tested averaged 95 to 100%. Fisher's Exact Test with Bonferroni-Holm's Adjustment

determined no significant differences in perc ent of reproducing females among mysids exposed

to any of the treatment levels tested, compared to the control.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 53



# 6.0 CONCLUSIONS

For the F 0 

### exposure, male and female mysid dry we ight (day 14) were the most sensitive

endpoints in the mysid two-gene ration exposure to lindane. Li ndane was found to significantly

increase F 0 

### 14-day male and female dry weight at 200, 500 and 1250 ng/L; 14-day female length

at 200 and 500 ng/L and offspring per female produced in the fi rst brood release at 500 ng/L.

Significant reductions (p ≤ 0.05) were observed in male term ination (day 27) dry weight at

1250 ng/L; and time to first and second brood release at 500 ng/L.

F 1 

### generation survival, day 27 male dry weight and total body length were the most sensitive

endpoints in the mysid two-gene ration exposure to lindane. Li ndane was found to significantly

increase F 1 

### female termination (day 27) length at 500 ng/L; and time to first brood release at 32,

80 and 200 ng/L. Significant reductions (p ≤ 0.05) were observed in ma le 14-day dry weight at

500 ng/L; 14-day male length, male termination (day 27) length and male termination (day 27)

dry weight at 1250 ng/L; offspring per female pr oduced in the first a nd second brood release at

200 ng/L; and total offspring per female dur ing first and second brood release at 80 and

200 ng/L.

Table 10 summarizes the F 0 

### and F 1 

### endpoints and the statistically significant differences from the

controls determined during this study.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 54



## PROTOCOL DEVIATIONS

1. The protocol states that at exposure initiation, water quality variables will be measured in each test vessel, and in alterna ting replicates daily thereafter . For this study, at exposure initiation, dissolved oxygen and temperatur e were measured, but not recorded for replicate C of the control group. For this exposure system, one solution is split three ways to each replicate of the treatment/con trol vessels using a common splitter; the F 0 

### control animals exceeded control acceptability cr iteria, therefore, it can be assumed that the water quality variables in this vessel were acceptable for propagation of healthy mysids. Therefore, this deviation did not have a negative impact on the results or interpretation of the study.

2. The protocol states th at at exposure initiation, forty-five , day 0 mysids will be collected from the test population. For this study, at exposure initiation, day 0 measurements were inadvertently not performed. These measur ements were performed 7 days following exposure initiation. For this exposure system, three sets of forty-five, < 24-hour old mysids were collected one week from each other to demonstrate that there was little variation in size of a mysid this age from week to week. Ther efore, this deviation did not have a negative impact on the resu lts or interpretation of the study.

3. The protocol states that the temperature of test solutions will be maintained at 26 ± 1 oC. On test days 37 and 38, the heater in the upper bath failed and measurements of daily temperature extremes were outside of the prescribed range. On both days, minimum temperatures of 23 and 24 oC were recorded. The heater was replaced and temperatures were maintained with the prescribed ra nge for the remainder of the study. Since temperatures maintained within this study ar e well within established culture and testing temperatures for this species and since control organisms exceeded the acceptance criteria, this deviation did not have a negative impact on the results or interpretation of the study.

4. The protocol states that the F 0 

### exposure will continue until seven days after the median day of release of the second brood in the control treatment s. For this study, the F 0 

### exposure was terminated six days after the median day of release of the second brood

release in the control treatmen ts. The data generated for this study examines the impact

on the first two broods. Since animals that survived long enough to release two broods released two broods and since for these animals, all sub-replicates fo r all concentrations released two broods, this deviation did not have a negative impact on the results or interpretation of the study.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 55



## REFERENCES

ASTM, 2002. Standard practice for conducti ng acute toxicity test with fishes, macroinvertebrates, and amphibians. Standa rd E729-96. American Society for Testing and Materials, 100 Barr Harbor Dr ive, West Conshohocken, PA 19428.

ASTM, 2008. Committee E-47 on Biol ogical Effects and Environmental Fate. Standard Guide for Conducting Life-Cycle Toxicity Tests with Mysids. ASTM Designation E 1191-039 (re-approved 2008). American Society for Test ing and Materials, 100 Barr Harbor Drive, West Conshohocken, Pennsylvania.

Ives, Michael, 2009. CETIS, Comprehensive Environmental Toxicity Information System TM



### . User’s Guide. Tidepool Scientific Software, McKinleyville, California.

McKenney CL, Jr., 1986. Influence 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 temper ature in an estuarine mysid ( Mysidopsis bahia ) in relation to its life cycle. Comp Biochem Physiol 109A: 199-208.

McKenney CL, Jr. and Celestial DM., 1995. Interactions among salinity, temperature, and age on growth of the estuarine mysid Mysidopsis bahia reared in the laboratory through a complete life cycle. I. Body mass and age-sp ecific growth rate. J Crust Biol 15: 169-

178.

McKenney CL, Jr., 1996. The combined effects of salinity and temperature on various aspects of the reproductive biology of the estuarine mysid, Mysidopsis bahia

. Invertebr Reprod

Dev 29(1): 9-18.

Mount, D.I. and W.A. Brungs, 1967. A simplifie d dosing apparatus for fish toxicological

studies. Water Research . 1: 21-29.

Nimmo DR, Bahner LH, Rigby RA, She ppard JM and Wilson AJ, Jr., 1977. Mysidopsis bahia : an estuarine species suitable for life-cycle to xicity tests to determine the effects of a pollutant. In: Aquatic Toxicology and Hazard Evaluation, ASTM STP 634 . FL Mayer and JL Hamelink, Eds., American Society for Te sting and Materials, Philadelphia, PA, pp.

109-116.

Nimmo DR, Hamaker TL and Sommers CA., 1978. En tire life cycle to xicity test using mysids ( Mysidopsis bahia ) in flowing water. In: Bioassay Procedures for the Ocean Disposal
Permit Program , EPA-600/9-78-010, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL, pp. 64-68.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 56

Verslycke TA, N Fockedey, CL McKenney, Jr., SD Roast, MB Jones, J Mees, and CR Janssen, 2004. Mysid crustaceans as potential test orga nisms for the evaluation of environmental endocrine disruption: a review. Environ Tox Chem 23 (5): 1219-34.

U.S. EPA. Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice Standards; Final Rule (40 CFR, Part 160). U.S. Environmental Protection Agency, Washington, D.C.

U.S. EPA, 2002. Short-term Methods for Estim ating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. 3 rd 

### Edition. EPA-821-R-02-014, U.S. Environmental Protection Agency, Washington, DC.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 57



## Table 1. Summary of water quality pa rameters measured in the exposure solutions during the mysid ( Americamysis bahia ) two-generation test

with lindane.

Nominal

Concentration (ng/L) Ranges a

Dissolved

Oxygen pH Temperature b

( o C) Salinity (‰) mg/L %

Saturation

Control 5.7 - 7.1 78 - 97 7.7 - 8.1 24 - 26 19 - 21

32 5.7 - 7.1 79 - 97 7.8 - 8.1 24 - 27 19 - 21

80 5.9 - 7.1 81 - 97 7.8 - 8.1 24 - 27 19 - 22

200 5.9 - 8.0 81 - 110 7.7 - 8.1 24 - 27 19 - 21

500 5.6 - 7.3 79 - 99 7.7 - 8.1 24 - 27 19 - 22

1250 5.9 - 7.1 81 - 97 7.7 - 8.1 24 - 27 19 - 21

a Dissolved oxygen, pH, temperat ure and salinity were measured daily in each replicate.

b Continuous temperature monitoring of the controls established a temperature range of 23 to 27 oC (see Protocol

Deviations ) throughout the exposure period.

NOTE: Measurements have been r ounded to two significant figures.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 58Table 2. Concentrations of lindane measuredin the exposure solutions during the mysid (Americamysis bahia)two-generation test.NominalConcentration(ng/L)Measured Concentration (ng/L)%CVPercentof Nominal(%)aDay 0 Day 5 Day 8 Day 11 Day14 Day 22 Day 29 Day 35 Day 42 Day 45/46 Day 47 Day 48Mean(SD)aControl< 8.0b< 8.0< 8.0< 7.8< 7.8< 7.8< 7.7 NSc< 8.0 < 7.8 < 8.0 < 7.8 < 8.0 NS< 7.8< 7.8< 8.1NAd(NA)NA NA32313115e35312734 NS 33 37 30 30 25 NS252825NS30(3.8)13 9480100120110NS 130 NS 100 120 97 94 96 868382NS100(15)15 130200180190190NS 190 NS 190 220 180 140 150 NS170130150NS170(27)16 86500420420380NS 430 NS 400 440 360 310 350330400350360 NS380(42)11 76125010001200920NS 550e1300130014001100 1200 850 980 1100 NS100010001000NS1100(150)14 88aMean measured values, standard deviations (SD) and percent ofnominal were calculated using the actual analytical results andnot the rounded values (two significant figures) presented in this table.bConcentrations expressed as less than values were below thelimit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area ofthe low standards and the dilutionfactor of the controls.cNS = Not Sampled.dNA = Not Applicable.eLow recoveries attributed to sample processing error. Value not used in calculation of mean measured concentration.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 59



## Table 3. Results of the quality contro l (QC) samples analyzed concurrently

with test solution sa mples during the mysid ( Americamysis bahia ) two-generation test

.

Quality Control Sample (ng/L)

20.0 a 200 a 1250 a

Test Day Measured

Concentration (ng/L) Percent of

Nominal Measured

Concentration (ng/L) Percent of

Nominal Measured

Concentration (ng/L) Percent of

Nominal

0 25.5 127 b 235 117 1470 118

5 26.0 130 b 233 117 1350 108

8 21.6 108 204 102 1410 113

11 29.1 145 b 216 108 1440 115

14 21.6 108 197 98.6 1260 100

22 16.3 81.7 180 89.8 1280 102

29 18.1 90.4 183 91.4 983 78.7

35 23.4 117 163 81.3 1140 91.0

42 21.9 110 196 98.0 1200 96.3

45/46 17.8 89.2 210 105 1290 103

47 18.1 90.4 215 108 1240 99.6

48 17.3 86.6 209 105 1190 95.0

a QC sample fortified concentrations.

b Percent recovery for this QC sample is outsi de of the acceptable range (i.e., 70 to 120%, Appendix 3 ).RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 60Table 4. F0mysid survival and growth (lengthand dry weight) during the mysid (Americamysis bahia) twogeneration testwith lindane.NominalConcentration(ng/L)ReplicateSurvival(%)p-ValueMean Total Body Length (mm)aDay 7 p-ValueDay 14 Day 27Male p-Value Female p-ValueMale p-Value Female p-ValueControl A 81 4.33 5.93 6.07 7.21 7.30B 87 4.67 6.49 6.57 7.30 7.51C 77 4.38 6.33 6.37 7.47 7.72Mean(SD) 82(5.0) NAc4.46(0.19) NA 6.25(0.29) NA 6.34(0.25) NA 7.32(0.13) NA 7.51(0.21) NA32 A 84 4.44 6.58 6.67 7.16 7.47B 77 4.44 6.45 6.74 7.37 7.66C 83 4.51 6.81 6.66 7.51 7.89Mean(SD) 81(3.9) 0.7977 4.46(0.04) 0.8272 6.61(0.18) 0.2293 6.69(0.040) 0.0963 7.35(0.18) 0.7982 7.67(0.21) 0.406080 A 85 4.56 6.43 6.61 7.11 7.35B 85 4.99 7.16 7.09 7.07 7.68C 90 4.42 6.64 6.47 7.40 7.75Mean(SD) 87(2.7) 0.9820 4.66(0.30) 0.3316 6.74(0.38) 0.1028 6.73(0.33) 0.0696 7.19(0.18) 0.9785 7.60(0.21) 0.6342200 A 89 4.87 6.56 6.64 6.97 7.47B 75 5.01 6.92 7.15 7.34 7.83C 72 4.49 6.91 6.78 7.00 7.52Mean(SD) 79(9.3) 0.6630 4.79(0.27) 0.1036 6.80(0.20) 0.0716 6.86(0.26)d0.0176 7.10(0.21) 0.9957 7.61(0.19) 0.5964500 A 69 4.68 6.87 7.19 7.05 7.75B 86 5.03 6.71 7.00 NA 7.94C 86 4.81 6.96 7.06 7.41 7.88Mean(SD) 80(9.8) 0.7749 4.84(0.18) 0.0622 6.85(0.13) 0.0504 7.08(0.10)d0.0016 7.23(0.26) 0.9532 7.86(0.10) 0.08011250 A 76 4.65 6.07 6.68 7.07 7.68B 83 4.53 6.50 6.77 7.02 7.86C 76 4.58 6.96 6.66 6.79 7.47Mean(SD) 79(4.2) 0.6059 4.58(0.06) 0.5189 6.51(0.45) 0.3881 6.70(0.057) 0.0847 6.96(0.15) 0.9997 7.67(0.19) 0.4152aMean total body length for the control on day 0 = 1.84 mm; standard deviation = 0.018.bMean dry body weight for the control on day 0 = 0.035 mg; standard deviation = 0.0028.cNA = Not Applicable.dStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.eStatistically different compared to the control, based on Bonferroni's Adjusted t-Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 61Table 4. Continued. F0mysid survival and growth (length and dry weight) during the mysid (Americamysisbahia) two-generation test with lindane.NominalConcentration(ng/L)ReplicateMean Dry Body Weight (mg)bDay 7 p-ValueDay 14 Day 27Male p-Value Female p-ValueMale p-Value Female p-ValueControl A 0.31 0.55 0.69 1.06 1.24B 0.33 0.58 0.84 0.92 1.26C 0.25 0.62 0.58 0.90 1.40Mean(SD) 0.30(0.04) NA 0.58(0.03) NA 0.70(0.13) NA 0.96(0.09) NA 1.30(0.09) NA32 A 0.34 0.59 0.77 0.95 1.43B 0.40 0.56 0.73 0.88 1.30C 0.33 0.57 0.70 1.06 1.52Mean(SD) 0.36(0.04) 0.1168 0.57(0.01) 0.8797 0.73(0.03) 0.7409 0.97(0.09) 1.0000 1.42(0.11) 0.338280 A 0.25 0.54 0.81 0.86 1.09B 0.32 0.70 1.04 0.88 1.45C 0.27 0.69 0.55 0.90 1.26Mean(SD) 0.28(0.04) 0.9453 0.64(0.09) 0.4002 0.80(0.24) 0.4775 0.88(0.02) 0.6270 1.27(0.18) 0.9145200 A 0.33 0.66 0.88 0.85 1.30B 0.28 0.74 1.06 0.88 1.45C 0.33 0.84 1.05 0.81 1.26Mean(SD) 0.32(0.03) 0.6265 0.75(0.09)d0.0233 0.99(0.10)d0.0369 0.84(0.03) 0.2847 1.34(0.10) 0.6989500 A 0.32 0.87 1.14 0.89 1.47B 0.37 0.89 1.18 NA 1.34C 0.39 0.90 1.01 1.02 1.55Mean(SD) 0.36(0.03) 0.0982 0.89(0.01)d0.0004 1.11(0.08)d0.0059 0.95(0.09) 1.0000 1.45(0.11) 0.20821250 A 0.35 0.75 1.03 0.77 1.20B 0.30 0.94 1.17 0.80 1.30C 0.27 0.90 0.96 0.58 1.10Mean(SD) 0.30(0.04) 0.7189 0.86(0.10)d0.0007 1.05(0.11)d0.0149 0.72(0.12)e0.0089 1.20(0.10) 0.9833aMean total body length for the control on day 0 = 1.84 mm; standard deviation = 0.018.bMean dry body weight for the control on day 0 = 0.035 mg; standard deviation = 0.0028.cNA = Not Applicable.dStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.eStatistically different compared to the control, based on Bonferroni's Adjusted t-Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 62Table 5. F0mysid time to maturation, timeto first brood release, time tosecond brood release, offspringreleased per female and interbrood duration during the mysid (Americamysis bahia) two-generationtest with lindane.NominalConcentration(ng/L)ReplicateTime toMaturation(Days)pValueFirst Brood Second BroodInterbroodDuration(Days)pValueTime toRelease(Days)pValueOffspring/Femalep-ValueTime toRelease(Days)pValueOffspring/Femalep-ValueControl A 10 15.7 4.7 21.1 9.9 5.4B 10 15.3 5.9 20.3 7.1 5.0C 11 16.4 5.9 22.3 9.5 5.8Mean(SD) 10.3(0.58) NAa15.8(0.68) NA 5.5(0.66) NA 21.3(1.0) NA 8.8(1.5) NA 5.4(0.42) NA32 A 9 16.1 6.0 20.3 9.2 4.2B 10 16.4 8.0 20.9 11.4 4.4C 10 15.6 5.9 20.6 10.6 5.0Mean(SD) 9.7(0.58) 0.9742 16.0(1.8) 0.9379 6.6(1.2) 0.3031 20.6(0.26) 0.4544 10.4(1.1) 0.3304 4.5(0.43) 0.154180 A 10 15.9 6.4 21.1 8.6 5.3B 9 14.3 8.3 19.3 10.3 5.0C 10 15.3 4.4 20.3 8.7 5.0Mean(SD) 9.7(0.58) 0.9742 15.2(1.4) 0.2888 6.4(1.9) 0.4272 20.2(0.93) 0.2435 9.2(1.0) 0.7355 5.1(0.16) 0.5944200 A 10 15.0 6.0 20.0 6.7 5.0B 9 14.9 6.7 19.8 11.2 5.0C 9 16.1 6.4 21.1 7.9 5.0Mean(SD) 9.3(0.58) 0.9974 15.3(0.7) 0.4305 6.4(0.36) 0.4272 20.3(0.72) 0.2765 8.6(2.4) 0.8812 5.0(0) 0.5006500 A 9 14.6 8.4 20.1 10.3 5.6B 9 13.9 8.3 18.8 11.5 5.3C 9 14.1 7.4 19.1 10.3 5.0Mean(SD) 9.0(0)0.9997 14.2(0.92)b0.0105 8.1(0.54)b0.0245 19.3(0.72)b0.0297 10.7(0.70) 0.2463 5.3(0.29) 0.76531250 A 9 15.9 6.0 21.3 7.7 6.7B 10 15.0 6.4 22.0 10.0 7.5C 10 14.9 76 19.8 11.8 5.0Mean(SD) 9.7(0.58) 0.9742 15.2(0.80) 0.3706 6.7(0.81) 0.2891 21.0(1.1) 0.7328 9.8(2.1) 0.5212 6.4(1.3) 0.9990aNA = Not Applicable.bStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 63



## Table 6. F 0 

## mysid total number of young in the first two broods, sex ratio and percentage of reproductively acti ve females during the mysid ( Americamysis bahia ) two-generation test with lindane

.

Nominal

Concentration (ng/L) Replicate Total

Number of Young

in First

Two

Broods pValue Sex Ratio

Percentage of Reproductively

Active Females p-Value Total

Number

of Males Total

Number

of

Females Male:Female

Ratio pValue

Control A 14.6 13 15 0.87 100

B 13.0 15 16 0.94 100

C 15.5 20 10 2.0 100 Mean(SD) 14.4(1.3) NA a 1.3(0.63) NA 100(0) NA

32 A 15.3 18 13 1.4 100

B 19.4 17 11 1.6 100

C 16.8 15 14 1.1 100 Mean(SD) 17.2(2.1) 0.1909 1.3(0.24) 0.8790 100(0) 1.0000

80 A 15.0 21 11 1.9 100

B 18.6 18 13 1.4 100

C 13.8 14 18 0.78 86 Mean(SD) 15.8(2.5) 0.4938 1.4(0.57) 0.8927 95(8.2) 1.0000

200 A 13.2 13 17 0.76 100

B 17.4 14 15 0.93 100

C 14.3 7 18 0.39 100 Mean(SD) 15.0(2.2) 0.7095 0.70(0.28) 0.2323 100(0) 1.0000

500 A 18.7 15 11 1.4 100

B 20.0 9 22 0.41 100

C 17.7 18 13 1.4 100 Mean(SD) 18.8(1.1) 0.0410 1.1(0.56) 0.6186 100(0) 1.0000

1250 A 14.0 18 15 1.2 100

B 17.0 16 13 1.2 100

C 19.5 11 18 0.61 100 Mean(SD) 16.8(2.8) 0.2496 1.0(0.35) 0.5732 100(0) 1.0000

a NA = Not Applicable.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 64Table 7. F1mysid survival and growth (lengthand dry weight) during the mysid (Americamysis bahia) twogeneration testwith lindane.NominalConcentration(ng/L)ReplicateSurvival(%)p-ValueMean Total Body Length (mm)Day 0p-ValueDay 7p-ValueDay 14 Day 27Malep-ValueFemalep-ValueMalep-ValueFemalep-ValueControl A 71 1.67 4.74 6.54 6.45 7.29 7.58B 96 1.69 4.63 5.96 6.18 7.22 7.40C 89 1.55 4.78 6.47 6.75 7.39 7.58Mean(SD) 85(13) NAa1.63(0.07) NA 4.72(0.08) NA 6.32(0.32) NA6.46(0.28) NA 7.30(0.09) NA 7.52(0.19) NA32 A 75 1.65 4.71 6.45 6.34 7.29 7.22B 77 1.66 4.24 6.29 6.08 7.17 7.37C 87 1.70 4.92 6.25 6.62 7.51 7.51Mean(SD) 80(6.3) 0.9940 1.67(0.03) 0.4874 4.62(0.35) 0.7378 6.33(0.10) 1.0000 6.35(0.27) 0.6443 7.32(0.17) 0.8645 7.37(0.21) 0.988980 A 96 1.56 4.15 5.79 6.36 6.89 7.23B 95 1.66 4.91 5.98 6.08 7.08 7.49C 86 1.70 4.88 6.17 6.62 7.24 7.44Mean(SD) 92(5.3) 0.6525 1.64(0.07) 0.8069 4.64(0.43) 0.7640 5.98(0.19) 0.2108 6.35(0.27) 0.6572 7.07(0.17) 0.3720 7.39(0.21) 0.9834200 A 95 1.73 3.90 5.52 6.06 6.69 7.11B 92 1.73 4.74 6.06 6.25 7.03 7.35C 79 1.66 5.19 6.13 6.39 6.99 7.52Mean(SD) 89(8.6) 0.8585 1.70(0.04) 0.1527 4.61(0.65) 0.7222 5.90(0.33) 0.1089 6.23(0.16) 0.4168 6.91(0.19) 0.1242 7.33(0.16) 0.9952500 A 100 1.69 4.59 6.02 6.26 6.85 7.73B 88 1.70 4.80 6.01 6.81 7.87 8.06C 79 1.68 5.19 6.26 6.68 7.48 7.90Mean(SD) 89(11) 0.9773 1.69(0.01) 0.2765 4.86(0.30) 0.9290 6.10(0.14) 0.5343 6.59(0.29) 0.9459 7.40(0.51) 0.9373 7.90(0.28)b0.02341250 A 69 1.71 4.72 NA7.04 6.84 7.71B 68 1.69 3.94 5.74 6.46 6.67 7.56C 74 1.69 4.35 NA 6.49 6.72 7.38Mean(SD) 70(2.9)0.9997 1.70(0.01) 0.2206 4.33(0.39) 0.3634 5.74(NA)bNA 6.66(0.32) 0.9772 6.74(0.09)b0.0339 7.55(0.28) 0.7558aNA = Not Applicable.bStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 65Table 7. Continued. F1mysid survival and growth (length and dry weight) during the mysid (Americamysisbahia) two-generation test with lindane.NominalConcentration(ng/L)ReplicateMean Dry Body Weight (mg)Day 0 p-Value Day 7 p-ValueDay 14 Day 27Male p-Value Female p-Value Male p-Value Femalep-ValueControl A 0.028 0.30 0.77 0.79 0.83 1.31B 0.034 0.32 0.64 0.77 0.83 1.20C 0.024 0.52 0.76 1.23 0.91 1.23Mean(SD) 0.029(0.0050) NA 0.38(0.12) NA 0.72(0.08)NA 0.93(0.26) NA 0.86(0.05) NA 1.25(0.06) NA32 A 0.038 0.44 0.89 0.88 0.89 1.06B 0.018 0.23 0.59 0.75 0.83 1.10C 0.034 0.49 0.79 1.06 0.99 1.29Mean(SD) 0.030(0.011) 0.8801 0.38(0.14) 0.8592 0.76(0.15) 1.0000 0.90(0.16) 0.7375 0.90(0.08) 0.9758 1.15(0.12) 0.302880 A 0.028 0.22 0.56 0.66 0.79 1.18B 0.020 0.31 0.55 0.69 0.82 1.00C 0.034 0.35 0.46 0.91 0.84 1.13Mean(SD) 0.027(0.0070) 0.7758 0.29(0.06) 0.3363 0.52(0.06) 0.0796 0.75(0.14) 0.2363 0.81(0.02) 0.5308 1.10(0.10) 0.1262200 A 0.022 0.23 0.38 0.66 0.68 1.12B 0.042 0.33 0.55 0.77 0.88 1.15C 0.036 0.36 0.46 0.77 0.78 1.23Mean(SD) 0.033(0.010) 0.9548 0.31(0.06) 0.4133 0.47(0.09) 0.0237 0.73(0.06) 0.1871 0.78(0.10) 0.2468 1.17(0.06) 0.3919500 A 0.020 0.29 0.48 0.74 0.85 1.33B 0.018 0.34 0.72 0.93 0.94 1.38C 0.038 0.37 0.64 0.84 0.83 1.17Mean(SD) 0.025(0.011) 0.6717 0.34(0.04) 0.5804 0.61(0.12)b0.0237 0.84(0.09) 0.5186 0.87(0.06) 0.9095 1.29(0.11) 0.95431250 A 0.014 0.30 NA 0.77 0.73 1.19B 0.028 0.30 0.45 0.69 0.65 1.02C 0.032 0.36 NA 0.72 0.72 1.10Mean(SD) 0.025(0.0095) 0.6334 0.32(0.03) 0.4959 0.45(NA) NA 0.73(0.04) 0.1725 0.70(0.05)b0.0213 1.10(0.09) 0.1262aNA = Not Applicable.bStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 66Table 8. F1mysid time to maturation, timeto first brood release, time tosecond brood release, offspringreleased per female and interbrood duration during the mysid (Americamysis bahia) two-generationtest with lindane.NominalConcentration(ng/L)ReplicateTime toMaturation(Days)pValueFirst Brood Second BroodInterbroodDuration(Days)pValueTime toRelease(Days)pValueOffspring/Femalep-ValueTime toRelease(Days)pValueOffspring/Femalep-ValueControl A 10 14.7 6.9 20.4 10.4 5.7B 10 15.0 5.1 20.0 8.3 5.0C 9.3 14.9 7.7 19.7 10.2 4.8Mean(SD) 9.7(0.58) NAa14.9(1.0) NA 6.6(1.3) NA 20.0(0.38) NA 9.6(1.2) NA 5.2(0.47) NA32 A 12 16.9 5.6 21.0 7.0 5.0B 10 16.0 5.6 20.3 10.3 4.2C 11 16.9 7.0 21.5 9.8 5.2Mean(SD) 8.7(1.2) 0.9934 16.6(2.4)b0.0436 6.1(0.82) 0.5737 20.9(0.59) 0.3228 9.1(1.8) 0.5688 4.8(0.54) 0.964980 A 13 16.9 4.3 21.9 6.7 5.0B 12 17.0 5.9 22.7 8.4 5.7C 11 16.1 4.3 20.8 7.2 5.0Mean(SD) 11(1.2) 0.2725 16.7(1.4)b0.0367 4.8(0.91) 0.0677 21.8(0.96) 0.0542 7.4(0.88) 0.0506 5.2(0.41) 0.7909200 A 13 17.7 4.1 23.0 6.2 4.8B 10 15.0 4.7 20.0 4.5 5.0C 11 17.3 3.0 21.8 5.3 5.0Mean(SD) 9.7(0.58) 0.8333 16.7(1.6)b0.0367 3.9(0.85)b0.0083 21.6(1.5) 0.0859 5.3(0.83)b0.0008 4.9(0.10) 0.9347500 A 11 16.3 5.7 21.3 10.0 5.0B 12 14.4 4.1 20.7 11.3 6.3C 12 16.3 4.4 21.3 10.5 5.0Mean(SD) 10(1.0) 0.6646 15.7(1.8) 0.3457 4.8(0.81) 0.0541 21.1(0.36) 0.2413 10.6(0.65) 0.9856 5.4(0.74) 0.63201250 A 13 16.5 5.0 21.0 10.2 5.0B 13 15.7 5.9 20.5 8.8 5.0C 13 15.8 5.0 22.3 9.0 6.7Mean(SD) 11(0.58) 0.2725 16.0(1.5) 0.1816 5.3(0.49) 0.1795 21.3(0.95) 0.1751 9.3(0.78) 0.7151 5.6(0.96) 0.5112aNA = Not Applicable.bStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 67



## Table 9. F 1 

## mysid total number of young in the first two broods, sex ratio and percentage of reproductively acti ve females during the mysid ( Americamysis bahia ) two-generation test with lindane

.

Nominal

Concentration (ng/L) Replicate Total

Number of Young

in First

Two

Broods p-Value Sex Ratio

Percentage of Reproductively

Active Females p-Value Total

Number

of Males Total

Number

of

Females Male:Female

Ratio pValue

Control A 17.3 19 9 2.1 100

B 13.4 11 14 0.79 100

C 17.3 12 11 1.1 100 Mean(SD) 16.0(2.2) NA 1.3(0.69) NA a 100(0) NA

32 A 12.0 16 12 1.3 100

B 16.2 18 8 2.3 100

C 16.5 16 10 1.6 100 Mean(SD) 14.9(2.5) 0.5107 1.7(0.47) 0.3718 100(0) 1.0000

80 A 11.0 12 13 0.92 100

B 14.3 15 9 1.7 100

C 11.4 17 10 1.7 100 Mean(SD) 12.2(1.8) b 0.0315 1.4(0.44) 0.7367 100(0) 1.0000

200 A 10.5 13 12 1.1 100

B 9.2 11 15 0.73 100

C 8.0 15 12 1.3 100 Mean(SD) 9.2(1.3) b 0.0007 1.0(0.26) 0.9743 100(0) 1.0000

500 A 15.7 8 15 0.53 100

B 15.4 11 11 1.0 100

C 15.2 13 12 1.1 100 Mean(SD) 15.4(0.25) 0.6840 0.87(0.30) 0.9914 100(0) 1.0000

1250 A 14.2 12 11 1.1 86

B 13.8 14 13 1.1 100

C 15.0 10 16 0.63 100 Mean(SD) 14.3(0.63) 0.3325 0.93(0.27) 0.9867 95(8.2) 1.0000

a NA = Not Applicable.

b Statistically different compared to the contro l, based on Dunnett's Multiple Comparison Test.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 68



## Table 10. Summary of F 0 

## and F 1 

## endpoints, No-Observed-Effect Concentration (NOEC) and Lowes t-Observed-Effect Concentration (LOEC) values determin ed during the mysid ( Americamysis bahia ) two-generation test with lindane

.

F 0 Endpoints Measured Concentration (ng/L)

30 100 170 380 1100

Survival Day 7 Dry Weight Day 14 Male Dry Weight ↑ ↑ ↑ Day 14 Female Dry Weight ↑ ↑ ↑ Termination (Day 27) Male Dry Weight ↓ Termination (Day 27) Female Dry Weight Day 7 Length Day 14 Male Length Day 14 Female Length ↑ ↑ Termination (Day 27) Male Length Termination (Day 27) Female Length

Time to Maturation

Time to 1 s t Brood Release ↓

1 s t

Brood Offspring/Female ↑

Time to 2 nd Brood Release ↓

2 nd Brood Offspring/Female

Interbrood Duration Total Offspring/Female

Sex Ratio % Reproducing Females
↓ Endpoint determined to be sta tistically significantly reduced (p ≤ 0.05) compared to control ↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control

F 1 Endpoints Measured Concentration (ng/L)

30 100 170 380 1100

Survival Day 0 Dry Weight Day 7 Dry Weight Day 14 Male Dry Weight ↓ Day 14 Female Dry Weight Termination (Day 27) Male Dry Weight ↓ Termination (Day 27) Female Dry Weight Day 0 Length Day 7 Length Day 14 Male Length ↓ Day 14 Female Length Termination (Day 27) Male Length ↓ Termination (Day 27) Female Length ↑

Time to Maturation

Time to 1 s t Brood Release ↑ ↑ ↑

1 s t

Brood Offspring/Female ↓

Time to 2 nd Brood Release

2 nd Brood Offspring/Female ↓

Interbrood Duration Total Offspring/Female ↓ ↓

Sex Ratio % Reproducing Females
↓ Endpoint determined to be sta tistically significantly reduced (p ≤ 0.05) compared to control

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 69



## Figure 1. Schematic of biological methods during the mysid ( Americamysis

bahia ) two-generation test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 70



## Figure 2. Relationship between mean measured concentrations and the nominal treatment levels during the mysid ( Americamysis bahia ) two-generation test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 71



## Figure 3. Mean percent survival of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 72



## Figure 4. Day 7 mean total body length of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 73



## Figure 5. Day 7 mean dry body weight of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 74



## Figure 6. Day 14 male and female mean total body length of F 0 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 75



## Figure 7. Day 14 male and female mean dry body weight of F 0 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 76



## Figure 8. Day 27 male and female mean total body length of F 0 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

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## Figure 9. Day 27 male and female mean dry body weight of F 0 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

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## Figure 10. Mean time to maturation of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 11. Mean time to first brood release of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 12. F 0 

## Mean number of offspring/fema le produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane.

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## Figure 13. Mean time to second brood release of F 0 

## mysids ( Americamysis

bahia ) during the two-generati on test with lindane.

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## Figure 14. F 0 

## Mean number of offspring/female produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane.

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## Figure 15. F 0 

## Mean interbrood du ration for mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 16. F 0 

## Mean total number of offsprin g released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test with

lindane.

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## Figure 17. F 0 

## Mean male:female se x ratio for mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 18. F 0 

## Mean percentage of reproductiv ely active females for mysids ( Americamysis bahia ) during the two-generati on test with lindane.

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## Figure 19. Mean percent survival of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

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## Figure 20. Day 0 mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

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## Figure 21. Day 0 mean dr y body weight of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

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## Figure 22. Day 7 mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

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## Figure 23. Day 7 mean dr y body weight of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane

.

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## Figure 24. Day 14 male and female mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

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## Figure 25. Day 14 male and female mean dry body weight of F 1 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

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## Figure 26. Day 27 male and female mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

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## Figure 27. Day 27 male and female mean dry body weight of F 1 

## mysids ( Americamysis bahia ) during the two-genera tion test with lindane

.

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## Figure 28. Mean time to maturation of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 29. Mean time to first brood release of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 30. F 1 

## Mean number of offspring/fema le produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane.

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## Figure 31. Mean time to second brood release of F 1 

## mysids ( Americamysis

bahia ) during the two-generati on test with lindane.

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## Figure 32. F 1 

## Mean number of offspring/female produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation

test with lindane.

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## Figure 33. F 1 

## Mean interbrood du ration for mysids ( Americamysis bahia ) during the two-generation test with lindane.

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## Figure 34. F 1 

## Mean total number of offsprin g released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test with

lindane.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 103



## Figure 35. F 1 

## Mean male:female se x ratio for mysids ( Americamysis bahia ) during the two-generation test with lindane.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 104



## Figure 36. F 1 

## Mean percentage of reproductiv ely active females for mysids ( Americamysis bahia ) during the two-generati on test with lindane.

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## APPENDIX 1 - QAPP

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## APPENDIX 2 - STUDY PLAN

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## APPENDIX 3 - ANALYTICAL METHODOLOGY - LINDANE

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SUMMARY

Methodology was validated (22 February 2011) to quantify the amount of lindane present in

recovery samples prepared in filtered seawater. The recovery samples were adjusted to pH 3

with phosphoric acid, extracted twice with ethy l acetate, reduced in volume to approximately

2.0 mL by rotary evaporation at 35 oC, taken to n ear-dryness under a gentle st ream of nitrogen at

room temperature and reconstitute d with 0.1% peanut oil in iso-oc tane. Recovery samples were

then analyzed by gas chromatography using micro electron capture detection (GC/μECD).

This method was validated by fortification of filter ed seawater with lindane at concentrations of

0.0200, 0.200 and 2.00 μg a.i./L. Recoveries averag ed 98.1% with a standard deviation of

6.59% and a limit of quantification of 0.00392 μg a.i./L. Defined limits for acceptance of quality

control sample performance in subseque nt studies were set at 70 to 120%.

EXPERIMENTAL

Equipment

1. Instrument: Hewlett Packard M odel 6890 Series gas chromatograph equipped with a micro electr on capture detector, Hewlett Packard autosampler and injector Series 7683, and an Agilent ChemStation ECM Version B.04.02 for data acquisition

2. Balance: Orion Model 0960 3. Laboratory equipment: Positive displacement pipets, disposable Pasteur pipets, volumetric flasks, autosampler vials, amber glass bottles and separatory funnels with Teflon ® 

### -lined caps, rotary evaporator, round-bottom flasks

Reagents

1. Ethyl acetate: Burdick & Jackson, reagent grade 2. Phosphoric acid: EMD, reagent grade 3. Acetone: Burdick & Jackson, reagent grade 4. Peanut oil: Loriva, reagent grade

5. Iso-octane: Burdick & Jackson, reagent grade 6. pH paper: EMD, reagent grade

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 187

Test Substance

The test substance, lindane, was received on 19 January 2011 from Battelle Marine Science

Laboratory, Sequim, Washington. The fo llowing information was provided:

Name: Lindane, 97% Synonyms: Lindane; 1 α ,2 α ,3 β ,4 α ,5 α ,6 β -Hexachlorocyclohexane; y-BHC

Product No.: 233390

Lot No.: 14419eb

CAS No.: 58-89-9 Purity: 99.6% Expiry Date: December 2012

Upon receipt at Smithers Viscient, the test s ubstance (SMV No. 4841) was stored at room

temperature in the original contai ner in a dark ventilated cabinet. Concentrations were adjusted

for the purity of the test substance.

PROCEDURES

Preparation of Stock Solutions

A 1.00 mg a.i./mL primary stock solution was prepared by placing 0.1008 g of lindane (0.1004 g

as active ingredient) in a 100-mL volumetric fl ask and bringing it to volume with acetone. A

secondary stock solution (1.00 mg a.i./L ) was prepared by placing 0.0500 mL of the

1.00 mg a.i./mL primary stock so lution in a 50.0-mL volumetric fl ask and bringing it to volume

with acetone. The 1.00 mg a.i./L secondary stock solution was used to fo rtify all the recovery

samples.

An additional 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0503 g of

lindane (0.0501 g as active ingr edient) in a 50.0-mL volumetric flask and bringing it to volume

with acetone. A 10.0 mg a.i./L secondary stoc k solution was prepared by placing 0.500 mL of

the 1.00 mg a.i./mL primary stock solution in a 50.0-mL volumetric flask and bringing it to

volume with iso-octane. This 10.0 mg a.i./L secondary stock solution was used to prepare

calibration standards.

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All stock solutions were stored in a refrigerator in amber glass bottles fitted with Teflon ® 

### -lined

caps.

Preparation of Calibration Standards

Calibration standards were prepar ed in 0.1% peanut oil in iso- octane by fortifying with the

10.0 mg a.i./L secondary stock solution to yield concentrations of 2.00, 5.00, 10.0, 25.0, 35.0 and

50.0 μg a.i./L.

Preparation of Reagent Solution

A reagent solution of 0.1% peanut oil in iso-oc tane (v:v) was prepared by adding 0.500 mL of

peanut oil to 500 mL of iso-octane. This soluti on was mixed using a magne tic stir plate and stir

bar.

Sample Fortification and Preparation

Fortification of Recovery Samples:

The recovery samples were prepared by for tifying filtered seawater with lindane at

concentrations of 0.0200, 0.200 and 2.00 μg a.i./L in separatory funnels as per the table below.

Sample

ID Stock

Concentration

(mg a.i./L) Volume of

Stock Solution (mL) Volume (mL) of

Filtered Seawater Fortified Sample

Concentration

(μg a.i./L)

Control
A, B & C NA a NA 1000 NA

Low A, B & C 1.00 0.0200 1000 0.0200

Mid
A, B & C 1.00 0.0600 300 0.200

High
A, B & C 1.00 0.100 50.0 2.00

a NA = Not Applicable

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Extraction of Recovery Samples :

Concentrated phosphoric acid was added to all recove ry samples prior to extraction to adjust the

samples to pH 3. All recovery and control sa mples were extracted twice with ethyl acetate

(200 mL each time). Sodium sulfate was not used to remove residual water to prevent potential

losses and the extracts were transferred into round-bottom flasks. Following extraction and

removal of the post-extraction aqueous sample, th e separatory funnels were rinsed with 50.0 mL

of ethyl acetate and the rinsate was added to th e round-bottom flasks. The extracts were taken to

low volume (approximately 2 mL) by rotary eva poration using minimal h eating (<35 oC). A

solvent transfer step with acetone was then perf ormed to remove any residual water from the

round-bottom flasks. An aliquot of acetone (100 mL) was added to each round-bottom and

mixed well. The extracts were again taken to low volume (approximately 5 mL) by rotary

evaporation using minimal heating (<35 oC). The concentrated extracts were then transferred,

using ethyl acetate followed by acetone, to glass cen trifuge tubes (or larger glass vials depending

on the final volume) and taken to incipien t dryness (approximately 200 μL) under a gentle

stream of nitrogen at room temperature. Sa mples were not allowed to go dry or approach

dryness to reduce losses of the test substance. An aliquot of 0.1% peanut oil in iso-octane was

added to the low (2.00 mL), mid (2.00 mL) and high (3.00 mL) recovery samples, which were

then vortexed for 30 seconds and sonicated for five minutes to mix and aid in dissolution of the

concentrated extract. All samples were then tr ansferred to autosampler vials to be analyzed by

GC/μECD.

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ANALYSIS

Instrumental Conditions

The gas chromatographic analysis with micr o electron capture detection (GC/μECD) was

conducted utilizing the following instrumental conditions:

Column: Agilent DB-5, 30 m x 0.32 mm internal diameter, 0.25-μm film thickness, back inlet Gas flows: Carrier gas – Helium held at a constant flow of 2.0 mL/minute Detector make-up gas - Argon/methan e (5%) at a combined flow of

60.0 mL/minute Temperatures: Injector - 260 °C

Detector - 300 oC Oven: Initial temp - 50 oC

Initial time - 1.00 minute Ramp at 20 oC/min to 320 oC (hold for 2.50 minutes) Injection Volume: 2.0 μL; splitless (purge flow on at 60.0 mL/minute at 1.00 minute) Retention Time: Approximately 9.6 minutes

Preparation of Standard Curve

Two sets of calibration standards were analyzed with each recovery sample set; one set prior to

analysis of the recovery samples, and the sec ond set immediately following the analysis of the

recovery samples. Injecti on of recovery samples and calibration standards onto the

chromatographic system was performe d by programmed automated injection.

CALCULATIONS

A calibration curve was constructed by plotting the natural logarithm (ln) of the analyte

concentration (μg a.i./L) of the calibration standards against the natural logarithm (ln) of the sum

of the peak area of the three peak s of the analyte in the calibration standards. The equation of the

line (equation 1) was algebraically manipulated to give equation 2. The concentration of test

substance in each recovery sample was calculated using the slope and intercept of the regression

analysis, and the natural logar ithm of the peak area and the dilution factor of the recovery

sample. Equations 2, 3 and 4 were then used to calculate measured concentrations and analytical

results.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 191

(1) lny m(lnx) b

(2) lnx (lny b)

m

(3) DC (x) inverse(lnx)

(4) A DC x DF  

 







### where:

lnx = natural logarithm of sample concentration lny = natural logarithm of detector response (sum of the areas of the three peaks) m = slope from regression analysis b = y-intercept from regression analysis DC (x) = detected concentrati on (μg a.i./L) in the sample DF = dilution factor (final volume of the sample divided by the original sample volume) A = analytical result (μg a.i./L)

The limit of quantitation (LOQ) was cal culated using the following equation:

CNTL INST INST INST LS

INST



### DF x LOQ LOQ (7) ) (LOQ (ln inverse LOQ (6) m b ) (A ln ) (LOQ ln (5)

  



### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 192

where:

A LS 

### = mean detector response (peak area) of the low concentration calibration standard (two injections) b = y-intercept from regression analysis m = slope from regression analysis LOQ INST 

### = limit of quantitation on the instrument

DF CNTL 

### = dilution factor of the control samples (smallest dilution factor used) LOQ = limit of quantitation reported for the analysis

RESULTS AND DISCUSSION

Recoveries averaged 98.1% with a standard devi ation of 6.59% and a lim it of quantification of

0.00392 μg a.i./L. The LOQ will vary from one analysis interval to another, since it is dependent

upon the regression of the calibration standards, the peak area of the low standards, and dilution

factor of the controls. These parameters, wh ile relatively constant, do vary somewhat among

runs and produce small variations in the LOQ. Defined limits for acceptance of quality control

sample performance in subsequent st udies were set at 70 to 120%.

Analytical results for the recovery of lindane from filtered seawater are presented in Table 3A

. Representative chromatograms from the analysis of a calibration standard, recovery sample and a

control sample are presented in Figure 3A

, Figure 3B and Figure 3C , respectively. A typical

regression analysis is presented in Figure 3D

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 193



## Table 3A. Analytical results for the re covery of lindane present in filtered seawater during the method validation study.

Sample ID Fortified

Concentration

(μg a.i./L) Dilution

Factor Analytical Result (μg a.i./L) Percent

of Fortified

Control A 0.00 0.00200 < 0.00392 a NA b

Control B 0.00 0.00200 < 0.00392 NA

Control C 0.00 0.00200 < 0.00392 NA

Low A 0.0200 0.00200 0.0193 96.3

Low B 0.0200 0.00200 0.0228 114

Low C 0.0200 0.00200 0.0200 99.8

Mid A 0.200 0.00667 0.199 99.5

Mid B 0.200 0.00667 0.190 95.1

Mid C 0.200 0.00667 0.194 97.1

High A 2.00 0.0600 1.87 93.6

High B 2.00 0.0600 1.82 91.1

High C 2.00 0.0600 1.92 96.1

Mean: 98.1

SD: 6.59

RSD (%): 6.72

N: 9

a Concentrations expressed as less than values were be low the limit of quantitati on (LOQ). The LOQ for each analysis is dependent upon the linear regression, the ar ea of the low standards and the dilution factor of the controls. The limit of quantitation for the method validation was 0.00392 μg a.i./L.

b NA = Not Applicable.

Note: Results were calculated using the actual analytical (unrounded) results and not the rounded values presented

in this table.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 194



## Figure 3A. Representative chromatogram of a 25.0 μg a.i./L calibration standard used for quan tification during the method validation study

with lindane.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 195



## Figure 3B. Representative chromatogram of a 0.200 μg a.i./L recovery sample fortified with lindane during the method validation study.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 196



## Figure 3C. Representative chromatogram of a control sample during the method validation study with lindane.

Note: Lindane is normally detected at a retention time of approximately 9.6 minutes. The peak present in the control samples at the retention time of the test material is below the LOQ for this analysis.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 197



## Figure 3D. A typical regression analysis for the calibration standards used to quantitate lindane in recovery samples during the method validation study.

Regression Analysis

R 2 = 0.99929 y = 1.084294x + 6.6570

ln Standard Standard ln Standard

Concentration Concentration Response Response

μg a.i./L μg a.i./L Area Area

0.6931 2.00 7.4021 1639.36743

1.6094 5.00 8.4519 4684.19287

2.3026 10.0 9.1334 9259.65430

3.2189 25.0 10.1947 26762.3

3.5553 35.0 10.5297 37411.9

3.9120 50.0 10.9124 54853.5

0.6931 2.00 7.3710 1589.18213

1.6094 5.00 8.4499 4674.53662

2.3026 10.0 9.1177 9114.81738

3.2189 25.0 10.1092 24567.4

3.5553 35.0 10.4959 36168.0

3.9120 50.0 10.8764 52910.4

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6109 Page 198



## APPENDIX 4 - POST-PAIRING SURVIVAL DATA - LINDANERTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 199Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

ControlReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total #% Survival % Survival Post-PairingMean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival% Survival0 20 201001 20 201002 18 2 20903 18 20904 18 20905 18 20906 18 20907 18 20908 18 20909 18 209010 14 16 4 49011 13 15 5 59012e8 10 10 10100 100 1009013 8 10 10 10100 100 1009014 7 1 9 10 10100 100 1008915 7 9 10 10100 100 1008916 7 9 10 10100 100 1008917 7 9 10 10100 100 1008918 7 9 10 10100 100 1008919 7 9 10 10100 100 1008920 7 9 9 1 9100 100 1008921 7 9 9 9100 100 1008922 7 9 8 1 8100 100 1008823 7 9 8 8100 100 1008824 7 9 7 1 8100 88 938225 7 9 7 8100 88 938226 6 1 8 7 8100 88 938127 6 8 6 1 7100 86 9280aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 200Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

ControlReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 20 20 10010 16 16 4 4 10011 13 13 7 7 10012e10 1 11 9 9 100 100 100 9513 8 9 11 11 100 100 100 9514 8 9 11 11 100 100 100 9515 8 9 11 11 100 100 100 9516 8 9 10 1 10 100 100 100 9517 7 1 8 10 10 100 100 100 9418 7 8 10 10 100 100 100 9419 7 8 10 10 100 100 100 9420 7 8 10 10 100 100 100 9421 7 8 10 10 100 100 100 9422 7 8 10 10 100 100 100 9423 6 1 8 9 1 10 86 90 88 8324 6 8 9 10 86 90 88 8325 6 8 9 10 86 90 88 8326 6 8 9 10 86 90 88 8327 5 1 7 9 10 83 90 88 82aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 201Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

ControlReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 20 20 10010 20 20 10011 18 18 2 2 10012e12 4 16 6 6 100 100 100 8213 13 17 7 7 100 100 100 8314 12 1 16 7 7 100 100 100 8315 12 16 7 7 100 100 100 8316 12 16 7 7 100 100 100 8317 12 16 7 7 100 100 100 8318 11 1 16 6 1 6 92 100 94 7719 10 1 15 6 6 91 100 94 7620 10 15 6 6 91 100 94 7621 10 15 6 6 91 100 94 7622 10 15 6 6 91 100 94 7623 10 15 6 6 91 100 94 7624 10 15 5 1 6 91 83 88 7125 10 15 5 6 91 83 88 7126 9 1 14 5 6 90 83 88 7027 8 1 14 5 6 80 83 81 65aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 202Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

32 ng/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 19 1 20 957 19 20 958 19 20 959 18 19 1 1 9510 16 17 3 3 9511 13 14 6 6 9512e10 11 9 9 100 100 100 9513 10 11 9 9 100 100 100 9514 10 11 9 9 100 100 100 9515 10 11 9 9 100 100 100 9516 10 11 9 9 100 100 100 9517 9 1 11 8 1 9 90 89 89 8518 9 11 8 9 90 89 89 8519 9 11 8 9 90 89 89 8520 9 11 8 9 90 89 89 8521 9 11 8 9 90 89 89 8522 8 1 10 7 1 8 89 88 88 8323 8 10 7 8 89 88 88 8324 8 10 7 8 89 88 88 8325 8 10 7 8 89 88 88 8326 7 1 9 7 8 88 88 88 8227 7 9 7 8 88 88 88 82aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 203Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

32 ng/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 19 1 20 953 18 1 20 904 18 20 905 18 20 906 18 20 907 17 1 20 858 16 1 20 809 16 20 8010 11 15 5 5 8011 9 13 7 7 8012e7 1 12 8 8 100 100 100 7513 8 13 7 7 100 100 100 7514 7 1 12 7 7 100 100 100 7415 7 12 7 7 100 100 100 7416 7 12 7 7 100 100 100 7417 7 12 7 7 100 100 100 7418 7 12 7 7 100 100 100 7419 7 12 7 7 100 100 100 7420 7 12 7 7 100 100 100 7421 6 1 12 7 7 86 100 93 6822 6 12 7 7 86 100 93 6823 6 12 7 7 86 100 93 6824 6 12 6 1 6 86 100 92 6725 6 12 5 1 5 86 100 92 6526 6 12 5 5 86 100 92 6527 5 1 11 5 5 83 100 91 63aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 204Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

32 ng/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 18 2 20 902 18 20 903 18 20 904 18 20 905 18 20 906 18 20 907 18 20 908 18 20 909 18 20 9010 14 16 4 4 9011 13 15 5 5 9012e10 1 13 7 7 100 100 100 8513 10 13 7 7 100 100 100 8514 9 1 12 7 7 100 100 100 8415 9 12 7 7 100 100 100 8416 9 12 7 7 100 100 100 8417 9 12 6 1 6 100 100 100 8318 9 12 6 6 100 100 100 8319 9 12 5 1 5 100 100 100 8220 9 12 5 5 100 100 100 8221 8 1 11 5 5 100 100 100 8122 8 11 5 5 100 100 100 8123 8 11 5 5 100 100 100 8124 8 11 5 5 100 100 100 8125 7 11 5 5 88 100 92 7526 7 11 5 5 88 100 92 7527 6 1 10 5 5 86 100 92 73aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 205Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

80 ng/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 19 1 20 959 19 20 9510 16 17 3 3 9511 14 15 5 5 9512e14 15 5 5 100 100 100 9513 14 15 7 7 100 100 100 9514 14 15 7 7 100 100 100 9515 14 15 7 7 100 100 100 9516 14 15 7 7 100 100 100 9517 13 1 15 7 7 93 100 95 9118 13 15 7 7 93 100 95 9119 13 15 7 7 93 100 95 9120 13 15 7 7 93 100 95 9121 13 15 7 7 93 100 95 9122 13 15 7 7 93 100 95 9123 13 15 7 7 93 100 95 9124 13 15 7 7 93 100 95 9125 12 1 15 7 7 86 100 90 8626 12 15 7 7 86 100 90 8627 12 15 7 7 86 100 90 86aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 206Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

80 ng/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 19 20 953 19 20 954 19 20 955 19 20 956 19 20 957 19 20 958 19 20 959 18 19 1 1 9510 13 14 6 6 9511 11 12 8 8 9512e10 11 9 9 100 100 100 9513 9 1 11 9 9 90 100 95 9014 9 11 9 9 90 100 95 9015 9 11 9 9 90 100 95 9016 9 11 9 9 90 100 95 9017 9 11 9 9 90 100 95 9018 9 11 9 9 90 100 95 9019 8 1 10 9 9 89 100 94 8920 8 10 9 9 89 100 94 8921 8 10 9 9 89 100 94 8922 8 10 9 9 89 100 94 8923 8 10 9 9 89 100 94 8924 8 10 9 9 89 100 94 8925 8 10 9 9 89 100 94 8926 8 10 9 9 89 100 94 8927 8 10 9 9 89 100 94 89aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 207Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

80 ng/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 19 1 20 959 19 20 9510 16 17 3 3 9511 15 16 4 4 9512e8 1 10 10 10 100 100 100 9013 8 10 10 10 100 100 100 9014 7 9 11 11 100 100 100 9015 7 9 11 11 100 100 100 9016 7 9 11 11 100 100 100 9017 7 9 11 11 100 100 100 9018 7 9 11 11 100 100 100 9019 7 9 11 11 100 100 100 9020 6 1 8 10 1 10 100 100 100 8921 6 8 10 10 100 100 100 8922 6 8 10 10 100 100 100 8923 6 8 10 10 100 100 100 8924 6 8 10 10 100 100 100 8925 6 8 9 1 10 100 90 94 8326 6 8 8 1 9 100 89 93 8227 5 1 7 7 1 8 100 88 92 80aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 208Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

200 ng/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 19 1 20 953 19 20 954 19 20 955 19 20 956 19 20 957 18 1 20 908 18 20 909 18 20 9010 11 13 7 7 9011 10 12 8 8 9012e7 1 10 9 9 100 100 100 8413 7 1 10 9 9 100 100 100 8414 7 10 9 9 100 100 100 8415 7 10 9 9 100 100 100 8416 7 10 9 9 100 100 100 8417 7 10 9 9 100 100 100 8418 7 10 9 9 100 100 100 8419 6 1 9 9 9 100 100 100 8320 6 9 9 9 100 100 100 8321 6 9 9 9 100 100 100 8322 6 9 9 9 100 100 100 8323 6 9 8 1 8 100 100 100 8224 6 9 8 8 100 100 100 8225 6 9 8 8 100 100 100 8226 5 1 8 8 8 100 100 100 8127 5 8 7 1 7 100 100 100 80aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 209Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

200 ng/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 19 1 20 957 18 1 20 908 18 20 909 16 18 2 2 9010 13 15 5 5 9011 12 14 6 6 9012e10 12 8 8 100 100 100 9013 9 1 11 8 8 100 100 100 8914 9 11 8 8 100 100 100 8915 9 11 6 1 1 7 100 86 94 8316 9 11 6 7 100 86 94 8317 9 11 6 7 100 86 94 8318 9 11 6 7 100 86 94 8319 9 11 6 7 100 86 94 8320 9 11 5 1 7 100 71 88 7821 9 11 5 7 100 71 88 7822 9 11 5 7 100 71 88 7823 8 1 11 5 7 89 71 81 7224 8 11 5 7 89 71 81 7225 8 11 5 7 89 71 81 7226 8 11 5 7 89 71 81 7227 8 11 5 7 89 71 81 72aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 210Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

200 ng/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 18 2 20 903 16 2 20 804 16 20 805 17 20 856 17 20 857 17 20 858 15 20 759 14 19 1 1 7510 11 16 4 4 7511 9 14 6 6 7512e7 9 11 11 100 100 100 9013 7 9 11 11 100 100 100 9014 7 9 11 11 100 100 100 9015 7 9 11 11 100 100 100 9016 7 9 11 11 100 100 100 9017 7 9 11 11 100 100 100 9018 7 9 10 1 10 100 100 100 8919 6 1 8 10 10 100 100 100 8920 6 8 10 10 100 100 100 8921 6 8 10 10 100 100 100 8922 6 8 10 10 100 100 100 8923 6 8 10 10 100 100 100 8924 6 8 10 10 100 100 100 8925 6 8 10 10 100 100 100 8926 6 8 10 10 100 100 100 8927 6 8 10 10 100 100 100 89aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 211Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

500 ng/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 18 2 20 902 18 20 903 18 20 904 18 20 905 18 20 906 18 20 907 18 20 908 18 20 909 13 1 16 4 4 8510 10 13 7 7 8511 10 13 7 7 8512e8 11 9 9 100 100 100 8513 7 1 10 8 1 9 100 89 94 7914 7 10 8 9 100 89 94 7915 7 10 8 9 100 89 94 7916 7 10 8 9 100 89 94 7917 7 10 8 9 100 89 94 7918 7 10 7 1 8 100 88 93 7819 6 1 9 7 8 100 88 93 7620 6 9 6 1 7 100 86 92 7521 6 9 6 7 100 86 92 7522 6 9 6 7 100 86 92 7523 6 9 6 7 100 86 92 7524 6 9 6 7 100 86 92 7525 6 9 6 7 100 86 92 7526 6 9 6 7 100 86 92 7527 6 9 5 1 6 100 83 92 73aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 212Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

500 ng/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 19 20 953 18 20 904 18 20 905 18 20 906 19 20 957 19 20 958 19 20 959 13 14 6 6 9510 9 1 11 9 9 9011 6 8 12 12 9012e7 9 13 13 100 100 100 9113 7 9 13 13 100 100 100 9114 7 9 13 13 100 100 100 9115 7 9 12 1 12 100 100 100 9016 7 9 12 12 100 100 100 9017 6 1 8 12 12 100 100 100 9018 5 1 7 12 12 100 100 100 8919 4 1 6 10 2 10 100 100 100 8820 4 6 9 1 9 100 100 100 8721 4 6 9 9 100 100 100 8722 4 6 9 9 100 100 100 8723 3 1 5 9 9 100 100 100 8624 1 1 1 4 9 9 50 100 91 7725 0 1 3 9 9 0 100 90 7526 0 3 9 9 0 100 90 7527 0 3 9 9 0 100 90 75aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 213Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

500 ng/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 16 16 4 4 10010 13 13 7 7 10011 12 12 8 8 10012e10 2 12 8 8 100 100 100 9013 10 12 8 8 100 100 100 9014 10 12 8 8 100 100 100 9015 10 12 8 8 100 100 100 9016 10 12 8 8 100 100 100 9017 9 1 11 8 8 100 100 100 8918 9 11 8 8 100 100 100 8919 9 11 8 8 100 100 100 8920 9 11 8 8 100 100 100 8921 8 1 10 8 8 100 100 100 8922 6 1 1 9 8 8 86 100 93 8223 6 9 8 8 86 100 93 8224 5 1 8 8 8 83 100 93 8125 4 1 7 8 8 80 100 92 8026 4 7 8 8 80 100 92 8027 4 7 8 8 80 100 92 80aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 214Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

1250 ng/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 19 19 1 1 10010 15 15 5 5 10011 15 15 5 5 10012e12 1 12 7 7 100 100 100 10013 11 1 12 7 7 92 100 95 9514 8 3 12 7 7 67 100 79 7915 8 12 7 7 67 100 79 7916 8 12 6 1 6 67 100 78 7817 8 12 6 6 67 100 78 7818 7 1 11 6 6 64 100 76 7619 7 11 6 6 64 100 76 7620 5 2 9 5 1 5 56 100 71 7121 5 9 5 5 56 100 71 7122 5 9 4 1 4 56 100 69 6923 3 2 7 4 4 43 100 64 6424 3 7 4 4 43 100 64 6425 3 7 4 4 43 100 64 6426 3 7 3 1 4 43 75 55 5527 3 7 3 4 43 75 55 55aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 215Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

1250 ng/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 19 1 20 956 19 20 957 19 20 958 18 1 20 909 18 20 9010 13 15 5 5 9011 12 14 6 6 9012e11 13 7 7 100 100 100 9013 11 13 7 7 100 100 100 9014 11 13 7 7 100 100 100 9015 10 1 12 7 7 100 100 100 8916 10 12 7 7 100 100 100 8917 10 12 7 7 100 100 100 8918 9 1 11 7 7 100 100 100 8919 7 2 9 6 1 6 100 100 100 8720 7 9 6 6 100 100 100 8721 7 9 4 2 4 100 100 100 8522 6 1 8 4 4 100 100 100 8323 6 8 4 4 100 100 100 8324 6 8 4 4 100 100 100 8325 5 1 7 4 4 100 100 100 8226 5 7 4 4 100 100 100 8227 5 7 4 4 100 100 100 82aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 216Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF0 Reproduction Basket Survival

1250 ng/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 20 20 1003 19 20 954 19 20 955 18 1 20 906 18 20 907 18 20 908 18 20 909 18 20 9010 12 14 6 6 9011 10 12 8 8 9012e8 10 12 12 100 100 100 9113 8 10 11 1 11 100 100 100 9014 8 10 11 11 100 100 100 9015 8 10 10 1 11 100 91 95 8616 8 10 10 11 100 91 95 8617 6 2 8 10 11 100 91 94 8418 3 3 5 10 11 100 91 93 8119 3 5 10 11 100 91 93 8120 3 5 10 11 100 91 93 8121 3 5 9 1 10 100 90 92 8022 3 5 7 2 8 100 88 91 7723 3 5 7 8 100 88 91 7724 2 1 4 7 8 100 88 90 7525 2 4 7 8 100 88 90 7526 1 1 3 7 8 100 88 89 7327 1 3 7 8 100 88 89 73aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc).dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 217Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

ControlReplicate AfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009c15 15 10010ab9 9 6 6 100 100 100 10011 8 1 9 6 6 89 100 93 9312 8 9 7 7 89 100 94 9413 8 9 7 7 89 100 94 9414 8 9 7 7 89 100 94 9415 8 9 7 7 89 100 94 9416 8 9 7 7 89 100 94 9417 7 1 8 7 7 88 100 93 9318 7 8 7 7 88 100 93 9319 7 8 7 7 88 100 93 9320 7 8 7 7 88 100 93 9321 7 8 6 1 6 88 100 93 9322 7 8 6 6 88 100 93 9323 7 8 6 6 88 100 93 9324 7 8 6 6 88 100 93 9325 7 8 6 6 88 100 93 9326 5 1 1 7 6 6 71 100 85 8527 5 7 6 6 71 100 85 85aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 218Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

ControlReplicate BfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009c15 15 10010ab7 7 9 9 100 100 100 10011 7 7 9 9 100 100 100 10012 7 7 9 9 100 100 100 10013 7 7 9 9 100 100 100 10014 7 1 7 9 9 100 100 100 10015 7 7 9 9 100 100 100 10016 7 7 9 9 100 100 100 10017 7 7 9 9 100 100 100 10018 7 7 9 9 100 100 100 10019 7 7 9 9 100 100 100 10020 7 7 9 9 100 100 100 10021 7 7 7 2 7 100 100 100 10022 7 7 7 7 100 100 100 10023 7 7 7 7 100 100 100 10024 7 7 7 7 100 100 100 10025 7 7 7 7 100 100 100 10026 7 7 7 7 100 100 100 10027 7 7 7 7 100 100 100 100aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 219Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

ControlReplicate CfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 14 1 15 937 14 15 938 14 15 939c8 9 6 6 100 100 100 9310ab8 9 6 6 100 100 100 9311 7 8 8 8 100 100 100 9412 6 8 9 9 86 100 94 8813 7 8 9 9 100 100 100 9414 7 8 9 9 100 100 100 9415 7 8 9 9 100 100 100 9416 6 1 7 9 9 100 100 100 9417 6 7 9 9 100 100 100 9418 6 7 8 1 8 100 100 100 9319 6 7 8 8 100 100 100 9320 6 7 8 8 100 100 100 9321 6 7 8 8 100 100 100 9322 6 7 7 1 7 100 100 100 9323 6 7 7 7 100 100 100 9324 6 7 7 7 100 100 100 9325 6 7 7 7 100 100 100 9326 5 1 6 7 7 100 100 100 9227 5 6 7 7 100 100 100 92aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 220Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

32 ng/LReplicate AfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010bc12 12 3 3 10011 12 12 3 3 10012a10 1 11 7 7 100 100 100 9413 9 1 11 7 1 8 90 88 89 8414 9 11 7 8 90 88 89 8415 9 11 7 8 90 88 89 8416 9 11 7 8 90 88 89 8417 9 11 7 8 90 88 89 8418 9 11 7 8 90 88 89 8419 9 11 7 8 90 88 89 8420 9 11 7 8 90 88 89 8421 9 11 7 8 90 88 89 8422 9 11 6 1 8 90 75 83 7923 9 11 6 8 90 75 83 7924 9 11 6 8 90 75 83 7925 9 11 6 8 90 75 83 7926 9 11 6 8 90 75 83 7927 8 1 11 6 8 80 75 78 74aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 221Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

32 ng/LReplicate BfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 14 1 15 932 14 15 933 14 15 934 14 15 935 14 15 936 14 15 937 14 15 938 12 13 2 2 939 12 13 2 2 9310bc8 9 6 6 100 100 100 9311 8 9 7 7 100 100 100 9412a8 9 7 7 100 100 100 9413 8 9 7 7 100 100 100 9414 8 9 7 7 100 100 100 9415 8 9 7 7 100 100 100 9416 8 9 7 7 100 100 100 9417 8 9 7 7 100 100 100 9418 8 9 7 7 100 100 100 9419 8 9 7 7 100 100 100 9420 8 9 6 1 6 100 100 100 9321 8 9 6 6 100 100 100 9322 8 9 6 6 100 100 100 9323 8 9 6 6 100 100 100 9324 8 9 6 6 100 100 100 9325 6 2 9 6 6 75 100 86 8026 5 1 8 6 6 71 100 85 7927 5 8 6 6 71 100 85 79aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 222Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

32 ng/LReplicate CfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 14 1 15 933 13 1 15 874 13 15 875 13 15 876 13 15 877 13 15 878 11 13 2 2 879 11 13 2 2 8710bc7 9 8 8 100 100 100 8811 7 9 8 8 100 100 100 8812a7 9 8 8 100 100 100 8813 7 9 7 1 7 100 100 100 8814 7 9 7 7 100 100 100 8815 7 1 10 7 7 88 100 93 8216 7 10 7 7 88 100 93 8217 7 10 7 7 88 100 93 8218 7 10 7 7 88 100 93 8219 7 10 7 7 88 100 93 8220 7 10 7 7 88 100 93 8221 7 10 7 7 88 100 93 8222 6 1 9 7 7 86 100 93 8123 6 9 7 7 86 100 93 8124 6 9 7 7 86 100 93 8125 6 9 7 7 86 100 93 8126 6 9 7 7 86 100 93 8127 6 9 7 7 86 100 93 81aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 223Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

80 ng/LReplicate AfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010 13 13 2 2 10011c12 12 3 3 10012b11 11 4 4 10013a7 7 8 8 100 100 100 10014 7 7 8 8 100 100 100 10015 7 7 8 8 100 100 100 10016 7 7 8 8 100 100 100 10017 7 7 8 8 100 100 100 10018 7 7 8 8 100 100 100 10019 7 7 8 8 100 100 100 10020 7 7 8 8 100 100 100 10021 7 7 8 8 100 100 100 10022 7 7 8 8 100 100 100 10023 7 7 8 8 100 100 100 10024 7 7 8 8 100 100 100 10025 7 7 8 8 100 100 100 10026 7 7 8 8 100 100 100 10027 7 7 8 8 100 100 100 100aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 224Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

80 ng/LReplicate BfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 14 1 15 935 14 15 936 14 15 937 14 15 938 14 15 939 14 15 9310 14 15 9311c14 15 9312b7 8 8 8 100 100 100 9413a7 8 8 8 100 100 100 9414 7 8 8 8 100 100 100 9415 7 8 8 8 100 100 100 9416 7 8 8 8 100 100 100 9417 7 8 8 8 100 100 100 9418 7 8 8 8 100 100 100 9419 6 1 7 8 8 100 100 100 9320 6 7 8 8 100 100 100 9321 6 7 8 8 100 100 100 9322 6 7 7 1 7 100 100 100 9323 5 1 6 7 7 100 100 100 9224 5 6 7 7 100 100 100 9225 5 6 7 7 100 100 100 9226 5 6 7 7 100 100 100 9227 5 6 7 7 100 100 100 92aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 225Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

80 ng/LReplicate CfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010 11 11 4 4 10011c7 7 8 8 100 100 100 10012b7 7 8 8 100 100 100 10013a7 7 8 8 100 100 100 10014 7 7 8 8 100 100 100 10015 7 7 8 8 100 100 100 10016 7 7 8 8 100 100 100 10017 7 7 8 8 100 100 100 10018 7 7 8 8 100 100 100 10019 7 7 8 8 100 100 100 10020 7 7 8 8 100 100 100 10021 7 7 7 1 7 100 100 100 10022 6 1 7 7 7 86 100 93 9323 6 7 6 1 6 86 100 92 9224 5 1 7 6 6 71 100 85 8525 4 1 7 6 6 57 100 77 7726 4 7 6 6 57 100 77 7727 4 7 6 6 57 100 77 77aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 226Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

200 ng/LReplicate AfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010b13 13 2 2 10011c12 12 3 3 10012 12 12 3 3 10013a8 8 7 7 100 100 100 10014 8 8 7 7 100 100 100 10015 8 8 7 7 100 100 100 10016 7 1 7 6 1 7 100 86 93 9317 7 7 6 7 100 86 93 9318 7 7 6 7 100 86 93 9319 7 7 6 7 100 86 93 9320 7 7 6 7 100 86 93 9321 7 7 6 7 100 86 93 9322 7 7 6 7 100 86 93 9323 7 7 6 7 100 86 93 9324 7 7 6 7 100 86 93 9325 7 7 6 7 100 86 93 9326 6 1 6 6 7 100 86 92 9227 6 6 6 7 100 86 92 92aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 227Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

200 ng/LReplicate BfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 12 12 3 3 10010b7 7 8 8 100 100 100 10011c7 7 8 8 100 100 100 10012 7 7 8 8 100 100 100 10013a6 6 9 9 100 100 100 10014 6 6 9 9 100 100 100 10015 6 6 9 9 100 100 100 10016 6 6 9 9 100 100 100 10017 6 6 9 9 100 100 100 10018 6 6 9 9 100 100 100 10019 6 6 9 9 100 100 100 10020 6 6 9 9 100 100 100 10021 6 6 9 9 100 100 100 10022 5 1 6 9 9 83 100 93 9323 5 6 9 9 83 100 93 9324 5 6 8 1 9 83 89 87 8725 5 6 8 9 83 89 87 8726 5 6 8 9 83 89 87 8727 5 6 8 9 83 89 87 87aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 228Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

200 ng/LReplicate CfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010b12 12 3 3 10011c9 9 6 6 100 100 100 10012 9 9 6 6 100 100 100 10013a9 9 6 6 100 100 100 10014 9 9 6 6 100 100 100 10015 9 9 6 6 100 100 100 10016 9 9 6 6 100 100 100 10017 9 9 6 6 100 100 100 10018 8 1 8 6 6 100 100 100 10019 8 8 5 1 5 100 100 100 10020 8 8 5 5 100 100 100 10021 8 8 5 5 100 100 100 10022 7 1 7 5 5 100 100 100 10023 7 7 4 1 4 100 100 100 10024 6 1 7 4 4 86 100 91 9125 6 7 4 4 86 100 91 9126 6 7 4 4 86 100 91 9127 5 1 7 4 4 71 100 82 82aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 229Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

500 ng/LReplicate AfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 14 1 15 934 14 15 935 14 15 936 14 15 937 15 15 1008 15 15 1009 12 12 3 3 10010 9 9 6 6 10011a5 1 6 9 9 100 100 100 9312bc7 8 9 9 100 100 100 9413 7 8 9 9 100 100 100 9414 7 8 9 9 100 100 100 9415 7 8 8 1 8 100 100 100 9416 7 8 8 8 100 100 100 9417 7 8 8 8 100 100 100 9418 7 8 8 8 100 100 100 9419 7 8 8 8 100 100 100 9420 7 8 8 8 100 100 100 9421 7 8 8 8 100 100 100 9422 5 2 6 8 8 100 100 100 9323 5 6 8 8 100 100 100 9324 5 6 8 8 100 100 100 9325 5 6 8 8 100 100 100 9326 5 6 8 8 100 100 100 9327 5 6 7 1 7 100 100 100 92aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 230Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

500 ng/LReplicate BfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010 10 1 11 4 4 9311a9 10 5 5 9312bc8 1 10 7 7 100 100 100 8813 8 10 7 7 100 100 100 8814 8 10 7 7 100 100 100 8815 7 1 9 7 7 100 100 100 8816 7 9 7 7 100 100 100 8817 7 9 7 7 100 100 100 8818 6 1 8 7 7 100 100 100 8719 6 8 7 7 100 100 100 8720 6 8 7 7 100 100 100 8721 5 1 8 7 7 83 100 92 8022 5 8 7 7 83 100 92 8023 5 8 6 1 6 83 100 92 7924 3 2 6 6 6 75 100 90 7525 3 6 6 6 75 100 90 7526 3 6 6 6 75 100 90 7527 3 6 6 6 75 100 90 75aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 231Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

500 ng/LReplicate CfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010 15 15 10011a10 10 5 5 10012bc8 8 7 7 100 100 100 10013 8 8 7 7 100 100 100 10014 8 8 7 7 100 100 100 10015 7 1 7 7 7 100 100 100 10016 6 1 7 7 7 86 100 93 9317 6 7 7 7 86 100 93 9318 6 7 7 7 86 100 93 9319 6 7 7 7 86 100 93 9320 6 7 7 7 86 100 93 9321 6 7 6 1 6 86 100 92 9222 6 7 6 6 86 100 92 9223 6 7 6 6 86 100 92 9224 5 1 6 6 6 83 100 92 9225 4 1 5 6 6 80 100 91 9126 3 1 4 6 6 75 100 90 9027 3 4 6 6 75 100 90 90aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 232Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

1250 ng/LReplicate AfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 14 1 15 938 14 15 939 14 15 9310 11 12 3 3 9311 10 11 4 4 9312ac10 11 4 4 100 100 100 9313b8 9 6 6 100 100 100 9314 8 9 7 7 100 100 100 9415 7 8 8 8 100 100 100 9416 6 1 7 7 1 7 100 100 100 9317 6 7 7 7 100 100 100 9318 6 7 7 7 100 100 100 9319 6 7 7 7 100 100 100 9320 6 7 7 7 100 100 100 9321 6 7 7 7 100 100 100 9322 5 1 7 7 7 83 100 92 8623 5 7 7 7 83 100 92 8624 4 1 7 7 7 67 100 85 7925 4 7 7 7 67 100 85 7926 3 1 6 6 1 6 60 100 82 7527 3 6 5 1 5 60 100 80 73aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 233Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

1250 ng/LReplicate BfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010 14 1 15 9311 9 1 11 4 4 8712ac9 11 4 4 8713b7 9 7 7 100 100 100 8814 7 9 7 7 100 100 100 8815 7 9 7 7 100 100 100 8816 6 1 9 7 7 86 100 93 8117 6 9 7 7 86 100 93 8118 6 9 7 7 86 100 93 8119 5 1 8 7 7 83 100 92 8020 5 8 6 1 6 83 100 92 7921 5 8 5 1 5 83 100 91 7722 4 1 7 4 1 5 80 80 80 6723 3 1 6 4 5 75 80 78 6424 2 1 6 4 5 50 80 67 5525 2 6 4 5 50 80 67 5526 2 6 4 5 50 80 67 5527 2 6 4 5 50 80 67 55aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6109Page 234Study Number: 13888.6109Mysid Two Generation Exposure with LindaneF1 Reproduction Basket Survival

1250 ng/LReplicate CfgTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMe♂/Immatureb♀Dead/Missing NTRMe♀b♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 14 1 15 937 14 15 938 14 15 939 14 15 9310 14 15 9311 11 15 3 3 7812ac6 8 7 7 86 100 100 8713b6 8 7 7 86 100 100 8714 7 8 8 8 100 100 107 9415 7 8 8 8 100 100 107 9416 7 8 8 8 100 100 107 9417 7 8 8 8 100 100 107 9418 7 8 8 8 100 100 107 9419 7 8 7 1 7 100 100 108 9320 5 2 6 7 7 100 100 109 9221 5 6 6 1 7 100 86 100 8522 5 6 5 1 7 100 71 91 7723 5 6 5 7 100 71 91 7724 5 6 4 1 6 100 67 90 7525 5 6 4 6 100 67 90 7526 5 6 3 1 5 100 60 89 7327 5 6 3 5 100 60 89 73aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.eNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)fSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)gFollowing test initiation until mysid pairing,counts are considered to be estimations.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 235



## SUMMARY, Part B

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

SPONSOR: RTI International

SMITHERS VISCIENT

STUDY PLAN TITLE: Two-Generation Life-Cycle T oxicity Test with Mysids ( Americamysis bahia ) Inter-Laboratory Comparison, Smithers

Viscient Protocol No.: 0 91310/OECD/Mysid, two-generation

SMITHERS VISCIENT

STUDY NUMBER: 13888.6111

RTI

PROJECT NUMBER: 0210114.030

EPA CONTRACT

NUMBER: EP-W-06-026

TEST SUBSTANCE: 4-tert-octylphenol (4-tOP), Lot No. 08118jhv, CAS No. 140-66-9, reported to have a purity of 98.0% (tested as 100%), was received from Battelle Marine Science Laboratory on 10 November 2011.

DEFINITIVE

TEST DATES: 25 May to 17 July 2012

TEST ORGANISM: Americamysis bahia ( ≤ 21 hours old post-release) Origin: Smithers Viscient culture

DILUTION WATER: Dilute, filtered, natural seawater Salinity: 19 to 21‰ pH: 7.6 to 8.1

TEST CONDITIONS: Exposure duration: 53 days Temperature range: 25 to 28 oC Salinity range: 19 to 21 ‰ Dissolved oxygen range: 5.4 to 9.0 mg/L

75 to 130% of saturation pH range: 7.6 to 8.3

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 236

Photoperiod: 14 hours light: 10 hours dark Light intensity range: 71 to 96 footcandles (see Protocol

Deviations )

760 to 1000 lux

NOMINAL TEST

CONCENTRATIONS: 0.26, 0.64, 1.6, 4.0 and 10 μg/L

MEAN MEASURED

CONCENTRATIONS: 0.36, 0.98, 2.0, 3.9 and 11 μg/L

ENDPOINTS: Parental Generation (F 0 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 7, 14, 30)

o Dry weight to the nearest 0.01 mg (days 7, 14, 30)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

First Filial Generation (F 1 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 27)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 27)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 237

ENDPOINTS AND

RESULTS: For this exposure, the F 0 

### generation appeared to be more sensitive (seven endpoints impacted) to the concentrations of 4-tOP than the

F 1 

### generation (one endpoint impacted). A summary of the responses for each endpoint in each generation is presented below.

The most sensitive endpoints in the F 0 

### generation were determined

to be 1 st 

### brood (offspring/female), total offspring/female and Day 7 length (0.36 μg/L). While standard statistical analys is determined that these endpoints were the most sensitive, a hormetic response

was observed in a number of F 0 

### endpoints (F 0 

### 1 st 

### brood, offspring per female; F 0 

### Day 14 male dry weight, F 0 

### Day 14 female dry weight, F 0 

### interbrood duration, F 0 

### sex ratio, F 0 

### time to 1 st 

### brood, F 0 

### time to 2 nd 

### brood). Hormesis best e xplains why some endpoints are determined to be significantly different from the controls at a high and low dose, but not in the mid doses.

The most sensitive endpoint in the F 1 

### generation was determined to be total offspring per female (0.36 μg/L). A hormetic response was observed in this genera tion, but in fewer endpoints (F 1 

### Day 7 dry weight, sex ratio, time to matu ration, total young). Also, this response was not determined to be statistically significant at any dose for any endpoint.

While a dose-response rela tionship was not observed in these endpoints, thereby establishing a clear NOEC and LOEC, it is clear that all concentrations of 4-tOP had an impact on the F 0 

### generation in some fashion. This response was not carried on to the second generation, indicating that these animals can recover from these

concentrations of 4-tOP after one generation.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 238

F 0 Endpoints Measured Concentration (μg/L)

0.36 0.98 2.0 3.9 11

Survival Day 7 Dry Weight Day 14 Male Dry Weight ↑ ↑ Day 14 Female Dry Weight Termination (Day 27) Male Dry Weight Termination (Day 27) Female Dry Weight Day 7 Length ↑ ↑ ↑ ↑ Day 14 Male Length ↑ ↑ Day 14 Female Length ↑ Termination (Day 27) Male Length Termination (Day 27) Female Length

Time to Maturation

Time to 1 s t Brood Release

1 s t

Brood Offspring/Female ↓ ↓ ↓ ↓

Time to 2 nd Brood Release

2 nd Brood Offspring/Female

Interbrood Duration ↑ ↑ Total Offspring/Female ↓ ↓ ↓ ↓ ↓

Sex Ratio % Reproducing Females
↓ Endpoint determined to be sta tistically significantly reduced (p ≤ 0.05) compared to control ↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control

F 1 Endpoints Measured Concentration (μg/L)

0.36 0.98 2.0 3.9 11

Survival Day 0 Dry Weight Day 7 Dry Weight Day 14 Male Dry Weight Day 14 Female Dry Weight Termination (Day 27) Male Dry Weight Termination (Day 27) Female Dry Weight Day 0 Length Day 7 Length Day 14 Male Length Day 14 Female Length Termination (Day 27) Male Length Termination (Day 27) Female Length

Time to Maturation

Time to 1 s t Brood Release

1 s t

Brood Offspring/Female

Time to 2 nd Brood Release

2 nd Brood Offspring/Female

Interbrood Duration Total Offspring/Female ↑

Sex Ratio % Reproducing Females
↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 239



# 7.0 INTRODUCTION

The U.S. Environmental Protection Agency's En docrine Disruptor Screening Program (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 endoc rine effects and provide dose re sponse information needed for

risk assessment. One of the longer term Tier2 assays being developed is the mysid twogeneration test with Americamysis bahia . This test contrasts with existing mysid life-cycle

methods (that represent a single ge neration life-cycle) in that an additional generation is included

to allow evaluation of potential trans-generational effects. The inclusion of the invertebrate

assay in Tier 2 is important because invertebrate s make up the majority of animal species and the

endocrine systems of invertebrate s often are not directly analogous to those of vertebrates.

Additionally, a method for testi ng crustaceans for effects of endocrine disrupting chemicals

(EDCs) is relevant to assessing the adverse consequences of chem icals indicated to be endocrine

active in Tier 1 assays.

The purpose of work to be conducted under this assignment is to obtain data on the interlaboratory reproducibility and comparability of the mysid two-generation test method in support

of the validation of the mysi d two-generation test method being considered for the EDSP.

This study was conducted under the Quality Assurance Project Plan (QAPP, Appendix 5 ), the

Smithers Viscient study plan entitled "Study Plan on Two-Generation Life-Cycle Toxicity Test

with Mysids ( Americamysis bahia ) Inter-Laboratory Comparison," Smithers Viscient protocol

No. 091310/OECD/Mysid, two-generation ( Appendix 6 ), relevant program and facility standard

operating procedures (SOPs), and guidance documents.

The experimental phase of the study was conduc ted from 25 May to 17 July 2012 at Smithers

Viscient (SMV), located in Wareham, Massachuse tts. The original raw data, protocol, and the

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 240

original final report produced during this study are stored at Smithers Viscient, Wareham,

Massachusetts.



# 8.0 MATERIALS AND METHODS

8.1 QAPP/Study Plan

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

Plan (QAPP) for Task Order 30 ( Appendix 5 ). The study plan ( Appendix 6 ) describes a twogeneration test with an estuarine mysid that considers reproductive fitness in parents and

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

of other endpoints that allow for diagnostic and definitive evaluation of EDCs or other types of

reproductive toxicants ( Verslycke et al., 2004 ). The mysid two-generati on test is a relatively

long-term (normally 60 days or longer) assay th at assesses early development, growth and

reproduction in two generations (F 0 

### and F 1 

### ). The two-generation test described in the guideline

is an extension of existing standard pract ice for conducting a mysid life-cycle test ( ASTM, 2008 ;

McKenney, 1986, 1996 ; and Nimmo et al., 1977, 1978 ).



## 8.2 Test Substance

The test substance, 4-tert-oct ylphenol (4-tOP), was received on 10 November 2011 from Battelle

Marine Science Laboratory, Sequim, Washingto n. The following information was provided:

Name: 4-tert-octylphenol Synonym: 4-(1,1,3,3-tetra methyl butyl) phenol Lot No.: 08118jhv

CAS No.: 140-66-9 Purity: 98.0% Expiration Date: November 2015

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 241

Upon receipt at Smithers Viscient, the test s ubstance (SMV No. 5305) was stored at room

temperature in the original container in a dark, ventilated cabinet. Analytical and QC stock

concentrations were corrected for th e purity of the test substance.



## 8.3 Test Organism

Juvenile mysids ( ≤ 21 hours old), Americamysis bahia (formerly Mysidopsis bahia ) were used to

initiate the life-cycle test. The adult mysids (SMV Lot No. 11A168) used to produce juvenile

mysids for testing were obtaine d from SMV cultures. The brood stock was originally obtained

from MBL Aquaculture, a commercial supplie r located in Sarasota, Florida, and are

supplemented with animals from reputable source s or suppliers periodica lly. Culture organisms

were received on 29 December 2011 and were main tained in-house for approximately 5 months

prior to exposure initiation. Mysids were cultur ed using dilute, filtered, natural seawater and six

80-L glass aquaria under closed-l oop, recirculating conditions. Standard aquarium undergravel

filters were used to provide aeration, biological filtration and a current conducive to feeding.

The seawater used for culturing was characterize d as having a temperature range of 24 to 26  C,

salinity range of 21 to 22‰ and a pH range of 7.6 to 7.8 during the 14-day period prior to

exposure initiation. Dissolved oxygen concentration ranged from 5.7 to 6.7 mg/L. The area in

which the mysids were cultured received a re gulated photoperiod of 16 hour s of light and 8 hours

of darkness. Culture conditions and test conditio ns are based on previously determined optimal

salinity-temperature conditions for survival ( McKenney, 1994 ), growth ( McKenney and

Celestial, 1995 ) and reproduction ( McKenney, 1996 ).

Juvenile mysids were obtained by isolating mature gravid females in isolation baskets

approximately 21 hours prior to exposure initia tion. Young produced by these organisms were

harvested from designated mysid breeding tanks usi ng a variation of the method described in the

Short-Term Methods for Estimating the Chronic T oxicity of Effluents and Receiving Waters to

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 242

Marine and Estuarine Organisms ( U.S. EPA, 2002 ). Based on these procedures, the post-release

age of the juveniles used to initiate this assay was ≤ 21 hours.



## 8.4 Feeding

During this exposure with the exception of test day 1 (see Protocol Deviations ), mysids (both F 0



### and F 1 

### ) were fed twice daily with brine shrimp ( Artemia salina , SMV Lot No. 100209, supplied

by INVE Aquaculture) nauplii ≤ 48 hours old (post-hydration). At least one of these feedings

was with brine shrimp nauplii enriched with Selco ® 

### , a substance high in uns aturated fatty acids.

Mysid survival, growth and reproducti on are improved with high levels of Ω -3 highly

unsaturated fatty acids. Selco ® 

### is added to newly hatched brine shrimp. The newly hatched

Artemia become enriched by feeding on the lipid-coat ed micro particles. Densities per mL of

concentrated collections of nauplii from Artemia cultures created from daily hydrated cysts were

determined by triplicate counts of aliquots pipetted onto filter pape r. Based on these densities,

the following pre-determined volumes of nauplii were fed to mysids at each feeding: rations for

retention/brood chambers were approximately 1800 nauplii/retention chamber on days 0 to 3,

2700 on days 4 to 6, 3600 on days 7 to 9, 4500 on days 10 to 13, and 3600 on days > 12, while

brood chambers (containing individually paired mysids) were fed approximately

900 nauplii/brood chamber throughout the test. All treatments and co ntrols received, as near as

reasonably possible, the same rati on of food. Excess brine shrimp a nd organic debris in the test

chambers were removed daily prior to the addition of new nauplii.

Prior to use of a new batch of feed, a represen tative sample of the br ine shrimp nauplii was

analyzed for the presence of pesticides, PCBs, and toxic metals, by GeoLabs, Inc., Braintree,

Massachusetts ( Appendix 10 ). None of these compounds were detected at concentrations that

are considered toxic in any of the fo od samples analyzed in agreement with ASTM (2002)

standard practice. Based on these analyses and the performance of the control organisms, the

food source was considered to be of acceptable qu ality since all analyte concentrations were

below levels of concern and control organisms exceeded guideline criteria.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 243



## 8.5 Dilution Water

Dilute, filtered, natural seawater was used as dilution and control wa ter during this study.

Seawater was collected from the Cape Cod Cana l in Bourne, Massachusetts from about 1 to

4 meters offshore at a depth of approximately 0.5 meters. The seawater was transferred with a

pump (fiberglass reinforced thermoplastic housin g) and a polyvinyl chloride (PVC) pipe and was

then transported to the testing facility in a 3400-L fi berglass tank. At the testing facility, the

seawater was transferred to an 8500-L epoxy-coat ed concrete tank, wher e it was diluted to a

salinity of 20 ± 3‰ with laborat ory well water using an actuated valve controlled by a Signet

Controller. The diluted seawater was mixed thoroughly prior to characterization. Following

characterization, the water was then pumped into holding tanks where it was recirculated for a

minimum of 48 hours prior to use. The seawater was pumped through a se ries of polypropylene

core filters (20-, 5- and 1-μm) to the laboratory under constant pressure through PVC pipe to a

head tank, where it was aerated to ensure pr oper equilibration of disso lved gases with the

laboratory atmosphere and heated to test temper ature prior to delivery to the intermittent-flow

proportional diluter system. The seawater used fo r this exposure had a salinity range of 19 to

21‰ and a pH range of 7.6 to 8.1.

Representative samples of the dilution water we re analyzed periodically for the presence of

pesticides, PCBs and toxic metals by GeoLabs, In c., Braintree, Massachusetts. None of these

compounds have been detected at concentrations that are considered toxic in any of the water

samples analyzed, in agreement with ASTM (2002) standard practice.

In addition, representative sample s of the dilution water were anal yzed monthly for total organic

carbon (TOC) concentration. The TOC concentrati on in the seawater source ranged from 1.0 to

1.4 mg/L for May through July 2012. Based on these analyses and the performance of the

control organisms, the dilution water source was considered to be of acceptable quality since all

analyte concentrations were be low levels of concern and control organisms exceeded guideline

performance criteria.

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## 8.6 Test Substance Concentrations

Nominal concentrations of 0.26, 0.64, 1.6, 4.0 and 10 μg/L, and a control were selected by the

EPA for the definitive exposure.



## 8.7 Stock Preparation

A saturation vessel was prepared weekly th roughout the exposure by first weighing out

approximately 5 g of 4-tert-octylphenol in a 100-mL beaker, crushing the test substance and

finally dissolving the material in 100 mL of acetone (CAS No. 67-64-1). The acetone solution

was then added to a 500-mL bulb-type prepara tive vessel containing 3-mm glass beads. The

bottom end of the preparative vesse l was sealed with a solid Teflon ® 

### plug. The vessel was then

connected to a rotary evaporator (rotovap). The rotovap was used to remove the acetone within

the vessel, thereby leaving a coat ing of 4-tert-octylphenol on the gl ass beads within the vessel.

Once the acetone was removed from the vessel, it was connected to a di lution water source at a

flow rate of 8 mL/min and allowed to flush at this flow rate for 24 hour s prior to use. Water

flowed continuously through this vessel until it wa s replaced (e.g., every 7 days). This saturation

vessel was used to prepare the daily diluter stocks . Each day, the effluent from this vessel was

used to fill a 2000-mL graduated stock bottle. Th is solution was used as the diluter stock. Prior

to exposure initiation, the concen tration of the outflow solution from the saturation vessel was

measured to be approximately 5.0 mg/L. Th e solution produced by th e saturation vessel was

observed to be clear and colorless.



## 8.8 Exposure System

8.8.1 Exposure Vessels

The exposure vessels were 24-L glass aquaria me asuring 20 x 10 x 12 cm (L x W x H) equipped

with side drain at 4.5 cm. The total so lution volume maintained in each vessel was

approximately 9 L. The treatment aquaria were randomly distributed be tween two tiers (upper

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and lower tier) of water baths, th e upper tier maintained two rep licates (A and B) and the lower

tier maintained one replicate (C). The control ve ssels were placed in a water bath of a separate

exposure system (see Section 8.8.4 ). Each aquarium was labeled to identify the nominal test

substance treatment concentration and the replicate designation.



## 8.8.2 Retention Chambers

Retention chambers were construc ted of 10-cm glass Petri dishes to which a 15-cm high cylinder

of nylon mesh screen (250-μm mesh) had been attached w ith aquarium grade (without

fungicides) silicone adhesive. There were two retention chambers in each exposure vessel prior

to reaching sexual maturity. One chamber, the reproduction chamber, was used to rear mysids

which were used for reproductive ev aluation; these mysids were held in these retention chambers

until sexual maturity was reached. The second cham ber, the growth chamber, was used to rear

mysids which were used for collecting growth metr ics; these mysids were held in the retention

chamber until Day 14.



## 8.8.3 Brood Chambers

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

constructed of 6-cm glass Petri dishes to wh ich a 15-cm high cylinder of nylon mesh screen

(250-μm mesh) was attached.



## 8.8.4 Diluter System

Two separate, 2-L intermittent-flow proportional diluters (e.g., Mount and Brungs, 1967 ) were

employed for this exposure. One diluter was used to deliver the five test substance

concentrations; the other diluter was used to maintain the negative control.

During the period prior to exposure initiation, sampling of the contro l vessels indicated

inconsistent contamination of control vessels wi th 4-tOP. Since 4-tOP is a highly volatile

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substance and controlling this contamination was difficult, the control vessels were maintained in

a system separate from the 4-tOP treatment tanks.

For the treatment diluter, an FMI pump was initi ally calibrated to deliver 6.6 mL/cycle of an

8.5 mg/L stock solution into the diluter's chemi cal mixing chamber, which also received 3.3 L of

dilution water per cycle (for a high concentration of 10 μg/L). However, during the pre-exposure

period analytical samples removed from the expos ure vessels indicated th at approximately 40%

loss at each concentration was occurring due to the volatility of 4-tert-octylphenol under aqueous

conditions. In order to compensa te for these losses, the stock delivery was increased. For this

adjustment, the FMI pump was recalibrated to de liver 13.2 mL/cycle of a 5 mg/L stock solution

into the mixing cell chamber. The mixing cham ber was positioned over a magnetic stir plate

which aided in mixing the stock solution with the dilution water. The solution contained in the

mixing chamber constituted the highest nominal concentration (10 μg/L). This solution was

subsequently diluted (40%) to provide the re maining nominal exposure concentrations (4.0, 1.6,

0.64 and 0.26 μg/L). For the control diluter, a se t of control vessels we re established which

contained the same dilution water. The control diluter was maintained und er the same conditions

as the treatment level vessels, but contained no 4-tert-octylphenol. Th ree replicates were

maintained for all treatments and the control.

For both exposure systems, a flow-splitting cham ber was used between the diluter cells and the

aquaria to split solutions equally between the test aquaria. For the treatment diluter, the flowsplitting chambers were used to pr omote mixing of the test substa nce solution and diluent water.

In each chamber, three standpipes with umbre lla siphons were employed to split the test

solutions. The volume of test solution delivered to each of three replicate aquaria was 670 mL

with a flow splitting accuracy of ± 5%. Solution was delivered to the exposure vess els at a rate

of approximately seven exposure vessel volum e additions per 24 hours and an approximate

7-hour 90% replacement time.

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The calibration of the diluter systems was checked and recorded prior to exposure initiation. If

there was any indication during th e test that the diluter calib ration had changed (e.g., diluter

malfunction or unexplained differences in water quality or measured concentrations in the

aquaria), calibration of the necessary diluter components was checked. During the exposure, a

complete check of diluter function (e.g. diluter cy cle rate, toxicant stock flow, etc.) and a visual

inspection was performed at the begi nning and end of the work day.



## 8.9 Test Conditions

The test area was illuminated with fluorescent bulbs at an intensity range of 71 to 96 footcandles

(760 to 1000 lux) at the solutions' surface (see Protocol Deviations ). The photoperiod for each

exposure system was set to be maintained at 14 hours light:10 hours dark . The photoperiod was

gradual (i.e., began and ended with 30 minutes of reduced lighting, and 13 hours of full light

intensity in between the reduced lighting periods ). Photoperiods were maintained by using an

automatic timer. Light intensity was meas ured with a VWR light meter once during the

exposure. Two Process Technology heaters were se t to maintain the protocol specified test

solution temperature at 26 ± 1 oC inside each of the temperature-controlled water baths. The

salinity of the test solutions was set to maintain the protocol specified at 20 ± 2‰ and monitored

prior to use to ensure these parameters we re maintained. The total dissolved oxygen was

maintained between 80 to 100% of saturation at exposure initiation and > 60% saturation

(4.3 mg/L at 26 oC and 20‰ sa linity) throughout the test.



## 8.10 Exposure Conduct

8.10.1 Failed Exposures

Prior to initiating the exposure reported below, there were at least two attempts to initiate

exposure with the same test chemical. These ex posures were terminated prematurely due to low

and variable measured concentration and cont rol contamination with 4-tert-octylphenol.

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## 8.10.2 Exposure Initiation

The exposure was not initiated unt il the diluters and test substa nce delivery device had been

properly functioning for at least 48 hours prior. This allowed the test solution to reach an

equilibrium concentration of test substance in the exposure system and ensured concentrations

were maintained at exposure in itiation. The exposure was initia ted when the retention chambers

(growth and reproduction) containing F 0 

### mysids were added to the respective test vessels (see

Section 8.10.3.1.1 ).



## 8.10.3 Biological Methods



## 8.10.3.1 Exposed Parental Generation (F 0 

## )

8.10.3.1.1 F 0 

## Exposure Initiation

Three replicate aquaria per treatment were us ed for each test concentration (0, 0.26, 0.64, 1.6, 4.0

and 10 μg/L). Each replicate tank housed one growth and one reproduction chamber (days 0

through 14) or seven brood chambers (days 14 th rough 7 days after releas e of the second brood

in the controls; Figure 37 ).

At exposure initiation, 20 juvenile mysids ( ≤ 21 hours old, post-release) were impartially

transferred into both the growth and reproduction chambers in the following manner: (1) a pool

of > 630 juvenile mysids were removed from the cu ltures; (2) mysids were distributed, four at a

time, to unlabeled intermediate beakers (two per replicate) containing dilution water until each

beaker contains four mysids. This process was repeated five more times until each intermediate

beaker contained 20 mysids; (3) following distribu tion, mysids in each intermediate beaker were

then transferred to a labeled rete ntion chamber. Care was exercise d to avoid injury to juvenile

mysids during counting. Mysids were transferre d using either a smooth-e dge pipette or a piece

of fine, nylon mesh screen. Mysids that were dropped or injured during transfer were removed

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and replaced with a new individual from the same cohort. The exposure was initiated when each

labeled growth and reproduction basket was ad ded to the respective exposure vessel.

At exposure initiation, 45 additi onal mysids (from the cohort used for initiating the exposure),

were to be obtained from the culture for grow th metrics. These 45 mysids were pooled then

divided into 9 groups of 5 mysids and were placed in ice-cold, deionized water, blotted dry, and

digital images were taken of each group of mysi ds for total length measurements. Each group

was then placed on pre-weighed aluminum tins, and placed in an oven at approximately 100  C

for approximately 24 hours. Following the dr ying period, mysid samples were kept in a

desiccator for a minimum of 30 minutes (to allow for cooling of the sample) and weighed to the

nearest 0.01 mg on an analytical balance.

Length measurements were obtained from digita l photographs using the following software and

hardware: Zeiss Stemi-2000 CS, a Zeiss AxioCa m ICc 3 and Zeiss AxioVision image analyzing

software.



## 8.10.3.1.2 F 0 

## Growth Chambers

Following 7 and 14 days of exposure, mysids were sampled from each growth chamber for

survival, length and dry weight determinations . On day 7, six mysids were removed from each

growth chamber, separated into three groups of two mysids, euthanized in ice-cold deionized

water, blotted dry and photographed for total le ngth measurements. Each group was then placed

on pre-weighed aluminum tins and placed in an oven at approximately 100  C for approximately

24 hours. Following the drying period, mysid sample s were kept in a desiccator for a minimum

of 30 minutes (to allow for cooling of the sa mple) and weighed to the nearest 0.01 mg on an

analytical balance.

On day 14, the remaining mysids were sampled from each growth chamber for survival, length

and dry weight determinations. Prior to th e day 14 termination, mysids from the growth

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

the reproduction chamber was skewed pr eventing obtaining seven pairs (see Section 8.10.3.1.3 ).

The remaining mysids were removed, identified as male, female or immature (following the

criteria outlined in U.S. EPA, 2002 ), euthanized in ice-cold deionized water, blotted dry and

photographed for total length measurements. E ach mysid was then placed on pre-weighed

aluminum tins and placed in an oven at approximately 100  C for approximately 24 hours.

Following the drying period, mysid samples were kept in a desiccator for a minimum of

30 minutes (to allow for cooling of the samp le) and weighed to the nearest 0.01 mg on an

analytical balance.



## 8.10.3.1.3 F 0 

## Reproduction Chambers

Mysids maintained within reproduction cham bers were observed daily for survival, sex

determination and reproductive output. Begi nning on day 10, developing embryos could be

discerned within individual females at most tr eatment levels. By day 12 all females observed

had developed marsupial pouches. At sexual ma turity (designated by th e appearance of the

marsupial pouch), one ovigerous female was paired with one male, and the pair was transferred

to a brood chamber housed in the replicate tank. For each exposure concentration, 3 replicate

tanks housed up to seven brood chambers each ( Figure 37 ). Mysids that were not paired

remained in reproduction chambers until three or fewer remained; if three or fewer remained,

these mysids were transferred to a separate br ood chamber. Remaining male mysids (e.g., those

not used in the initial male/female pairings) were held and used to replace male mysids that died

in brood chambers during the reproductive phas e of the exposure. Prior to the day 14

terminations, mysids from growth chambers were recruited to form br ood chamber pairs in the

event that the sex ratio in the reproduction chamber was skewed, preventing obtaining seven

pairs.

During the reproductive phase until F 0 

### termination, offspring were enumerated daily. Offspring

from the F 0 

### mysids (used to establish the F 1 

### generation) were collected and transferred to growth

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and reproduction chambers exposed to the same test substance concentration from which they

originated. The F 0 

### exposure was terminated seven days after the median day of release of the

second brood release in the control treatments.

At exposure termination, F 0 

### mysids were removed, identified as male, female or immature,

euthanized in ice-cold deionized water, blotted dry and photographed for total length

measurements. Each mysid was then placed on i ndividual aluminum tins and placed in an oven

at approximately 100  C for approximately 24 hours. Following the drying period, mysid

samples were kept in a desiccator for a mini mum of 30 minutes (to allow for cooling of the

sample) and weighed to the nearest 0.01 mg on an analytical balance.



## 8.10.3.2 Exposed First Filial Generation (F 1 

## )

8.10.3.2.1 Initiation of F 1 

## Generation

Between exposure days 16 and 27, groups of 15 < 24-hour old offspring (F 1 

### ), from either the first

(F 1 

### ') or second (F 1 " 

### ) brood of the F 0 

### mysids, were isolated in gr owth and reproduction chambers

(as previously described). F 1 

### offspring were exposed to the sa me test substance concentrations

as their F 0 

### parents. Mixing of young from replicate exposure tanks released on the same day

across replicate chambers was encouraged to mi nimize reproduction between siblings. When F 1



### mysids were limited, priority was given to popula ting all replicate reprod uction chambers first,

then growth chambers.

In addition, day 0 F 1 

### mysids (nine groups of five organisms) were collected from each

F 0 

### exposure concentration, euthaniz ed in cold-water deionized wa ter, blotted dry and digital

images were taken for total length measurements. Each group was then placed on pre-weighed

aluminum tins, and placed in an oven at approximately 100  C for approximately 24 hours.

Following the drying period, mysid samples were kept in a desiccator for a minimum of

30 minutes (to allow for cooling of the samp le) and weighed to the nearest 0.01 mg on an

analytical balance.

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## 8.10.3.2.2 F 1 

## Growth Chambers

For each growth chamber, on their respective day 7 and day 14, mysids were sampled for length

and dry weight determinations.

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

mysids, euthanized in ice-cold deionized wate r, blotted dry and photographed for total length

measurements. Each group was then placed on pre-weighed aluminum tins and placed in an

oven at approximately 100  C for approximately 24 hours. Following the drying period, mysid

samples were kept in a desiccator for a mini mum of 30 minutes (to allow for cooling of the

sample) and weighed to the nearest 0.01 mg on an analytical balance.

On day 14, mysids remaining in the growth cham ber, (e.g., those not sampled on day 7 and those

not used to complete reproduc tive pairings) were removed, identified as male, female or

immature (following the criteria outlined in U.S. EPA, 2002 ), euthanized in ice-cold deionized

water, blotted dry and photographed for total le ngth measurements. Each mysid was then placed

on pre-weighed aluminum tins and placed in an oven at approximately 100  C for approximately

24 hours. Following the drying period, mysid sample s were kept in a desiccator for a minimum

of 30 minutes (to allow for cooling of the sa mple) and weighed to the nearest 0.01 mg on an

analytical balance.



## 8.10.3.2.3 F 1 

## Reproduction Chambers

Mysids maintained within reproduction cham bers were observed daily for survival, sex

determination and reproductive output. Betw een the respective days 9 to 13 for the F 1



### generation, developing embryos coul d be discerned within individua l females at most treatment

levels. When developed marsupial pouches could be discerned within an individual female, the

ovigerous female was paired with one male, a nd the pair was isolated in a separate brood

chamber housed within the replicate tank for that specific exposure conc entration. A maximum

of seven pairs were isolated in br ood chambers for each replicate tank ( Figure 37 ). Mysids that

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were not paired remained in reproduction chambe rs until three or fewer remained; if three or

fewer remained, these mysids were transferred to a separate brood chamber. Remaining male

mysids (e.g., those not used in the initial male /female pairings) were held and used to replace

male mysids that died in brood chambers during th e reproductive phase of the exposure. Prior to

their respective day 14 terminations, mysids from growth chambers were recruited to form brood

chamber pairs in the event that the sex ratio in the reproduction chamber was skewed, preventing

obtaining seven pairs.

During the reproductive phase , offspring from the F 1 

### mysids were removed, enumerated, and

recorded from their respective brood chamber daily until exposu re termination. The exposure

was terminated seven days after the median day of release of the second brood in the F 1 

### control

tanks.

At exposure termination, F 1 

### mysids were removed, identified as male, female or immature,

euthanized in ice-cold deionized water, blotted dry and photographed for total length

measurements. Each mysid was then placed on i ndividual aluminum tins and placed in an oven

at approximately 100  C for approximately 24 hours. Following the drying period, mysid

samples were kept in a desiccator for a mini mum of 30 minutes (to allow for cooling of the

sample) and weighed to the nearest 0.01 mg on an analytical balance.



## 8.10.4 Analytical Measurements

At exposure initiation (day 0), water samples from each replicate of each concentration and

control were removed and analyzed for 4-tert-o ctylphenol. Following day 0, water samples were

removed weekly from alternating replicates (e .g., replicate A sampled in week 2, replicate B

sampled in week 3, replicate C sampled in week 4, etc.). At exposure termination (seven days

after the median day of release of the second brood release in the F 1 

### control vessels), each

replicate vessel was sampled. Since the initiat ion of each reproduction chamber was not uniform

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across the treatment and control replicates, each exposure vessel was sampled on its respective

termination day.

Water samples were removed from a point appr oximately midway between the water surface,

bottom, and sides of each test vessel and processe d for analysis immediately. A sample of the

stock solution was analyzed at each sampling interval, with one exception. Since exposure

termination encompassed multiple days, the stock sample was not analyzed at each sampling

interval. The diluter stock was typically prepar ed about once per week, therefore the stock was

sampled once during the termination period.

Three quality control (QC) samples were prep ared at each sampling interval at nominal

concentrations of 0.200, 1.67 and 10.0 μg/L. These c oncentrations were intended to approximate

the test concentration range. The QC samples were processed and analyzed with the exposure

solution samples and remained with the exposur e solution samples thr oughout the analytical

process. Results of the analyses of the QC samples were used to evaluate the precision and the

quality control maintained during the an alysis of exposure solution samples.

All exposure solutions and QC samples were analyzed for 4-tert-o ctylphenol using liquid

chromatography with mass spectrometry (LC/ MS/MS) based on methodology validated at

Smithers Viscient. The method validation was conduc ted prior to the initiation of the definitive

test and established an average recovery of 103% ± 7.97% for 4-tert-octylphenol from 20 ppt

filtered seawater ( Appendix 7 ). Defined limits for acceptance of quality control sample

performance in subsequent studies were set at 70 to 120%. Conditions and procedures used

throughout the analysis of exposure solutions and QC samples during this study were similar to

those used in the method validati on with the exception of days 6 and 10. On these days, due to

excessive variability in the recovery of the inte rnal standard, the result s were calculated on the

response areas of the calibration standards rather than the ratio of the response areas. This

exception did not have a negative impact on the study since the rec overies of the six QC samples

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were all acceptable for those intervals and in dicate that the calibration standards were

quantifying properly.



## 8.10.5 Water Quality Measurements

On day 0, temperature, dissolved oxygen concentr ation, pH and salinity were measured in each

replicate of each treatment and the control solutions. Following day 0, these parameters were

measured in alternating replic ates of each concentration and the control. Exposure solution

temperature was continuously monitored in one representative vessel within each water bath by

using a VWR digital minimum/maximum thermometer. These vessels were: replicate A of the

control, in the control diluter; replicate B of the 0.26 μg/L nomin al treatment in the upper water

bath of the treatment diluter; replicate C of the 0.26 μg/L nominal treatment in the lower water

bath of the treatment diluter. Salinity was m easured with a Yellow Springs Instrument (YSI)

Model 30 conductivity/salin ity meter. Dissolved oxygen conc entration and daily temperature

were measured using a YSI Model 550A dissolv ed oxygen meter/temperature probe. The pH

was measured with a YSI Model pH100 pH meter.



## 8.11 Biological Observations

Biological observations of the mysids were reco rded at exposure initia tion and daily thereafter

until termination of the F 1 

### generation (day 53). Dead orga nisms were recorded and removed

when observed and discarded at each observation interval. The cr iteria used for determining

death were the absence of mobility and failure to respond to gentle prodding. Survival was

based on the number of mysids init ially present in each test vessel.

The primary biological observations made during the exposure were:

Parental Generation (F 0 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 30)

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o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 30)

 Time to maturation, appearance of marsupial pouch

 Time to first brood release, number of days after test initiatio n to release of young

in the first brood

 Time to second brood release, number of da ys after exposure initiation to release of young in the second brood

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

First Filial Generation (F 1 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 27)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 27)

 Time to maturation, appearance of marsupial pouch

 Time to first brood release, number of days after test initiatio n to release of young

in the first brood

 Time to second brood release, number of da ys after exposure initiation to release of young in the second brood

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active



# 9.0 Statistical Methods

9.1 Endpoints

Parental Generation (F 0 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 7, 14, 30)

o Dry weight to the nearest 0.01 mg (days 7, 14, 30)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active

First Filial Generation (F 1 

### )

 Survival

 Growth:

o Total length to the nearest 0.1 mm (days 0, 7, 14, 27)

o Dry weight to the nearest 0.01 mg (days 0, 7, 14, 27)

 Time to first brood release

 Time to second brood release

 Interbrood duration

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

 Total number of young in the first tw o broods in each replicate population

 Sex ratio

 Percentage of females that were reproductively active



## 9.2 Statistical Procedures

All statistical conclusions were made at the 95% level of certa inty except in the cases of

Modified Levene's Test ( U.S. EPA, 2002 ), Bartlett's Test ( U.S. EPA, 2002 ) and Shapiro-Wilks’

Test ( U.S. EPA, 2002 ), in which the 99% level of cer tainty was applied. The following

procedures were used:

 Prior to statistical analysis, data repr esenting endpoints obtained as proportions (e.g., percentage of females that are repr oductively active, survival, etc.) were transformed using the arcsine square-root transformation.

 As a check on the assumption of homogene ity of variance implicit in parametric statistics, data for each endpoint were analyzed by using Bartlett's Test ( U.S. EPA, 2002 )

, 

### which can be used for equal and unequal replicates as long as the populations are normally distri buted. Since Bartlett’s Te st can be overly sensitive to non-normality, modified Levene's Test ( U.S. EPA, 2002 ) was also utilized for homogeneity of variance.

 Shapiro-Wilks’ Test for normality ( U.S. EPA, 2002 ) was conducted and compared the observed sample distribu tion with a normal distribution. The

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 258

assumption that observations are normally distributed must be validated before subsequent analyses, following parametr ic procedures, can be performed.

 All endpoints were analyzed using parametr ic procedures with one exception: F 0 

### time to maturation. The data for F 0 

### time to maturation had no variance for any concentration or control; th erefore, parametric proce dures could not be used. These data were compared with the perf ormance of the control data using Dunn's Test with Bonferroni-Holm Adjustment ( U.S. EPA, 2002 ).

 Significant treatment effects for all rema ining endpoints were determined from Dunnett's Multiple Comparison Test ( U.S. EPA, 2002 ).

CETIS TM 

### version 1.8.4.20 ( Ives, 2011 ) was used to perform the statistical computations.



# 10.0 PERFORMANCE CRITERIA

The following performance criteria were us ed for judging test data acceptability:

 Water quality characteristics should remain within the tolerance limits illustrated in Tables 1 and 2 of the study plan ( Appendix 6 ).

 There should be more than 70% survival of control animals in all test phases over the duration of the chemical exposure.

 At least 75% of the females in the F 0 

### or F 1 

### controls must produce young.

 The average number of young produced per co ntrol female in the first two broods combined must be at least eight.



# 11.0 RESULTS

11.1 Water Quality Measurements

Water quality parameters measured during the te st were unaffected by the concentrations of

4-tert-octylphenol tested and remained within a cceptable limits for the survival, reproduction and

growth of mysids ( Table 11 ). Daily measurements establis hed dissolved oxygen concentrations

that were ≥ 91% of saturation at exposure initiation and maintained at ≥ 75% of saturation

throughout the remainder of the test. These measur ements also determined a pH range of 7.6 to

8.3; a temperature range of 25 to 27 oC; a nd a salinity range of 19 to 21‰. Continuous

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temperature monitoring of the control establis hed a temperature range of 26 to 28 oC (see

Protocol Deviations ) during the exposure period.



## 11.2 Analytical Results

The diluter systems which prepared and delivered the test solutions to the exposure vessels

functioned properly during the pre-exposure peri od and throughout the definitive study. During

the in-life phase of the definitive study, no undissolved test substance (e.g., precipitate) was

observed in the diluter system (e.g., mixing cham ber and chemical cells) or exposure solutions

throughout the exposure period.

During the in-life exposure, test solutions were sampled for concen trations of 4-tert-octylphenol

on day 0, 6, 10, 13, 20, 27, 34, 41, 43, 45, 46, 48, 49, 50 and 53. The results of these analyses

are presented in Table 12 and Table 13 . Throughout the exposure period, the measured

recoveries at each exposur e concentration approximated the nominal doses.

Analysis of the test solutions throughout the e xposure period demonstrated that the expected

concentration-gradient was generally maintained during the exposure. Exposure solutions were

generally consistent throughout the exposure period and were maintained at a CV of ≤ 22%.

Mean measured concentrations of 4-tert-octylphenol ranged from 98 to 150% of nominal. Based

on mean measured concentrations of 4-tert-octylphenol (as activ e ingredient), the treatment

levels tested (and measured) were defined as 0.36, 0.98, 2.0, 3.9 and 11 μg/L. The relationship

between the nominal treatment levels and the m ean measured concentrations established during

the exposure period is illustrated in Figure 38

.

Forty-three of the forty-five QC samples ranged from 79.3% to 118% ( Table 13 ) of the nominal

fortified concentrations (0.200, 1.67 and 10.0 μg/L) a nd were consistent with the predetermined

recovery range (i.e., set at 70 to 120%, Appendix 7 ). Based on these results, it was determined

that the appropriate quality control was maintained during the analyses of the 4-tert-octylphenol

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exposure solution samples. Two of the forty- five QC samples had r ecoveries outside the

predetermined recovery range at 10.2 and 224% recovery. QC sa mples can be out of range due

to a number of factors, some of which are spiking errors, handling e rrors and instrument errors.



## 11.3 Biological Results

11.3.1 F 0 

## Generation



## 11.3.1.1 F 0 

## Survival

Mean percent survival in the control for the F 0 

### generation averaged 79% ( Table 14 and

Figure 39 ).

Mean percent survival among F 0 

### mysids exposed to the treatment levels tested ranged from 70 to

81%. Dunnett’s Multiple Comparison Test (c ontrol > treatments) de termined no significant

differences in survival among mysids exposed to a ny of the treatment levels tested, compared to

the control.



## 11.3.1.2 F 0 

## Growth

Growth measurements were determined at three time points during the F 0 

### exposure. Mysid dry

body weight and mysid total length were measured at day 0, 7, 14 and 30.



## 11.3.1.2.1 Day 0 (Exposure Initiation) – F 0 

## Total Body Length and Dry Body Weight

Day 0 mean total body length for the F 0 

### generation was 1.70 mm and mean dry body weight for

the F 0 

### generation was 0.034 mg ( Table 14 ).

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## 11.3.1.2.2 Day 7 - F 0 

## Total Body Length and Dry Body Weight

Day 7 mean total body length in the control for the F 0 

### generation averaged 3.47 mm ( Table 14

and Figure 40 ). Mean total body length among F 0 

### mysids exposed to the 0.26, 0.64, 1.6, 4.0 and

10 μg/L nominal treatment levels averag ed 4.06, 3.93, 3.89, 3.81 and 3.93 mm, respectively.

Dunnett’s Multiple Comparison Test (control < tr eatments) determined a significant difference

(p ≤ 0.05) in total body length among mysids e xposed to the 0.26, 0.64, 1.6 and 10 μg/L nominal

treatment levels compared to the control.

Day 7 mean dry body weight in the control for the F 0 

### generation averaged 0.08 mg ( Table 14 and

Figure 41 ). Mean dry body weight among F 0 

### mysids exposed to the treatment levels tested

ranged from 0.12 to 0.16 mg. Dunnett’s Multiple Comparison Test (control < treatments)

determined no significant differences in dry bod y weight among mysids exposed to any of the

treatment levels tested, compared to the control.



## 11.3.1.2.3 Day 14 - F 0 

## Total Body Length and Dry Body Weight

Day 14 mean male total body lengt h in the control for the F 0 

### generation averaged 5.33 mm

( Table 14 and Figure 42 ). Mean total body length among F 0 

### male mysids exposed to the 0.26,

0.64, 1.6, 4.0 and 10 μg/L nominal treatment levels averaged 5.78, 5.71, 5.61, 5.55 and 5.88 mm,

respectively. Dunnett’s Multiple Comparison Te st (control < treatments) determined a

significant difference (p ≤ 0.05) in male total body length among mysids exposed to the 0.26 and

10 μg/L nominal treatment levels test ed compared to the control.

Day 14 mean female total body length in the control for the F 0 

### generation averaged 5.37 mm

( Table 14 and Figure 42 ). Mean total body length among F 0 

### female mysids exposed to the 0.26,

0.64, 1.6, 4.0 and 10 μg/L nominal treatment levels averaged 5.85, 5.71, 5.67, 5.67 and 5.77 mm,

respectively. Dunnett's Multiple Comparison Te st (control < treatments) determined a

significant difference (p ≤ 0.05) in female total body lengt h among mysids exposed to the

0.26 μg/L nominal treatment leve l compared to the control.

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Day 14 mean male dry body weight in the control for the F 0 

### generation averaged 0.42 mg

( Table 14 and Figure 43 ). Mean dry body weight among F 0 

### male mysids exposed to the 0.26,

0.64, 1.6, 4.0 and 10 μg/L nominal treatment levels averaged 0.53, 0.44, 0.39, 0.44 and 0.53 mg,

respectively. Dunnett's Multiple Comparison Te st (control < treatments) determined a

significant difference (p ≤ 0.05) in male dry body weight am ong mysids exposed to the 0.26 and

10 μg/L nominal treatment levels compared to the control.

Day 14 mean female dry body weight in the control for the F 0 

### generation averaged 0.50 mg

( Table 14 and Figure 43 ). Mean dry body weight among F 0 

### female mysids exposed to the

treatment levels tested ranged from 0.45 to 0.60 mg. Dunnett's Multiple Comparison Test

(control < treatments) determined no significan t differences in female dry body weight among

mysids exposed to any of the treatment le vels tested, compared to the control.



## 11.3.1.2.4 Day 30 (F 0 

## Termination) - Total Body Length and Dry Body Weight

Day 30 mean male total body lengt h in the control for the F 0 

### generation averaged 7.23 mm

( Table 14 and Figure 44 ). Mean total body length among F 0 

### male mysids exposed to the

treatment levels tested ranged from 7.00 to 7.28 mm. Dunnett’s Multiple Comparison Test

(control > treatments) determined no significan t differences in male total body length among

mysids exposed to any of the treatment leve ls tested, compared to the control.

Day 30 mean female total body length in the control for the F 0 

### generation averaged 7.61 mm

( Table 14 and Figure 44 ). Mean total body length among F 0 

### female mysids exposed to the

treatment levels tested ranged from 7.24 to 7.56 mm. Dunnett’s Multiple Comparison Test

(control > treatments) determined no significan t differences in female total body length among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 30 mean male dry body weight in the control for the F 0 

### generation averaged 0.82 mg

( Table 14 and Figure 45 ). Mean dry body weight among F 0 

### male mysids exposed to the

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 263

treatment levels tested ranged from 0.83 to 0.85 mg. Dunnett's Multiple Comparison Test

(control < treatments) determined no significan t differences in male dry body weight among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 30 mean female dry body weight in the control for the F 0 

### generation averaged 1.11 mg

( Table 14 and Figure 45 ). Mean dry body weight among F 0 

### female mysids exposed to the

treatment levels tested ranged from 1.14 to 1.17 mg. Dunnett’s Multiple Comparison Test

(control < treatments) determined no significan t differences in female dry body weight among

mysids exposed to any of the treatment leve ls tested, compared to the control.



## 11.3.1.3 F 0 

## Time to Maturation

Mean time to maturation for both the F 0 

### control mysids and all treatment levels tested averaged

10 days ( Table 15 and Figure 46 ). Dunn’s Test with Bonferroni-Holm's Adjustment determined

(control > treatments) no signifi cant differences in time to maturation among mysids exposed to

any of the treatment levels teste d, compared to the control.



## 11.3.1.4 F 0 

## Time to First Brood Release

Mean time to first brood release for the F 0 

### control mysids averaged 19.0 days ( Table 15 and

Figure 47 ). Mean time to first brood release for the F 0 

### mysids exposed to the treatment levels

tested ranged from 17.5 to 19.9 days. Dunnett's Mu ltiple Comparison Test (control < treatments)

determined no significant difference in time to first brood release among mysids exposed to any

of the treatment levels tested, compared to the control.



## 11.3.1.5 F 0 

## Number of Offspring in the First Brood

The mean total number produced in the first brood from the F 0 

### control mysids averaged

6.4 offspring/female ( Table 15 and Figure 48 ). The mean total number produced in the first

brood from the F 0 

### mysids exposed to the 0.26, 0.64, 1.6, 4.0 and 10 μg/L nominal treatment

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levels averaged 5.1, 4.4, 4.5, 6.0 and 4.8 offspring/female, respectively. Dunnett's Multiple

Comparison Test (control > treatments) determined a significant difference (p ≤ 0.05) in number

of offspring in the first brood among mysids exposed to the 0.26, 0.64, 1.6 and 10 μg/L nominal

treatment levels compared to the control.



## 11.3.1.6 F 0 

## Time to Second Brood Release

Mean time to second brood release for the F 0 

### control mysids averaged 23.5 days ( Table 15 and

Figure 49 ). Mean time to second brood release for the F 0 

### mysids exposed to the treatment levels

tested ranged from 23.0 to 25.6 days. Dunnett' s Multiple Comparison Test determined no

significant difference in time to second brood release (control < treatments) among mysids

exposed to any of the treatment levels tested, compared to the control.



## 11.3.1.7 F 0 

## Number of Offspring in the Second Brood

The mean total number of offspring produced in the second brood from the F 0 

### control mysids

averaged 8.7 offspring/female ( Table 15 and Figure 50 ). The mean total number produced in the

second brood from the F 0 

### mysids exposed to the treatment levels tested ranged from 5.5 to

7.4 offspring/female. Dunnett’s Multiple Comparison Test (control > treatments) determined no

significant differences in total number of offspr ing produced in the second brood among mysids

exposed to any of the treatment levels tested, compared to the control.



## 11.3.1.8 F 0 

## Interbrood Duration

Mean F 0 

### interbrood duration for the cont rol mysids averaged 4.5 days ( Table 15 and Figure 51 ).

Mean interbrood duration for mysids exposed to the 0.26, 0.64, 1.6, 4.0 and 10 μg/L nominal

treatment levels averaged 6.4, 4.5, 5.2, 6.1 and 5.5 days, respectively. Dunnett’s Multiple

Comparison Test (control < treatments) determined a significant difference (p ≤ 0.05) in

interbrood duration among mysids exposed to the 0.26 and 4.0 μg/L nomin al treatment levels

compared to the control.

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## 11.3.1.9 F 0 

## Total Number of Offspring in First Two Broods

The total mean number of offspring re leased in the first two broods for F 0 

### control mysids

averaged 14.3 offspring/female ( Table 16 and Figure 52 ). The total mean number released in the

first two broods for F 0 

### mysids exposed to the 0.26, 0.64, 1.6, 4.0 and 10 μg/L nominal treatment

levels tested averaged 9.9, 9.7, 10.7, 11.2 and 9.6 offspring/female, respectively. Dunnett’s

Multiple Comparison Test (control > treatments) determined a significant difference (p ≤ 0.05) in

number of offspring released am ong mysids exposed to all treatment levels tested compared to

the control.



## 11.3.1.10 F 0 

## Sex Ratio (Male:Female)

The F 0 

### male to female sex ratio in the control averaged 1.8 ( Table 16 and Figure 53 ). The F 0



### male to female sex ratio among mysids exposed to the treatment levels tested ranged from 1.5 to

2.3. Dunnett’s Multiple Comparison Test (control > treatments) determined no significant

differences in sex ratio among mysids exposed to a ny of the treatment levels tested, compared to

the control.



## 11.3.1.11 F 0 

## Percent of Reproducti vely Active Females

The mean percent of reproducing females in the F 0 

### control averaged 95% ( Table 16 and

Figure 54 ). Mean percent of reproducing F 0 

### females among mysids exposed to the treatment

levels tested ranged from 90 to 100%. Dunnett’s Multiple Comparison Test

(control > treatments) determined no significant differences in m ean percent of reproducing F 0



### females among mysids exposed to any of the treatm ent levels tested, compared to the control.

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## 11.3.2 F 1 

## Generation



## 11.3.2.1 F 1 

## Survival

Mean percent survival in the control for the F 1 

### generation averaged 78% ( Table 17 and

Figure 55 ). Mean percent survival among F 1 

### mysids exposed to the treatment levels tested

ranged from 69 to 86%. Dunnett’s Multiple Compar ison Test (control > treatments) determined

no significant differences in survival among mysids exposed to any of the treatment levels tested,

compared to the control.



## 11.3.2.2 F 1 

## Growth

Growth measurements were determin ed at four time points during the F 1 

### exposure. Mysid dry

body weight and mysid total length were measured at the respective day 0, 7, 14 and 27 for the

F 1 

### generation.



## 11.3.2.2.1 Day 0 (Exposure Initiation) – F 1 

## Total Body Length and Dry Body Weight

Day 0 mean total body length in the control for the F 1 

### generation averaged 1.61 mm ( Table 17

and Figure 56 ). Mean total body length among F 1 

### mysids exposed to the treatment levels tested

ranged from 1.58 to 1.61 mm. Dunn ett’s Multiple Comparison Te st (control > treatments)

determined no significant differences in total body length among mysids exposed to any of the

treatment levels tested, compared to the control.

Day 0 mean dry body weight in the control for the F 1 

### generation averaged 0.023 mg ( Table 17

and Figure 57 ). Mean dry body weight among F 1 

### mysids exposed to the treatment levels tested

ranged from 0.020 to 0.026 mg. Dunnett’s Multiple Comparison Test (control > treatments)

determined no significant differences in dry bod y weight among mysids exposed to any of the

treatment levels tested, compared to the control.

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## 11.3.2.2.2 Day 7 - F 1 

## Total Body Length and Dry Body Weight

Day 7 mean total body length in the control for the F 1 

### generation averaged 4.45 mm ( Table 17

and Figure 58 ). Mean total body length among F 1 

### mysids exposed to the treatment levels tested

ranged from 4.47 to 4.94 mm. Dunn ett’s Multiple Comparison Te st (control < treatments)

determined no significant differences in total body length among mysids exposed to any of the

treatment levels tested, compared to the control.

Day 7 mean dry body weight in the control for the F 1 

### generation averaged 0.25 mg ( Table 17 and

Figure 59 ). Mean dry body weight among F 1 

### mysids exposed to the treatment levels tested

ranged from 0.23 to 0.29 mg. Dunnett’s Multiple Comparison Test (control < treatments)

determined no significant differences in dry bod y weight among mysids exposed to any of the

treatment levels tested, compared to the control.



## 11.3.2.2.3 Day 14 - F 1 

## Total Body Length and Dry Body Weight

Day 14 mean male total body lengt h in the control for the F 1 

### generation averaged 5.96 mm

( Table 17 and Figure 60 ). Mean total body length among F 1 

### male mysids exposed to the

treatment levels tested ranged from 6.04 to 6.88 mm. Dunnett's Multiple Comparison Test

(control < treatments) determin ed no significant differences in total body length among mysids

exposed to any of the treatment levels tested, compared to the control.

Day 14 mean female total body length in the control for the F 1 

### generation averaged 6.11 mm

( Table 17 and Figure 60 ). Mean total body length among F 1 

### female mysids exposed to the

treatment levels tested ranged from 6.34 to 6.78 mm. Dunnett’s Multiple Comparison Test

(control < treatments) determined no significan t differences in female total body length among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 14 mean male dry body weight in the control for the F 1 

### generation averaged 0.51 mg

( Table 17 and Figure 61 ). Mean dry body weight among F 1 

### male mysids exposed to the

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treatment levels tested ranged from 0.45 to 0.57 mg. Dunnett's Multiple Comparison Test

(control < treatments) determined no significan t differences in male dry body weight among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 14 mean female dry body weight in the control for the F 1 

### generation averaged 0.62 mg

( Table 17 and Figure 61 ). Mean dry body weight among F 1 

### female mysids exposed to the

treatment levels tested ranged from 0.63 to 0.68 mg. Dunnett’s Multiple Comparison Test

(control < treatments) determined no significan t differences in female dry body weight among

mysids exposed to any of the treatment le vels tested, compared to the control.



## 11.3.2.2.4 Day 27 (Exposure Termination) - Total Body Length and Dry Body

Weight

Day 27 mean male total body lengt h in the control for the F 1 

### generation averaged 7.03 mm

( Table 17 and Figure 62 ). Mean total body length among F 1 

### male mysids exposed to the

treatment levels tested ranged from 6.87 to 7.30 mm. Dunnett's Multiple Comparison Test

(control < treatments) determined no significan t differences in male total body length among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 27 mean female total body length in the control for the F 1 

### generation averaged 7.52 mm

( Table 17 and Figure 62 ). Mean total body length among F 1 

### female mysids exposed to the

treatment levels tested ranged from 7.27 to 7.67 mm. Dunnett's Multiple Comparison Test

(control > treatments) determined no significan t differences in female total body length among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 27 mean male dry body weight in the control for the F 1 

### generation averaged 0.83 mg

( Table 17 and Figure 63 ). Mean dry body weight among F 1 

### male mysids exposed to the

treatment levels tested ranged from 0.82 to 0.89 mg. Dunnett's Multiple Comparison Test

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(control < treatments) determined no significan t differences in male dry body weight among

mysids exposed to any of the treatment le vels tested, compared to the control.

Day 27 mean female dry body weight in the control for the F 1 

### generation averaged 1.25 mg

( Table 17 and Figure 63 ). Mean dry body weight among F 1 

### female mysids exposed to the

treatment levels tested ranged from 1.09 to 1.21 mg. Dunnett’s Multiple Comparison Test

(control > treatments) determined no significan t differences in female dry body weight among

mysids exposed to any of the treatment le vels tested, compared to the control.



## 11.3.2.3 F 1 

## Time to Maturation

Mean time to maturation for the F 1 

### control mysids averaged 9.0 days ( Table 18 and Figure 64 ).

Mean time to maturation among mysids exposed to the treatment levels test ed ranged from 8.0 to

10.3 days. Dunnett’s Multiple Comparison Test (c ontrol < treatments) determined no significant

differences in time to maturation among mysids e xposed to any of the treatment levels tested,

compared to the control.



## 11.3.2.4 F 1 

## Time to First Brood Release

Mean time to first brood release for the F 1 

### control mysids averaged 16.3 days ( Table 18 and

Figure 65 ). Mean time to first brood release for the F 1 

### mysids exposed to the treatment levels

tested ranged from 15.7 to 18.0 days. Dunnett's Mu ltiple Comparison Test (control < treatments)

determined no significant differences in time to first brood release among mysids exposed to any

of the treatment levels tested, compared to the control.



## 11.3.2.5 F 1 

## Number of Offspring in the First Brood

The mean total number produced in the first brood from the F 1 

### control mysids averaged

6.9 offspring/female ( Table 18 and Figure 66 ). The mean total number produced in the first

brood from the F 1 

### mysids exposed to the treatment levels tested ranged from 4.7 to

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8.1 offspring/female. Dunnett's Multiple Compar ison Test (control > trea tments) determined no

significant difference in number of offspring in the first brood among mysids exposed to any of

the treatment levels tested, compared to the control.



## 11.3.2.6 F 1 

## Time to Second Brood Release

Mean time to second brood release for the F 1 

### control mysids averaged 21.1 days ( Table 18 and

Figure 67 ). Mean time to second brood release for the F 1 

### mysids exposed to the treatment levels

tested ranged from 20.6 to 23.1 days. Dunnett’s Multiple Comparison Test

(control < treatments) determined no significan t difference in time to second brood release

among mysids exposed to any of the treatment levels tested, compared to the control.



## 11.3.2.7 F 1 

## Number of Offspring in the Second Brood

The mean total number produced in the second brood from the F 1 

### control mysids averaged

8.6 offspring/female ( Table 18 and Figure 68 ). The mean total number produced in the second

brood from the F 1 

### mysids exposed to the treatment levels tested ranged from 7.4 to

11 offspring/female. Dunnett's Multiple Comparis on Test (control < treatments) determined no

significant difference in number of offspring in the first brood among mysids exposed to any of

the treatment levels tested, compared to the control.



## 11.3.2.8 F 1 

## Interbrood Duration

Mean F 1 

### interbrood duration for the cont rol mysids averaged 5.3 days ( Table 18 and Figure 69 ).

Mean interbrood duration for mysids exposed to the treatmen t levels tested ranged from 4.5 to

5.3 days. Dunnett’s Multiple Comparison Test (control > treatments) determined no significant

difference in interbrood duration among mysids exposed to any of the treatment levels tested,

compared to the control.

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## 11.3.2.9 F 1 

## Total Number of Offspring in First Two Broods

The total mean number released in the first two broods for F 1 

### control mysids averaged

12.6 offspring/female ( Table 19 and Figure 70 ). The total mean number released in the first two

broods for F 1 

### mysids exposed to the 0.26, 0.64, 1.6, 4.0 and 10 μg/L nominal treatment levels

averaged 18.7, 12.7, 12.7, 15.0 and 9.9 offspring/female, respectively. Dunnett's Multiple

Comparison Test (control < treatments) determined a significant difference (p ≤ 0.05) in number

offspring among mysids exposed to the 0.26 μg/L treatment level compar ed to the control.



## 11.3.2.10 F 1 

## Sex Ratio (Male:Female)

The F 1 

### male to female sex ratio in the control averaged 1.2 ( Table 19 and Figure 71 ). The F 1



### male to female sex ratio among mysids exposed to the treatment levels tested ranged from 0.64

to 1.1. Dunnett’s Multiple Comparison Test (c ontrol > treatments) determined no significant

differences in sex ratio among mysids exposed to a ny of the treatment levels tested, compared to

the control.



## 11.3.2.11 F 1 

## Percent of Reproducti vely Active Females

The mean percent of reproducing females in the F 1 

### control averaged 89% ( Table 19 and

Figure 72 ). Mean percent of reproducing F 1 

### females among mysids exposed to the treatment

levels tested ranged from 94 to 100%. Dunnett's Multiple Comparison Test

(control < treatments) determin ed no significant difference in percent of reproducing females

among mysids exposed to any of the treatment levels tested, compared to the control.



# 12.0 CONCLUSIONS

For this exposure, the F 0 

### generation appeared to be more sensitive (seven endpoints impacted) to

the concentrations of 4-tOP than the F 1 

### generation (one endpoint impacted). A summary of the

responses for each endpoint in each generation is presented below.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 272

The most sensitive endpoints in the F 0 

### generation were determined to be 1 st 

### brood

(offspring/female), total offspring/female and Da y 7 length. While standard statistical analysis

determined that these endpoints were the most se nsitive, a hormetic response was observed in a

number of F 0 

### endpoints (F 0 

### 1 st 

### brood, offspring per female; F 0 

### Day 14 male dry weight, F 0 

### Day

14 female dry weight, F 0 

### interbrood duration, F 0 

### sex ratio, F 0 

### time to 1 st 

### brood, F 0 

### time to 2 nd



### brood). Hormesis best explains why some endpoint s are determined to be significantly different

from the controls at a high and low dose, but not in the mid doses.

The most sensitive endpoint in the F 1 

### generation was determined to be total offspring per female

(0.36 μg/L). A hormetic response was observed in this generation, but in fewer endpoints (F 1



### Day 7 dry weight, sex ratio, time to maturati on, total young). Also, this response was not

determined to be statistically signifi cant at any dose for any endpoint.

While a dose-response relationship was not obser ved in these endpoints, thereby establishing a

clear NOEC and LOEC, it is clear that all con centrations of 4-tOP had an impact on the F 0



### generation in some fashion. This response wa s not carried on to the second generation,

indicating that these animals can recover from these concentrations of 4-tOP after one

generation.

Table 20 summarizes the F 0 

### and F 1 

### endpoints and the statistically significant differences from the

controls determined during this study.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 273



## PROTOCOL DEVIATIONS

1. The protocol states th at during the test, mysids from bot h generations will be fed at least

twice daily with brine shrimp 24 or 48 hours old. On 26 May 2012, the mysids were not

fed due to an aeration failure in the brine shrimp culture. Since all replicates were treated

equally and control mysids met the performan ce criteria, this deviation did not have a

negative impact on the results or interpretation of the study.

2. The protocol states that the te mperature of test solutions will be maintained at 26 ± 1 oC.

On test days 2, 4, 28, 29, 32, 33, 36, 39, 40, 48, 50 and 52, the minimum/maximum

readings were recorded to be greater than 27 oC. Temp eratures exceeding 27.6 oC were

not observed. Since the temperatures were with in the range tolerated by this species, this

deviation did not have a negative impact on the results or interp retation of the study.

3. The protocol states that light intensity will be measured above each test vessel once

during the test. During this study, light inte nsity was only measured above the treatment

vessels; light intensity was not measured above the control vessels, which were housed in

a separate exposure system. The light intensit y was measured in the control dilu ter after

exposure termination. These readings indicated that the light intensities should have been

within the specified range during testing. Si nce the control animals exceeded guideline

criteria and the post-hoc readings were within specification, it should be assumed that this

deviation had no negative impact on the resu lts or interpretation of this study.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 274



## REFERENCES

ASTM, 2002. Standard practice for conducti ng acute toxicity test with fishes, macroinvertebrates, and amphibians. Standa rd E729-96. American Society for Testing and Materials, 100 Barr Harbor Dr ive, West Conshohocken, PA 19428.

ASTM, 2008. Committee E-47 on Biol ogical Effects and Environmental Fate. Standard Guide for Conducting Life-Cycle Toxicity Tests with Mysids. ASTM Designation E 1191-039 (re-approved 2008). American Society for Test ing and Materials, 100 Barr Harbor Drive, West Conshohocken, Pennsylvania.

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information System TM



### . User’s Guide. Tidepool Scientific Software, McKinleyville, California.

McKenney CL, Jr., 1986. Influence 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 temper ature in an estuarine mysid ( Mysidopsis bahia ) in relation to its life cycle. Comp Biochem Physiol 109A: 199-208.

McKenney CL, Jr. and Celestial DM., 1995. Interactions among salinity, temperature, and age on growth of the estuarine mysid Mysidopsis bahia reared in the laboratory through a complete life cycle. I. Body mass and age-sp ecific growth rate. J Crust Biol 15: 169-

178.

McKenney CL, Jr., 1996. The combined effects of salinity and temperature on various aspects of the reproductive biology of the estuarine mysid, Mysidopsis bahia

. Invertebr Reprod

Dev 29(1): 9-18.

Mount, D.I. and W.A. Brungs, 1967. A simplifie d dosing apparatus for fish toxicological

studies. Water Research . 1: 21-29.

Nimmo DR, Bahner LH, Rigby RA, She ppard JM and Wilson AJ, Jr., 1977. Mysidopsis bahia : an estuarine species suitable for life-cycle to xicity tests to determine the effects of a pollutant. In: Aquatic Toxicology and Hazard Evaluation, ASTM STP 634 . FL Mayer and JL Hamelink, Eds., American Society for Te sting and Materials, Philadelphia, PA, pp.

109-116.

Nimmo DR, Hamaker TL and Sommers CA., 1978. En tire life cycle to xicity test using mysids ( Mysidopsis bahia ) in flowing water. In: Bioassay Procedures for the Ocean Disposal
Permit Program , EPA-600/9-78-010, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL, pp. 64-68.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 275

Verslycke TA, N Fockedey, CL McKenney, Jr., SD Roast, MB Jones, J Mees, and CR Janssen, 2004. Mysid crustaceans as potential test orga nisms for the evaluation of environmental endocrine disruption: a review. Environ Tox Chem 23 (5): 1219-34.

U.S. EPA. Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice Standards; Final Rule (40 CFR, Part 160). U.S. Environmental Protection Agency, Washington, D.C.

U.S. EPA, 2002. Short-term Methods for Estim ating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. 3 rd 

### Edition. EPA-821-R-02-014, U.S. Environmental Protection Agency, Washington, DC.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 276



## Table 11. Summary of water quality para meters measured in the exposure solutions during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol.

Nominal

Concentration (μg/L) Ranges a

Dissolved

Oxygen pH Temperature b

( o C) Salinity (‰) mg/L %

Saturation

Control 5.4 - 7.3 75 - 100 7.6 - 8.1 26 - 27 19 - 21

0.26 5.7 - 7.6 79 - 110 7.7 - 8.2 25 - 27 19 - 21

0.64 5.8 - 7.4 81 - 100 7.7 - 8.2 25 - 27 19 - 21

1.6 5.8 - 9.0 81 - 130 7.8 - 8.2 25 - 27 19 - 21

4.0 5.8 - 8.1 81 - 110 7.7 - 8.3 25 - 27 19 - 21

10 5.9 - 7.4 83 - 100 7.7 - 8.3 25 - 27 19 - 21

a Dissolved oxygen, pH, temperature and salinity were measured in each replicate on day 0 and in alternating replicates daily thereafter.

b Continuous temperature monitoring of replicate A of th e control vessel in the control diluter and the water baths containing the treatment aquaria (replicate B of the 0.26 μg/L nominal treatment in the upper water bath; replicate C of the 0.26 μg/L nominal treatment in the lower water bath) established a temperature range of 26 to 28 oC (see Protocol Deviations ) throughout the exposure period.

NOTE: Measurements have been r ounded to two significant figures.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 277Table 12. Concentrations of 4-tert-octylphenol measured in the exposure solutions during the mysid(Americamysis bahia) two-generation test.NominalConcentration(μg/L)Measured Concentration (μg/L)%CVPercentof Nominal(%)aDay 0 Day 6 Day 10 Day 13 Day 20Day 27 Day 34 Day 41 Day 43 Day 45 Day 46 Day 48 Day 49 Day 50 Day 53Mean(SD)aControl<0.070b<0.070<0.070<0.082 <0.092 <0.077 <0.083 <0.069 <0.095 <0.082 NS NSc<0.089<0.089NS <0.077 NS NSNAd(NA)NA NA0.260.400.440.370.42 0.18 0.27 0.28 0.36 0.38 0.48 NS0.340.36NS NS NS NS 0.340.36(0.079)22 1400.641.11.21.21.2 0.63 0.75 0.78 1.0 0.85 1.1 0.90 0.87 NS 1.1 NS NS NS0.98(0.19)20 1501.61.92.12.12.0 1.3 1.5 1.5 1.8 2.4 2.4 NS 1.8 NS 2.4 2.3 NS NS2.0(0.37)19 1204.04.34.64.14.4 2.8 3.3 3.2 4.0 4.3 3.83.93.5NS NS NS 4.6 NS NS3.9(0.56)14 981012111112 8.0 7.6 8.1 11 12 12 NS 12 NS NS 12 10 NS11(1.7)16 110aMean measured values, standard deviations (SD) and percent ofnominal were calculated using the actual analytical results andnot the rounded values (two significant figures) presented in this table.bConcentrations expressed as less than values were below thelimit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area ofthe low standards and the dilutionfactor of the controls.cNS = Not Sampled.dNA = Not Applicable.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 278



## Table 13. Results of the quality contro l (QC) samples analyzed concurrently

with test solution sa mples during the mysid ( Americamysis bahia ) two-generation test

.

Quality Control Sample (μg/L)

0.200 a 1.67 a 10.0 a

Test Day Measured

Concentration (μg/L) Percent of

Nominal Measured

Concentration (μg/L) Percent of

Nominal Measured

Concentration (μg/L) Percent of

Nominal

0 0.180 90.1 1.60 96.0 10.2 102

6 0.226 113 1.97 118 11.0 110

10 0.188 94.1 1.38 82.4 8.68 86.8

13 0.193 96.3 1.59 95.2 9.52 95.2

20 0.209 104 1.53 91.7 9.99 99.9

27 0.212 106 1.79 107 11.0 110

34 0.199 99.6 1.32 79.3 8.41 84.1

41 0.231 116 1.83 110 10.7 107

43 0.214 107 1.82 109 10.7 107

45 0.218 109 1.66 99.7 10.2 102

46 0.447 224 b 1.78 107 1.18 118

48 0.226 113 1.71 102 1.02 10.2 b

49 0.212 106 1.68 101 10.7 107

50 0.235 118 1.82 109 11.0 110

53 0.207 103 1.64 98.3 10.6 106

a QC sample fortified concentrations.

b Percent recovery for this QC sample is outsi de of the acceptable range (i.e., 70 to 120%, Appendix 7 ).RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 279Table 14. F0mysid survival and growth (lengthand dry weight) during the mysid (Americamysis bahia) twogeneration test with4-tert-octylphenol.NominalConcentration(μg/L)ReplicateSurvival(%)p-ValueMean Total Body Length (mm)aDay 7 p-ValueDay 14 Day 30Male p-Value Female p-ValueMale p-Value Female p-ValueControl A 79 3.40 5.25 5.43 7.09 7.55B 70 3.70 5.57 5.30 7.33 7.55C 88 3.31 5.16 5.37 7.25 7.74Mean(SD) 79(8.9) NAc3.47(0.21) NA 5.33(0.22) NA 5.37(0.07) NA 7.23(0.12) NA 7.61(0.11) NA0.26 A 76 3.82 5.69 5.64 7.06 7.37B 70 4.31 5.77 5.60 7.42 7.43C 65 4.05 5.88 6.33 7.35 7.90Mean(SD) 70(5.2) 0.3539 4.06(0.24)d0.0027 5.78(0.09)d0.0236 5.85(0.41)d0.0229 7.28(0.19) 0.9121 7.56(0.29) 0.72860.64 A 81 4.11 5.60 5.64 7.07 7.35B 83 3.78 5.73 5.69 7.19 7.35C 67 3.90 5.79 5.80 7.33 7.84Mean(SD) 77(9.1) 0.7545 3.93(0.17)d0.0138 5.71(0.09) 0.0537 5.71(0.09) 0.1008 7.20(0.13) 0.7800 7.51(0.28) 0.58011.6 A 79 3.99 5.39 5.64 6.98 7.19B 83 3.86 5.75 5.85 7.34 7.34C 76 3.83 5.69 5.52 7.35 7.33Mean(SD) 79(3.5) 0.8434 3.89(0.09)d0.0218 5.61(0.19) 0.1467 5.67(0.16) 0.1464 7.22(0.21) 0.8333 7.28(0.08) 0.09104.0 A 79 3.75 5.30 5.55 7.34 7.30B 70 4.02 5.92 5.73 6.81 7.40C 94 3.66 5.43 5.73 7.33 7.49Mean(SD) 81(12.1) 0.9275 3.81(0.19) 0.0600 5.55(0.33) 0.2531 5.67(0.10) 0.1464 7.16(0.31) 0.6928 7.40(0.09) 0.261710 A 66 4.01 5.83 NA 6.86 7.15B 81 3.82 5.96 5.81 6.98 7.18C 68 3.95 5.83 5.73 7.18 7.39Mean(SD) 71(8.2) 0.4127 3.93(0.09)d0.0144 5.88(0.07)d0.0081 5.77(0.06) 0.0852 7.00(0.16) 0.2850 7.24(0.13) 0.0549aMean total body length for the control on day 0 = 1.70 mm; standard deviation = 0.084.bMean dry body weight for the control on day 0 = 0.034 mg; standard deviation = 0.0048.cNA = Not Applicable.dStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 280Table 14. Continued. F0mysid survival and growth (length and dry weight)during the mysid (Americamysisbahia) two-generation test with 4-tert-octylphenol.NominalConcentration(μg/L)ReplicateMean Dry Body Weight (mg)bDay 7 p-ValueDay 14 Day 30Male p-Value Female p-ValueMale p-Value Female p-ValueControl A 0.06 0.37 0.44 0.81 0.99B 0.12 0.52 0.49 0.85 1.18C 0.08 0.37 0.58 0.80 1.18Mean(SD) 0.08(0.03) NA 0.42(0.09) NA 0.50(0.07) NA 0.82(0.03) NA 1.11(0.11) NA0.26 A 0.12 0.51 0.56 0.82 1.20B 0.18 0.57 0.56 0.87 1.06C 0.14 0.52 0.58 0.84 1.21Mean(SD) 0.14(0.03) 0.2113 0.53(0.04)d0.0265 0.57(0.01) 0.2757 0.84(0.03) 0.5284 1.16(0.09) 0.58490.64 A 0.16 0.40 0.60 0.82 1.09B 0.07 0.45 0.49 0.85 1.18C 0.14 0.48 0.50 0.86 1.24Mean(SD) 0.12(0.05) 0.4406 0.44(0.04) 0.6089 0.53(0.06) 0.6187 0.84(0.02) 0.5284 1.17(0.08) 0.48781.6 A 0.22 0.37 0.48 0.83 1.05B 0.12 0.39 0.56 0.86 1.21C 0.08 0.43 0.48 0.87 1.19Mean(SD) 0.14(0.07) 0.2669 0.39(0.03) 0.9501 0.51(0.04) 0.8185 0.85(0.02) 0.3832 1.15(0.08) 0.63254.0 A 0.13 0.46 0.38 0.86 1.18B 0.22 0.41 0.50 0.77 1.15C 0.12 0.45 0.47 0.88 1.13Mean(SD) 0.16(0.05) 0.1446 0.44(0.03) 0.6459 0.45(0.06) 0.9893 0.84(0.06) 0.6270 1.15(0.02) 0.608810 A 0.17 0.55 NA 0.82 1.12B 0.12 0.57 0.64 0.79 1.12C 0.14 0.48 0.55 0.89 1.19Mean(SD) 0.15(0.03) 0.2380 0.53(0.05)d0.0265 0.60(0.07) 0.1572 0.83(0.05) 0.6743 1.14(0.04) 0.6785aMean total body length for the control on day 0 = 1.70 mm; standard deviation = 0.084.bMean dry body weight for the control on day 0 = 0.034 mg; standard deviation = 0.0048.cNA = Not Applicable.dStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 281Table 15. F0mysid time to maturation, timeto first brood release, time tosecond brood release, offspringreleased per female and interbrood duration during the mysid (Americamysis bahia) two-generationtest with 4-tert-octylphenol.NominalConcentration(μg/L)ReplicateTime toMaturation(Days)p-ValueFirst Brood Second BroodInterbroodDuration(Days)pValueTime toRelease(Days)pValueOffspring/Femalep-ValueTime toRelease(Days)pValueOffspring/Femalep-ValueControl A 10 19.7 6.1 24.4 8.7 4.7B 10 18.4 6.6 22.6 7.7 4.1C 10 18.8 6.3 23.5 9.7 4.7Mean(SD) 10(0) NAa19.0(0.66) NA 6.4(0.21) NA 23.5(0.90) NA 8.7(1.0) NA 4.5(0.32) NA0.26 A 10 17.4 4.6 22.6 4.9 5.1B 10 16.8 5.7 23.8 3.3 7.0C 11 18.1 5.0 25.4 10.4 7.0Mean(SD) 10(0.58) 0.9783 17.5(0.70) 0.9960 5.1(0.55)b0.0489 23.9(1.4) 0.6849 6.2(3.7) 0.2244 6.4(1.1)b0.00920.64 A 10 18.7 4.2 22.6 6.0 3.8B 10 16.7 5.0 21.6 4.6 4.9C 10 20.0 4.1 24.9 8.8 4.9Mean(SD) 10(0) 1.0000 18.5(1.7) 0.9405 4.4(0.49)b0.0045 23.0(1.7) 0.9310 6.5(2.2) 0.2780 4.5(0.61) 0.81461.6 A 10 17.6 4.6 23.1 6.4 5.6B 10 20.7 4.0 25.2 9.6 5.0C 10 19.4 5.0 24.8 6.2 5.0Mean(SD) 10(0) 1.0000 19.2(1.6) 0.7409 4.5(0.50)b0.0064 24.4(1.1) 0.5017 7.4(1.9) 0.5152 5.2(0.33) 0.30434.0 A 10 16.9 5.4 23.4 5.3 6.6B 10 17.7 7.3 23.1 4.9 5.4C 10 19.9 5.3 25.7 6.5 6.2Mean(SD) 10(0) 1.0000 18.1(1.6) 0.9712 6.0(1.1) 0.5691 24.1(1.4) 0.6305 5.5(0.85) 0.1277 6.1(0.58)b0.024710 A 10 20.2 5.0 26.6 6.2 6.0B 10 19.7 4.9 25.4 5.9 5.7C 10 19.8 4.7 24.7 6.3 4.8Mean(SD) 10(0) 1.0000 19.9(0.23) 0.4440 4.8(0.17)b0.0214 25.6(1.0) 0.1180 6.1(0.20) 0.2122 5.5(0.60) 0.1418aNA = Not Applicable.bStatistically different compared to the control, based on Dunnett's Multiple Comparison Test.

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## Table 16. F 0 

## mysid total number of young in the first two broods, sex ratio and percentage of reproductively ac tive females during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol

.

Nominal

Concentration (μg/L) Replicate Total

Number of Young

in First

Two

Broods p-Value Sex Ratio

Percentage of Reproductively

Active Females p-Value Total

Number

of

Males Total

Number

of

Females Male:Female

Ratio pValue

Control A 14.9 19 12 1.6 100

B 14.3 16 11 1.5 100

C 13.7 32 14 2.3 86 Mean(SD) 14.3(0.6) NA a 1.8(0.45) NA 95(8.2) NA

0.26 A 9.4 34 12 2.8 100

B 7.7 16 14 1.1 86

C 12.4 44 15 2.9 100 Mean(SD) 9.9(2.4) b 0.0038 2.3(1.0) 0.9691 95(8.2) 0.8333

0.64 A 7.9 34 13 2.6 86

B 9.6 19 11 1.7 100

C 11.7 13 13 1.0 100 Mean(SD) 9.7(1.9) b 0.0032 1.8(0.81) 0.7369 95(8.2) 0.8333

1.6 A 11.0 13 16 0.81 100

B 10.9 36 13 2.6 100

C 10.3 15 16 0.94 100 Mean(SD) 10.7(0.4) b 0.0156 1.5(1.0) 0.6901 100(0) 0.9742

4.0 A 10.7 16 12 1.3 100

B 12.1 16 9 1.9 100

C 10.9 21 13 1.6 100 Mean(SD) 11.2(0.8) b 0.0340 1.6(0.28) 0.7315 100(0) 0.9742

10 A 8.7 19 9 2.1 86

B 10.7 18 11 1.6 100

C 9.4 14 14 1.1 86 Mean(SD) 9.6(1.0) b 0.0026 1.6(0.52) 0.7355 90(8.2) 0.4850

a NA = Not Applicable.

b Statistically different compared to the contro l, based on Dunnett's Multiple Comparison Test.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 283Table 17. F1mysid survival and growth (lengthand dry weight) during the mysid (Americamysis bahia) twogeneration test with4-tert-octylphenol.NominalConcentration(μg/L)ReplicateSurvival(%)p-ValueMean Total Body Length (mm)Day 0ap-ValueDay 7p-ValueDay 14 Day 27Malep-ValueFemalep-ValueMalep-ValueFemalep-ValueControl A 74 4.13 5.63 6.23 7.15 7.41B 86 4.58 6.12 6.38 6.95 7.66C 75 4.63 6.14 5.73 7.00 7.50Mean(SD) 78(7.0) NAb1.61(0.09) NA 4.45(0.28) NA 5.96(0.29) NA6.11(0.34) NA 7.03(0.11) NA 7.52(0.13) NA0.26 A 91 4.94 5.72 5.99 7.30 7.54B 75 5.23 6.46 7.04 7.08 7.31C 86 4.66 7.32 7.08 7.52 8.08Mean(SD) 84(8.2) 0.9826 1.58(0.07) 0.5962 4.94(0.29) 0.1107 6.50(0.80) 0.2994 6.70(0.62) 0.1443 7.30(0.22) 0.3263 7.64(0.40) 0.95050.64 A 81 4.61 5.84 6.10 7.08 7.53B 80 4.42 NA 6.37 6.95 7.62C 68 4.37 6.27 6.55 7.48 7.86Mean(SD) 76(7.0) 0.7253 1.59(0.06) 0.6555 4.47(0.13) 0.8084 6.06(0.30) 0.7819 6.34(0.23) 0.5559 7.17(0.28) 0.5886 7.67(0.17) 0.96371.6 A 78 5.09 6.08 6.55 6.81 7.34B 63 5.02 NA 6.76 7.03 7.44C 67 4.11 6.01 6.14 7.30 7.54Mean(SD) 69(8.2) 0.2677 1.61(0.11) 0.8630 4.74(0.54) 0.3458 6.04(0.05) 0.7898 6.48(0.31) 0.3596 7.05(0.24) 0.8145 7.44(0.10) 0.68554.0 A 87 4.91 6.86 7.25 7.47 7.85B 86 5.07 7.02 6.57 6.67 7.17C 86 4.62 6.76 6.51 7.42 7.62Mean(SD) 86(0.7) 0.9916 1.61(0.12) 0.8411 4.86(0.23) 0.1770 6.88(0.13) 0.0772 6.78(0.41) 0.1012 7.19(0.45) 0.5537 7.55(0.34) 0.864910 A 95 4.80 6.176.33 6.80 7.44B 87 4.51 5.72 6.55 7.03 7.31C 78 4.64 6.96 6.96 6.79 7.07Mean(SD) 86(8.5)0.9955 1.58(0.07) 0.5841 4.65(0.14) 0.5029 6.28(0.63)0.5291 6.61(0.32) 0.2162 6.87(0.14) 0.9653 7.27(0.18) 0.3272aMean values presented are based on measurement of nine replicates. Individual measurements for these replicates are presentedinAppendix 8.bNA = Not Applicable.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 284Table 17. Continued. F1mysid survival and growth (length and dry weight)during the mysid (Americamysisbahia) two-generation test with 4-tert-octylphenol.NominalConcentration(μg/L)ReplicateMean Dry Body Weight (mg)Day 0ap-Value Day 7 p-ValueDay 14 Day 27Male p-Value Female p-Value Male p-Value Femalep-ValueControl A 0.30 0.58 0.71 0.79 1.21B 0.24 0.45 0.52 0.81 1.16C 0.22 0.48 0.64 0.89 1.38Mean(SD) 0.023(0.0036) NAb0.25(0.04) NA 0.51(0.07) NA 0.62(0.09) NA 0.83(0.05) NA 1.25(0.11) NA0.26 A 0.30 0.46 0.72 0.88 1.20B 0.32 0.44 0.56 0.82 1.20C 0.25 0.45 0.67 0.89 1.22Mean(SD) 0.020(0.0043) 0.2820 0.29(0.03) 0.4094 0.45(0.01) 0.9763 0.65(0.08) 0.7091 0.86(0.04) 0.6827 1.21(0.01) 0.69520.64 A 0.22 0.45 0.57 0.78 1.17B 0.34 NA 0.71 0.89 1.20C 0.23 0.50 0.65 0.90 1.25Mean(SD) 0.020(0.0044) 0.2283 0.26(0.06) 0.7374 0.48(0.04) 0.9323 0.64(0.07) 0.7437 0.86(0.07) 0.7168 1.20(0.04) 0.69521.6 A 0.28 0.60 0.73 0.71 1.01B 0.27 NA 0.53 0.89 1.09C 0.21 0.44 0.61 0.86 1.18Mean(SD) 0.020(0.0033) 0.1721 0.25(0.04) 0.8333 0.52(0.11) 0.7719 0.63(0.10) 0.8333 0.82(0.09) 0.8675 1.09(0.09) 0.26574.0 A 0.21 0.50 0.63 0.83 1.15B 0.28 0.49 0.70 0.66 1.04C 0.21 0.48 0.59 1.02 1.37Mean(SD) 0.026(0.0077) 0.8690 0.23(0.04) 0.9472 0.49(0.01) 0.8986 0.64(0.06) 0.7602 0.84(0.18) 0.8076 1.19(0.17) 0.618510 A 0.25 0.65 0.82 0.92 1.29B 0.30 0.41 0.57 0.80 0.89C 0.29 0.64 0.64 0.95 1.34Mean(SD) 0.021(0.012) 0.2459 0.28(0.03) 0.5352 0.57(0.13) 0.4431 0.68(0.13) 0.5553 0.89(0.08)0.5347 1.17(0.25) 0.5649aMean values presented are based on measurement of nine replicates. Individual measurements for these replicates are presentedinAppendix 8.bNA = Not Applicable.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 285Table 18. F1mysid time to maturation, timeto first brood release, time tosecond brood release, offspringreleased per female and interbrood duration during the mysid (Americamysis bahia) two-generationtest with 4-tert-octylphenol.NominalConcentration(μg/L)ReplicateTime toMaturation(Days)pValueFirst Brood Second BroodInterbroodDuration(Days)pValueTime toRelease(Days)pValueOffspring/Femalep-ValueTime toRelease(Days)p-ValueOffspring/Femalep-ValueControl A 9 16.8 8.5 20.7 10.3 4.3B 9 15.6 6.2 23.0 5.8 7.0C 9 16.7 6.0 19.8 9.8 4.5Mean(SD) 9.0 (0) NAa16.3(0.64) NA 6.9(1.4) NA 21.1(1.7) NA 8.6(2.5) NA 5.3(1.5) NA0.26 A 9 17.1 6.3 22.2 9.2 4.7B 8 16.9 7.3 22.0 11.6 5.1C 8 20.0 11 25.0 12.4 5.0Mean(SD) 8.3 (0.58) 0.9536 18.0(1.7)0.1607 8.1(2.4) 0.9842 23.1(1.7) 0.1639 11(1.7) 0.1513 4.9(0.24) 0.65670.64 A 9 15.2 6.0 20.0 7.2 4.8B 13 15.4 5.4 21.4 8.0 6.0C 9 16.6 6.6 22.0 11.3 5.0Mean(SD) 10.3(1.3) 0.3253 15.7(0.80) 0.9602 6.0(0.59) 0.5267 21.1(1.0) 0.8422 8.8(2.2) 0.7860 5.3(0.63) 0.83331.6 A 6 17.0 4.4 21.3 9.0 6.3B 8 17.0 7.2 21.0 10.0 4.0C 10 16.4 5.6 21.6 10.6 5.0Mean(SD) 8.0 (1.2) 0.9782 16.8(0.30) 0.6614 5.7(1.4)0.4245 21.3(0.30) 0.7946 9.9(0.80) 0.4612 5.1(1.2) 0.75334.0 A 8 17.5 6.7 21.2 9.8 4.8B 9 16.3 4.9 21.2 9.3 4.8C 9 16.6 7.9 21.7 11.0 5.0Mean(SD) 8.7 (0.58) 0.9080 16.8(0.63) 0.6614 6.5(1.5) 0.7137 21.3(0.28) 0.7734 10(0.86) 0.4070 4.9(0.11) 0.614710 A 8 14.5 4.8 18.5 6.2 4.0B 9 18.0 4.8 22.3 7.8 4.5C 9 16.6 4.4 21.0 8.3 5.0Mean(SD) 8.7 (0.58)0.9080 16.4(1.8) 0.8333 4.7(0.24) 0.1289 20.6(1.9) 0.9420 7.4(1.1) 0.9759 4.5(0.50) 0.3801aNA = Not Applicable.

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## Table 19. F 1 

## mysid total number of young in the first two broods, sex ratio and percentage of reproductively ac tive females during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol

.

Nominal

Concentration (μg/L) Replicate Total

Number of Young

in First

Two

Broods pValue Sex Ratio

Percentage of Reproductively

Active Females pValue Total

Number

of

Males Total

Number

of

Females Male:Female

Ratio pValue

Control A 16.3 14 8 1.8 80

B 10.8 7 15 0.47 100

C 10.7 12 9 1.3 86 Mean(SD) 12.6(3.2) NA 1.2(0.65) NA a 89(10) NA

0.26 A 14.1 12 11 1.1 100

B 18.9 10 10 1.0 100

C 23.2 9 13 0.69 100 Mean(SD) 18.7(4.5) b 0.9998 0.93(0.21) 0.5172 100(0) 0.0804

0.64 A 13.2 11 23 0.48 100

B 13.4 5 15 0.33 100

C 11.4 10 9 1.1 100 Mean(SD) 12.7(1.1) 0.8416 0.64(0.41) 0.2426 100(0) 0.0905

1.6 A 9.8 7 13 0.54 83

B 15.2 6 11 0.55 100

C 13.1 12 9 1.3 100 Mean(SD) 12.7(2.7) 0.8457 0.81(0.46) 0.3893 94(9.6) 0.3696

4.0 A 14.8 10 11 0.91 100

B 12.9 11 10 1.1 100

C 17.3 11 11 1.0 100 Mean(SD) 15.0(2.2) 0.9834 1.0(0.10) 0.9104 100(0) 0.0689

10 A 11.0 15 9 1.7 100

B 11.0 8 13 0.62 100

C 7.8 11 10 1.1 83 Mean(SD) 9.9(1.8) 0.3602 1.1(0.53) 0.7236 94(9.6) 0.4000

a NA = Not Applicable.

b Statistically different compared to the contro l, based on Dunnett's Multiple Comparison Test.

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## Table 20. Summary of F 0 

## and F 1 

## endpoints, No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) values determined during the mysid ( Americamysis bahia ) two-generation test with 4-tert-octylphenol

.

F 0 Endpoints Measured Concentration (μg/L)

0.36 0.98 2.0 3.9 11

Survival Day 7 Dry Weight Day 14 Male Dry Weight ↑ ↑ Day 14 Female Dry Weight Termination (Day 27) Male Dry Weight Termination (Day 27) Female Dry Weight Day 7 Length ↑ ↑ ↑ ↑ Day 14 Male Length ↑ ↑ Day 14 Female Length ↑ Termination (Day 27) Male Length Termination (Day 27) Female Length

Time to Maturation

Time to 1 s t Brood Release

1 s t

Brood Offspring/Female ↓ ↓ ↓ ↓

Time to 2 nd Brood Release

2 nd Brood Offspring/Female

Interbrood Duration ↑ ↑ Total Offspring/Female ↓ ↓ ↓ ↓ ↓

Sex Ratio % Reproducing Females
↓ Endpoint determined to be sta tistically significantly reduced (p ≤ 0.05) compared to control ↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control

F 1 Endpoints Measured Concentration (μg/L)

0.36 0.98 2.0 3.9 11

Survival Day 0 Dry Weight Day 7 Dry Weight Day 14 Male Dry Weight Day 14 Female Dry Weight Termination (Day 27) Male Dry Weight Termination (Day 27) Female Dry Weight Day 0 Length Day 7 Length Day 14 Male Length Day 14 Female Length Termination (Day 27) Male Length Termination (Day 27) Female Length

Time to Maturation

Time to 1 s t Brood Release

1 s t

Brood Offspring/Female

Time to 2 nd Brood Release

2 nd Brood Offspring/Female

Interbrood Duration Total Offspring/Female ↑

Sex Ratio % Reproducing Females
↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control

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## Figure 37. Schematic of biological methods during the mysid ( Americamysis

bahia ) two-generation test with 4-tert-octylphenol

.

Growth Retention Chamber (G)

▪ Daily Observations: survival, mature, gravid

▪ Sampled for growth measurements on Day 0, 7 and 14

Reproduction Retention Chamber (R) & Brood Chamber (B)

▪ Daily Observations: survival, mature, gravid, # young

▪ Upon sexual maturity (e.g., day 13), 1 ♀ & 1 ♂ paired in Brood Chamber (B)

Replicate 1 Replicate 2 Replicate 3

Collect F1 and Repeat Exposure Exposure Dose

Exposure Dose

Replicate 1 Replicate 2 Replicate 3 G

R G

R G

R

B

R B B B B B B B

R B B B B B B B

R B B B B B B

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## Figure 38. Relationship between mean measured concentrations and the nominal treatment levels during the mysid ( Americamysis bahia ) two-generation test wi th 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 290



## Figure 39. Mean percent survival of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 291



## Figure 40. Day 7 mean total body length of F 0 

## mysids ( Americamysis bahia ) during the two-generation te st with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 292



## Figure 41. Day 7 mean dr y body weight of F 0 

## mysids ( Americamysis bahia ) during the two-generation te st with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 293



## Figure 42. Day 14 male and female mean total body length of F 0 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 294



## Figure 43. Day 14 male and female mean dry body weight of F 0 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 295



## Figure 44. Day 30 male and female mean total body length of F 0 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 296



## Figure 45. Day 30 male and female mean dry body weight of F 0 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 297



## Figure 46. Mean time to maturation of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 298



## Figure 47. Mean time to first brood release of F 0 

## mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 299



## Figure 48. F 0 

## Mean number of offspring/female produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 300



## Figure 49. Mean time to second brood release of F 0 

## mysids ( Americamysis

bahia ) during the two-generation te st with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 301



## Figure 50. F 0 

## Mean number of offspring/female produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 302



## Figure 51. F 0 

## Mean interbrood du ration for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 303



## Figure 52. F 0 

## Mean total number of offspring released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 304



## Figure 53. F 0 

## Mean male:female se x ratio for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 305



## Figure 54. F 0 

## Mean percentage of reproductive ly active females for mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 306



## Figure 55. Mean percent survival of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 307



## Figure 56. Day 0 mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-generation te st with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 308



## Figure 57. Day 0 mean dr y body weight of F 1 

## mysids ( Americamysis bahia ) during the two-generation te st with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 309



## Figure 58. Day 7 mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-generation te st with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 310



## Figure 59. Day 7 mean dr y body weight of F 1 

## mysids ( Americamysis bahia ) during the two-generation te st with 4-tert-octylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 311



## Figure 60. Day 14 male and female mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 312



## Figure 61. Day 14 male and female mean dry body weight of F 1 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 313



## Figure 62. Day 27 male and female mean total body length of F 1 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 314



## Figure 63. Day 27 male and female mean dry body weight of F 1 

## mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol

.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 315



## Figure 64. Mean time to maturation of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

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## Figure 65. Mean time to first brood release of F 1 

## mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

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## Figure 66. F 1 

## Mean number of offspring/female produced in the first brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 318



## Figure 67. Mean time to second brood release of F 1 

## mysids ( Americamysis

bahia ) during the two-generation te st with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 319



## Figure 68. F 1 

## Mean number of offspring/female produced in the second brood release of mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 320



## Figure 69. F 1 

## Mean interbrood du ration for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 321



## Figure 70. F 1 

## Mean total number of offspring released in the first two broods for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 322



## Figure 71. F 1 

## Mean male:female se x ratio for mysids ( Americamysis bahia ) during the two-generation test with 4-tert-octylphenol.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 323



## Figure 72. F 1 

## Mean percentage of reproductive ly active females for mysids ( Americamysis bahia ) during the two-generati on test with 4-tertoctylphenol.

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## APPENDIX 5 - QAPP

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## APPENDIX 6 - STUDY PLAN

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## APPENDIX 7 - ANALYTICAL METHODOLOGY - 4-TERTOCTYLPHENOL

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SUMMARY

Methodology was validated (11 to 12 January 201 2) to quantify the amount 4-tert-octylphenol

present in recovery samples prepared in 20 ppt filtered seawater. The recovery samples were

extracted once with hexane, taken to near-dryness under a gentle stream of nitrogen at room

temperature and reconstituted with 50:50 acetoni trile:purified reagent water. All recovery

samples were analyzed by automated inje ction by liquid chro matography with mass

spectrometry (LC/MS/MS).

The method was validated by fortification of 20 ppt filtered seawater with 4-tert-octylphenol at

concentrations of 0.100, 1.00 and 10.0 μg/L. Rec overies for the LC/MS/MS method analysis

averaged 103% ± 7.97%, with a limit of quan titation (LOQ) of 0.0397 μg/L. Defined limits for

acceptance of quality control sample performance in subsequent studies were set at 70 to 120%.

EXPERIMENTAL

Equipment

1. Instrument: LEAP HTS PAL autosampler, Agilent 1200 SL vacuum degasser, an Agilent SL 1200 binary pump, an Agilent 1200 SL column thermostat, an MDS Sciex/API 5000Q mass spectrometer equipped with a MDS Sciex 4000Q Turbo V Source APCI and Analyst 1.5 software for data acquisition

2. Balance: O’Haus Model G160 3. Laboratory equipment: syringes, volumetr ic flasks, disposable glass/plastic pipets, positive displacement pipets, autosampler vials, and amber glass bottles with Teflon®-lined caps Reagents

1. Acetonitrile: Burdick & Jackson, reagent grade 2. Hexane: Burdick & Jackson, reagent grade

3. Methanol: Burdick & Jackson, reagent grade 4. Reagent grade water: Prepared from a Millipore ® 

### MilliQ Direct 8 water purification system (meets AS TM Type II requirements)

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Test Substance

The test substance, 4-tert-octylphenol, was re ceived on 10 November 2011 from Battelle Marine

Science Laboratory, Sequim, Washington. The following information was provided:

Name: 4-tert-octylphenol Synonym: 4-(1,1,3,3-tetra methyl butyl) phenol Lot No.: 08118jhv

CAS No.: 140-66-9 Purity: 98.0% Expiration Date: November 2015

Upon receipt at Smithers Viscient, the test s ubstance (SMV No. 5305) was stored at room

temperature in the original contai ner in a dark ventilated cabinet. Concentrations were adjusted

for the purity of the test substance.

Internal Standard

The internal standard, 4-tert-octylphenol-ring 13 

### C6, was received on 5 January 2012 from SigmaAldrich, Inc., Allentown, Pennsylvania. The following information was provided:

Name: 4-tert-octylphenol -ring 13 

### C6

Batch No.: SZBA203X

CAS No.: 1173020-24-0 Purity: 99.3% (tested as 100%) Expiration Date: 22 July 2013

Upon receipt at Smithers Viscient, the internal standard (SMV No. 5380) was stored frozen in

the original container. Concentr ations were not adjusted for the purity of the internal standard.

PROCEDURES

Preparation of Stock Solutions

A 1.00 mg/mL primary stock solution was pr epared by placing 0.1020 g (0.1000 g as active

ingredient) of 4-tert-o ctylphenol in a 100-mL volumetric fl ask and bringing it to volume with

acetonitrile. A 10.0 mg/L secondary stock solu tion was prepared by placing 0.500 mL of the

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1.00 mg/mL primary stock solution in a 50.0-mL volum etric flask and bringing it to volume with

acetonitrile. The 10.0 mg/L secondary stock so lution was used to prepare the high-level

recovery samples. Two sub-stock solutions (0.100 and 1.00 mg/L) we re prepared by placing

0.100 and 1.00 mL, respectively, of the 10.0 mg/L sec ondary stock solutions in separate 10.0-mL

volumetric flasks and bringing each to volume with acetonitrile. The 0.100 and 1.00 mg/L substock solutions were used to prepare the low- an d mid-level recovery samp les, respectively.

An additional 1.00 mg/mL primary stock solution was prepared by placing 0.1021 g (0.1001 g as

active ingredient) of 4-tert-oct ylphenol in a 100-mL volumetric flask and bringing it to volume

with acetonitrile. A 10.0 mg/L secondary stock solution was pr epared by placing 0.500 mL of

the 1.00 mg/mL primary stock so lution into a 50.0-mL volumetri c flask and bringing it to

volume with acetonitrile. A 1.00 mg/L sub-stock solution was prepared by placing 1.00 mL of

the 10.0 mg/L secondary stock solution in a 10.0- mL volumetric flask an d bringing it to volume

with acetonitrile. The 1.00 mg /L sub-stock solution was used to prepare the calibration

standards.

Preparation of Internal Stock Solutions

A 1.00 mg/L internal standard stock solution was prepared by transferring 1.00 mL of internal

standard stock solution received (10 mg/L) to a 10.0-mL volumetric flask and bringing it to

volume with acetonitrile. This 1.00 mg/L stock solution was used to fortify the high-level

recovery samples and calibration standards. A 0.100 mg/L sub-stock solution was prepared by

bringing 0.200 mL of the internal standard stock solution to a final volume of 2.00 mL with

acetonitrile. This 0.100 mg/L sub-stock solution was used to fortify the low- and mid-level

recovery samples.

All primary and secondary stock so lutions were stored in a refrig erator in amber glass bottles

fitted with Teflon ® 

### -lined caps. All sub-stock solutions were prepared on the day of use and

discarded after use.

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Preparation of Calibration Standards

Calibration standards were prepar ed in 50:50 acetonitrile:purifi ed reagent water by fortifying

with the 1.00 mg/L sub-stoc k solution to yield concentr ations of 0.500, 1.00, 5.00, 15.0, 20.0 and

25.0 μg/L. Internal standard (IS) was also fortif ied in these standards at a concentration of

5.0 μg/L according to the table below.

Standard

ID Stock

Concentration (mg/L) Fortification

Volume

(mL) Internal Standard (IS)

Stock

Concentration (mg/L) IS Volume Spiked (mL) Final

Volume

(mL) Standard

Concentration (μg/L)

Std 1 1.00 0.0250 1.00 0.250 50.0 0.500

Std 2 1.00 0.0500 1.00 0.250 50.0 1.00

Std 3 1.00 0.0500 1.00 0.0500 10.0 5.00

Std 4 1.00 0.150 1.00 0.0500 10.0 15.0

Std 5 1.00 0.200 1.00 0.0500 10.0 20.0

Std 6 1.00 0.250 1.00 0.0500 10.0 25.0



### Sample Fortification and Preparation

A set of recovery samples was prepared by fortifying 20 ppt filtered seawater at nominal

concentrations of 0.100, 1.00 and 10.0 μg/L according to the table below. Internal standard was

also fortified into these sample s according to the table below. Each concentration level was

produced in triplicate. In additi on, three samples were left unfor tified to serve as controls and

were processed in a similar fashion as the low concentration recovery samples:

Sample

ID Stock

Concentration (mg/L) Fortification

Volume

(mL) Internal Standard (IS)

Stock

Concentration (mg/L) IS Volume Spiked (mL) Final

Volume

(mL) Sample

Concentration (μg/L)

Control NA a NA 0.100 0.125 30.0 0.00

Low 0.100 0.0300 0.100 0.125 30.0 0.100

Mid 1.00 0.0300 0.100 0.125 30.0 1.00 High 10.0 0.0200 1.00 0.0500 20.0 10.0

a NA = Not Applicable

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Extraction Procedure

All recovery samples and standard s were prepared in 45-mL glass vials with PTFE-lined caps.

Each sample was fortified in the glass vials a nd extracted once with hexane. The table below

lists the volume of water and hexane in each sample. The total volumes were chosen to

minimize headspace in the vials. The samples were shaken on a shaker table for 60 minutes at

150 rpm. A portion of the hexane extract (see table below) was th en transferred to disposable

glass vials and reduced to in cipient dryness (approximately 25 μL) under a gentle stream of

nitrogen at room temperature. An aliquot of 50:50 acetonitrile:purified reagent water was added

to each sample. The extracts were then vortexe d for approximately 30 seconds and sonicated for

approximately five minutes to mix well and aid in dissolution of the concentrated extract. An

aliquot of each extract was then quantitatively tran sferred to autosampler vials to be analyzed by

LC/MS/MS. The extraction procedure is described in the table below.

Sample

ID Nominal

Concentration (μg/L) Sample

Volume

(mL) Extraction

Solvent

Volume 1

(mL) Secondary

Volume

(mL) Final

Volume 2

(mL) Dilution

Factor

Control
A, B & C 0.00 30.0 5.00 4.00 2.00 0.0833

Low A, B & C 0.100 30.0 5.00 4.00 2.00 0.0833

Mid
A, B & C 1.00 30.0 5.00 4.00 2.00 0.0833

High
A, B & C 10.0 20.0 10.0 5.00 5.00 0.500

1- Extraction solvent: Hexane

2 -Final dilution solvent : 50:50 acetonitrile:purif ied reagent water



### ANALYSIS

Instrumental Conditions

The LC/MS/MS analysis was conducted utilizi ng the following instrumental conditions:

LC Parameters

Column: XBridge C18, 2.5 μm, 2.1 x 50 mm Mobile phases: A: 100% purified reagent water

B: 100% methanol

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Gradient: Time Solvent Solvent (min) A B

0.0 35.0 65.0

1.50 35.0 65.0

5.00 0.00 100.0

7.00 0.00 100.0

7.50 35.0 65.0

9.50 35.0 65.0

Flow Rate: 300 μL/min Injection Volume: 100 μL Retention Time: approximately 5 minutes

Divert Valve: LC/MS: 0.9 min, Waste: 7.5 min

MS Parameters Instrument: MDS Sciex API 5000Q mass spectrometer equipped with a MDS Sciex 4000Q Turbo V Source APCI Ionization Mode: Negative Q1/Q3 Mass: 205.10/133.0 amu (4-tert-octylphenol) 211.20/139.0 amu (internal standard) Dwell time: 500 milliseconds (4-tert-octylphenol) 250 milliseconds (internal standard) Source temperature: 300 oC Scan type: MRM

Preparation of Standard Curve

Two sets of calibration standards were analyzed with each recovery sample set; one set prior to

analysis of the recovery samples, and the sec ond set immediately following the analysis of the

recovery samples. Injecti on of recovery samples and calibration standards onto the

chromatographic system was performe d by programmed automated injection.

CALCULATIONS

A calibration curve was constructed by plotting the analyte concentration (μg/L) in the

calibration standards against the peak area of the calibration standards. The equation of the line

(equation 1) was algebraically ma nipulated to give equation 2. The concentration of the test

substance within each recovery sample was determ ined using the regression coefficients from the

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quadratic equation, the peak area of the recovery sample, and th e dilution factor. Equations 2

and 3 were then used to calculate measur ed concentrations and analytical results.

where:

y = detector response (peak area) for analyte a, b and c = regression constants DC (x) = detected concentration (μg/L) in the sample C = constant c minus the peak area; C = (c - y) DF = dilution factor (the fina l sample volume divided by the original sample volume) A = concentration of the analyt e in the original sample

The limit of quantitation (LOQ) was cal culated using the following equation:

CTRL INST INST



### DF x LOQ LOQ (5) 4aC b b LOQ (4) 2



###   

 

where:

Area LS 

### = mean detector response (peak area) of the low concentration calibration standard (two injections) a, b, c = regression constants C = regression constant ; C = (c - Area LS 

### ) LOQ INST 

### = limit of quantitation on the instrument

DF CTRL 

### = dilution factor of the control samples (smallest dilution factor used) LOQ = limit of quantitation reported for the analysis 

#   DF x DC A 3 2a 4aC 2 b b (x) DC (2) c bx 2
ax y (1)

  



 

 

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RESULTS AND DISCUSSION



### The mean recovery was 103% with a standard de viation of 7.97%. The limit of quantitation was

0.0397 μg/L. The LOQ will vary from one analysis interval to another, since it is dependent

upon the regression of the calibration standards, the peak area of the low standards, and dilution

factor of the controls. These parameters, wh ile relatively constant, do vary somewhat among

runs and produce small variations in the LOQ. Defined limits for acceptance of quality control

sample performance in subsequent studies were set at 70 to 120%.

Analytical results for the recovery of 4-tert -octylphenol from 20 ppt filtered seawater are

presented in Table 7A . Representative chromatograms fr om the analysis of a calibration

standard, recovery sample and a control sample are presented in Figure 7A through Figure 7C

, respectively. A typical regr ession analysis for 4-tert-o ctylphenol is presented in Figure 7D

.

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## Table 7A. Analytical results for the recovery of 4-tert-octylphenol from 20 ppt filtered seawater during the method validation study.

Sample

ID Fortified Concentration (μg/L) Dilution

Factor Analytical Result (μg/L) Percent

of Fortified (%)

Control A 0.00 0.0833 < 0.0397 a NA b

Control B 0.00 0.0833 < 0.0397 NA

Control C 0.00 0.0833 < 0.0397 NA

Low A 0.100 0.0833 0.112 112

Low B 0.100 0.0833 0.111 111

Low C 0.100 0.0833 0.116 116

Mid A 1.00 0.0833 1.00 100

Mid B 1.00 0.0833 0.988 98.8

Mid C 1.00 0.0833 0.982 98.2

High A 10.0 0.500 95.4 95.4 High B 10.0 0.500 95.6 95.6 High C 10.0 0.500 95.8 95.8

Mean: 103

SD: 7.97

RSD: 7.78

N: 9

a Concentrations expressed as less than values were be low the limit of quantitati on (LOQ). The LOQ for each analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls. The limit of quantitation for the method validation was 0.0397 μg/L.

b NA = Not Applicable.

Note: Results were calculated using the actual analy tical (unrounded) results and not the rounded values presented in this table.

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## Figure 7A. Representative chromatogram of a 5.00 μg/L calibration standard and internal standard during the method validation study with 4- tert-octylphenol.

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## Figure 7B. Representative chromatogram of a 1.00 μg/L recovery sample fortified with 4-tert-octylphenol and internal standard during the method validation study.

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## Figure 7C. Representative chromatogram of a control sample and internal standard during the method validation study with 4-tertoctylphenol.

Note: The peak present at the retention time of 4-tert-octylphenol (approximately 5.0 minutes) is below the LOQ for this analysis.

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## Figure 7D. A typical regression analysis for the calibration standards used to quantitate 4-tert-octylphenol in the recovery samples.

0.00000 0.50000 1.00000 1.50000 2.00000 2.50000 3.00000 3.50000 4.00000 4.50000 5.00000

0.00 5.00 10.00 15.00 20.00 25.00 30.00Detector Response



### Concentration 

## Polynomial Regression Analysis

Polynomial Regression Analysis

R 2 = 0.99990 y = 1.8702E-04x 2 + 1.6861E-01x + 1.0455E-02

Standard Ratio Standard IS

Concentration Response Response Response

μg/L ratio Area Area

0.500 0.09218 45575 494410

1.00 0.17390 87881 505340

5.00 0.86698 437060 504120

15.0 2.57068 1290200 501890

20.0 3.45550 1715900 496570

25.0 4.31262 2177400 504890

0.500 0.089484 48112 537660

1.00 0.17471 87575 501250

5.00 0.87863 465770 530110

15.0 2.58295 1337400 517780

20.0 3.44783 1850900 536830

25.0 4.38207 2316800 528700

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## APPENDIX 8 - INDIVIDUAL F 1 

## DAY 0 LENGTH AND WEIGHT

MEASUREMENTS

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Study Number: 13888.6111 Mysid Two Generation Exposure with 4-tert-octylphenol

F 1 Day 0 Measurements

Nominal

Concentration Set ID Sample ID Total Length (mm) Tin Tare Weight

(g) Tin Total Weight

(g) Mean Dry Weight Per

Mysid (mg)Control A 6A1 1.55

1.31155 1.31166 0.022 6A2 1.62

6A3 1.73

6A4 1.77

6A5 1.78

B 6B1 1.38

1.29204 1.29214 0.020 6B2 1.52

6B3 1.56

6B4 1.74

6B5 1.54

C 6C1 1.82

1.29894 1.29907 0.026 6C2 1.48

6C3 1.79

6C4 1.77

6C5 1.43

D 6D1 1.39

1.32636 1.32647 0.022 6D2 1.77

6D3 1.25

6D4 1.52

6D5 1.79

E 6E.1 1.73

1.30092 1.30103 0.022 6E.2 1.54

6E.3 1.64

6E.4 1.23

6E.5 1.70

F 6F1 1.44

1.30692 1.30707 0.030 6F2 1.60

6F3 1.59

6F4 1.51

6F5 1.46

G 6G1 1.48

1.32700 1.32710 0.020 6G2 1.59

6G3 1.52

6G4 1.63

6G5 1.34

H 6H1 1.49

1.31831 1.31840 0.018 6H2 1.53

6H3 1.56

6H4 1.58

6H5 2.04

I 6I1 1.76

1.33021 1.33033 0.024 6I2 1.69

6I3 2.01

6I4 1.69

6I5 1.77

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 420

Study Number: 13888.6111 Mysid Two Generation Exposure with 4-tert-octylphenol

F 1 Day 0 Measurements

Nominal

Concentration Set ID Sample ID Total Length (mm) Tin Tare Weight

(g) Tin Total Weight

(g) Mean Dry Weight Per

Mysid (mg)0.26 μg/L A 5A1 1.65

1.32232 1.32245 0.026 5A2 1.85

5A3 1.45

5A4 1.73

5A5 1.65

B 5B1 1.69

1.32602 1.32610 0.016 5B2 1.41

5B3 1.40

5B4 1.68

5B5 1.48

C 5C1 1.47

1.33056 1.33065 0.018 5C2 1.58

5C3 1.52

5C4 1.54

5C5 1.48

D 5D1 1.35

1.29579 1.29590 0.022 5D2 1.37

5D3 1.61

5D4 1.38

5D5 1.51

E 5E.1 1.46

1.30796 1.30809 0.026 5E.2 1.51

5E.3 1.67

5E.4 1.40

5E.5 1.75

F 5F1 1.55

1.28575 1.28584 0.018 5F2 1.63

5F3 1.55

5F4 1.75

5F5 1.66

G 5G1 1.52

1.28992 1.28999 0.014 5G2 1.66

5G3 1.79

5G4 1.63

5G5 1.54

H 5H1 1.54

1.28866 1.28876 0.020 5H2 1.59

5H3 1.70

5H4 1.69

5H5 1.60

I 5I1 1.63

1.27982 1.27990 0.016 5I2 1.64

5I3 1.65

5I4 1.49

5I5 1.74

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 421

Study Number: 13888.6111 Mysid Two Generation Exposure with 4-tert-octylphenol

F 1 Day 0 Measurements

Nominal

Concentration Set ID Sample ID Total Length (mm) Tin Tare Weight

(g) Tin Total Weight

(g) Mean Dry Weight Per

Mysid (mg)0.64 μg/L A 4A1 1.35

1.32882 1.32892 0.020 4A2 1.59

4A3 1.48

4A4 1.62

4A5 1.58

B 4B1 1.40

1.32727 1.32740 0.026 4B2 1.64

4B3 1.72

4B4 1.70

4B5 1.63

C 4C1 1.54

1.32869 1.32878 0.018 4C2 1.78

4C3 1.64

4C4 1.52

4C5 1.55

D 4D1 1.76

1.32982 1.32991 0.018 4D2 1.42

4D3 1.54

4D4 1.64

4D5 1.57

E 4E.1 1.52

1.32553 1.32559 0.012 4E.2 1.54

4E.3 1.74

4E.4 1.58

4E.5 1.57

F 4F1 1.68

1.32605 1.32615 0.020 4F2 1.65

4F3 1.63

4F4 1.43

4F5 1.71

G 4G1 1.53

1.32702 1.32710 0.016 4G2 1.66

4G3 1.70

4G4 1.73

4G5 1.68

H 4H1 1.56

1.33050 1.33060 0.020 4H2 1.48

4H3 1.59

4H4 1.29

4H5 1.44

I 4I1 1.55

1.28855 1.28868 0.026 4I2 1.50

4I3 1.74

4I4 1.62

4I5 1.60

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 422

Study Number: 13888.6111 Mysid Two Generation Exposure with 4-tert-octylphenol

F 1 Day 0 Measurements

Nominal

Concentration Set ID Sample ID Total Length (mm) Tin Tare Weight

(g) Tin Total Weight

(g) Mean Dry Weight Per

Mysid (mg)1.6 μg/L A 3A1 1.64

1.30703 1.30715 0.024 3A2 1.60

3A3 1.55

3A4 1.53

3A5 1.52

B 3B1 1.30

1.30983 1.30992 0.018 3B2 1.67

3B3 1.49

3B4 1.59

3B5 1.43

C 3C1 1.81

1.32740 1.32750 0.020 3C2 1.76

3C3 1.77

3C4 1.81

3C5 1.95

D 3D1 1.81

1.32714 1.32723 0.018 3D2 1.69

3D3 1.49

3D4 1.65

3D5 1.77

E 3E.1 1.48

1.32634 1.32647 0.026 3E.2 1.80

3E.3 1.39

3E.4 1.63

3E.5 1.60

F 3F1 1.63

1.32578 1.32589 0.022 3F2 1.79

3F3 1.61

3F4 1.56

3F5 1.61

G 3G1 1.83

1.33014 1.33023 0.018 3G2 1.65

3G3 1.71

3G4 1.61

3G5 1.72

H 3H1 1.53

1.33621 1.33629 0.016 3H2 1.44

3H3 1.45

3H4 1.65

3H5 1.56

I 3I1 1.44

1.31890 1.31899 0.018 3I2 1.36

3I3 1.59

3I4 1.63

3I5 1.37

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 423

Study Number: 13888.6111 Mysid Two Generation Exposure with 4-tert-octylphenol

F 1 Day 0 Measurements

Nominal

Concentration Set ID Sample ID Total Length (mm) Tin Tare Weight

(g) Tin Total Weight

(g) Mean Dry Weight Per

Mysid (mg)4.0 μg/L A 2A1 1.69

1.30424 1.30436 0.024 2A2 1.72

2A3 1.73

2A4 1.56

2A5 1.49

B 2B1 1.52

1.27310 1.27326 0.032 2B2 1.65

2B3 1.44

2B4 1.33

2B5 1.61

C 2C1 1.62

1.31482 1.31489 0.014 2C2 1.39

2C3 1.70

2C4 1.57

2C5 1.56

D 2D1 1.51

1.29238 1.29254 0.032 2D2 1.28

2D3 1.45

2D4 1.50

2D5 1.39

E 2E.1 1.81

1.29693 1.29712 0.038 2E.2 1.70

2E.3 1.88

2E.4 1.69

2E.5 1.46

F 2F1 1.92

1.30174 1.30187 0.026 2F2 1.84

2F3 1.75

2F4 1.94

2F5 1.72

G 2G1 1.61

1.32052 1.32065 0.026 2G2 1.60

2G3 1.57

2G4 1.66

2G5 1.73

H 2H1 1.59

1.30837 1.30848 0.022 2H2 1.58

2H3 1.53

2H4 1.62

2H5 1.54

I 2I1 1.56

1.29916 1.29924 0.016 2I2 1.50

2I3 1.50

2I4 1.67

2I5 1.67

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 424

Study Number: 13888.6111 Mysid Two Generation Exposure with 4-tert-octylphenol

F 1 Day 0 Measurements

Nominal

Concentration Set ID Sample ID Total Length (mm) Tin Tare Weight

(g) Tin Total Weight

(g) Mean Dry Weight Per

Mysid (mg)10 μg/L A 1A1 1.68

1.32531 1.32543 0.024 1A2 1.65

1A3 1.60

1A4 1.68

1A5 1.66

B 1B1 1.57

1.29274 1.29298 0.048 1B2 1.62

1B3 1.56

1B4 1.64

1B5 1.75

C 1C1 1.48

1.29257 1.29264 0.014 1C2 1.49

1C3 1.60

1C4 1.56

1C5 1.42

D 1D1 1.34

1.28413 1.28420 0.014 1D2 1.62

1D3 1.66

1D4 1.43

1D5 1.50

E 1E.1 1.32

1.28682 1.28688 0.012 1E.2 1.53

1E.3 1.74

1E.4 1.78

1E.5 1.74

F 1F1 1.69

1.31042 1.31047 0.010 1F2 1.82

1F3 1.45

1F4 1.43

1F5 1.53

G 1G1 1.52

1.30218 1.30230 0.024 1G2 1.48

1G3 1.72

1G4 1.55

1G5 1.79

H 1H1 1.56

1.28828 1.28839 0.022 1H2 1.42

1H3 1.38

1H4 1.34

1H5 1.61

I 1I1 1.55

1.28479 1.28488 0.018 1I2 1.65

1I3 1.61

1I4 1.81

1I5 1.65

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 425



## APPENDIX 9 - POST-PAIRING SURVIVAL DATA - 4-TERTOCTYLPHENOLRTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 426Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

ControlReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 19 20 955 20 20 1006 20 20 1007 20 20 1008 20 20 1009 19 1 20 9510 17 18 2 2 9511 17 18 2 2 9512 16 1 18 2 2 9013* 10 12 8 8 100 100 100 9014 9 11 9 9 100 100 100 9015 9 11 9 9 100 100 100 9016 9 11 9 9 100 100 100 9017 9 11 9 9 100 100 100 9018 9 11 9 9 100 100 100 9019 9 11 9 9 100 100 100 9020 9 11 9 9 100 100 100 9021 9 11 9 9 100 100 100 9022 9 11 9 9 100 100 100 9023 9 11 9 9 100 100 100 9024 9 11 8 1 9 100 89 94 8525 8 1 11 8 9 89 89 89 8026 8 11 8 9 89 89 89 8027 8 11 8 9 89 89 89 8028 8 11 8 9 89 89 89 8029 6 1 1 10 8 9 75 89 82 7430 5 1 10 8 9 63 89 76 68aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 427Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

ControlReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 19 1 20 955 19 20 956 19 20 957 19 20 958 19 20 959 17 2 20 8510 15 18 2 2 8511 14 17 3 3 8512 13 16 4 4 8513* 9 12 8 8 100 100 100 8514 9 12 8 8 100 100 100 8515 9 12 8 8 100 100 100 8516 9 12 8 8 100 100 100 8517 9 12 8 8 100 100 100 8518 9 12 8 8 100 100 100 8519 9 12 8 8 100 100 100 8520 8 1 12 8 8 89 100 94 8021 8 12 8 8 89 100 94 8022 8 12 8 8 89 100 94 8023 8 12 8 8 89 100 94 8024 7 1 12 8 8 78 100 88 7525 7 12 7 1 8 78 88 82 7026 7 12 7 8 78 88 82 7027 7 12 7 8 78 88 82 7028 7 12 6 1 7 78 86 81 6829 7 12 6 7 78 86 81 6830 7 12 6 7 78 86 81 68aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 428Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

ControlReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 16 4 20 805 16 20 806 16 20 807 16 20 808 16 20 809 16 20 8010 14 18 2 2 8011 14 18 2 2 8012 14 18 2 2 8013* 7 10 10 10 100 100 100 8514 7 10 10 10 100 100 100 8515 7 10 10 10 100 100 100 8516 7 10 10 10 100 100 100 8517 7 10 10 10 100 100 100 8518 7 10 10 10 100 100 100 8519 7 10 10 10 100 100 100 8520 7 10 10 10 100 100 100 8521 7 10 10 10 100 100 100 8522 7 10 10 10 100 100 100 8523 7 10 10 10 100 100 100 8524 7 10 10 10 100 100 100 8525 7 10 10 10 100 100 100 8526 7 10 10 10 100 100 100 8527 6 1 9 10 10 100 100 100 8428 6 9 10 10 100 100 100 8429 6 9 10 10 100 100 100 8430 5 1 8 10 10 100 100 100 83aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 429Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

0.26 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival% Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 20 20 10010 17 17 3 3 10011 16 16 4 4 10012 15 1 16 4 4 9513* 11 12 8 8 100 100 100 9514 11 12 8 8 100 100 100 9515 11 12 8 8 100 100 100 9516 11 12 8 8 100 100 100 9517 10 1 12 7 1 8 91 88 89 8518 10 12 7 8 91 88 89 8519 9 1 12 7 8 82 88 84 8020 9 12 7 8 82 88 84 8021 8 1 12 7 8 73 88 79 7522 8 12 7 8 73 88 79 7523 8 12 7 8 73 88 79 7524 8 12 7 8 73 88 79 7525 8 12 7 8 73 88 79 7526 8 12 7 8 73 88 79 7527 8 12 7 8 73 88 79 7528 7 1 11 7 8 70 88 78 7429 7 11 7 8 70 88 78 7430 7 11 7 8 70 88 78 74aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 430Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

0.26 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 19 1 20 953 19 20 954 18 1 20 905 18 20 906 18 20 907 18 20 908 18 20 909 18 20 9010 16 18 2 2 9011 15 17 3 3 9012 15 17 3 3 9013* 10 1 13 7 7 100 100 100 8514 9 12 8 8 100 100 100 8515 9 12 8 8 100 100 100 8516 9 12 8 8 100 100 100 8517 9 12 7 1 8 100 88 94 8018 7 1 11 8 9 88 89 88 7519 7 11 8 9 88 89 88 7520 6 1 10 8 9 86 89 88 7421 6 10 8 9 86 89 88 7422 6 10 8 9 86 89 88 7423 6 10 8 9 86 89 88 7424 5 1 9 8 9 83 89 87 7225 4 1 9 8 9 67 89 80 6726 4 9 7 1 9 67 78 73 6127 4 9 7 9 67 78 73 6128 3 1 8 6 1 9 60 67 64 5329 2 1 7 6 9 50 67 62 5030 2 7 6 9 50 67 62 50aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 431Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

0.26 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 18 1 20 903 18 20 904 18 20 905 18 20 906 18 20 907 18 20 908 18 20 909 18 20 9010 18 19 1 1 9511 17 18 2 2 9512 13 14 6 6 9513* 10 11 9 9 100 100 100 9514 9 10 10 10 100 100 100 9515 9 10 10 10 100 100 100 9516 9 10 10 10 100 100 100 9517 6 1 8 11 1 12 86 92 89 8518 6 8 11 12 86 92 89 8519 6 8 11 12 86 92 89 8520 6 8 10 1 11 86 91 89 8421 6 8 10 11 86 91 89 8422 6 8 10 11 86 91 89 8423 6 8 10 11 86 91 89 8424 5 7 11 12 83 92 89 8425 5 7 9 2 10 83 90 88 8226 5 7 9 10 83 90 88 8227 4 1 6 9 10 80 90 87 8128 4 6 8 1 9 80 89 86 8029 4 6 8 9 80 89 86 8030 3 1 6 6 2 7 60 86 75 69aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 432Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

0.64 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 19 20 953 19 20 954 19 20 955 19 20 956 19 20 957 19 20 958 19 20 959 19 20 9510 16 17 3 3 9511 16 17 3 3 9512 15 16 4 4 9513* 8 9 11 11 100 100 100 9514 8 9 11 11 100 100 100 9515 8 9 11 11 100 100 100 9516 8 9 11 11 100 100 100 9517 8 9 10 1 11 100 91 95 9018 8 9 10 11 100 91 95 9019 8 9 10 11 100 91 95 9020 7 1 9 10 11 88 91 89 8521 7 9 9 1 11 88 82 84 8022 7 9 9 11 88 82 84 8023 7 9 9 11 88 82 84 8024 7 9 9 11 88 82 84 8025 7 9 9 11 88 82 84 8026 7 9 9 11 88 82 84 8027 6 1 8 8 1 10 86 80 82 7828 6 8 8 10 86 80 82 7829 6 8 8 10 86 80 82 7830 6 8 8 10 86 80 82 78aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 433Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

0.64 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 18 1 20 909 18 20 9010 18 20 9011 16 18 2 2 9012 16 17 3 3 9513* 9 10 10 10 100 100 100 9514 10 11 9 9 100 100 100 9515 9 1 10 9 9 100 100 100 9516 9 10 9 9 100 100 100 9517 9 10 9 9 100 100 100 9518 9 10 8 1 9 100 89 94 8919 9 10 8 9 100 89 94 8920 9 10 8 9 100 89 94 8921 9 10 8 9 100 89 94 8922 9 10 8 9 100 89 94 8923 9 10 8 9 100 89 94 8924 9 10 8 9 100 89 94 8925 9 10 8 9 100 89 94 8926 9 10 8 9 100 89 94 8927 9 10 7 1 8 100 88 94 8928 8 1 9 7 8 100 88 94 8829 8 9 7 8 100 88 94 8830 7 1 8 7 8 100 88 93 88aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 434Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

0.64 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 18 1 20 903 17 1 20 854 17 20 855 17 20 856 17 20 857 17 20 858 15 2 20 759 15 20 7510 12 1 18 2 2 7011 11 1 18 2 2 6512 11 18 2 2 6513* 7 10 10 10 100 100 100 8514 7 10 10 10 100 100 100 8515 7 10 10 10 100 100 100 8516 7 10 10 10 100 100 100 8517 7 10 10 10 100 100 100 8518 7 10 10 10 100 100 100 8519 7 10 10 10 100 100 100 8520 7 10 10 10 100 100 100 8521 7 10 10 10 100 100 100 8522 7 10 10 10 100 100 100 8523 7 10 10 10 100 100 100 8524 7 10 10 10 100 100 100 8525 7 10 10 10 100 100 100 8526 7 10 10 10 100 100 100 8527 6 1 10 9 1 10 86 90 88 7528 6 10 8 1 10 86 80 82 7029 5 1 9 8 10 83 80 81 6830 5 9 8 10 83 80 81 68aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 435Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

1.6 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 18 1 20 903 18 20 904 17 1 20 855 17 20 856 17 20 857 17 20 858 17 20 859 16 20 8010 15 18 2 2 8511 15 18 2 2 8512 14 17 3 3 8513* 8 11 9 9 100 100 100 8514 8 11 9 9 100 100 100 8515 8 11 9 9 100 100 100 8516 8 11 9 9 100 100 100 8517 8 11 9 9 100 100 100 8518 8 11 9 9 100 100 100 8519 8 11 9 9 100 100 100 8520 8 11 9 9 100 100 100 8521 8 11 9 9 100 100 100 8522 8 11 9 9 100 100 100 8523 7 1 11 9 9 88 100 94 8024 7 11 9 9 88 100 94 8025 7 11 9 9 88 100 94 8026 7 11 9 9 88 100 94 8027 7 11 8 1 9 88 89 88 7528 7 11 8 9 88 89 88 7529 7 11 8 9 88 89 88 7530 7 11 8 9 88 89 88 75aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 436Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

1.6 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 20 20 10010 18 18 2 2 10011 18 18 2 2 10012 16 16 4 4 10013* 12 12 8 8 100 100 100 10014 11 11 9 9 100 100 100 10015 11 11 9 9 100 100 100 10016 11 11 9 9 100 100 100 10017 10 1 11 9 9 91 100 95 9518 10 11 9 9 91 100 95 9519 9 1 10 8 1 8 90 100 94 9420 9 10 8 8 90 100 94 9421 9 10 8 8 90 100 94 9422 8 1 9 8 8 89 100 94 9423 7 1 8 7 1 7 88 100 93 9324 5 2 8 7 7 63 100 80 8025 5 8 7 7 63 100 80 8026 5 8 7 7 63 100 80 8027 5 8 7 7 63 100 80 8028 5 8 6 1 7 63 86 73 7329 5 8 6 7 63 86 73 7330 5 8 6 7 63 86 73 73aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 437Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

1.6 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 19 20 953 19 20 954 19 20 955 19 20 956 18 1 20 907 18 20 908 18 20 909 18 20 9010 18 20 9011 16 18 2 2 9012 15 17 3 3 9013* 7 9 11 11 100 100 100 9014 7 9 11 11 100 100 100 9015 7 9 11 11 100 100 100 9016 7 9 11 11 100 100 100 9017 7 9 10 1 11 100 91 94 8518 7 9 9 1 11 100 82 89 8019 7 9 9 11 100 82 89 8020 6 1 9 9 11 86 82 83 7521 6 9 9 11 86 82 83 7522 6 9 8 1 11 86 73 78 7023 6 9 8 11 86 73 78 7024 6 9 8 11 86 73 78 7025 5 1 8 8 11 83 73 76 6826 5 8 8 11 83 73 76 6827 5 8 8 11 83 73 76 6828 5 8 7 1 11 83 64 71 6329 5 8 7 11 83 64 71 6330 5 8 7 11 83 64 71 63aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 438Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

4.0 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 19 1 20 952 19 20 953 19 20 954 19 20 955 19 20 956 19 20 957 19 20 958 19 20 959 19 20 9510 16 1 18 2 2 9011 15 17 3 3 9012 13 2 17 3 3 8013* 8 12 8 8 100 100 100 8014 8 12 8 8 100 100 100 8015 8 12 8 8 100 100 100 8016 8 12 8 8 100 100 100 8017 8 12 8 8 100 100 100 8018 7 1 12 8 8 88 100 94 7519 7 12 8 8 88 100 94 7520 7 12 8 8 88 100 94 7521 7 12 8 8 88 100 94 7522 7 12 8 8 88 100 94 7523 7 12 8 8 88 100 94 7524 7 12 8 8 88 100 94 7525 7 12 8 8 88 100 94 7526 7 12 8 8 88 100 94 7527 7 12 7 1 7 88 100 93 7428 7 12 7 7 88 100 93 7429 7 12 7 7 88 100 93 7430 7 12 7 7 88 100 93 74aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 439Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

4.0 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 17 3 20 852 16 1 20 803 16 20 804 16 20 805 16 20 806 16 20 807 16 20 808 15 1 20 759 15 20 7510 11 16 4 4 7511 13 18 2 2 7512 11 16 4 4 7513* 7 1 13 7 7 100 100 100 7014 7 13 7 7 100 100 100 7015 7 13 7 7 100 100 100 7016 7 13 7 7 100 100 100 7017 7 13 7 7 100 100 100 7018 7 13 7 7 100 100 100 7019 6 1 13 7 7 86 100 93 6520 6 13 7 7 86 100 93 6521 6 13 7 7 86 100 93 6522 6 13 7 7 86 100 93 6523 6 13 7 7 86 100 93 6524 6 13 7 7 86 100 93 6525 6 13 7 7 86 100 93 6526 6 13 7 7 86 100 93 6527 5 1 12 7 7 83 100 92 6328 5 12 7 7 83 100 92 6329 5 12 7 7 83 100 92 6330 5 12 7 7 83 100 92 63aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 440Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

4.0 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 20 20 1006 20 20 1007 20 20 1008 20 20 1009 20 20 10010 17 17 3 3 10011 18 18 2 2 10012 15 1 16 4 4 9513* 9 9 11 11 100 100 100 10014 8 8 12 12 100 100 100 10015 8 8 12 12 100 100 100 10016 8 8 12 12 100 100 100 10017 8 8 12 12 100 100 100 10018 8 8 12 12 100 100 100 10019 8 8 11 1 12 100 92 95 9520 8 8 11 12 100 92 95 9521 7 1 7 11 12 100 92 95 9522 7 7 11 12 100 92 95 9523 7 7 11 12 100 92 95 9524 7 7 11 12 100 92 95 9525 7 7 11 12 100 92 95 9526 7 7 11 12 100 92 95 9527 7 7 11 12 100 92 95 9528 7 7 11 12 100 92 95 9529 7 7 10 1 12 100 83 89 8930 7 7 9 1 11 100 82 89 89aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 441Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

10 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 20 20 1005 19 1 20 956 19 20 957 19 20 958 19 20 959 19 20 9510 16 1 18 2 2 9011 16 18 2 2 9012 16 18 2 2 9013* 10 12 8 8 100 100 100 9014 10 12 8 8 100 100 100 9015 9 1 11 8 8 100 100 100 8916 9 11 8 8 100 100 100 8917 8 10 9 9 100 100 100 8918 8 10 9 9 100 100 100 8919 8 10 8 1 9 100 89 94 8420 8 10 7 1 9 100 78 88 7921 8 10 7 9 100 78 88 7922 8 10 7 9 100 78 88 7923 8 10 7 9 100 78 88 7924 8 10 6 1 9 100 67 82 7425 8 10 5 1 8 100 63 81 7226 8 10 5 8 100 63 81 7227 8 10 5 8 100 63 81 7228 6 1 1 9 5 8 86 63 73 6529 5 1 8 5 8 83 63 71 6330 5 8 4 1 7 83 57 69 60aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 442Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

10 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 19 1 20 953 19 20 954 19 20 955 19 20 956 19 20 957 19 20 958 19 20 959 19 20 9510 16 1 18 2 2 9011 16 18 2 2 9012 14 1 17 3 3 8513* 10 13 7 7 100 100 100 8514 10 13 7 7 100 100 100 8515 10 13 7 7 100 100 100 8516 10 13 7 7 100 100 100 8517 10 13 7 7 100 100 100 8518 10 13 7 7 100 100 100 8519 9 1 12 7 7 100 100 100 8420 9 12 7 7 100 100 100 8421 8 1 11 7 7 100 100 100 8322 8 11 7 7 100 100 100 8323 7 1 10 7 7 100 100 100 8224 7 10 7 7 100 100 100 8225 6 1 10 7 7 86 100 93 7626 6 10 7 7 86 100 93 7627 6 10 7 7 86 100 93 7628 6 10 7 7 86 100 93 7629 6 10 7 7 86 100 93 7630 6 10 7 7 86 100 93 76aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 443Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF0 Reproduction Basket Survival

10 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRMa♂/Immatureb♀Dead/Missing NTRMa♀b♂/Immature♀% Survival % Survival0 20 20 1001 20 20 1002 20 20 1003 20 20 1004 19 1 20 955 17 2 20 856 17 20 857 17 20 858 17 20 859 17 20 8510 14 17 3 3 8511 14 17 3 3 8512 13 16 4 4 8513* 7 10 10 10 100 100 100 8514 7 10 10 10 100 100 100 8515 7 10 10 10 100 100 100 8516 7 10 10 10 100 100 100 8517 7 10 10 10 100 100 100 8518 6 1 10 9 1 10 86 90 88 7519 6 10 8 1 9 86 89 88 7420 6 10 8 9 86 89 88 7421 6 10 7 1 9 86 78 81 6822 6 10 7 9 86 78 81 6823 5 1 9 7 9 83 78 80 6724 4 1 9 7 9 67 78 73 6125 4 9 7 9 67 78 73 6126 4 9 6 1 8 67 75 71 5927 4 9 6 8 67 75 71 5928 4 9 6 8 67 75 71 5929 4 9 6 8 67 75 71 5930 4 9 6 8 67 75 71 59aNTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)bSurvival adjusted for non-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.eDay of Pairing. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gender.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 444Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

ControlReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 14 1 15 933 14 15 934 14 15 935 14 15 936 14 15 937 14 15 938 14 15 939 12 13 2 2 9310c11 12 3 3 9311ab11 12 3 3 100 100 100 9312 8 1 10 5 5 89 100 93 8713 8 10 5 5 89 100 93 8714 8 10 4 1 5 89 80 86 8015 8 10 4 5 89 80 86 8016 8 10 4 5 89 80 86 8017 8 10 4 5 89 80 86 8018 8 10 3 1 5 89 60 79 7319 6 2 8 3 5 86 60 75 6920 6 8 3 5 86 60 75 6921 6 8 3 5 86 60 75 6922 6 8 3 5 86 60 75 6923 6 8 3 5 86 60 75 6924 4 2 6 3 5 80 60 70 6425 3 1 5 3 5 75 60 67 6026 3 5 3 5 75 60 67 6027 3 5 3 5 75 60 67 60NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 445Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

ControlReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 11 11 4 4 10010c8 8 7 7 10011ab8 8 7 7 100 100 100 10012 5 5 10 10 100 100 100 10013 5 5 10 10 100 100 100 10014 5 5 10 10 100 100 100 10015 5 5 9 1 9 100 100 100 10016 5 5 9 9 100 100 100 10017 5 5 9 9 100 100 100 10018 5 5 9 9 100 100 100 10019 5 5 9 9 100 100 100 10020 5 5 9 9 100 100 100 10021 5 5 9 9 100 100 100 10022 5 5 7 1 1 8 100 88 92 9223 5 5 7 8 100 88 92 9224 5 5 7 8 100 88 92 9225 5 5 7 8 100 88 92 9226 5 5 7 8 100 88 92 9227 5 5 7 8 100 88 92 92NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 446Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

ControlReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 14 1 15 937 14 15 938 14 15 939 9 10 5 5 9310c9 10 4 1 5 100 100 100 8711ab8 1 9 7 8 100 100 100 8812 8 9 7 8 100 100 100 8813 8 9 7 8 100 100 100 8814 8 9 7 8 100 100 100 8815 7 1 9 7 8 88 100 93 8216 7 9 7 8 88 100 93 8217 7 9 7 8 88 100 93 8218 6 1 8 7 8 86 100 93 8119 6 8 7 8 86 100 93 8120 5 1 7 7 8 83 100 92 8021 5 7 6 1 7 83 100 92 7922 5 7 6 7 83 100 92 7923 5 7 6 7 83 100 92 7924 5 7 6 7 83 100 92 7925 5 7 6 7 83 100 92 7926 5 7 6 7 83 100 92 7927 5 7 6 7 83 100 92 79NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 447Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

0.26 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 14 1 15 932 14 15 933 14 15 934 14 15 935 14 15 936 14 15 937* 14 15 938 14 15 939 14 15 9310 9 10 5 5 9311 9 10 5 5 9312abc8 9 6 6 100 100 100 9313 7 8 7 7 100 100 100 9314 7 8 7 7 100 100 100 9315 7 8 7 7 100 100 100 9316 7 8 7 7 100 100 100 9317 7 8 7 7 100 100 100 9318 7 8 7 7 100 100 100 9319 7 8 7 7 100 100 100 9320 7 8 6 1 7 100 86 93 8721 7 8 5 1 6 100 83 92 8622 6 1 7 5 6 100 83 92 8523 6 7 5 6 100 83 92 8524 6 7 5 6 100 83 92 8525 6 7 5 6 100 83 92 8526 6 7 5 6 100 83 92 8527 6 7 5 6 100 83 92 85NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 448Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

0.26 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007* 14 15 938 14 15 939 14 15 9310 14 15 9311 14 15 9312abc7 1 7 7 7 100 100 100 10013 7 7 7 7 100 100 100 10014 7 7 7 7 100 100 100 10015 7 7 7 7 100 100 100 10016 7 7 7 7 100 100 100 10017 7 7 7 7 100 100 100 10018 7 7 7 7 100 100 100 10019 7 7 7 7 100 100 100 10020 7 7 7 7 100 100 100 10021 7 7 7 7 100 100 100 10022 7 7 6 1 6 100 100 100 10023 7 7 5 1 5 100 100 100 10024 6 1 6 4 1 4 100 100 100 10025 5 1 5 4 4 100 100 100 10026 5 5 3 1 3 100 100 100 10027 5 5 2 1 2 100 100 100 100NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 449Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

0.26 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007* 15 15 1008 15 15 1009 14 1 15 9310 9 10 5 5 9311 9 10 5 5 9312abc5 6 9 9 100 100 100 9313 5 6 9 9 100 100 100 9314 5 6 9 9 100 100 100 9315 5 6 9 9 100 100 100 9316 5 6 9 9 100 100 100 9317 5 6 9 9 100 100 100 9318 5 6 9 9 100 100 100 9319 5 6 9 9 100 100 100 9320 5 6 9 9 100 100 100 9321 4 1 6 9 9 80 100 93 8722 4 6 9 9 80 100 93 8723 4 6 9 9 80 100 93 8724 4 6 9 9 80 100 93 8725 4 6 9 9 80 100 93 8726 4 6 9 9 80 100 93 8727 3 1 5 8 1 8 75 100 92 85NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedb Day Rep B was pairedc Day Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 450Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

0.64 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 14 1 15 934 14 15 935 14 15 936 14 15 937 14 15 938 14 15 939 14 15 9310 14 15 9311c14 15 9312a6 1 8 7 7 8713 6 8 7 7 8714b6 8 7 7 100 100 100 8715 6 8 7 7 100 100 100 8716 6 8 7 7 100 100 100 8717 6 8 7 7 100 100 100 8718 6 8 7 7 100 100 100 8719 6 8 7 7 100 100 100 8720 6 8 7 7 100 100 100 8721 6 8 7 7 100 100 100 8722 6 8 7 7 100 100 100 8723 6 8 7 7 100 100 100 8724 4 2 6 7 7 100 100 100 8525 4 6 6 1 6 100 100 100 8326 4 6 6 6 100 100 100 8327 4 6 6 6 100 100 100 83NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 451Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

0.64 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 14 1 15 933 14 15 934 14 15 935 12 2 15 806 12 15 807 12 15 808 12 15 809 12 15 8010 12 15 8011c12 15 8012a12 15 8013 12 15 8014b5 8 7 7 100 100 100 8015 5 8 7 7 100 100 100 8016 5 8 7 7 100 100 100 8017 5 8 7 7 100 100 100 8018 4 1 7 7 7 100 100 100 7919 4 7 7 7 100 100 100 7920 4 7 7 7 100 100 100 7921 4 7 7 7 100 100 100 7922 4 7 7 7 100 100 100 7923 3 1 6 6 1 6 100 100 100 7524 3 6 6 6 100 100 100 7525 3 6 6 6 100 100 100 7526 3 6 6 6 100 100 100 7527 2 1 5 6 6 100 100 100 73NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 452Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

0.64 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 14 1 15 933 14 15 934 14 15 935 12 2 15 806 12 15 807 12 15 808 12 15 809 12 15 8010 8 11 4 4 8011c5 8 7 7 100 100 100 8012a8 11 7 7 100 100 100 8313 7 1 10 7 7 100 100 100 8214b7 10 7 7 100 100 100 8215 7 10 7 7 100 100 100 8216 7 10 7 7 100 100 100 8217 7 10 5 1 1 6 100 83 92 7518 7 10 5 6 100 83 92 7519 7 10 5 6 100 83 92 7520 7 10 5 6 100 83 92 7521 6 1 9 5 6 100 83 92 7322 6 9 4 1 5 100 80 91 7123 5 1 8 4 5 100 80 90 6924 5 8 4 5 100 80 90 6925 5 8 4 5 100 80 90 6926 5 8 4 5 100 80 90 6927 5 8 4 5 100 80 90 69NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 453Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

1.6 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 14 1 15 934 14 15 935 14 15 936 14 15 937 14 15 938 14 15 939 14 15 9310 10 1 12 3 3 8711c8 10 5 5 8712ab6 8 7 7 100 100 100 8713 5 7 8 8 100 100 100 8714 5 7 8 8 100 100 100 8715 5 7 8 8 100 100 100 8716 5 7 8 8 100 100 100 8717 5 7 8 8 100 100 100 8718 5 7 8 8 100 100 100 8719 5 7 8 8 100 100 100 8720 5 7 8 8 100 100 100 8721 5 7 8 8 100 100 100 8722 4 1 7 8 8 80 100 92 8023 4 7 8 8 80 100 92 8024 4 7 8 8 80 100 92 8025 4 7 8 8 80 100 92 8026 4 7 8 8 80 100 92 8027 3 1 6 8 8 75 100 92 79NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 454Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

1.6 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 14 1 15 932 14 15 933 14 15 934 13 1 15 875 12 1 15 806 12 15 807 12 15 808 12 15 809 12 15 8010 9 12 3 3 8011c6 9 6 6 8012ab3 1 7 8 8 100 100 100 7313 5 9 8 8 100 100 100 7614 5 9 8 8 100 100 100 7615 4 1 9 8 8 80 100 92 7116 4 9 8 8 80 100 92 7117 4 9 8 8 80 100 92 7118 4 9 8 8 80 100 92 7119 4 9 8 8 80 100 92 7120 4 9 8 8 80 100 92 7121 4 9 8 8 80 100 92 7122 4 9 8 8 80 100 92 7123 4 9 8 8 80 100 92 7124 4 9 8 8 80 100 92 7125 4 9 8 8 80 100 92 7126 4 9 8 8 80 100 92 7127 4 9 8 8 80 100 92 71NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 455Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

1.6 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 14 1 15 933 14 15 934 14 15 935 14 15 936 14 15 937 14 15 938 14 15 939 13 1 15 8710 10 12 3 3 8711c6 8 7 7 100 100 100 8712ab7 9 8 8 100 100 100 8813 6 1 9 8 8 86 100 93 8214 6 9 8 8 86 100 93 8215 6 9 8 8 86 100 93 8216 6 9 8 8 86 100 93 8217 6 9 8 8 86 100 93 8218 6 9 8 8 86 100 93 8219 6 9 8 8 86 100 93 8220 5 1 9 8 8 71 100 87 7621 3 1 1 8 7 1 7 50 100 77 6722 3 8 7 7 50 100 77 6723 3 8 7 7 50 100 77 6724 2 1 7 7 7 40 100 75 6425 2 7 7 7 40 100 75 6426 2 7 5 2 5 40 100 70 5827 2 7 4 1 4 40 100 67 55NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 456Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

4.0 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 14 1 15 934 14 15 935 14 15 936 14 15 937 14 15 938 14 15 939 14 15 9310 11 12 3 3 9311c6 7 8 8 9312 6 7 8 8 9313 6 7 8 8 9314ab6 7 8 8 100 100 100 9315 6 7 8 8 100 100 100 9316 6 7 8 8 100 100 100 9317 6 7 8 8 100 100 100 9318 6 7 8 8 100 100 100 9319 6 7 8 8 100 100 100 9320 6 7 8 8 100 100 100 9321 6 7 8 8 100 100 100 9322 6 7 8 8 100 100 100 9323 5 1 7 8 8 83 100 93 8724 5 7 8 8 83 100 93 8725 5 7 8 8 83 100 93 8726 5 7 8 8 83 100 93 8727 5 7 8 8 83 100 93 87NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 457Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

4.0 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010 11 11 4 4 10011c8 1 9 6 6 9312 8 9 6 6 9313 8 9 6 6 9314ab7 8 7 7 100 100 100 9315 7 8 7 7 100 100 100 9316 6 1 7 7 7 100 100 100 9317 5 1 6 7 7 100 100 100 9218 5 6 7 7 100 100 100 9219 5 6 6 1 6 100 100 100 9220 5 6 6 6 100 100 100 9221 5 6 6 6 100 100 100 9222 5 6 6 6 100 100 100 9223 5 6 6 6 100 100 100 9224 5 6 6 6 100 100 100 9225 5 6 6 6 100 100 100 9226 5 6 6 6 100 100 100 9227 5 6 6 6 100 100 100 92NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 458Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

4.0 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 14 15 932 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 14 1 15 938 14 15 939 11 12 3 3 9310 10 11 4 4 9311c7 8 7 7 100 100 100 9312 7 8 7 7 100 100 100 9313 7 8 7 7 100 100 100 9314ab7 8 7 7 100 100 100 9315 7 8 7 7 100 100 100 9316 7 8 7 7 100 100 100 9317 7 8 6 1 6 100 100 100 9318 7 8 6 6 100 100 100 9319 7 8 6 6 100 100 100 9320 7 8 6 6 100 100 100 9321 7 8 6 6 100 100 100 9322 7 8 6 6 100 100 100 9323 7 8 6 6 100 100 100 9324 7 8 6 6 100 100 100 9325 7 8 5 1 6 100 83 92 8626 7 8 5 6 100 83 92 8627 7 8 4 1 5 100 80 92 85NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 459Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

10 μg/LReplicate ATest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 12 12 3 3 1009 12 12 3 3 10010a9 9 6 6 100 100 100 10011c9 9 6 6 100 100 100 10012b9 9 6 6 100 100 100 10013 9 9 6 6 100 100 100 10014 9 9 6 6 100 100 100 10015 9 9 6 6 100 100 100 10016 9 9 6 6 100 100 100 10017 9 9 6 6 100 100 100 10018 8 1 8 6 6 100 100 100 10019 8 8 6 6 100 100 100 10020 7 1 7 6 6 100 100 100 10021 7 7 6 6 100 100 100 10022 7 7 6 6 100 100 100 10023 7 7 5 1 5 100 100 100 10024 7 7 5 5 100 100 100 10025 6 1 6 5 5 100 100 100 10026 6 6 4 1 4 100 100 100 10027 5 1 6 4 4 83 100 90 90NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 460Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

10 μg/LReplicate BTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 15 15 10010a15 15 10011c11 11 4 4 10012b5 5 10 10 100 100 100 10013 5 5 10 10 100 100 100 10014 5 5 10 10 100 100 100 10015 5 5 10 10 100 100 100 10016 5 5 10 10 100 100 100 10017 5 5 10 10 100 100 100 10018 5 5 10 10 100 100 100 10019 5 5 10 10 100 100 100 10020 5 5 10 10 100 100 100 10021 5 5 10 10 100 100 100 10022 5 5 10 10 100 100 100 10023 5 5 10 10 100 100 100 10024 5 5 10 10 100 100 100 10025 5 5 10 10 100 100 100 10026 4 1 4 10 10 100 100 100 10027 4 4 10 10 100 100 100 100NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026Smithers Viscient Study No. 13888.6111Page 461Study Number: 13888.6111Mysid Two Generation Exposure with 4-tert-octylphenolF1 Reproduction Basket Survival

10 μg/LReplicate CTest # Live # of♂♂Total # # Live # of♀♀Total # % Survival % Survival Post-Pairing Mean replicateDay♂/Immature Dead/Missing NTRM♂/Immature ^♀Dead/Missing NTRM♀^♂/Immature♀% Survival % Survival0 15 15 1001 15 15 1002 15 15 1003 15 15 1004 15 15 1005 15 15 1006 15 15 1007 15 15 1008 15 15 1009 14 14 1 1 10010a11 11 4 4 10011c9 9 6 6 100 100 100 10012b9 9 6 6 100 100 100 10013 8 1 9 6 6 89 100 93 9314 8 9 6 6 89 100 93 9315 8 9 5 1 5 89 100 93 9316 8 9 5 5 89 100 93 9317 8 9 5 5 89 100 93 9318 8 9 5 5 89 100 93 9319 8 9 5 5 89 100 93 9320 8 9 5 5 89 100 93 9321 7 1 8 5 5 88 100 92 9222 6 1 7 3 2 3 86 100 90 9023 6 7 3 3 86 100 90 9024 6 7 3 3 86 100 90 9025 6 7 3 3 86 100 90 9026 6 7 3 3 86 100 90 9027 5 1 6 3 3 83 100 89 89NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.)^Survival adjusted fornon-toxicant related mortalities (i.e. impingement, etc.)dFollowing test initiation until mysid pairing,counts are considered to be estimations.aDay Rep A was pairedbDay Rep B was pairedcDay Rep C was paired. Prior to pairing, mysid counts are estimated; follwing mysid pairing through test termination, mysid survival will also be determined by gender.* Mysid was recorded as missing in 0.26 μg/L Rep B on Day 7. 15 Mysids were observed on pairing day. It is assumed mysids were miscounted from day 8-11. Missing mysid will not be noted inspreadsheet.

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 462



## APPENDIX 10 - BRINE SHRI MP NAUPLII ANALYSIS

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 463

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 464

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 465

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 466

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 467

### RTI Project No. 0210114.030 EPA C ontract No. EP-W-06-026 Smithers Viscient Study No. 13888.6111 Page 468