Study Title 

Lindane and 4-Tert-octylphenol - Two-Generation 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 2 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIM 

No claim of confidentiality, on any 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 registered or previously registered pesticide is 

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

regarding disclosure to multinational entities under FIFRA 10(g). 

Company: 

Company Agent: Date: 

 (Typed Name) 

 (Title) (Signature) 
RTI Project No. 0210114.030 EPA Contract 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-octylphenol - 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 metals were conducted using standard U.S. EPA 

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

Smithers Viscient’s Standard Operating Procedures. Since the analyses were conducted 

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

SMITHERS VISCIENT 

________________________________________________ ________________________ 

Michael R. Lee Date 

Study Director 

RTI INTERNATIONAL 

________________________________________________ ________________________ 

Study Sponsor Date 

________________________________________________ ________________________ 

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

Smithers Viscient Study No. 13888.6109 Page 4 

QUALITY ASSURANCE STATEMENT 

The study conduct, raw data and report for "Lindane and 4-Tert-octylphenol - TwoGeneration Life-Cycle Toxicity 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 and laboratory standard operating procedures. 

Dates of study inspections, inspection type, and dates reported 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, F0 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, F1 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 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 5 

KEY STUDY PERSONNEL 

The following Smithers Viscient personnel were responsible 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 Director, Ecotoxicology 
RTI Project No. 0210114.030 EPA Contract 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 (F0) .............................................29

2.10.2.1.1 F0 Exposure Initiation ...............................................................29

2.10.2.1.2 F0 Growth Chambers .................................................................30

2.10.2.1.3 F0 Reproduction Chambers .......................................................31

2.10.2.2 Exposed First Filial Generation (F1) .........................................32

2.10.2.2.1 Initiation of F1 Generation .........................................................32

2.10.2.2.2 F1 Growth Chambers .................................................................32
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2.10.2.2.3 F1 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 F0 Generation .............................................................................................41

5.3.1.1 F0 Survival .................................................................................41

5.3.1.2 F0 Growth ..................................................................................41

5.3.1.2.1 Day 0 (Exposure Initiation) – F0 Total Body Length and 

Dry Body Weight ......................................................................42

5.3.1.2.2 Day 7 - F0 Total Body Length and Dry Body Weight ..............42

5.3.1.2.3 Day 14 - F0 Total Body Length and Dry Body Weight ............42

5.3.1.2.4 Day 27 (F0 Termination) - Total Body Length and Dry 

Body Weight .............................................................................43

5.3.1.3 F0 Time to Maturation ...............................................................44

5.3.1.4 F0 Time to First Brood Release .................................................44

5.3.1.5 F0 Number of Offspring in the First Brood ...............................45

5.3.1.6 F0 Time to Second Brood Release ............................................45

5.3.1.7 F0 Number of Offspring in the Second Brood ..........................45

5.3.1.8 F0 Interbrood Duration ..............................................................46

5.3.1.9 F0 Total Number of Offspring in First Two Broods .................46

5.3.1.10 F0 Sex Ratio (Male:Female) ......................................................46

5.3.1.11 F0 Percent of Reproductively Active Females ..........................46

5.3.2 F1 Generation .............................................................................................47

5.3.2.1 F1 Survival .................................................................................47

5.3.2.2 F1 Growth ..................................................................................47

5.3.2.2.1 Day 0 (Exposure Initiation) – F1 Total Body Length and 

Dry Body Weight ......................................................................47

5.3.2.2.2 Day 7 - F1 Total Body Length and Dry Body Weight ..............48

5.3.2.2.3 Day 14 - F1 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 F1 Time to Maturation ...............................................................50

5.3.2.4 F1 Time to First Brood Release .................................................50

5.3.2.5 F1 Number of Offspring in the First Brood ...............................51

5.3.2.6 F1 Time to Second Brood Release ............................................51

5.3.2.7 F1 Number of Offspring in the Second Brood ..........................51

5.3.2.8 F1 Interbrood Duration ..............................................................51

5.3.2.9 F1 Total Number of Offspring in First Two Broods .................52

5.3.2.10 F1 Sex Ratio (Male:Female) ......................................................52

5.3.2.11 F1 Percent of Reproductively Active Females ..........................52

6.0 CONCLUSIONS ..................................................................................................................53

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. F0 mysid survival and growth (length and dry weight) during the 

mysid (Americamysis bahia) two-generation test with lindane. ................60

Table 5. F0 mysid time to maturation, time to first brood release, time to 

second brood release, offspring released per female and interbrood 

duration during the mysid (Americamysis bahia) two-generation test 

with lindane. ...............................................................................................62

Table 6. F0 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. F1 mysid survival and growth (length and dry weight) during the 

mysid (Americamysis bahia) two-generation test with lindane. ................64

Table 8. F1 mysid time to maturation, time to first brood release, time to 

second brood release, offspring released per female and interbrood 

duration during the mysid (Americamysis bahia) two-generation test 

with lindane. ...............................................................................................66
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Table 9. F1 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 F0 and F1 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 F0 mysids (Americamysis bahia) during 

the two-generation test with lindane. .........................................................71

Figure 4. Day 7 mean total body length of F0 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................72

Figure 5. Day 7 mean dry body weight of F0 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................73

Figure 6. Day 14 male and female mean total body length of F0 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........74

Figure 7. Day 14 male and female mean dry body weight of F0 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........75

Figure 8. Day 27 male and female mean total body length of F0 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........76

Figure 9. Day 27 male and female mean dry body weight of F0 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........77

Figure 10. Mean time to maturation of F0 mysids (Americamysis bahia) during 

the two-generation test with lindane. .........................................................78

Figure 11. Mean time to first brood release of F0 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................79

Figure 12. F0 Mean number of offspring/female 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 F0 mysids (Americamysis 

bahia) during the two-generation test with lindane. ..................................81

Figure 14. F0 Mean number of offspring/female produced in the second brood 

release of mysids (Americamysis bahia) during the two-generation 

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

Figure 15. F0 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................83
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Figure 16. F0 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. F0 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................85

Figure 18. F0 Mean percentage of reproductively active females for mysids 

(Americamysis bahia) during the two-generation test with lindane. .........86

Figure 19. Mean percent survival of F1 mysids (Americamysis bahia) during 

the two-generation test with lindane. .........................................................87

Figure 20. Day 0 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................88

Figure 21. Day 0 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................89

Figure 22. Day 7 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................90

Figure 23. Day 7 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................91

Figure 24. Day 14 male and female mean total body length of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........92

Figure 25. Day 14 male and female mean dry body weight of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........93

Figure 26. Day 27 male and female mean total body length of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........94

Figure 27. Day 27 male and female mean dry body weight of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane. .........95

Figure 28. Mean time to maturation of F1 mysids (Americamysis bahia) during 

the two-generation test with lindane. .........................................................96

Figure 29. Mean time to first brood release of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. ..............................................97

Figure 30. F1 Mean number of offspring/female 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 F1 mysids (Americamysis 

bahia) during the two-generation test with lindane. ..................................99

Figure 32. F1 Mean number of offspring/female produced in the second brood 

release of mysids (Americamysis bahia) during the two-generation 

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

Figure 33. F1 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with lindane. ............................................101
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Figure 34. F1 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. F1 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with lindane. ............................................103

Figure 36. F1 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 quantification 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 samples during the method validation 

study. ........................................................................................................197

APPENDIX 4 - POST-PAIRING SURVIVAL 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 (F0) ...........................................248

8.10.3.1.1 F0 Exposure Initiation .............................................................248

8.10.3.1.2 F0 Growth Chambers ...............................................................249

8.10.3.1.3 F0 Reproduction Chambers .....................................................250

8.10.3.2 Exposed First Filial Generation (F1) .......................................251

8.10.3.2.1 Initiation of F1 Generation .......................................................251

8.10.3.2.2 F1 Growth Chambers ...............................................................252

8.10.3.2.3 F1 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 F0 Generation ...........................................................................................260

11.3.1.1 F0 Survival ...............................................................................260

11.3.1.2 F0 Growth ................................................................................260

11.3.1.2.1 Day 0 (Exposure Initiation) – F0 Total Body Length and 

Dry Body Weight ....................................................................260

11.3.1.2.2 Day 7 - F0 Total Body Length and Dry Body Weight ............261

11.3.1.2.3 Day 14 - F0 Total Body Length and Dry Body Weight ..........261

11.3.1.2.4 Day 30 (F0 Termination) - Total Body Length and Dry 

Body Weight ...........................................................................262

11.3.1.3 F0 Time to Maturation .............................................................263
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11.3.1.4 F0 Time to First Brood Release ...............................................263

11.3.1.5 F0 Number of Offspring in the First Brood .............................263

11.3.1.6 F0 Time to Second Brood Release ..........................................264

11.3.1.7 F0 Number of Offspring in the Second Brood ........................264

11.3.1.8 F0 Interbrood Duration ............................................................264

11.3.1.9 F0 Total Number of Offspring in First Two Broods ...............265

11.3.1.10 F0 Sex Ratio (Male:Female) ....................................................265

11.3.1.11 F0 Percent of Reproductively Active Females ........................265

11.3.2 F1 Generation ...........................................................................................266

11.3.2.1 F1 Survival ...............................................................................266

11.3.2.2 F1 Growth ................................................................................266

11.3.2.2.1 Day 0 (Exposure Initiation) – F1 Total Body Length and 

Dry Body Weight ....................................................................266

11.3.2.2.2 Day 7 - F1 Total Body Length and Dry Body Weight ............267

11.3.2.2.3 Day 14 - F1 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 F1 Time to Maturation .............................................................269

11.3.2.4 F1 Time to First Brood Release ...............................................269

11.3.2.5 F1 Number of Offspring in the First Brood .............................269

11.3.2.6 F1 Time to Second Brood Release ..........................................270

11.3.2.7 F1 Number of Offspring in the Second Brood ........................270

11.3.2.8 F1 Interbrood Duration ............................................................270

11.3.2.9 F1 Total Number of Offspring in First Two Broods ...............271

11.3.2.10 F1 Sex Ratio (Male:Female) ....................................................271

11.3.2.11 F1 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-octylphenol 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. F0 mysid survival and growth (length and dry weight) during the 

mysid (Americamysis bahia) two-generation test with 4-tertoctylphenol. ..............................................................................................279

Table 15. F0 mysid time to maturation, time to first brood release, time to 

second brood release, offspring released per female and interbrood 

duration during the mysid (Americamysis bahia) two-generation test 

with 4-tert-octylphenol. ............................................................................281

Table 16. F0 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 4-tert-octylphenol. ........282

Table 17. F1 mysid survival and growth (length and dry weight) during the 

mysid (Americamysis bahia) two-generation test with 4-tertoctylphenol. ..............................................................................................283

Table 18. F1 mysid time to maturation, time to first brood release, time to 

second brood release, offspring released per female and interbrood 

duration during the mysid (Americamysis bahia) two-generation test 

with 4-tert-octylphenol. ............................................................................285

Table 19. F1 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 4-tert-octylphenol. ........286

Table 20. Summary of F0 and F1 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. ............................................287

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

bahia) two-generation test with 4-tert-octylphenol. ................................288

Figure 38. Relationship between mean measured concentrations and the 

nominal treatment levels during the mysid (Americamysis bahia) 

two-generation test with 4-tert-octylphenol. ............................................289

Figure 39. Mean percent survival of F0 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol. ......................................290

Figure 40. Day 7 mean total body length of F0 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................291

Figure 41. Day 7 mean dry body weight of F0 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 F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................293

Figure 43. Day 14 male and female mean dry body weight of F0 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 F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................295

Figure 45. Day 30 male and female mean dry body weight of F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................296

Figure 46. Mean time to maturation of F0 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol. ......................................297

Figure 47. Mean time to first brood release of F0 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................298

Figure 48. F0 Mean number of offspring/female 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 F0 mysids (Americamysis 

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

Figure 50. F0 Mean number of offspring/female produced in the second brood 

release of mysids (Americamysis bahia) during the two-generation 

test with 4-tert-octylphenol. .....................................................................301

Figure 51. F0 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................302

Figure 52. F0 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. F0 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................304

Figure 54. F0 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 F1 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol. ......................................306

Figure 56. Day 0 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................307

Figure 57. Day 0 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................308

Figure 58. Day 7 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................309

Figure 59. Day 7 mean dry body weight of F1 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 F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................311

Figure 61. Day 14 male and female mean dry body weight of F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................312

Figure 62. Day 27 male and female mean total body length of F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................313

Figure 63. Day 27 male and female mean dry body weight of F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. ..............................................................................................314

Figure 64. Mean time to maturation of F1 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol. ......................................315

Figure 65. Mean time to first brood release of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................316

Figure 66. F1 Mean number of offspring/female 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 F1 mysids (Americamysis 

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

Figure 68. F1 Mean number of offspring/female produced in the second brood 

release of mysids (Americamysis bahia) during the two-generation 

test with 4-tert-octylphenol. .....................................................................319

Figure 69. F1 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................320

Figure 70. F1 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. F1 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. ..........................322

Figure 72. F1 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 validation 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 F1 DAY 0 LENGTH AND WEIGHT MEASUREMENTS .....418

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

APPENDIX 10 - BRINE SHRIMP 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 Toxicity Test with Mysids 

(Americamysis bahia) Inter-Laboratory Comparison, Smithers 

Viscient Protocol No.: 091310/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 (F0) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

 First Filial Generation (F1) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

ENDPOINTS AND 

RESULTS: For the F0 exposure, male and female mysid dry weight (day 14) 

were the most sensitive endpoints in the mysid two-generation 

exposure to lindane. F1 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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F0 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 1st Brood Release ↓ 

1st Brood Offspring/Female ↑ 

Time to 2nd Brood Release ↓ 

2nd Brood Offspring/Female 

Interbrood Duration 

Total Offspring/Female 

Sex Ratio 

% Reproducing Females 

↓ Endpoint determined to be statistically significantly reduced (p ≤ 0.05) compared to control 

↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control 

F1 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 1st Brood Release ↑ ↑ ↑ 

1st Brood Offspring/Female ↓ 

Time to 2nd Brood Release 

2nd Brood Offspring/Female ↓ 

Interbrood Duration 

Total Offspring/Female ↓ ↓ 

Sex Ratio 

% Reproducing Females 

↓ Endpoint determined to be statistically significantly reduced (p ≤ 0.05) compared to control 
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1.0 INTRODUCTION 

The U.S. Environmental Protection Agency's Endocrine 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 endocrine effects and provide dose response information needed for 

risk assessment. One of the longer term Tier 2 assays being developed is the mysid twogeneration test with Americamysis bahia. This test contrasts with existing mysid life-cycle 

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

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

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

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

Additionally, a method for testing crustaceans for effects of endocrine disrupting chemicals 

(EDCs) is relevant to assessing the adverse consequences of chemicals 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 mysid 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 conducted from 17 August to 4 October 2011 at 

Smithers Viscient (SMV), located in Wareham, Massachusetts. The original raw data, protocol, 
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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 procedures 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 exposure. 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-generation test is a relatively 

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

reproduction in two generations (F0 and F1). The two-generation test described in the guideline 

is an extension of existing standard practice 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 following information was provided: 

 Name: Lindane, 97% 

 Synonyms: Lindane; 1α,2α,3β,4α,5α,6β-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 substance (SMV No. 4841) was stored at room 

temperature in the original container 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 testing were obtained from SMV cultures. The 

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

located in Fort Collins, Colorado, and are refreshed 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 closed-loop, 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 characterized 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 initiation. 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 hours of light and 8 hours of darkness. 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 initiation. Young produced by these organisms were 

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

Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to 
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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 F0 and F1) were fed twice daily with brine shrimp (Artemia 

salina, SMV Lot No. 100209, supplied by INVE Aquaculture, 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 concentrated 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 nauplii/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 nauplii 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 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 quality since all 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 water during this study. 

Seawater was collected from the Cape Cod Canal 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 housing) and a polyvinyl chloride (PVC) pipe and was 

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

seawater was transferred to an 8500-L epoxy-coated concrete tank, where it was diluted to a 

salinity of 20 ± 3‰ with laboratory 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 

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 constant pressure through PVC pipe to a head tank, 

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

proportional diluter system. The seawater used for 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, Inc., 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 analyzed monthly for total organic carbon (TOC) 

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

August through October 2011. 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 below levels of concern 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 the 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 dilute, natural seawater. The stock was mixed overnight using a 

3⁄4 horsepower mixer with a stainless steel shaft 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 maintained in each vessel was approximately 21 L. 

The aquaria were randomly distributed 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 constructed of 10-cm glass Petri dishes to which a 15-cm high cylinder 

of nylon mesh screen (250-μm mesh) had been attached with 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 evaluation; these mysids were held in these retention chambers 

until sexual maturity was reached. The second chamber, the growth chamber, was used to rear 
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mysids which were used for collecting growth metrics; these mysids were held in the retention 

chamber until Day 14. 

2.8.3 Brood Chambers 

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

constructed of 6-cm glass Petri dishes to which 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 dilution 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 mixing 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 lindane. Three replicates were 

maintained for all treatments and the control. 

A flow-splitting chamber was used between the diluter 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 equally between test aquaria. 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 vessels at a rate of approximately six exposure 

vessel volume additions per 24 hours and an approximate 9-hour 90% replacement time. 
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The calibration of the diluter system was checked and recorded prior to exposure initiation. If 

there was any indication during the test that the diluter calibration 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 test, a 

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

inspection was performed at the beginning 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. The photoperiod 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 measured with a 

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

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‰ salinity) throughout the 

test. 

2.10 Exposure Conduct 

2.10.1 Exposure Initiation 

The exposure was not initiated until the diluter and test substance 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 initiation. The exposure was initiated when the retention chambers 
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(growth and reproduction) containing F0 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 (F0) 

2.10.2.1.1F0 Exposure Initiation 

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

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

through 14) or seven brood chambers (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 removed from the cultures; (2) mysids (≤ 22 hours old, postrelease) 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 distribution, 

mysids in each intermediate beaker were then transferred to a labeled retention 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 screen 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 additional 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 
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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 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 drying 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 digital photographs using the following software and 

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

software. 

2.10.2.1.2F0 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 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 sample) 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 the 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 preventing 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. Each mysid was then placed on pre-weighed 
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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 sample) and weighed to the nearest 0.01 mg on an 

analytical balance. 

2.10.2.1.3F0 Reproduction Chambers 

Mysids maintained within reproduction chambers were observed daily for survival, sex 

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

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

had developed marsupial pouches. At sexual maturity (designated by the 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 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 the reproductive phase of the exposure. Prior to the 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 until F0 termination, offspring were enumerated daily. Offspring 

from the F0 mysids (used to establish the F1 generation) were collected and transferred to growth 

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

originated. The F0 exposure was terminated six days after the median day of release of the 

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

At exposure termination, F0 mysids were removed, identified as male, female or immature, 

euthanized in ice-cold deionized water, blotted dry and photographed for total length 
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measurements. Each mysid was then placed on individual 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 

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

2.10.2.2 Exposed First Filial Generation (F1) 

2.10.2.2.1 Initiation of F1 Generation 

Between exposure days 18 and 24, groups of 15 < 24-hour old offspring (F1), from either the first 

(F1') or second (F1

"

) brood of the F0 mysids, were isolated in growth and reproduction chambers 

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

concentration, only 12 offspring were available for initiation of the growth chamber prior to F0

exposure termination. F1 offspring were exposed to the same test substance concentrations as 

their F0 parents. Mixing of young from replicate exposure tanks released on the same day across 

replicate chambers was encouraged to minimize reproduction between siblings. When F1 mysids 

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

growth chambers. 

In addition, five day 0 F1 mysids were collected from each F0 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 minimum of 30 minutes (to allow 

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

2.10.2.2.2F1 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, 
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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 sample) and weighed to the nearest 0.01 mg on an 

analytical balance. 

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

not used to complete reproductive 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 length 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 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. 

2.10.2.2.3F1 Reproduction Chambers 

Mysids maintained within reproduction chambers were observed daily for survival, sex 

determination and reproductive output. Between the respective days 9 to 13 for the F1

generation, developing embryos could be discerned within individual females at most treatment 

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

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

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

of seven pairs were isolated in brood chambers for each replicate tank (Figure 1). 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 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 the reproductive phase of the exposure. Prior to 
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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 F1 mysids were removed, enumerated, and 

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

was terminated seven days after the median day of release of the second brood in the F1 control 

tanks. 

At exposure termination, F1 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 individual 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 

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 replicates (e.g., replicate B sampled 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 second brood release in the F1 control vessels), each replicate vessel was 

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

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

Water samples were removed from a point approximately 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 
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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 prepared at each sampling interval at nominal 

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

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

solution samples and remained with the exposure solution samples throughout the analytical 

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

quality control maintained during the analysis of exposure solution samples. 

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

with micro electron capture detection (GC/μECD) based on methodology validated at Smithers 

Viscient. The method validation was conducted prior to the initiation of the 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 procedures used throughout the 

analysis of exposure solutions and QC samples during 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 control at exposure initiation (see Protocol Deviations). Exposure 

solution temperature was continuously monitored in one replicate of the control vessel 

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

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

(YSI) Model 30 conductivity/salinity meter. The dissolved oxygen concentration and daily 
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temperature were measured using a YSI Model 550A 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 recorded at exposure initiation and daily thereafter 

until termination of the F1 generation (day 48). Dead organisms were recorded and removed 

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

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

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

The primary biological observations made during the exposure were: 

Parental Generation (F0) 

 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 initiation to release of young 

in the first brood 

 Time to second brood release, number of days 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

First Filial Generation (F1) 

 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 initiation to release of young 

in the first brood 

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

of young in the second brood 
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 Interbrood duration 

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

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

 Sex ratio 

 Percentage of females that were reproductively active 

3.0 Statistical Methods 

3.1 Endpoints 

 Parental Generation (F0) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

 First Filial Generation (F1) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 
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3.2 Statistical Procedures

All statistical conclusions were made at the 95% level of certainty 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 certainty was applied. The following 

procedures were used: 

 Prior to statistical analysis, data representing endpoints obtained as proportions 

(e.g., percentage of females that are reproductively active, survival, etc.) were 

transformed using the arcsine square-root transformation.

 As a check on the assumption of homogeneity 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 distributed. Since Bartlett’s Test 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 distribution 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 (F0, 

F1) did not meet the assumptions of normality. Therefore these data were 

compared with the performance of the control data using Fisher's Exact Test with 

Bonferroni-Holm's Adjustment (U.S. EPA, 2002). 

 Significant treatment effects for all remaining endpoints were determined from 

either Dunnett's Multiple Comparison Test (U.S. EPA, 2002) or Bonferroni's 

Adjusted t-Test (U.S. EPA, 2002). 

CETISTM 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 used 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 F0 or F1 controls must produce young. 

 The average number of young produced per control 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 test were unaffected by the concentrations of 

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

mysids (Table 1). Daily measurements established dissolved 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 period 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 chamber and chemical cells) or exposure solutions 

throughout the exposure period. 
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During the in-life exposure, test solutions were sampled for concentrations 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 measured recoveries at each exposure 

concentration approximated the nominal doses and were generally consistent, with the following 

exceptions: 

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

were inconsistent between replicates. Therefore, all replicates 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. Therefore, 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 dose were similar; the anomalous measurements 

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

calculation of mean measured concentrations. 

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

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

and all remaining exposure concentrations approximated nominal, this measurement was 

believed to be erroneous. On day 11, all replicates 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 day 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 exposure 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 ranged from 76 to 130% of nominal. Based on mean 

measured concentrations of lindane, the treatment levels tested (and measured) were defined as 
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30, 100, 170, 380 and 1100 ng/L. The relationship between the nominal treatment levels and the 

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

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

fortified concentrations (20.0, 200 and 1250 ng/L) and 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 maintained 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 are spiking errors, handling errors and instrument errors. 

5.3 Biological Results 

5.3.1 F0 Generation 

5.3.1.1 F0 Survival 

Mean percent survival in the control for the F0 generation averaged 82% (Table 4 and Figure 3). 

Mean percent survival among F0 mysids exposed to the treatment levels tested averaged 79 to 

87%. Dunnett’s Multiple Comparison Test determined no significant differences in survival 

among mysids exposed to any of the treatment levels tested, compared to the control. 

5.3.1.2 F0 Growth 

Growth measurements were determined at three time points during the F0 exposure. Mysid dry 

body weight and mysid total length were measured at day 0, 7, 14 and 27 (see Protocol 

Deviations). 
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5.3.1.2.1 Day 0 (Exposure Initiation) – F0 Total Body Length and Dry Body 

Weight 

Day 0 mean total body length for the F0 generation was 1.84 mm and mean dry body weight for 

the F0 generation was 0.035 mg (Table 4). 

5.3.1.2.2 Day 7 - F0 Total Body Length and Dry Body Weight 

Day 7 mean total body length in the control for the F0 generation averaged 4.46 mm (Table 4 and 

Figure 4). Mean total body length among F0 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 exposed to any of the treatment levels tested, 

compared to the control. 

Day 7 mean dry body weight in the control for the F0 generation averaged 0.30 mg (Table 4 and 

Figure 5). Mean dry body weight among F0 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 among mysids exposed to any of the treatment levels tested, 

compared to the control. 

5.3.1.2.3 Day 14 - F0 Total Body Length and Dry Body Weight 

Day 14 mean male total body length in the control for the F0 generation averaged 6.25 mm 

(Table 4 and Figure 6). Mean total body length among F0 male mysids exposed to the treatment 

levels tested averaged 6.51 to 6.85 mm. Dunnett’s Multiple Comparison Test determined no 

significant differences in male 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 F0 generation averaged 6.34 mm 

(Table 4 and Figure 6). Mean total body length among F0 female mysids exposed to the 32, 80, 
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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 Test determined a significant difference (p ≤ 0.05) 

in female total body length among mysids exposed 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 F0 generation averaged 0.58 mg 

(Table 4 and Figure 7). Mean dry body weight among F0 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 Test determined a significant difference (p ≤ 0.05) 

in male dry body weight among mysids exposed 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 F0 generation averaged 0.70 mg 

(Table 4 and Figure 7). Mean dry body weight among F0 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 Test determined a significant difference (p ≤ 0.05) 

in female dry body weight among mysids exposed to the 200, 500 and 1250 ng/L nominal 

treatment levels compared to the control. 

5.3.1.2.4 Day 27 (F0 Termination) - Total Body Length and Dry Body Weight 

Day 27 mean male total body length in the control for the F0 generation averaged 7.32 mm 

(Table 4 and Figure 8). Mean total body length among F0 male mysids exposed to the treatment 

levels tested averaged 6.96 to 7.35 mm. Dunnett’s Multiple Comparison Test determined no 

significant differences in male total body length among mysids exposed to any of the treatment 

levels tested, compared to the control. 

Day 27 mean female total body length in the control for the F0 generation averaged 7.51 mm 

(Table 4 and Figure 8). Mean total body length among F0 female mysids exposed to the 
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treatment levels tested averaged 7.60 to 7.86 mm. Dunnett’s Multiple Comparison Test 

determined no significant differences in female total body length among mysids exposed to any 

of the treatment levels tested, compared to the control. 

Day 27 mean male dry body weight in the control for the F0 generation averaged 0.96 mg 

(Table 4 and Figure 9). Mean dry body weight among F0 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-Test determined a significant difference (p ≤ 0.05) in male 

dry body weight among mysids exposed to the 1250 ng/L nominal treatment level compared to 

the control. 

Day 27 mean female dry body weight in the control for the F0 generation averaged 1.30 mg 

(Table 4 and Figure 9). Mean dry body weight among F0 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 female dry body weight among mysids exposed to any 

of the treatment levels tested, compared to the control. 

5.3.1.3 F0 Time to Maturation 

Mean time to maturation for the F0 control mysids averaged 10.3 days (Table 5 and Figure 10). 

Mean time to maturation among mysids exposed to the treatment levels averaged 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 levels tested, compared to the control. 

5.3.1.4 F0 Time to First Brood Release 

Mean time to first brood release for the F0 control mysids averaged 15.8 days (Table 5 and 

Figure 11). Mean time to first brood release for the F0 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 Test determined a significant difference (p ≤ 0.05) 
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in time to first brood release among mysids exposed to the 500 ng/L nominal treatment level 

compared to the control. 

5.3.1.5 F0 Number of Offspring in the First Brood 

The mean total number produced in the first brood from the F0 control mysids averaged 

5.5 offspring/female (Table 5 and Figure 12). The mean total number produced in the first brood 

from the F0 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/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 500 ng/L treatment level compared to the control. 

5.3.1.6 F0 Time to Second Brood Release 

Mean time to second brood release for the F0 control mysids averaged 21.3 days (Table 5 and 

Figure 13). Mean time to second brood release for the F0 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 Test determined a significant difference (p ≤ 0.05) 

in time to second brood release among mysids exposed to the 500 ng/L nominal treatment level 

compared to the control. 

5.3.1.7 F0 Number of Offspring in the Second Brood 

The mean total number of offspring produced in the second brood from the F0 control mysids 

averaged 8.8 offspring/female (Table 5 and Figure 14). The mean total number produced in the 

second brood from the F0 mysids exposed to the treatment levels averaged 8.6 to 

10.7 offspring/female. Dunnett’s Multiple Comparison Test determined no significant 

differences in total number of offspring produced in the second brood among mysids exposed to 

any of the treatment levels tested, compared to the control. 
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5.3.1.8 F0 Interbrood Duration 

Mean F0 interbrood duration for the control 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 F0 Total Number of Offspring in First Two Broods 

The total mean number of offspring released in the first two broods for F0 control mysids 

averaged 14.4 offspring/female (Table 6 and Figure 16). The total mean number released in the 

first two broods for F0 mysids exposed to the treatment levels tested averaged 15.0 to 

18.8 offspring/female. Dunnett’s Multiple Comparison Test determined no significant 

differences in number of offspring released among mysids exposed to any of the treatment levels 

tested, compared to the control. 

5.3.1.10 F0 Sex Ratio (Male:Female) 

The F0 male to female sex ratio in the control averaged 1.3 (Table 6 and Figure 17). The F0 male 

to female sex ratio among mysids exposed to the 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 levels tested, compared to the control. 

5.3.1.11 F0 Percent of Reproductively Active Females 

The mean percent of reproducing females in the F0 control averaged 100% (Table 6 and 

Figure 18). Mean percent of reproducing F0 females among mysids exposed to the treatment 

levels tested ranged from 95 to 100%. Dunnett’s Multiple Comparison Test determined no 
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Smithers Viscient Study No. 13888.6109 Page 47 

significant differences in mean percent of reproducing F0 females among mysids exposed to any 

of the treatment levels tested, compared to the control. 

5.3.2 F1 Generation 

5.3.2.1 F1 Survival 

Mean percent survival in the control for the F1 generation averaged 85% (Table 7 and Figure 19). 

Mean percent survival among F1 mysids exposed to the treatment levels tested ranged from 70 to 

92%. Dunnett’s Multiple Comparison Test determined no significant differences in survival 

among mysids exposed to any of the treatment levels tested, compared to the control. 

5.3.2.2 F1 Growth 

Growth measurements were determined at four time points during the F1 exposure. Mysid dry 

body weight and mysid total length were measured at the respective day 0, 7, 14 and 27 for the 

F1 generation. 

5.3.2.2.1 Day 0 (Exposure Initiation) – F1 Total Body Length and Dry Body Weight 

Day 0 mean total body length in the control for the F1 generation averaged 1.63 mm (Table 7 and 

Figure 20). Mean total body length among F1 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 exposed to any of the treatment levels tested, 

compared to the control. 

Day 0 mean dry body weight in the control for the F1 generation averaged 0.029 mg (Table 7 and 

Figure 21). Mean dry body weight among F1 mysids exposed to the treatment levels tested 

averaged 0.025 to 0.033 mg. Dunnett’s Multiple Comparison Test determined no significant 
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Smithers Viscient Study No. 13888.6109 Page 48 

differences in dry body weight among mysids exposed to any of the treatment levels tested, 

compared to the control. 

5.3.2.2.2 Day 7 - F1 Total Body Length and Dry Body Weight 

Day 7 mean total body length in the control for the F1 generation averaged 4.72 mm (Table 7 and 

Figure 22). Mean total body length among F1 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 exposed to any of the treatment levels tested, 

compared to the control. 

Day 7 mean dry body weight in the control for the F1 generation averaged 0.38 mg (Table 7 and 

Figure 23). Mean dry body weight among F1 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 among mysids exposed to any of the treatment levels tested, 

compared to the control. 

5.3.2.2.3 Day 14 - F1 Total Body Length and Dry Body Weight 

Day 14 mean male total body length in the control for the F1 generation averaged 6.32 mm 

(Table 7 and Figure 24). Mean total body length among F1 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 Test determined a significant difference (p ≤ 0.05) 

among mysids exposed to the 1250 ng/L nominal treatment level compared to the control. 

Day 14 mean female total body length in the control for the F1 generation averaged 6.46 mm 

(Table 7 and Figure 24). Mean total body length among F1 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 female total body length among mysids exposed to any 

of the treatment levels tested, compared to the control. 
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Smithers Viscient Study No. 13888.6109 Page 49 

Day 14 mean male dry body weight in the control for the F1 generation averaged 0.72 mg 

(Table 7 and Figure 25). Mean dry body weight among F1 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 Test 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 F1 generation averaged 0.93 mg 

(Table 7 and Figure 25). Mean dry body weight among F1 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 female 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 length in the control for the F1 generation averaged 7.30 mm 

(Table 7 and Figure 26). Mean total body length among F1 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 Test determined a significant difference (p ≤ 0.05) 

in male total body length among mysids exposed to the 1250 ng/L nominal treatment level 

compared to the control. 

Day 27 mean female total body length in the control for the F1 generation averaged 7.52 mm 

(Table 7 and Figure 26). Mean total body length among F1 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 Test determined a significant difference (p ≤ 0.05) 

in female total body length among mysids exposed to the 500 ng/L nominal treatment level 

compared to the control. 
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Smithers Viscient Study No. 13888.6109 Page 50 

Day 27 mean male dry body weight in the control for the F1 generation averaged 0.86 mg 

(Table 7 and Figure 27). Mean dry body weight among F1 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 Test determined a significant difference (p ≤ 0.05) 

in male dry body weight among mysids exposed to the 1250 ng/L nominal treatment level 

compared to the control. 

Day 27 mean female dry body weight in the control for the F1 generation averaged 1.25 mg 

(Table 7 and Figure 27). Mean dry body weight among F1 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 female dry body weight among mysids exposed to any 

of the treatment levels tested, compared to the control. 

5.3.2.3 F1 Time to Maturation 

Mean time to maturation for the F1 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 determined no significant differences in time to 

maturation among mysids exposed to any of the treatment levels tested, compared to the control. 

5.3.2.4 F1 Time to First Brood Release 

Mean time to first brood release for the F1 control mysids averaged 14.9 days (Table 8 and 

Figure 29). Mean time to first brood release for the F1 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. 
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5.3.2.5 F1 Number of Offspring in the First Brood 

The mean total number produced in the first brood from the F1 control mysids averaged 

6.6 offspring/female (Table 8 and Figure 30). The mean total number produced in the first brood 

from the F1 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/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 compared to the control. 

5.3.2.6 F1 Time to Second Brood Release 

Mean time to second brood release for the F1 control mysids averaged 20.0 days (Table 8 and 

Figure 31). Mean time to second brood release for the F1 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 F1 Number of Offspring in the Second Brood 

The mean total number produced in the second brood from the F1 control mysids averaged 

9.6 offspring/female (Table 8 and Figure 32). The mean total number produced in the second 

brood from the F1 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 compared to the control. 

5.3.2.8 F1 Interbrood Duration 

Mean F1 interbrood duration for the control 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 
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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 F1 Total Number of Offspring in First Two Broods 

The total mean number released in the first two broods for F1 control mysids averaged 

16.0 offspring/female (Table 9 and Figure 34). The total mean number released in the first two 

broods for F1 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 F1 Sex Ratio (Male:Female) 

The F1 male to female sex ratio in the control averaged 1.3 (Table 9 and Figure 35). The F1 male 

to female sex ratio among mysids exposed to the 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 levels tested, compared to the control. 

5.3.2.11 F1 Percent of Reproductively Active Females 

The mean percent of reproducing females in the F1 control averaged 100% (Table 9 and 

Figure 36). Mean percent of reproducing F1 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 percent of reproducing females among mysids exposed 

to any of the treatment levels tested, compared to the control. 
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Smithers Viscient Study No. 13888.6109 Page 53 

6.0 CONCLUSIONS 

For the F0 exposure, male and female mysid dry weight (day 14) were the most sensitive 

endpoints in the mysid two-generation exposure to lindane. Lindane was found to significantly 

increase F0 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 first brood release at 500 ng/L. 

Significant reductions (p ≤ 0.05) were observed in male termination (day 27) dry weight at 

1250 ng/L; and time to first and second brood release at 500 ng/L. 

F1 generation survival, day 27 male dry weight and total body length were the most sensitive 

endpoints in the mysid two-generation exposure to lindane. Lindane was found to significantly 

increase F1 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 male 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 produced in the first and second brood release at 

200 ng/L; and total offspring per female during first and second brood release at 80 and 

200 ng/L. 

Table 10 summarizes the F0 and F1 endpoints and the statistically significant differences from the 

controls determined during this study. 
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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 alternating replicates daily thereafter. For this study, at exposure 

initiation, dissolved oxygen and temperature 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/control vessels using a common splitter; the F0

control animals exceeded control acceptability criteria, 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 that 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 measurements 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. Therefore, this deviation did not 

have a negative impact on the results 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 range for the remainder of the study. Since 

temperatures maintained within this study are 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 F0 exposure will continue until seven days after the median 

day of release of the second brood in the control treatments. For this study, the F0

exposure was terminated six days after the median day of release of the second brood 

release in the control treatments. 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 for all concentrations 

released two broods, this deviation did not have a negative impact on the results or 

interpretation of the study. 
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Smithers Viscient Study No. 13888.6109 Page 55 

REFERENCES 

ASTM, 2002. Standard practice for conducting acute toxicity test with fishes, 

macroinvertebrates, and amphibians. Standard E729-96. American Society for Testing 

and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428. 

ASTM, 2008. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide 

for Conducting Life-Cycle Toxicity Tests with Mysids. ASTM Designation E 1191-039 

(re-approved 2008). American Society for Testing and Materials, 100 Barr Harbor Drive, 

West Conshohocken, Pennsylvania. 

Ives, Michael, 2009. CETIS, Comprehensive Environmental Toxicity Information SystemTM. 

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 temperature 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-specific 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 simplified dosing apparatus for fish toxicological 

studies. Water Research. 1: 21-29. 

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

an estuarine species suitable for life-cycle toxicity tests to determine the effects of a 

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

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

109-116. 

Nimmo DR, Hamaker TL and Sommers CA., 1978. Entire life cycle toxicity test using mysids 

(Mysidopsis bahia) in flowing water. In: Bioassay Procedures for the Ocean Disposal 

Permit Program, EPA-600/9-78-010, U.S. Environmental Protection Agency, 

Environmental Research Laboratory, Gulf Breeze, FL, pp. 64-68. 
RTI Project No. 0210114.030 EPA Contract 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 organisms 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 Estimating the Chronic Toxicity of Effluents and 

Receiving Waters to Marine and Estuarine Organisms. 3rd Edition. EPA-821-R-02-014, 

U.S. Environmental Protection Agency, Washington, DC. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 57 

Table 1. Summary of water quality parameters measured in the exposure 

solutions during the mysid (Americamysis bahia) two-generation test 

with lindane. 

Nominal 

Concentration 

(ng/L) 

Rangesa

Dissolved 

Oxygen pH Temperatureb

(

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, temperature 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 rounded to two significant figures. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Table 2. Concentrations of lindane measured in the exposure solutions during the mysid (A

two-generation test.

Nominal 

Concentration 

(ng/L) 

Measured Concentration (ng/L) 

Day 0 Day 5 Day 8 Day 11 Day 14 Day 22 Day 29 Day 35 Day 42 Day 45/46 Day 47 Day 48 

Mea

(SD

Control 

< 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 < 8.1 NA

(NA

32 

31 31 

15e

35 31 27 

34 NS 33 37 30 30 25 NS 

25 28 25 

NS 30 (3.8

80 

100 120 110 

NS 130 NS 100 120 97 94 96 86 83 82 NS 10

(15

200 

180 190 190 

NS 190 NS 190 220 180 140 150 NS 

170 130 150 

NS 17

(27

500 

420 420 380 

NS 430 NS 400 440 360 310 350 

330 400 350 

360 NS 38

(42

1250 

1000 1200 

920 

NS 550e

1300 1300 1400 

1100 1200 850 980 1100 NS 

1000 1000 1000 

NS 110

(15

a Mean measured values, standard deviations (SD) and percent of nominal were calculated using the actual analytical results and not the rounded values (two significab Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area of th

factor of the controls. 

c NS = Not Sampled. d NA = Not Applicable. e Low recoveries attributed to sample processing error. Value not used in calculation of mean measured concentration. 
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Smithers Viscient Study No. 13888.6109 Page 59 

Table 3. Results of the quality control (QC) samples analyzed concurrently 

with test solution samples during the mysid (Americamysis bahia) 

two-generation test.

Quality Control Sample (ng/L) 

20.0a 200a 1250a

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 127b 235 117 1470 118 

5 26.0 130b 233 117 1350 108 

8 21.6 108 204 102 1410 113 

11 29.1 145b 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 outside of the acceptable range (i.e., 70 to 120%, Appendix 3). 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Table 4. F0 mysid survival and growth (length and dry weight) during the mysid (Americam

generation test with lindane. 

Nominal 

Concentration 

(ng/L) 

Replicate Survival (%) p-Value 

Mean Total Body Length (mm)a

Day 7 p-Value 

Day 14 

Male p-Value Female p-Value Male p-Val

Control A 81 4.33 5.93 6.07 7.21 

 B 87 4.67 6.49 6.57 7.30  C 77 4.38 6.33 6.37 7.47 

Mean(SD) 82(5.0) NAc 4.46(0.19) NA 6.25(0.29) NA 6.34(0.25) NA 7.32(0.13) NA 

32 A 84 4.44 6.58 6.67 7.16 

 B 77 4.44 6.45 6.74 7.37  C 83 4.51 6.81 6.66 7.51 

Mean(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) 0.798

80 A 85 4.56 6.43 6.61 7.11 

 B 85 4.99 7.16 7.09 7.07  C 90 4.42 6.64 6.47 7.40 

Mean(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) 0.978

200 A 89 4.87 6.56 6.64 6.97 

 B 75 5.01 6.92 7.15 7.34  C 72 4.49 6.91 6.78 7.00 

Mean(SD) 79(9.3) 0.6630 4.79(0.27) 0.1036 6.80(0.20) 0.0716 6.86(0.26)d 0.0176 7.10(0.21) 0.995

500 A 69 4.68 6.87 7.19 7.05 

 B 86 5.03 6.71 7.00 NA  C 86 4.81 6.96 7.06 7.41 

Mean(SD) 80(9.8) 0.7749 4.84(0.18) 0.0622 6.85(0.13) 0.0504 7.08(0.10)d 0.0016 7.23(0.26) 0.953

1250 A 76 4.65 6.07 6.68 7.07 

 B 83 4.53 6.50 6.77 7.02  C 76 4.58 6.96 6.66 6.79 

Mean(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) 0.999

a Mean total body length for the control on day 0 = 1.84 mm; standard deviation = 0.018. b Mean dry body weight for the control on day 0 = 0.035 mg; standard deviation = 0.0028. c NA = Not Applicable. d Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. e Statistically different compared to the control, based on Bonferroni's Adjusted t-Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Table 4. Continued. F0 mysid survival and growth (length and dry weight) during the m

bahia) two-generation test with lindane. 

Nominal 

Concentration 

(ng/L) 

Replicate 

Mean Dry Body Weight (mg)b

Day 7 p-Value Day 14 D

Male p-Value Female p-Value Male p-Value 

Control A 0.31 0.55 0.69 1.06 

 B 0.33 0.58 0.84 0.92  C 0.25 0.62 0.58 0.90 

Mean(SD) 0.30(0.04) NA 0.58(0.03) NA 0.70(0.13) NA 0.96(0.09) NA 

32 A 0.34 0.59 0.77 0.95 

 B 0.40 0.56 0.73 0.88  C 0.33 0.57 0.70 1.06 

Mean(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 

80 A 0.25 0.54 0.81 0.86 

 B 0.32 0.70 1.04 0.88  C 0.27 0.69 0.55 0.90 

Mean(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 

200 A 0.33 0.66 0.88 0.85 

 B 0.28 0.74 1.06 0.88  C 0.33 0.84 1.05 0.81 

Mean(SD) 0.32(0.03) 0.6265 0.75(0.09)d 0.0233 0.99(0.10)d 0.0369 0.84(0.03) 0.2847 

500 A 0.32 0.87 1.14 0.89 

 B 0.37 0.89 1.18 NA  C 0.39 0.90 1.01 1.02 

Mean(SD) 0.36(0.03) 0.0982 0.89(0.01)d 0.0004 1.11(0.08)d 0.0059 0.95(0.09) 1.0000 

1250 A 0.35 0.75 1.03 0.77 

 B 0.30 0.94 1.17 0.80  C 0.27 0.90 0.96 0.58 

Mean(SD) 0.30(0.04) 0.7189 0.86(0.10)d 0.0007 1.05(0.11)d 0.0149 0.72(0.12)e 0.0089 

a Mean total body length for the control on day 0 = 1.84 mm; standard deviation = 0.018. b Mean dry body weight for the control on day 0 = 0.035 mg; standard deviation = 0.0028. c NA = Not Applicable. d Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. e Statistically different compared to the control, based on Bonferroni's Adjusted t-Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Table 5. F0 mysid time to maturation, time to first brood release, time to second brood relea

released per female and interbrood duration during the mysid (Americamysis bahitest with lindane.

Nominal 

Concentration 

(ng/L) 

Replicate 

Time to 

Maturation 

(Days) 

pValue 

First Brood Second Brood 

Time to Release 

(Days) 

pValue Offspring/Female p- Value 

Time to 

Release 

(Days) 

pValue Offspring/Female 

Control A 10 15.7 4.7 21.1 9.9 

 B 10 15.3 5.9 20.3 7.1  C 11 16.4 5.9 22.3 9.5 

Mean(SD) 10.3(0.58) NAa 15.8(0.68) NA 5.5(0.66) NA 21.3(1.0) NA 8.8(1.5) 

32 A 9 16.1 6.0 20.3 9.2 

 B 10 16.4 8.0 20.9 11.4  C 10 15.6 5.9 20.6 10.6 

Mean(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) 

80 A 10 15.9 6.4 21.1 8.6 

 B 9 14.3 8.3 19.3 10.3  C 10 15.3 4.4 20.3 8.7 

Mean(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) 

200 A 10 15.0 6.0 20.0 6.7 

 B 9 14.9 6.7 19.8 11.2  C 9 16.1 6.4 21.1 7.9 

Mean(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) 

500 A 9 14.6 8.4 20.1 10.3 

 B 9 13.9 8.3 18.8 11.5  C 9 14.1 7.4 19.1 10.3 

Mean(SD) 9.0(0) 0.9997 14.2(0.92)b 0.0105 8.1(0.54)b 0.0245 19.3(0.72)b 0.0297 10.7(0.70) 

1250 A 9 15.9 6.0 21.3 7.7 

 B 10 15.0 6.4 22.0 10.0  C 10 14.9 76 19.8 11.8 

Mean(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) 

a NA = Not Applicable. b Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 63 

Table 6. F0 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.

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) NAa 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-06-026 

Smithers Viscient Study No. 13888.6109 Table 7. F1 mysid survival and growth (length and dry weight) during the mysid (Americam

generation test with lindane. 

Nominal 

Concentration 

(ng/L) 

Replicate Survival (%) p- Value 

Mean Total Body Length (mm)

Day 0 p- Value Day 7 p- Value Day 14 

Male p- Value Female p- Value Male 

Control A 71 1.67 4.74 6.54 6.45 7.29 

 B 96 1.69 4.63 5.96 6.18 7.22  C 89 1.55 4.78 6.47 6.75 7.39 

Mean(SD) 85(13) NAa 1.63(0.07) NA 4.72(0.08) NA 6.32(0.32) NA 6.46(0.28) NA 7.30(0.09) 

32 A 75 1.65 4.71 6.45 6.34 7.29 

 B 77 1.66 4.24 6.29 6.08 7.17  C 87 1.70 4.92 6.25 6.62 7.51 

Mean(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) 

80 A 96 1.56 4.15 5.79 6.36 6.89 

 B 95 1.66 4.91 5.98 6.08 7.08  C 86 1.70 4.88 6.17 6.62 7.24 

Mean(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) 

200 A 95 1.73 3.90 5.52 6.06 6.69 

 B 92 1.73 4.74 6.06 6.25 7.03  C 79 1.66 5.19 6.13 6.39 6.99 

Mean(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) 

500 A 100 1.69 4.59 6.02 6.26 6.85 

 B 88 1.70 4.80 6.01 6.81 7.87  C 79 1.68 5.19 6.26 6.68 7.48 

Mean(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) 

1250 A 69 1.71 4.72 NA 7.04 6.84 

 B 68 1.69 3.94 5.74 6.46 6.67  C 74 1.69 4.35 NA 6.49 6.72 

Mean(SD) 70(2.9) 0.9997 1.70(0.01) 0.2206 4.33(0.39) 0.3634 5.74(NA)b NA 6.66(0.32) 0.9772 6.74(0.09)b 

a NA = Not Applicable. b Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Table 7. Continued. F1 mysid survival and growth (length and dry weight) during the m

bahia) two-generation test with lindane. 

Nominal 

Concentration 

(ng/L) 

Replicate 

Mean Dry Body Weight (mg)

Day 0 p-Value Day 7 p-Value 

Day 14 

Male p-Value Female p-Value Male 

Control A 0.028 0.30 0.77 0.79 0.83 

 B 0.034 0.32 0.64 0.77 0.83  C 0.024 0.52 0.76 1.23 0.91 

Mean(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) 

32 A 0.038 0.44 0.89 0.88 0.89 

 B 0.018 0.23 0.59 0.75 0.83  C 0.034 0.49 0.79 1.06 0.99 

Mean(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) 

80 A 0.028 0.22 0.56 0.66 0.79 

 B 0.020 0.31 0.55 0.69 0.82  C 0.034 0.35 0.46 0.91 0.84 

Mean(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) 

200 A 0.022 0.23 0.38 0.66 0.68 

 B 0.042 0.33 0.55 0.77 0.88  C 0.036 0.36 0.46 0.77 0.78 

Mean(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) 

500 A 0.020 0.29 0.48 0.74 0.85 

 B 0.018 0.34 0.72 0.93 0.94  C 0.038 0.37 0.64 0.84 0.83 

Mean(SD) 0.025(0.011) 0.6717 0.34(0.04) 0.5804 0.61(0.12)b 0.0237 0.84(0.09) 0.5186 0.87(0.06) 

1250 A 0.014 0.30 NA 0.77 0.73 

 B 0.028 0.30 0.45 0.69 0.65  C 0.032 0.36 NA 0.72 0.72 

Mean(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)b 

a NA = Not Applicable. b Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Table 8. F1 mysid time to maturation, time to first brood release, time to second brood relea

released per female and interbrood duration during the mysid (Americamysis bahitest with lindane.

Nominal 

Concentration 

(ng/L) 

Replicate 

Time to 

Maturation 

(Days) 

pValue 

First Brood Second Brood 

Time to Release 

(Days) 

pValue Offspring/Female p- Value 

Time to 

Release 

(Days) 

pValue Offspring/Female 

Control A 10 14.7 6.9 20.4 10.4 

 B 10 15.0 5.1 20.0 8.3  C 9.3 14.9 7.7 19.7 10.2 

Mean(SD) 9.7(0.58) NAa 14.9(1.0) NA 6.6(1.3) NA 20.0(0.38) NA 9.6(1.2) 

32 A 12 16.9 5.6 21.0 7.0 

 B 10 16.0 5.6 20.3 10.3  C 11 16.9 7.0 21.5 9.8 

Mean(SD) 8.7(1.2) 0.9934 16.6(2.4)b 0.0436 6.1(0.82) 0.5737 20.9(0.59) 0.3228 9.1(1.8) 

80 A 13 16.9 4.3 21.9 6.7 

 B 12 17.0 5.9 22.7 8.4  C 11 16.1 4.3 20.8 7.2 

Mean(SD) 11(1.2) 0.2725 16.7(1.4)b 0.0367 4.8(0.91) 0.0677 21.8(0.96) 0.0542 7.4(0.88) 

200 A 13 17.7 4.1 23.0 6.2 

 B 10 15.0 4.7 20.0 4.5  C 11 17.3 3.0 21.8 5.3 

Mean(SD) 9.7(0.58) 0.8333 16.7(1.6)b 0.0367 3.9(0.85)b 0.0083 21.6(1.5) 0.0859 5.3(0.83)b 

500 A 11 16.3 5.7 21.3 10.0 

 B 12 14.4 4.1 20.7 11.3  C 12 16.3 4.4 21.3 10.5 

Mean(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) 

1250 A 13 16.5 5.0 21.0 10.2 

 B 13 15.7 5.9 20.5 8.8  C 13 15.8 5.0 22.3 9.0 

Mean(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) 

a NA = Not Applicable. b Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 67 

Table 9. F1 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.

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) NAa

 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 control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 68 

Table 10. Summary of F0 and F1 endpoints, No-Observed-Effect 

Concentration (NOEC) and Lowest-Observed-Effect Concentration 

(LOEC) values determined during the mysid (Americamysis bahia) 

two-generation test with lindane.

F0 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 1st Brood Release ↓ 

1st Brood Offspring/Female ↑ 

Time to 2nd Brood Release ↓ 

2nd Brood Offspring/Female 

Interbrood Duration 

Total Offspring/Female 

Sex Ratio 

% Reproducing Females 

↓ Endpoint determined to be statistically significantly reduced (p ≤ 0.05) compared to control 

↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control 

F1 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 1st Brood Release ↑ ↑ ↑ 

1st Brood Offspring/Female ↓ 

Time to 2nd Brood Release 

2nd Brood Offspring/Female ↓ 

Interbrood Duration 

Total Offspring/Female ↓ ↓ 

Sex Ratio 

% Reproducing Females 

↓ Endpoint determined to be statistically significantly reduced (p ≤ 0.05) compared to control 
RTI Project No. 0210114.030 EPA Contract 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 Contract 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 71 

Figure 3. Mean percent survival of F0 mysids (Americamysis bahia) during the 

two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 72 

Figure 4. Day 7 mean total body length of F0 mysids (Americamysis bahia) 

during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 73 

Figure 5. Day 7 mean dry body weight of F0 mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 77 

Figure 9. Day 27 male and female mean dry body weight of F0 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 78 

Figure 10. Mean time to maturation of F0 mysids (Americamysis bahia) during 

the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 79 

Figure 11. Mean time to first brood release of F0 mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 80 

Figure 12. F0 Mean number of offspring/female produced in the first brood 

release of mysids (Americamysis bahia) during the two-generation 

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

Smithers Viscient Study No. 13888.6109 Page 81 

Figure 13. Mean time to second brood release of F0 mysids (Americamysis 

bahia) during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 82 

Figure 14. F0 Mean number of offspring/female produced in the second brood 

release of mysids (Americamysis bahia) during the two-generation 

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

Smithers Viscient Study No. 13888.6109 Page 83 

Figure 15. F0 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 84 

Figure 16. F0 Mean total number of offspring released in the first two broods 

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

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

Smithers Viscient Study No. 13888.6109 Page 85 

Figure 17. F0 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 86 

Figure 18. F0 Mean percentage of reproductively active females for mysids 

(Americamysis bahia) during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 87 

Figure 19. Mean percent survival of F1 mysids (Americamysis bahia) during the 

two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 88 

Figure 20. Day 0 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 89 

Figure 21. Day 0 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 90 

Figure 22. Day 7 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 91 

Figure 23. Day 7 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 92 

Figure 24. Day 14 male and female mean total body length of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 93 

Figure 25. Day 14 male and female mean dry body weight of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 94 

Figure 26. Day 27 male and female mean total body length of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 95 

Figure 27. Day 27 male and female mean dry body weight of F1 mysids 

(Americamysis bahia) during the two-generation test with lindane.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 96 

Figure 28. Mean time to maturation of F1 mysids (Americamysis bahia) during 

the two-generation test with lindane. 
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Figure 29. Mean time to first brood release of F1 mysids (Americamysis bahia) 

during the two-generation test with lindane. 
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Figure 30. F1 Mean number of offspring/female 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 F1 mysids (Americamysis 

bahia) during the two-generation test with lindane. 
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Figure 32. F1 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. F1 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with lindane. 
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Figure 34. F1 Mean total number of offspring released in the first two broods 

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

lindane. 
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Figure 35. F1 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with lindane. 
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Figure 36. F1 Mean percentage of reproductively active females for mysids 

(Americamysis bahia) during the two-generation 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 ethyl acetate, reduced in volume to approximately 

2.0 mL by rotary evaporation at 35 oC, taken to near-dryness under a gentle stream of nitrogen at 

room temperature and reconstituted with 0.1% peanut oil in iso-octane. Recovery samples were 

then analyzed by gas chromatography using micro electron capture detection (GC/μECD). 

This method was validated by fortification of filtered seawater with lindane at concentrations of 

0.0200, 0.200 and 2.00 μg a.i./L. Recoveries averaged 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 subsequent studies were set at 70 to 120%. 

EXPERIMENTAL 

Equipment 

1. Instrument: Hewlett Packard Model 6890 Series gas chromatograph 

equipped with a micro electron 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 
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Test Substance 

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

Laboratory, Sequim, Washington. The following information was provided: 

 Name: Lindane, 97% 

 Synonyms: Lindane; 1α,2α,3β,4α,5α,6β-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 substance (SMV No. 4841) was stored at room 

temperature in the original container 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 flask 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 solution in a 50.0-mL volumetric flask and bringing it to volume 

with acetone. The 1.00 mg a.i./L secondary stock solution was used to fortify 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 ingredient) in a 50.0-mL volumetric flask and bringing it to volume 

with acetone. A 10.0 mg a.i./L secondary stock 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 prepared 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-octane (v:v) was prepared by adding 0.500 mL of 

peanut oil to 500 mL of iso-octane. This solution was mixed using a magnetic stir plate and stir 

bar. 

Sample Fortification and Preparation 

Fortification of Recovery Samples: 

The recovery samples were prepared by fortifying 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 NAa 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 recovery samples prior to extraction to adjust the 

samples to pH 3. All recovery and control samples 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, the separatory funnels were rinsed with 50.0 mL 

of ethyl acetate and the rinsate was added to the round-bottom flasks. The extracts were taken to 

low volume (approximately 2 mL) by rotary evaporation using minimal heating (<35 oC). A 

solvent transfer step with acetone was then performed 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 centrifuge tubes (or larger glass vials depending 

on the final volume) and taken to incipient dryness (approximately 200 μL) under a gentle 

stream of nitrogen at room temperature. Samples 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 transferred to autosampler vials to be analyzed by 

GC/μECD. 
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ANALYSIS 

Instrumental Conditions

The gas chromatographic analysis with micro 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/methane (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 second set immediately following the analysis of the 

recovery samples. Injection of recovery samples and calibration standards onto the 

chromatographic system was performed 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 peaks 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 logarithm 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. 
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(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 concentration (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 calculated using the following equation: 

INST CNTL

INST INST

LS

INST

(7) LOQ LOQ x DF

(6) LOQ inverse (ln (LOQ )

m

ln (A ) b (5) ln (LOQ )





 
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where: 

ALS = mean detector response (peak area) of the low 

concentration calibration standard (two injections) 

b = y-intercept from regression analysis 

m = slope from regression analysis 

LOQINST = limit of quantitation on the instrument 

DFCNTL = 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 deviation of 6.59% and a limit 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, while 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 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. 
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Table 3A. Analytical results for the recovery 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 < 0.00392a NAb

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 below the limit of quantitation (LOQ). The LOQ for each 

analysis is dependent upon the linear regression, the area 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. 
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Figure 3A. Representative chromatogram of a 25.0 μg a.i./L calibration 

standard used for quantification during the method validation study 

with lindane. 
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Figure 3B. Representative chromatogram of a 0.200 μg a.i./L recovery sample 

fortified with lindane during the method validation study. 
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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. 
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Figure 3D. A typical regression analysis for the calibration standards used to 

quantitate lindane in recovery samples during the method validation 

study. 

Regression Analysis 

R2

 = 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Page 198 

APPENDIX 4 - POST-PAIRING SURVIVAL DATA - LINDANE 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

Control 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 18 2 20 3 18 20 4 18 20 5 18 20 6 18 20 7 18 20 8 18 20 9 18 20 

10 14 16 4 4 11 13 15 5 5 

12e 8 10 10 10 100 100 

13 8 10 10 10 100 100 14 7 1 9 10 10 100 100 15 7 9 10 10 100 100 16 7 9 10 10 100 100 17 7 9 10 10 100 100 18 7 9 10 10 100 100 19 7 9 10 10 100 100 20 7 9 9 1 9 100 100 21 7 9 9 9 100 100 22 7 9 8 1 8 100 100 23 7 9 8 8 100 100 24 7 9 7 1 8 100 88 25 7 9 7 8 100 88 26 6 1 8 7 8 100 88 27 6 8 6 1 7 100 86 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

Control 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 20 20 

10 16 16 4 4 11 13 13 7 7 

12e 10 1 11 9 9 100 100 

13 8 9 11 11 100 100 14 8 9 11 11 100 100 15 8 9 11 11 100 100 16 8 9 10 1 10 100 100 17 7 1 8 10 10 100 100 18 7 8 10 10 100 100 19 7 8 10 10 100 100 20 7 8 10 10 100 100 21 7 8 10 10 100 100 22 7 8 10 10 100 100 23 6 1 8 9 1 10 86 90 24 6 8 9 10 86 90 25 6 8 9 10 86 90 26 6 8 9 10 86 90 27 5 1 7 9 10 83 90 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

Control 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 20 20 

10 20 20 11 18 18 2 2 

12e 12 4 16 6 6 100 100 

13 13 17 7 7 100 100 14 12 1 16 7 7 100 100 15 12 16 7 7 100 100 16 12 16 7 7 100 100 17 12 16 7 7 100 100 18 11 1 16 6 1 6 92 100 19 10 1 15 6 6 91 100 20 10 15 6 6 91 100 21 10 15 6 6 91 100 22 10 15 6 6 91 100 23 10 15 6 6 91 100 24 10 15 5 1 6 91 83 25 10 15 5 6 91 83 26 9 1 14 5 6 90 83 27 8 1 14 5 6 80 83 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

32 ng/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 19 1 20 7 19 20 8 19 20 9 18 19 1 1 

10 16 17 3 3 11 13 14 6 6 

12e 10 11 9 9 100 100 

13 10 11 9 9 100 100 14 10 11 9 9 100 100 15 10 11 9 9 100 100 16 10 11 9 9 100 100 17 9 1 11 8 1 9 90 89 18 9 11 8 9 90 89 19 9 11 8 9 90 89 20 9 11 8 9 90 89 21 9 11 8 9 90 89 22 8 1 10 7 1 8 89 88 23 8 10 7 8 89 88 24 8 10 7 8 89 88 25 8 10 7 8 89 88 26 7 1 9 7 8 88 88 27 7 9 7 8 88 88 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

32 ng/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 19 1 20 3 18 1 20 4 18 20 5 18 20 6 18 20 7 17 1 20 8 16 1 20 9 16 20 

10 11 15 5 5 11 9 13 7 7 

12e 7 1 12 8 8 100 100 

13 8 13 7 7 100 100 14 7 1 12 7 7 100 100 15 7 12 7 7 100 100 16 7 12 7 7 100 100 17 7 12 7 7 100 100 18 7 12 7 7 100 100 19 7 12 7 7 100 100 20 7 12 7 7 100 100 21 6 1 12 7 7 86 100 22 6 12 7 7 86 100 23 6 12 7 7 86 100 24 6 12 6 1 6 86 100 25 6 12 5 1 5 86 100 26 6 12 5 5 86 100 27 5 1 11 5 5 83 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

32 ng/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 18 2 20 2 18 20 3 18 20 4 18 20 5 18 20 6 18 20 7 18 20 8 18 20 9 18 20 

10 14 16 4 4 11 13 15 5 5 

12e 10 1 13 7 7 100 100 

13 10 13 7 7 100 100 14 9 1 12 7 7 100 100 15 9 12 7 7 100 100 16 9 12 7 7 100 100 17 9 12 6 1 6 100 100 18 9 12 6 6 100 100 19 9 12 5 1 5 100 100 20 9 12 5 5 100 100 21 8 1 11 5 5 100 100 22 8 11 5 5 100 100 23 8 11 5 5 100 100 24 8 11 5 5 100 100 25 7 11 5 5 88 100 26 7 11 5 5 88 100 27 6 1 10 5 5 86 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

80 ng/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 19 1 20 9 19 20 

10 16 17 3 3 11 14 15 5 5 

12e 14 15 5 5 100 100 

13 14 15 7 7 100 100 14 14 15 7 7 100 100 15 14 15 7 7 100 100 16 14 15 7 7 100 100 17 13 1 15 7 7 93 100 18 13 15 7 7 93 100 19 13 15 7 7 93 100 20 13 15 7 7 93 100 21 13 15 7 7 93 100 22 13 15 7 7 93 100 23 13 15 7 7 93 100 24 13 15 7 7 93 100 25 12 1 15 7 7 86 100 26 12 15 7 7 86 100 27 12 15 7 7 86 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

80 ng/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 19 20 3 19 20 4 19 20 5 19 20 6 19 20 7 19 20 8 19 20 9 18 19 1 1 

10 13 14 6 6 11 11 12 8 8 

12e 10 11 9 9 100 100 

13 9 1 11 9 9 90 100 14 9 11 9 9 90 100 15 9 11 9 9 90 100 16 9 11 9 9 90 100 17 9 11 9 9 90 100 18 9 11 9 9 90 100 19 8 1 10 9 9 89 100 20 8 10 9 9 89 100 21 8 10 9 9 89 100 22 8 10 9 9 89 100 23 8 10 9 9 89 100 24 8 10 9 9 89 100 25 8 10 9 9 89 100 26 8 10 9 9 89 100 27 8 10 9 9 89 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

80 ng/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 19 1 20 9 19 20 

10 16 17 3 3 11 15 16 4 4 

12e 8 1 10 10 10 100 100 

13 8 10 10 10 100 100 14 7 9 11 11 100 100 15 7 9 11 11 100 100 16 7 9 11 11 100 100 17 7 9 11 11 100 100 18 7 9 11 11 100 100 19 7 9 11 11 100 100 20 6 1 8 10 1 10 100 100 21 6 8 10 10 100 100 22 6 8 10 10 100 100 23 6 8 10 10 100 100 24 6 8 10 10 100 100 25 6 8 9 1 10 100 90 26 6 8 8 1 9 100 89 27 5 1 7 7 1 8 100 88 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

200 ng/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 19 1 20 3 19 20 4 19 20 5 19 20 6 19 20 7 18 1 20 8 18 20 9 18 20 

10 11 13 7 7 11 10 12 8 8 

12e 7 1 10 9 9 100 100 

13 7 1 10 9 9 100 100 14 7 10 9 9 100 100 15 7 10 9 9 100 100 16 7 10 9 9 100 100 17 7 10 9 9 100 100 18 7 10 9 9 100 100 19 6 1 9 9 9 100 100 20 6 9 9 9 100 100 21 6 9 9 9 100 100 22 6 9 9 9 100 100 23 6 9 8 1 8 100 100 24 6 9 8 8 100 100 25 6 9 8 8 100 100 26 5 1 8 8 8 100 100 27 5 8 7 1 7 100 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

200 ng/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 19 1 20 7 18 1 20 8 18 20 9 16 18 2 2 

10 13 15 5 5 11 12 14 6 6 

12e 10 12 8 8 100 100 

13 9 1 11 8 8 100 100 14 9 11 8 8 100 100 15 9 11 6 1 1 7 100 86 16 9 11 6 7 100 86 17 9 11 6 7 100 86 18 9 11 6 7 100 86 19 9 11 6 7 100 86 20 9 11 5 1 7 100 71 21 9 11 5 7 100 71 22 9 11 5 7 100 71 23 8 1 11 5 7 89 71 24 8 11 5 7 89 71 25 8 11 5 7 89 71 26 8 11 5 7 89 71 27 8 11 5 7 89 71 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

200 ng/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 18 2 20 3 16 2 20 4 16 20 5 17 20 6 17 20 7 17 20 8 15 20 9 14 19 1 1 

10 11 16 4 4 11 9 14 6 6 

12e 7 9 11 11 100 100 

13 7 9 11 11 100 100 14 7 9 11 11 100 100 15 7 9 11 11 100 100 16 7 9 11 11 100 100 17 7 9 11 11 100 100 18 7 9 10 1 10 100 100 19 6 1 8 10 10 100 100 20 6 8 10 10 100 100 21 6 8 10 10 100 100 22 6 8 10 10 100 100 23 6 8 10 10 100 100 24 6 8 10 10 100 100 25 6 8 10 10 100 100 26 6 8 10 10 100 100 27 6 8 10 10 100 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

500 ng/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 18 2 20 2 18 20 3 18 20 4 18 20 5 18 20 6 18 20 7 18 20 8 18 20 9 13 1 16 4 4 

10 10 13 7 7 11 10 13 7 7 

12e 8 11 9 9 100 100 

13 7 1 10 8 1 9 100 89 14 7 10 8 9 100 89 15 7 10 8 9 100 89 16 7 10 8 9 100 89 17 7 10 8 9 100 89 18 7 10 7 1 8 100 88 19 6 1 9 7 8 100 88 20 6 9 6 1 7 100 86 21 6 9 6 7 100 86 22 6 9 6 7 100 86 23 6 9 6 7 100 86 24 6 9 6 7 100 86 25 6 9 6 7 100 86 26 6 9 6 7 100 86 27 6 9 5 1 6 100 83 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

500 ng/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 19 20 3 18 20 4 18 20 5 18 20 6 19 20 7 19 20 8 19 20 9 13 14 6 6 

10 9 1 11 9 9 11 6 8 12 12 

12e 7 9 13 13 100 100 

13 7 9 13 13 100 100 14 7 9 13 13 100 100 15 7 9 12 1 12 100 100 16 7 9 12 12 100 100 17 6 1 8 12 12 100 100 18 5 1 7 12 12 100 100 19 4 1 6 10 2 10 100 100 20 4 6 9 1 9 100 100 21 4 6 9 9 100 100 22 4 6 9 9 100 100 23 3 1 5 9 9 100 100 24 1 1 1 4 9 9 50 100 25 0 1 3 9 9 0 100 26 0 3 9 9 0 100 27 0 3 9 9 0 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

500 ng/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 16 16 4 4 

10 13 13 7 7 11 12 12 8 8 

12e 10 2 12 8 8 100 100 

13 10 12 8 8 100 100 14 10 12 8 8 100 100 15 10 12 8 8 100 100 16 10 12 8 8 100 100 17 9 1 11 8 8 100 100 18 9 11 8 8 100 100 19 9 11 8 8 100 100 20 9 11 8 8 100 100 21 8 1 10 8 8 100 100 22 6 1 1 9 8 8 86 100 23 6 9 8 8 86 100 24 5 1 8 8 8 83 100 25 4 1 7 8 8 80 100 26 4 7 8 8 80 100 27 4 7 8 8 80 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

1250 ng/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 19 19 1 1 

10 15 15 5 5 11 15 15 5 5 

12e 12 1 12 7 7 100 100 

13 11 1 12 7 7 92 100 14 8 3 12 7 7 67 100 15 8 12 7 7 67 100 16 8 12 6 1 6 67 100 17 8 12 6 6 67 100 18 7 1 11 6 6 64 100 19 7 11 6 6 64 100 20 5 2 9 5 1 5 56 100 21 5 9 5 5 56 100 22 5 9 4 1 4 56 100 23 3 2 7 4 4 43 100 24 3 7 4 4 43 100 25 3 7 4 4 43 100 26 3 7 3 1 4 43 75 27 3 7 3 4 43 75 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

1250 ng/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 19 1 20 6 19 20 7 19 20 8 18 1 20 9 18 20 

10 13 15 5 5 11 12 14 6 6 

12e 11 13 7 7 100 100 

13 11 13 7 7 100 100 14 11 13 7 7 100 100 15 10 1 12 7 7 100 100 16 10 12 7 7 100 100 17 10 12 7 7 100 100 18 9 1 11 7 7 100 100 19 7 2 9 6 1 6 100 100 20 7 9 6 6 100 100 21 7 9 4 2 4 100 100 22 6 1 8 4 4 100 100 23 6 8 4 4 100 100 24 6 8 4 4 100 100 25 5 1 7 4 4 100 100 26 5 7 4 4 100 100 27 5 7 4 4 100 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F0 Reproduction Basket Survival 

1250 ng/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 20 20 3 19 20 4 19 20 5 18 1 20 6 18 20 7 18 20 8 18 20 9 18 20 

10 12 14 6 6 11 10 12 8 8 

12e 8 10 12 12 100 100 

13 8 10 11 1 11 100 100 14 8 10 11 11 100 100 15 8 10 10 1 11 100 91 16 8 10 10 11 100 91 17 6 2 8 10 11 100 91 18 3 3 5 10 11 100 91 19 3 5 10 11 100 91 20 3 5 10 11 100 91 21 3 5 9 1 10 100 90 22 3 5 7 2 8 100 88 23 3 5 7 8 100 88 24 2 1 4 7 8 100 88 25 2 4 7 8 100 88 26 1 1 3 7 8 100 88 27 1 3 7 8 100 88 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc). d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

Control 

Replicate Afg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 

9c 15 15 

10ab 9 9 6 6 100 100 

11 8 1 9 6 6 89 100 12 8 9 7 7 89 100 13 8 9 7 7 89 100 14 8 9 7 7 89 100 15 8 9 7 7 89 100 16 8 9 7 7 89 100 17 7 1 8 7 7 88 100 18 7 8 7 7 88 100 19 7 8 7 7 88 100 20 7 8 7 7 88 100 21 7 8 6 1 6 88 100 22 7 8 6 6 88 100 23 7 8 6 6 88 100 24 7 8 6 6 88 100 25 7 8 6 6 88 100 26 5 1 1 7 6 6 71 100 27 5 7 6 6 71 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

Control 

Replicate Bfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 

9c 15 15 

10ab 7 7 9 9 100 100 

11 7 7 9 9 100 100 12 7 7 9 9 100 100 13 7 7 9 9 100 100 14 7 1 7 9 9 100 100 15 7 7 9 9 100 100 16 7 7 9 9 100 100 17 7 7 9 9 100 100 18 7 7 9 9 100 100 19 7 7 9 9 100 100 20 7 7 9 9 100 100 21 7 7 7 2 7 100 100 22 7 7 7 7 100 100 23 7 7 7 7 100 100 24 7 7 7 7 100 100 25 7 7 7 7 100 100 26 7 7 7 7 100 100 27 7 7 7 7 100 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

Control 

Replicate Cfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 14 1 15 7 14 15 8 14 15 

9c 8 9 6 6 100 100 

10ab 8 9 6 6 100 100 

11 7 8 8 8 100 100 12 6 8 9 9 86 100 13 7 8 9 9 100 100 14 7 8 9 9 100 100 15 7 8 9 9 100 100 16 6 1 7 9 9 100 100 17 6 7 9 9 100 100 18 6 7 8 1 8 100 100 19 6 7 8 8 100 100 20 6 7 8 8 100 100 21 6 7 8 8 100 100 22 6 7 7 1 7 100 100 23 6 7 7 7 100 100 24 6 7 7 7 100 100 25 6 7 7 7 100 100 26 5 1 6 7 7 100 100 27 5 6 7 7 100 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

32 ng/L 

Replicate Afg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10bc 12 12 3 3 

11 12 12 3 3 

12a 10 1 11 7 7 100 100 

13 9 1 11 7 1 8 90 88 14 9 11 7 8 90 88 15 9 11 7 8 90 88 16 9 11 7 8 90 88 17 9 11 7 8 90 88 18 9 11 7 8 90 88 19 9 11 7 8 90 88 20 9 11 7 8 90 88 21 9 11 7 8 90 88 22 9 11 6 1 8 90 75 23 9 11 6 8 90 75 24 9 11 6 8 90 75 25 9 11 6 8 90 75 26 9 11 6 8 90 75 27 8 1 11 6 8 80 75 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

32 ng/L 

Replicate Bfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 14 1 15 2 14 15 3 14 15 4 14 15 5 14 15 6 14 15 7 14 15 8 12 13 2 2 9 12 13 2 2 

10bc 8 9 6 6 100 100 

11 8 9 7 7 100 100 

12a 8 9 7 7 100 100 

13 8 9 7 7 100 100 14 8 9 7 7 100 100 15 8 9 7 7 100 100 16 8 9 7 7 100 100 17 8 9 7 7 100 100 18 8 9 7 7 100 100 19 8 9 7 7 100 100 20 8 9 6 1 6 100 100 21 8 9 6 6 100 100 22 8 9 6 6 100 100 23 8 9 6 6 100 100 24 8 9 6 6 100 100 25 6 2 9 6 6 75 100 26 5 1 8 6 6 71 100 27 5 8 6 6 71 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

32 ng/L 

Replicate Cfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 14 1 15 3 13 1 15 4 13 15 5 13 15 6 13 15 7 13 15 8 11 13 2 2 9 11 13 2 2 

10bc 7 9 8 8 100 100 

11 7 9 8 8 100 100 

12a 7 9 8 8 100 100 

13 7 9 7 1 7 100 100 14 7 9 7 7 100 100 15 7 1 10 7 7 88 100 16 7 10 7 7 88 100 17 7 10 7 7 88 100 18 7 10 7 7 88 100 19 7 10 7 7 88 100 20 7 10 7 7 88 100 21 7 10 7 7 88 100 22 6 1 9 7 7 86 100 23 6 9 7 7 86 100 24 6 9 7 7 86 100 25 6 9 7 7 86 100 26 6 9 7 7 86 100 27 6 9 7 7 86 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

80 ng/L 

Replicate Afg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10 13 13 2 2 

11c 12 12 3 3 

12b 11 11 4 4 

13a 7 7 8 8 100 100 

14 7 7 8 8 100 100 15 7 7 8 8 100 100 16 7 7 8 8 100 100 17 7 7 8 8 100 100 18 7 7 8 8 100 100 19 7 7 8 8 100 100 20 7 7 8 8 100 100 21 7 7 8 8 100 100 22 7 7 8 8 100 100 23 7 7 8 8 100 100 24 7 7 8 8 100 100 25 7 7 8 8 100 100 26 7 7 8 8 100 100 27 7 7 8 8 100 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

80 ng/L 

Replicate Bfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 14 1 15 5 14 15 6 14 15 7 14 15 8 14 15 9 14 15 

10 14 15 

11c 14 15 

12b 7 8 8 8 100 100 

13a 7 8 8 8 100 100 

14 7 8 8 8 100 100 15 7 8 8 8 100 100 16 7 8 8 8 100 100 17 7 8 8 8 100 100 18 7 8 8 8 100 100 19 6 1 7 8 8 100 100 20 6 7 8 8 100 100 21 6 7 8 8 100 100 22 6 7 7 1 7 100 100 23 5 1 6 7 7 100 100 24 5 6 7 7 100 100 25 5 6 7 7 100 100 26 5 6 7 7 100 100 27 5 6 7 7 100 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

80 ng/L 

Replicate Cfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10 11 11 4 4 

11c 7 7 8 8 100 100 

12b 7 7 8 8 100 100 

13a 7 7 8 8 100 100 

14 7 7 8 8 100 100 15 7 7 8 8 100 100 16 7 7 8 8 100 100 17 7 7 8 8 100 100 18 7 7 8 8 100 100 19 7 7 8 8 100 100 20 7 7 8 8 100 100 21 7 7 7 1 7 100 100 22 6 1 7 7 7 86 100 23 6 7 6 1 6 86 100 24 5 1 7 6 6 71 100 25 4 1 7 6 6 57 100 26 4 7 6 6 57 100 27 4 7 6 6 57 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

200 ng/L 

Replicate Afg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10b 13 13 2 2 

11c 12 12 3 3 

12 12 12 3 3 

13a 8 8 7 7 100 100 

14 8 8 7 7 100 100 15 8 8 7 7 100 100 16 7 1 7 6 1 7 100 86 17 7 7 6 7 100 86 18 7 7 6 7 100 86 19 7 7 6 7 100 86 20 7 7 6 7 100 86 21 7 7 6 7 100 86 22 7 7 6 7 100 86 23 7 7 6 7 100 86 24 7 7 6 7 100 86 25 7 7 6 7 100 86 26 6 1 6 6 7 100 86 27 6 6 6 7 100 86 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

200 ng/L 

Replicate Bfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 12 12 3 3 

10b 7 7 8 8 100 100 

11c 7 7 8 8 100 100 

12 7 7 8 8 100 100 

13a 6 6 9 9 100 100 

14 6 6 9 9 100 100 15 6 6 9 9 100 100 16 6 6 9 9 100 100 17 6 6 9 9 100 100 18 6 6 9 9 100 100 19 6 6 9 9 100 100 20 6 6 9 9 100 100 21 6 6 9 9 100 100 22 5 1 6 9 9 83 100 23 5 6 9 9 83 100 24 5 6 8 1 9 83 89 25 5 6 8 9 83 89 26 5 6 8 9 83 89 27 5 6 8 9 83 89 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

200 ng/L 

Replicate Cfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10b 12 12 3 3 

11c 9 9 6 6 100 100 

12 9 9 6 6 100 100 

13a 9 9 6 6 100 100 

14 9 9 6 6 100 100 15 9 9 6 6 100 100 16 9 9 6 6 100 100 17 9 9 6 6 100 100 18 8 1 8 6 6 100 100 19 8 8 5 1 5 100 100 20 8 8 5 5 100 100 21 8 8 5 5 100 100 22 7 1 7 5 5 100 100 23 7 7 4 1 4 100 100 24 6 1 7 4 4 86 100 25 6 7 4 4 86 100 26 6 7 4 4 86 100 27 5 1 7 4 4 71 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

500 ng/L 

Replicate Afg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 14 1 15 4 14 15 5 14 15 6 14 15 7 15 15 8 15 15 9 12 12 3 3 

10 9 9 6 6 

11a 5 1 6 9 9 100 100 

12bc 7 8 9 9 100 100 

13 7 8 9 9 100 100 14 7 8 9 9 100 100 15 7 8 8 1 8 100 100 16 7 8 8 8 100 100 17 7 8 8 8 100 100 18 7 8 8 8 100 100 19 7 8 8 8 100 100 20 7 8 8 8 100 100 21 7 8 8 8 100 100 22 5 2 6 8 8 100 100 23 5 6 8 8 100 100 24 5 6 8 8 100 100 25 5 6 8 8 100 100 26 5 6 8 8 100 100 27 5 6 7 1 7 100 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

500 ng/L 

Replicate Bfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10 10 1 11 4 4 

11a 9 10 5 5 

12bc 8 1 10 7 7 100 100 

13 8 10 7 7 100 100 14 8 10 7 7 100 100 15 7 1 9 7 7 100 100 16 7 9 7 7 100 100 17 7 9 7 7 100 100 18 6 1 8 7 7 100 100 19 6 8 7 7 100 100 20 6 8 7 7 100 100 21 5 1 8 7 7 83 100 22 5 8 7 7 83 100 23 5 8 6 1 6 83 100 24 3 2 6 6 6 75 100 25 3 6 6 6 75 100 26 3 6 6 6 75 100 27 3 6 6 6 75 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

500 ng/L 

Replicate Cfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10 15 15 

11a 10 10 5 5 

12bc 8 8 7 7 100 100 

13 8 8 7 7 100 100 14 8 8 7 7 100 100 15 7 1 7 7 7 100 100 16 6 1 7 7 7 86 100 17 6 7 7 7 86 100 18 6 7 7 7 86 100 19 6 7 7 7 86 100 20 6 7 7 7 86 100 21 6 7 6 1 6 86 100 22 6 7 6 6 86 100 23 6 7 6 6 86 100 24 5 1 6 6 6 83 100 25 4 1 5 6 6 80 100 26 3 1 4 6 6 75 100 27 3 4 6 6 75 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

1250 ng/L 

Replicate Afg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 14 1 15 8 14 15 9 14 15 

10 11 12 3 3 11 10 11 4 4 

12ac 10 11 4 4 100 100 

13b 8 9 6 6 100 100 

14 8 9 7 7 100 100 15 7 8 8 8 100 100 16 6 1 7 7 1 7 100 100 17 6 7 7 7 100 100 18 6 7 7 7 100 100 19 6 7 7 7 100 100 20 6 7 7 7 100 100 21 6 7 7 7 100 100 22 5 1 7 7 7 83 100 23 5 7 7 7 83 100 24 4 1 7 7 7 67 100 25 4 7 7 7 67 100 26 3 1 6 6 1 6 60 100 27 3 6 5 1 5 60 100 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

1250 ng/L 

Replicate Bfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10 14 1 15 11 9 1 11 4 4 

12ac 9 11 4 4 

13b 7 9 7 7 100 100 

14 7 9 7 7 100 100 15 7 9 7 7 100 100 16 6 1 9 7 7 86 100 17 6 9 7 7 86 100 18 6 9 7 7 86 100 19 5 1 8 7 7 83 100 20 5 8 6 1 6 83 100 21 5 8 5 1 5 83 100 22 4 1 7 4 1 5 80 80 23 3 1 6 4 5 75 80 24 2 1 6 4 5 50 80 25 2 6 4 5 50 80 26 2 6 4 5 50 80 27 2 6 4 5 50 80 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6109 Study Number: 13888.6109 

Mysid Two Generation Exposure with Lindane 

F1 Reproduction Basket Survival 

1250 ng/L 

Replicate Cfg

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMe ♂/Immature b ♀ Dead/Missing NTRMe ♀ b ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 14 1 15 7 14 15 8 14 15 9 14 15 

10 14 15 11 11 15 3 3 

12ac 6 8 7 7 86 100 

13b 6 8 7 7 86 100 

14 7 8 8 8 100 100 15 7 8 8 8 100 100 16 7 8 8 8 100 100 17 7 8 8 8 100 100 18 7 8 8 8 100 100 19 7 8 7 1 7 100 100 20 5 2 6 7 7 100 100 21 5 6 6 1 7 100 86 22 5 6 5 1 7 100 71 23 5 6 5 7 100 71 24 5 6 4 1 6 100 67 25 5 6 4 6 100 67 26 5 6 3 1 5 100 60 27 5 6 3 5 100 60 

a Day Rep A was paired b Day Rep B was paired c Day Rep C was paired. Prior to pairing, mysid counts are estimated; following mysid pairing through test termination, mysid survival will also be determined by gendee NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) f Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) g Following test initiation until mysid pairing, counts are considered to be estimations.
RTI Project No. 0210114.030 EPA Contract 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 Toxicity Test with Mysids 

(Americamysis bahia) Inter-Laboratory Comparison, Smithers 

Viscient Protocol No.: 091310/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 Contract 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 (F0) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

 First Filial Generation (F1) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 
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ENDPOINTS AND 

RESULTS: For this exposure, the F0 generation appeared to be more sensitive 

(seven endpoints impacted) to the concentrations of 4-tOP than the 

F1 generation (one endpoint impacted). A summary of the 

responses for each endpoint in each generation is presented below. 

 The most sensitive endpoints in the F0 generation were determined 

to be 1st brood (offspring/female), total offspring/female and Day 7 

length (0.36 μg/L). While standard statistical analysis determined 

that these endpoints were the most sensitive, a hormetic response 

was observed in a number of F0 endpoints (F0 1st brood, offspring 

per female; F0 Day 14 male dry weight, F0 Day 14 female dry 

weight, F0 interbrood duration, F0 sex ratio, F0 time to 1st brood, F0

time to 2nd brood). Hormesis best explains 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 F1 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 (F1 Day 7 

dry weight, sex ratio, time to maturation, total young). Also, this 

response was not determined to be statistically significant at any 

dose for any endpoint. 

 While a dose-response relationship 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 F0 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. 
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F0 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 1st Brood Release 

1st Brood Offspring/Female ↓ ↓ ↓ ↓

Time to 2nd Brood Release 

2nd Brood Offspring/Female 

Interbrood Duration ↑ ↑ 

Total Offspring/Female ↓ ↓ ↓ ↓ ↓

Sex Ratio 

% Reproducing Females 

↓ Endpoint determined to be statistically significantly reduced (p ≤ 0.05) compared to control 

↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control 

F1 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 1st Brood Release 

1st Brood Offspring/Female 

Time to 2nd Brood Release 

2nd 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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7.0 INTRODUCTION 

The U.S. Environmental Protection Agency's Endocrine 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 endocrine effects and provide dose response information needed for 

risk assessment. One of the longer term Tier 2 assays being developed is the mysid twogeneration test with Americamysis bahia. This test contrasts with existing mysid life-cycle 

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

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

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

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

Additionally, a method for testing crustaceans for effects of endocrine disrupting chemicals 

(EDCs) is relevant to assessing the adverse consequences of chemicals 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 mysid 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 conducted from 25 May to 17 July 2012 at Smithers 

Viscient (SMV), located in Wareham, Massachusetts. The original raw data, protocol, and the 
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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 procedures 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 exposure. 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-generation test is a relatively 

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

reproduction in two generations (F0 and F1). The two-generation test described in the guideline 

is an extension of existing standard practice 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-octylphenol (4-tOP), was received 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 
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Upon receipt at Smithers Viscient, the test substance (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 the 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 obtained from SMV cultures. The brood stock was originally obtained 

from MBL Aquaculture, a commercial supplier located in Sarasota, Florida, and are 

supplemented with animals from reputable sources or suppliers periodically. Culture organisms 

were received on 29 December 2011 and were maintained in-house for approximately 5 months 

prior to exposure initiation. Mysids were cultured using dilute, filtered, natural seawater and six 

80-L glass aquaria under closed-loop, 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 characterized 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 regulated photoperiod of 16 hours of light and 8 hours 

of darkness. Culture conditions and test conditions 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 initiation. Young produced by these organisms were 

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

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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 F0

and F1) 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 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 

concentrated 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 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 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. 

Prior to use of a new batch of feed, a representative sample of the brine 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 food 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 quality since all analyte concentrations were 

below levels of concern and control organisms exceeded guideline criteria. 
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8.5 Dilution Water 

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

Seawater was collected from the Cape Cod Canal 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 housing) and a polyvinyl chloride (PVC) pipe and was 

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

seawater was transferred to an 8500-L epoxy-coated concrete tank, where it was diluted to a 

salinity of 20 ± 3‰ with laboratory 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 series 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 proper equilibration of dissolved gases with the 

laboratory atmosphere and heated to test temperature prior to delivery to the intermittent-flow 

proportional diluter system. The seawater used for 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 were analyzed periodically for the presence of 

pesticides, PCBs and toxic metals by GeoLabs, Inc., 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 analyzed monthly for total organic 

carbon (TOC) concentration. The TOC concentration 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 below 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 throughout 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 preparative vessel containing 3-mm glass beads. The 

bottom end of the preparative vessel 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 coating of 4-tert-octylphenol on the glass beads within the vessel. 

Once the acetone was removed from the vessel, it was connected to a dilution water source at a 

flow rate of 8 mL/min and allowed to flush at this flow rate for 24 hours prior to use. Water 

flowed continuously through this vessel until it was 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. This solution was used as the diluter stock. Prior 

to exposure initiation, the concentration of the outflow solution from the saturation vessel was 

measured to be approximately 5.0 mg/L. The solution produced by the 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 measuring 20 x 10 x 12 cm (L x W x H) equipped 

with side drain at 4.5 cm. The total solution volume maintained in each vessel was 

approximately 9 L. The treatment aquaria were randomly distributed between two tiers (upper 
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and lower tier) of water baths, the upper tier maintained two replicates (A and B) and the lower 

tier maintained one replicate (C). The control vessels 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 constructed of 10-cm glass Petri dishes to which a 15-cm high cylinder 

of nylon mesh screen (250-μm mesh) had been attached with 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 evaluation; these mysids were held in these retention chambers 

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

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

chamber until Day 14. 

8.8.3 Brood Chambers 

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

constructed of 6-cm glass Petri dishes to which 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 control vessels indicated 

inconsistent contamination of control vessels with 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 initially calibrated to deliver 6.6 mL/cycle of an 

8.5 mg/L stock solution into the diluter's chemical 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 exposure vessels indicated that approximately 40% 

loss at each concentration was occurring due to the volatility of 4-tert-octylphenol under aqueous 

conditions. In order to compensate for these losses, the stock delivery was increased. For this 

adjustment, the FMI pump was recalibrated to deliver 13.2 mL/cycle of a 5 mg/L stock solution 

into the mixing cell chamber. 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 

mixing chamber constituted the highest nominal concentration (10 μg/L). This solution was 

subsequently diluted (40%) to provide the remaining nominal exposure concentrations (4.0, 1.6, 

0.64 and 0.26 μg/L). For the control diluter, a set of control vessels were established which 

contained the same dilution water. The control diluter was maintained under the same conditions 

as the treatment level vessels, but contained no 4-tert-octylphenol. Three replicates were 

maintained for all treatments and the control. 

For both exposure systems, a flow-splitting chamber 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 promote mixing of the test substance solution and diluent water. 

In each chamber, three standpipes with umbrella 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 vessels at a rate 

of approximately seven exposure vessel volume 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 the test that the diluter calibration 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 cycle rate, toxicant stock flow, etc.) and a visual 

inspection was performed at the beginning 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 measured with a VWR light meter once during the 

exposure. Two Process Technology heaters were set 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 were 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‰ salinity) 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 exposures were terminated prematurely due to low 

and variable measured concentration and control contamination with 4-tert-octylphenol. 
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8.10.2 Exposure Initiation 

The exposure was not initiated until the diluters and test substance 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 initiation. The exposure was initiated when the retention chambers 

(growth and reproduction) containing F0 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 (F0) 

8.10.3.1.1F0 Exposure Initiation 

Three replicate aquaria per treatment were used 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 through 7 days after release 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 cultures; (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 distribution, mysids in each intermediate beaker were 

then transferred to a labeled retention 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, 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 added to the respective exposure vessel. 

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

were to be obtained from the culture for growth 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 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 drying 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 digital photographs using the following software and 

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

software. 

8.10.3.1.2F0 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 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 sample) 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 the 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 preventing 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. 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 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. 

8.10.3.1.3F0 Reproduction Chambers 

Mysids maintained within reproduction chambers were observed daily for survival, sex 

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

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

had developed marsupial pouches. At sexual maturity (designated by the 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 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 the reproductive phase of the exposure. Prior to the 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 until F0 termination, offspring were enumerated daily. Offspring 

from the F0 mysids (used to establish the F1 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 F0 exposure was terminated seven days after the median day of release of the 

second brood release in the control treatments. 

At exposure termination, F0 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 individual 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 

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

8.10.3.2 Exposed First Filial Generation (F1) 

8.10.3.2.1 Initiation of F1 Generation 

Between exposure days 16 and 27, groups of 15 < 24-hour old offspring (F1), from either the first 

(F1') or second (F1

"

) brood of the F0 mysids, were isolated in growth and reproduction chambers 

(as previously described). F1 offspring were exposed to the same test substance concentrations 

as their F0 parents. Mixing of young from replicate exposure tanks released on the same day 

across replicate chambers was encouraged to minimize reproduction between siblings. When F1

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

then growth chambers. 

In addition, day 0 F1 mysids (nine groups of five organisms) were collected from each 

F0 exposure concentration, euthanized in cold-water deionized water, 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 sample) and weighed to the nearest 0.01 mg on an 

analytical balance. 
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8.10.3.2.2F1 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 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 

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

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

not used to complete reproductive 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 length 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 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. 

8.10.3.2.3F1 Reproduction Chambers 

Mysids maintained within reproduction chambers were observed daily for survival, sex 

determination and reproductive output. Between the respective days 9 to 13 for the F1

generation, developing embryos could be discerned within individual females at most treatment 

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

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

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

of seven pairs were isolated in brood chambers for each replicate tank (Figure 37). Mysids that 
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were not paired remained in reproduction chambers 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 the 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 F1 mysids were removed, enumerated, and 

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

was terminated seven days after the median day of release of the second brood in the F1 control 

tanks. 

At exposure termination, F1 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 individual 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 

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-octylphenol. 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 F1 control vessels), each 

replicate vessel was sampled. Since the initiation 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 approximately midway between the water surface, 

bottom, and sides of each test vessel and processed 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 prepared about once per week, therefore the stock was 

sampled once during the termination period. 

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

concentrations of 0.200, 1.67 and 10.0 μg/L. These concentrations were intended to approximate 

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

solution samples and remained with the exposure solution samples throughout the analytical 

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

quality control maintained during the analysis of exposure solution samples. 

All exposure solutions and QC samples were analyzed for 4-tert-octylphenol using liquid 

chromatography with mass spectrometry (LC/MS/MS) based on methodology validated at 

Smithers Viscient. The method validation was conducted 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 validation with the exception of days 6 and 10. On these days, due to 

excessive variability in the recovery of the internal standard, the results 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 recoveries of the six QC samples 
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were all acceptable for those intervals and indicate that the calibration standards were 

quantifying properly. 

8.10.5 Water Quality Measurements 

On day 0, temperature, dissolved oxygen concentration, pH and salinity were measured in each 

replicate of each treatment and the control solutions. Following day 0, these parameters were 

measured in alternating replicates 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 nominal 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 measured with a Yellow Springs Instrument (YSI) 

Model 30 conductivity/salinity meter. Dissolved oxygen concentration and daily temperature 

were measured using a YSI Model 550A dissolved 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 recorded at exposure initiation and daily thereafter 

until termination of the F1 generation (day 53). Dead organisms were recorded and removed 

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

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

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

The primary biological observations made during the exposure were: 

Parental Generation (F0) 

 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 initiation to release of young 

in the first brood 

 Time to second brood release, number of days 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

First Filial Generation (F1) 

 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 initiation to release of young 

in the first brood 

 Time to second brood release, number of days 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

9.0 Statistical Methods 

9.1 Endpoints 

 Parental Generation (F0) 

 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 two broods in each replicate population 

 Sex ratio 

 Percentage of females that were reproductively active 

 First Filial Generation (F1) 

 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 two 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 certainty 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 certainty was applied. The following 

procedures were used: 

 Prior to statistical analysis, data representing endpoints obtained as proportions 

(e.g., percentage of females that are reproductively active, survival, etc.) were 

transformed using the arcsine square-root transformation.

 As a check on the assumption of homogeneity 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 distributed. Since Bartlett’s Test 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 distribution with a normal distribution. The 
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assumption that observations are normally distributed must be validated before 

subsequent analyses, following parametric procedures, can be performed. 

 All endpoints were analyzed using parametric procedures with one exception: F0

time to maturation. The data for F0 time to maturation had no variance for any 

concentration or control; therefore, parametric procedures could not be used. 

These data were compared with the performance of the control data using Dunn's 

Test with Bonferroni-Holm Adjustment (U.S. EPA, 2002). 

 Significant treatment effects for all remaining endpoints were determined from 

Dunnett's Multiple Comparison Test (U.S. EPA, 2002). 

CETISTM version 1.8.4.20 (Ives, 2011) was used to perform the statistical computations. 

10.0 PERFORMANCE CRITERIA 

The following performance criteria were used 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 F0 or F1 controls must produce young. 

 The average number of young produced per control 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 test were unaffected by the concentrations of 

4-tert-octylphenol tested and remained within acceptable limits for the survival, reproduction and 

growth of mysids (Table 11). Daily measurements established dissolved oxygen concentrations 

that were ≥ 91% of saturation at exposure initiation and maintained at ≥ 75% of saturation 

throughout the remainder of the test. These measurements also determined a pH range of 7.6 to 

8.3; a temperature range of 25 to 27 oC; and a salinity range of 19 to 21‰. Continuous 
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temperature monitoring of the control established 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 period 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 chamber and chemical cells) or exposure solutions 

throughout the exposure period. 

During the in-life exposure, test solutions were sampled for concentrations 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 exposure concentration approximated the nominal doses. 

Analysis of the test solutions throughout the exposure 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 active 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 mean 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) and 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 recoveries outside the 

predetermined recovery range at 10.2 and 224% recovery. QC samples can be out of range due 

to a number of factors, some of which are spiking errors, handling errors and instrument errors. 

11.3 Biological Results 

11.3.1 F0 Generation 

11.3.1.1 F0 Survival 

Mean percent survival in the control for the F0 generation averaged 79% (Table 14 and 

Figure 39). 

Mean percent survival among F0 mysids exposed to the treatment levels tested ranged from 70 to 

81%. Dunnett’s Multiple Comparison Test (control > treatments) determined no significant 

differences in survival among mysids exposed to any of the treatment levels tested, compared to 

the control. 

11.3.1.2 F0 Growth 

Growth measurements were determined at three time points during the F0 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) – F0 Total Body Length and Dry Body Weight 

Day 0 mean total body length for the F0 generation was 1.70 mm and mean dry body weight for 

the F0 generation was 0.034 mg (Table 14). 
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11.3.1.2.2 Day 7 - F0 Total Body Length and Dry Body Weight 

Day 7 mean total body length in the control for the F0 generation averaged 3.47 mm (Table 14

and Figure 40). Mean total body length among F0 mysids exposed to the 0.26, 0.64, 1.6, 4.0 and 

10 μg/L nominal treatment levels averaged 4.06, 3.93, 3.89, 3.81 and 3.93 mm, respectively. 

Dunnett’s Multiple Comparison Test (control < treatments) determined a significant difference 

(p ≤ 0.05) in total body length among mysids exposed 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 F0 generation averaged 0.08 mg (Table 14 and 

Figure 41). Mean dry body weight among F0 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 body weight among mysids exposed to any of the 

treatment levels tested, compared to the control. 

11.3.1.2.3 Day 14 - F0 Total Body Length and Dry Body Weight 

Day 14 mean male total body length in the control for the F0 generation averaged 5.33 mm 

(Table 14 and Figure 42). Mean total body length among F0 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 Test (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 tested compared to the control. 

Day 14 mean female total body length in the control for the F0 generation averaged 5.37 mm 

(Table 14 and Figure 42). Mean total body length among F0 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 Test (control < treatments) determined a 

significant difference (p ≤ 0.05) in female total body length among mysids exposed to the 

0.26 μg/L nominal treatment level compared to the control. 
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Day 14 mean male dry body weight in the control for the F0 generation averaged 0.42 mg 

(Table 14 and Figure 43). Mean dry body weight among F0 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 Test (control < treatments) determined a 

significant difference (p ≤ 0.05) in male dry body weight among 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 F0 generation averaged 0.50 mg 

(Table 14 and Figure 43). Mean dry body weight among F0 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 significant differences in female dry body weight among 

mysids exposed to any of the treatment levels tested, compared to the control. 

11.3.1.2.4 Day 30 (F0 Termination) - Total Body Length and Dry Body Weight 

Day 30 mean male total body length in the control for the F0 generation averaged 7.23 mm 

(Table 14 and Figure 44). Mean total body length among F0 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 significant differences in male total body length among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 30 mean female total body length in the control for the F0 generation averaged 7.61 mm 

(Table 14 and Figure 44). Mean total body length among F0 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 significant differences in female total body length among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 30 mean male dry body weight in the control for the F0 generation averaged 0.82 mg 

(Table 14 and Figure 45). Mean dry body weight among F0 male mysids exposed to the 
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treatment levels tested ranged from 0.83 to 0.85 mg. Dunnett's Multiple Comparison Test 

(control < treatments) determined no significant differences in male dry body weight among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 30 mean female dry body weight in the control for the F0 generation averaged 1.11 mg 

(Table 14 and Figure 45). Mean dry body weight among F0 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 significant differences in female dry body weight among 

mysids exposed to any of the treatment levels tested, compared to the control. 

11.3.1.3 F0 Time to Maturation 

Mean time to maturation for both the F0 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 significant differences in time to maturation among mysids exposed to 

any of the treatment levels tested, compared to the control. 

11.3.1.4 F0 Time to First Brood Release 

Mean time to first brood release for the F0 control mysids averaged 19.0 days (Table 15 and 

Figure 47). Mean time to first brood release for the F0 mysids exposed to the treatment levels 

tested ranged from 17.5 to 19.9 days. Dunnett's Multiple 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 F0 Number of Offspring in the First Brood 

The mean total number produced in the first brood from the F0 control mysids averaged 

6.4 offspring/female (Table 15 and Figure 48). The mean total number produced in the first 

brood from the F0 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 F0 Time to Second Brood Release 

Mean time to second brood release for the F0 control mysids averaged 23.5 days (Table 15 and 

Figure 49). Mean time to second brood release for the F0 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 F0 Number of Offspring in the Second Brood 

The mean total number of offspring produced in the second brood from the F0 control mysids 

averaged 8.7 offspring/female (Table 15 and Figure 50). The mean total number produced in the 

second brood from the F0 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 offspring produced in the second brood among mysids 

exposed to any of the treatment levels tested, compared to the control. 

11.3.1.8 F0 Interbrood Duration 

Mean F0 interbrood duration for the control 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 nominal treatment levels 

compared to the control. 
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11.3.1.9 F0 Total Number of Offspring in First Two Broods 

The total mean number of offspring released in the first two broods for F0 control mysids 

averaged 14.3 offspring/female (Table 16 and Figure 52). The total mean number released in the 

first two broods for F0 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 among mysids exposed to all treatment levels tested compared to 

the control. 

11.3.1.10 F0 Sex Ratio (Male:Female) 

The F0 male to female sex ratio in the control averaged 1.8 (Table 16 and Figure 53). The F0

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 any of the treatment levels tested, compared to 

the control. 

11.3.1.11 F0 Percent of Reproductively Active Females 

The mean percent of reproducing females in the F0 control averaged 95% (Table 16 and 

Figure 54). Mean percent of reproducing F0 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 mean percent of reproducing F0

females among mysids exposed to any of the treatment levels tested, compared to the control. 
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11.3.2 F1 Generation 

11.3.2.1 F1 Survival 

Mean percent survival in the control for the F1 generation averaged 78% (Table 17 and 

Figure 55). Mean percent survival among F1 mysids exposed to the treatment levels tested 

ranged from 69 to 86%. Dunnett’s Multiple Comparison 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 F1 Growth 

Growth measurements were determined at four time points during the F1 exposure. Mysid dry 

body weight and mysid total length were measured at the respective day 0, 7, 14 and 27 for the 

F1 generation. 

11.3.2.2.1 Day 0 (Exposure Initiation) – F1 Total Body Length and Dry Body Weight 

Day 0 mean total body length in the control for the F1 generation averaged 1.61 mm (Table 17

and Figure 56). Mean total body length among F1 mysids exposed to the treatment levels tested 

ranged from 1.58 to 1.61 mm. Dunnett’s Multiple Comparison Test (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 F1 generation averaged 0.023 mg (Table 17

and Figure 57). Mean dry body weight among F1 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 body 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 - F1 Total Body Length and Dry Body Weight 

Day 7 mean total body length in the control for the F1 generation averaged 4.45 mm (Table 17

and Figure 58). Mean total body length among F1 mysids exposed to the treatment levels tested 

ranged from 4.47 to 4.94 mm. Dunnett’s Multiple Comparison Test (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 F1 generation averaged 0.25 mg (Table 17 and 

Figure 59). Mean dry body weight among F1 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 body weight among mysids exposed to any of the 

treatment levels tested, compared to the control. 

11.3.2.2.3 Day 14 - F1 Total Body Length and Dry Body Weight 

Day 14 mean male total body length in the control for the F1 generation averaged 5.96 mm 

(Table 17 and Figure 60). Mean total body length among F1 male mysids exposed to the 

treatment levels tested ranged from 6.04 to 6.88 mm. Dunnett's Multiple Comparison Test 

(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 14 mean female total body length in the control for the F1 generation averaged 6.11 mm 

(Table 17 and Figure 60). Mean total body length among F1 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 significant differences in female total body length among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 14 mean male dry body weight in the control for the F1 generation averaged 0.51 mg 

(Table 17 and Figure 61). Mean dry body weight among F1 male mysids exposed to the 
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Smithers Viscient Study No. 13888.6111 Page 268 

treatment levels tested ranged from 0.45 to 0.57 mg. Dunnett's Multiple Comparison Test 

(control < treatments) determined no significant differences in male dry body weight among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 14 mean female dry body weight in the control for the F1 generation averaged 0.62 mg 

(Table 17 and Figure 61). Mean dry body weight among F1 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 significant differences in female dry body weight among 

mysids exposed to any of the treatment levels 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 length in the control for the F1 generation averaged 7.03 mm 

(Table 17 and Figure 62). Mean total body length among F1 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 significant differences in male total body length among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 27 mean female total body length in the control for the F1 generation averaged 7.52 mm 

(Table 17 and Figure 62). Mean total body length among F1 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 significant differences in female total body length among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 27 mean male dry body weight in the control for the F1 generation averaged 0.83 mg 

(Table 17 and Figure 63). Mean dry body weight among F1 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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Smithers Viscient Study No. 13888.6111 Page 269 

(control < treatments) determined no significant differences in male dry body weight among 

mysids exposed to any of the treatment levels tested, compared to the control. 

Day 27 mean female dry body weight in the control for the F1 generation averaged 1.25 mg 

(Table 17 and Figure 63). Mean dry body weight among F1 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 significant differences in female dry body weight among 

mysids exposed to any of the treatment levels tested, compared to the control. 

11.3.2.3 F1 Time to Maturation 

Mean time to maturation for the F1 control mysids averaged 9.0 days (Table 18 and Figure 64). 

Mean time to maturation among mysids exposed to the treatment levels tested ranged from 8.0 to 

10.3 days. Dunnett’s Multiple Comparison Test (control < treatments) determined no significant 

differences in time to maturation among mysids exposed to any of the treatment levels tested, 

compared to the control. 

11.3.2.4 F1 Time to First Brood Release 

Mean time to first brood release for the F1 control mysids averaged 16.3 days (Table 18 and 

Figure 65). Mean time to first brood release for the F1 mysids exposed to the treatment levels 

tested ranged from 15.7 to 18.0 days. Dunnett's Multiple 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 F1 Number of Offspring in the First Brood 

The mean total number produced in the first brood from the F1 control mysids averaged 

6.9 offspring/female (Table 18 and Figure 66). The mean total number produced in the first 

brood from the F1 mysids exposed to the treatment levels tested ranged from 4.7 to 
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Smithers Viscient Study No. 13888.6111 Page 270 

8.1 offspring/female. Dunnett's Multiple Comparison 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. 

11.3.2.6 F1 Time to Second Brood Release 

Mean time to second brood release for the F1 control mysids averaged 21.1 days (Table 18 and 

Figure 67). Mean time to second brood release for the F1 mysids exposed to the treatment levels 

tested ranged from 20.6 to 23.1 days. Dunnett’s Multiple Comparison Test 

(control < treatments) determined no significant 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 F1 Number of Offspring in the Second Brood 

The mean total number produced in the second brood from the F1 control mysids averaged 

8.6 offspring/female (Table 18 and Figure 68). The mean total number produced in the second 

brood from the F1 mysids exposed to the treatment levels tested ranged from 7.4 to 

11 offspring/female. Dunnett's Multiple Comparison 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 F1 Interbrood Duration 

Mean F1 interbrood duration for the control mysids averaged 5.3 days (Table 18 and Figure 69). 

Mean interbrood duration for mysids exposed to the treatment 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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Smithers Viscient Study No. 13888.6111 Page 271 

11.3.2.9 F1 Total Number of Offspring in First Two Broods 

The total mean number released in the first two broods for F1 control mysids averaged 

12.6 offspring/female (Table 19 and Figure 70). The total mean number released in the first two 

broods for F1 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 compared to the control. 

11.3.2.10 F1 Sex Ratio (Male:Female) 

The F1 male to female sex ratio in the control averaged 1.2 (Table 19 and Figure 71). The F1

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 (control > treatments) determined no significant 

differences in sex ratio among mysids exposed to any of the treatment levels tested, compared to 

the control. 

11.3.2.11 F1 Percent of Reproductively Active Females 

The mean percent of reproducing females in the F1 control averaged 89% (Table 19 and 

Figure 72). Mean percent of reproducing F1 females among mysids exposed to the treatment 

levels tested ranged from 94 to 100%. Dunnett's Multiple Comparison Test 

(control < treatments) determined 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 F0 generation appeared to be more sensitive (seven endpoints impacted) to 

the concentrations of 4-tOP than the F1 generation (one endpoint impacted). A summary of the 

responses for each endpoint in each generation is presented below. 
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Smithers Viscient Study No. 13888.6111 Page 272 

The most sensitive endpoints in the F0 generation were determined to be 1st brood 

(offspring/female), total offspring/female and Day 7 length. While standard statistical analysis 

determined that these endpoints were the most sensitive, a hormetic response was observed in a 

number of F0 endpoints (F0 1st brood, offspring per female; F0 Day 14 male dry weight, F0 Day 

14 female dry weight, F0 interbrood duration, F0 sex ratio, F0 time to 1st brood, F0 time to 2nd

brood). Hormesis best explains 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 F1 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 (F1

Day 7 dry weight, sex ratio, time to maturation, total young). Also, this response was not 

determined to be statistically significant at any dose for any endpoint. 

While a dose-response relationship 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 F0

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. 

Table 20 summarizes the F0 and F1 endpoints and the statistically significant differences from the 

controls determined during this study. 
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Smithers Viscient Study No. 13888.6111 Page 273 

PROTOCOL DEVIATIONS 

1. The protocol states that during the test, mysids from both 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 performance criteria, this deviation did not have a 

negative impact on the results or interpretation of the study. 

2. The protocol states that the temperature 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. Temperatures exceeding 27.6 oC were 

not observed. Since the temperatures were within the range tolerated by this species, this 

deviation did not have a negative impact on the results or interpretation 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 intensity 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 intensity was measured in the control diluter after 

exposure termination. These readings indicated that the light intensities should have been 

within the specified range during testing. Since 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 results or interpretation of this study. 
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Smithers Viscient Study No. 13888.6111 Page 274 

REFERENCES 

ASTM, 2002. Standard practice for conducting acute toxicity test with fishes, 

macroinvertebrates, and amphibians. Standard E729-96. American Society for Testing 

and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428. 

ASTM, 2008. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide 

for Conducting Life-Cycle Toxicity Tests with Mysids. ASTM Designation E 1191-039 

(re-approved 2008). American Society for Testing and Materials, 100 Barr Harbor Drive, 

West Conshohocken, Pennsylvania. 

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. 

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 temperature 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-specific 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 simplified dosing apparatus for fish toxicological 

studies. Water Research. 1: 21-29. 

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

an estuarine species suitable for life-cycle toxicity tests to determine the effects of a 

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

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

109-116. 

Nimmo DR, Hamaker TL and Sommers CA., 1978. Entire life cycle toxicity test using mysids 

(Mysidopsis bahia) in flowing water. In: Bioassay Procedures for the Ocean Disposal 

Permit Program, EPA-600/9-78-010, U.S. Environmental Protection Agency, 

Environmental Research Laboratory, Gulf Breeze, FL, pp. 64-68. 
RTI Project No. 0210114.030 EPA Contract 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 organisms 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 Estimating the Chronic Toxicity of Effluents and 

Receiving Waters to Marine and Estuarine Organisms. 3rd Edition. EPA-821-R-02-014, 

U.S. Environmental Protection Agency, Washington, DC. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 276 

Table 11. Summary of water quality parameters measured in the exposure 

solutions during the mysid (Americamysis bahia) two-generation 

test with 4-tert-octylphenol. 

Nominal 

Concentration 

(μg/L) 

Rangesa

Dissolved 

Oxygen pH Temperatureb

(

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 the 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 rounded to two significant figures. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 12. Concentrations of 4-tert-octylphenol measured in the exposure solutions during t

(Americamysis bahia) two-generation test.

Nominal 

Concentration 

(μg/L) 

Measured Concentration (μg/L) 

Day 0 Day 6 Day 10 Day 13 Day 20 Day 27 Day 34 Day 41 Day 43 Day 45 Day 46 Day 48 Day 49 Day 50 Day 53 

M

(S

Control 

<0.070b

<0.070 <0.070 

<0.082 <0.092 <0.077 <0.083 <0.069 <0.095 <0.082 NS NSc <0.089 <0.089 NS <0.077 NS NS N

(N

0.26 

0.40 0.44 0.37 

0.42 0.18 0.27 0.28 0.36 0.38 0.48 NS 0.34 0.36 NS NS NS NS 0.34 0

(0

0.64 

1.1 1.2 1.2 

1.2 0.63 0.75 0.78 1.0 0.85 1.1 0.90 0.87 NS 1.1 NS NS NS 0

(0

1.6 

1.9 2.1 2.1 

2.0 1.3 1.5 1.5 1.8 2.4 2.4 NS 1.8 NS 2.4 2.3 NS NS 

(0

4.0 

4.3 4.6 4.1 

4.4 2.8 3.3 3.2 4.0 4.3 3.8 3.9 3.5 NS NS NS 4.6 NS NS 

(0

10 

12 11 11 

12 8.0 7.6 8.1 11 12 12 NS 12 NS NS 12 10 NS 

(

a Mean measured values, standard deviations (SD) and percent of nominal were calculated using the actual analytical results and not the rounded values (two significab Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area of th

factor of the controls. 

c NS = Not Sampled. d NA = Not Applicable. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 278 

Table 13. Results of the quality control (QC) samples analyzed concurrently 

with test solution samples during the mysid (Americamysis bahia) 

two-generation test.

Quality Control Sample (μg/L) 

0.200a 1.67a 10.0a

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 224b 1.78 107 1.18 118 

48 0.226 113 1.71 102 1.02 10.2b 

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 outside of the acceptable range (i.e., 70 to 120%, Appendix 7). 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 14. F0 mysid survival and growth (length and dry weight) during the mysid (America

generation test with 4-tert-octylphenol. 

Nominal 

Concentration 

(μg/L) 

Replicate Survival (%) p-Value 

Mean Total Body Length (mm)a

Day 7 p-Value 

Day 14 

Male p-Value Female p-Value Male p-Va

Control A 79 3.40 5.25 5.43 7.09 

 B 70 3.70 5.57 5.30 7.33  C 88 3.31 5.16 5.37 7.25 

Mean(SD) 79(8.9) NAc 3.47(0.21) NA 5.33(0.22) NA 5.37(0.07) NA 7.23(0.12) NA 

0.26 A 76 3.82 5.69 5.64 7.06 

 B 70 4.31 5.77 5.60 7.42  C 65 4.05 5.88 6.33 7.35 

Mean(SD) 70(5.2) 0.3539 4.06(0.24)d 0.0027 5.78(0.09)d 0.0236 5.85(0.41)d 0.0229 7.28(0.19) 0.912

0.64 A 81 4.11 5.60 5.64 7.07 

 B 83 3.78 5.73 5.69 7.19  C 67 3.90 5.79 5.80 7.33 

Mean(SD) 77(9.1) 0.7545 3.93(0.17)d 0.0138 5.71(0.09) 0.0537 5.71(0.09) 0.1008 7.20(0.13) 0.780

1.6 A 79 3.99 5.39 5.64 6.98 

 B 83 3.86 5.75 5.85 7.34  C 76 3.83 5.69 5.52 7.35 

Mean(SD) 79(3.5) 0.8434 3.89(0.09)d 0.0218 5.61(0.19) 0.1467 5.67(0.16) 0.1464 7.22(0.21) 0.833

4.0 A 79 3.75 5.30 5.55 7.34 

 B 70 4.02 5.92 5.73 6.81  C 94 3.66 5.43 5.73 7.33 

Mean(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) 0.692

10 A 66 4.01 5.83 NA 6.86 

 B 81 3.82 5.96 5.81 6.98  C 68 3.95 5.83 5.73 7.18 

Mean(SD) 71(8.2) 0.4127 3.93(0.09)d 0.0144 5.88(0.07)d 0.0081 5.77(0.06) 0.0852 7.00(0.16) 0.285

a Mean total body length for the control on day 0 = 1.70 mm; standard deviation = 0.084. b Mean dry body weight for the control on day 0 = 0.034 mg; standard deviation = 0.0048. c NA = Not Applicable. d Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 14. Continued. F0 mysid survival and growth (length and dry weight) during the mysi

bahia) two-generation test with 4-tert-octylphenol. 

Nominal 

Concentration 

(μg/L) 

Replicate 

Mean Dry Body Weight (mg)b

Day 7 p-Value Day 14 D

Male p-Value Female p-Value Male p-Value 

Control A 0.06 0.37 0.44 0.81 

 B 0.12 0.52 0.49 0.85  C 0.08 0.37 0.58 0.80 

Mean(SD) 0.08(0.03) NA 0.42(0.09) NA 0.50(0.07) NA 0.82(0.03) NA 

0.26 A 0.12 0.51 0.56 0.82 

 B 0.18 0.57 0.56 0.87  C 0.14 0.52 0.58 0.84 

Mean(SD) 0.14(0.03) 0.2113 0.53(0.04)d 0.0265 0.57(0.01) 0.2757 0.84(0.03) 0.5284 

0.64 A 0.16 0.40 0.60 0.82 

 B 0.07 0.45 0.49 0.85  C 0.14 0.48 0.50 0.86 

Mean(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 

1.6 A 0.22 0.37 0.48 0.83 

 B 0.12 0.39 0.56 0.86  C 0.08 0.43 0.48 0.87 

Mean(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 

4.0 A 0.13 0.46 0.38 0.86 

 B 0.22 0.41 0.50 0.77  C 0.12 0.45 0.47 0.88 

Mean(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 

10 A 0.17 0.55 NA 0.82 

 B 0.12 0.57 0.64 0.79  C 0.14 0.48 0.55 0.89 

Mean(SD) 0.15(0.03) 0.2380 0.53(0.05)d 0.0265 0.60(0.07) 0.1572 0.83(0.05) 0.6743 

a Mean total body length for the control on day 0 = 1.70 mm; standard deviation = 0.084. b Mean dry body weight for the control on day 0 = 0.034 mg; standard deviation = 0.0048. c NA = Not Applicable. d Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 15. F0 mysid time to maturation, time to first brood release, time to second brood rel

released per female and interbrood duration during the mysid (Americamysis bah

test with 4-tert-octylphenol.

Nominal 

Concentration 

(μg/L) 

Replicate 

Time to 

Maturation 

(Days) 

p-Value 

First Brood Second Brood 

Time to Release 

(Days) 

pValue Offspring/Female p- Value 

Time to 

Release 

(Days) 

pValue Offspring/Female 

Control A 10 19.7 6.1 24.4 8.7 

 B 10 18.4 6.6 22.6 7.7  C 10 18.8 6.3 23.5 9.7 

Mean(SD) 10(0) NAa 19.0(0.66) NA 6.4(0.21) NA 23.5(0.90) NA 8.7(1.0) 

0.26 A 10 17.4 4.6 22.6 4.9 

 B 10 16.8 5.7 23.8 3.3  C 11 18.1 5.0 25.4 10.4 

Mean(SD) 10(0.58) 0.9783 17.5(0.70) 0.9960 5.1(0.55)b 0.0489 23.9(1.4) 0.6849 6.2(3.7) 

0.64 A 10 18.7 4.2 22.6 6.0 

 B 10 16.7 5.0 21.6 4.6  C 10 20.0 4.1 24.9 8.8 

Mean(SD) 10(0) 1.0000 18.5(1.7) 0.9405 4.4(0.49)b 0.0045 23.0(1.7) 0.9310 6.5(2.2) 

1.6 A 10 17.6 4.6 23.1 6.4 

 B 10 20.7 4.0 25.2 9.6  C 10 19.4 5.0 24.8 6.2 

Mean(SD) 10(0) 1.0000 19.2(1.6) 0.7409 4.5(0.50)b 0.0064 24.4(1.1) 0.5017 7.4(1.9) 

4.0 A 10 16.9 5.4 23.4 5.3 

 B 10 17.7 7.3 23.1 4.9  C 10 19.9 5.3 25.7 6.5 

Mean(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) 

10 A 10 20.2 5.0 26.6 6.2 

 B 10 19.7 4.9 25.4 5.9  C 10 19.8 4.7 24.7 6.3 

Mean(SD) 10(0) 1.0000 19.9(0.23) 0.4440 4.8(0.17)b 0.0214 25.6(1.0) 0.1180 6.1(0.20) 

a NA = Not Applicable. b Statistically different compared to the control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 282 

Table 16. F0 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 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) NAa 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 control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 17. F1 mysid survival and growth (length and dry weight) during the mysid (America

generation test with 4-tert-octylphenol. 

Nominal 

Concentration 

(μg/L) 

Replicate Survival (%) p- Value 

Mean Total Body Length (mm)

Day 0a p- Value Day 7 p- Value Day 14 

Male p- Value Female p- Value Male 

Control A 74 4.13 5.63 6.23 7.15 

 B 86 4.58 6.12 6.38 6.95  C 75 4.63 6.14 5.73 7.00 

Mean(SD) 78(7.0) NAb 1.61(0.09) NA 4.45(0.28) NA 5.96(0.29) NA 6.11(0.34) NA 7.03(0.11) 

0.26 A 91 4.94 5.72 5.99 7.30 

 B 75 5.23 6.46 7.04 7.08  C 86 4.66 7.32 7.08 7.52 

Mean(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) 

0.64 A 81 4.61 5.84 6.10 7.08 

 B 80 4.42 NA 6.37 6.95  C 68 4.37 6.27 6.55 7.48 

Mean(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) 

1.6 A 78 5.09 6.08 6.55 6.81 

 B 63 5.02 NA 6.76 7.03  C 67 4.11 6.01 6.14 7.30 

Mean(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) 

4.0 A 87 4.91 6.86 7.25 7.47 

 B 86 5.07 7.02 6.57 6.67  C 86 4.62 6.76 6.51 7.42 

Mean(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) 

10 A 95 4.80 6.17 6.33 6.80 

 B 87 4.51 5.72 6.55 7.03  C 78 4.64 6.96 6.96 6.79 

Mean(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) 

a Mean values presented are based on measurement of nine replicates. Individual measurements for these replicates are presented in Appendix 8. b NA = Not Applicable. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 17. Continued. F1 mysid survival and growth (length and dry weight) during the mysi

bahia) two-generation test with 4-tert-octylphenol. 

Nominal 

Concentration 

(μg/L) 

Replicate 

Mean Dry Body Weight (mg)

Day 0a p-Value Day 7 p-Value 

Day 14 

Male p-Value Female p-Value Male 

Control A 0.30 0.58 0.71 0.79 

 B 0.24 0.45 0.52 0.81  C 0.22 0.48 0.64 0.89 

Mean(SD) 0.023(0.0036) NAb 0.25(0.04) NA 0.51(0.07) NA 0.62(0.09) NA 0.83(0.05) 

0.26 A 0.30 0.46 0.72 0.88 

 B 0.32 0.44 0.56 0.82  C 0.25 0.45 0.67 0.89 

Mean(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) 

0.64 A 0.22 0.45 0.57 0.78 

 B 0.34 NA 0.71 0.89  C 0.23 0.50 0.65 0.90 

Mean(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) 

1.6 A 0.28 0.60 0.73 0.71 

 B 0.27 NA 0.53 0.89  C 0.21 0.44 0.61 0.86 

Mean(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) 

4.0 A 0.21 0.50 0.63 0.83 

 B 0.28 0.49 0.70 0.66  C 0.21 0.48 0.59 1.02 

Mean(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) 

10 A 0.25 0.65 0.82 0.92 

 B 0.30 0.41 0.57 0.80  C 0.29 0.64 0.64 0.95 

Mean(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) 

a Mean values presented are based on measurement of nine replicates. Individual measurements for these replicates are presented in Appendix 8. b NA = Not Applicable. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Table 18. F1 mysid time to maturation, time to first brood release, time to second brood rel

released per female and interbrood duration during the mysid (Americamysis bah

test with 4-tert-octylphenol.

Nominal 

Concentration 

(μg/L) 

Replicate 

Time to 

Maturation 

(Days) 

pValue 

First Brood Second Brood 

Time to Release 

(Days) 

pValue Offspring/ Female p- Value Time to Release (Days) p- Value Offspring/ Female 

Control A 9 16.8 8.5 20.7 10.3 

 B 9 15.6 6.2 23.0 5.8  C 9 16.7 6.0 19.8 9.8 

Mean(SD) 9.0 (0) NAa 16.3(0.64) NA 6.9(1.4) NA 21.1(1.7) NA 8.6(2.5) 

0.26 A 9 17.1 6.3 22.2 9.2 

 B 8 16.9 7.3 22.0 11.6  C 8 20.0 11 25.0 12.4 

Mean(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) 

0.64 A 9 15.2 6.0 20.0 7.2 

 B 13 15.4 5.4 21.4 8.0  C 9 16.6 6.6 22.0 11.3 

Mean(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) 

1.6 A 6 17.0 4.4 21.3 9.0 

 B 8 17.0 7.2 21.0 10.0  C 10 16.4 5.6 21.6 10.6 

Mean(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) 

4.0 A 8 17.5 6.7 21.2 9.8 

 B 9 16.3 4.9 21.2 9.3  C 9 16.6 7.9 21.7 11.0 

Mean(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) 

10 A 8 14.5 4.8 18.5 6.2 

 B 9 18.0 4.8 22.3 7.8  C 9 16.6 4.4 21.0 8.3 

Mean(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) 

a NA = Not Applicable. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 286 

Table 19. F1 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 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) NAa

 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 control, based on Dunnett's Multiple Comparison Test. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 287 

Table 20. Summary of F0 and F1 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.

F0 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 1st Brood Release 

1st Brood Offspring/Female ↓ ↓ ↓ ↓

Time to 2nd Brood Release 

2nd Brood Offspring/Female 

Interbrood Duration ↑ ↑ 

Total Offspring/Female ↓ ↓ ↓ ↓ ↓

Sex Ratio 

% Reproducing Females 

↓ Endpoint determined to be statistically significantly reduced (p ≤ 0.05) compared to control 

↑ Endpoint determined to be statistically significantly increased (p ≤ 0.05) compared to control 

F1 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 1st Brood Release 

1st Brood Offspring/Female 

Time to 2nd Brood Release 

2nd 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 288 

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

B B B R

B B B B

B B B R

B B B B

B B B R

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

Smithers Viscient Study No. 13888.6111 Page 289 

Figure 38. Relationship between mean measured concentrations and the 

nominal treatment levels during the mysid (Americamysis bahia) 

two-generation test with 4-tert-octylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 290 

Figure 39. Mean percent survival of F0 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 291 

Figure 40. Day 7 mean total body length of F0 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 292 

Figure 41. Day 7 mean dry body weight of F0 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract 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 F0 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 297 

Figure 46. Mean time to maturation of F0 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 298 

Figure 47. Mean time to first brood release of F0 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 299 

Figure 48. F0 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 300 

Figure 49. Mean time to second brood release of F0 mysids (Americamysis 

bahia) during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 301 

Figure 50. F0 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 302 

Figure 51. F0 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 303 

Figure 52. F0 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 304 

Figure 53. F0 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 305 

Figure 54. F0 Mean percentage of reproductively active females for mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 306 

Figure 55. Mean percent survival of F1 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 307 

Figure 56. Day 0 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 308 

Figure 57. Day 0 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 309 

Figure 58. Day 7 mean total body length of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 310 

Figure 59. Day 7 mean dry body weight of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract 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 F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract 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 F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract 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 F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract 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 F1 mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 315 

Figure 64. Mean time to maturation of F1 mysids (Americamysis bahia) during 

the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 316 

Figure 65. Mean time to first brood release of F1 mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 317 

Figure 66. F1 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 318 

Figure 67. Mean time to second brood release of F1 mysids (Americamysis 

bahia) during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 319 

Figure 68. F1 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 320 

Figure 69. F1 Mean interbrood duration for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 321 

Figure 70. F1 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 322 

Figure 71. F1 Mean male:female sex ratio for mysids (Americamysis bahia) 

during the two-generation test with 4-tert-octylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 323 

Figure 72. F1 Mean percentage of reproductively active females for mysids 

(Americamysis bahia) during the two-generation test with 4-tertoctylphenol. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 324 

APPENDIX 5 - QAPP 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

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Smithers Viscient Study No. 13888.6111 Page 331 
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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 2012) 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 acetonitrile:purified reagent water. All recovery 

samples were analyzed by automated injection by liquid chromatography 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. Recoveries for the LC/MS/MS method analysis 

averaged 103% ± 7.97%, with a limit of quantitation (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, volumetric 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 ASTM Type II requirements) 
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Test Substance 

The test substance, 4-tert-octylphenol, was received 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 substance (SMV No. 5305) was stored at room 

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

for the purity of the test substance. 

Internal Standard 

The internal standard, 4-tert-octylphenol-ring13C6, was received on 5 January 2012 from SigmaAldrich, Inc., Allentown, Pennsylvania. The following information was provided: 

 Name: 4-tert-octylphenol -ring13C6 

 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. Concentrations were not adjusted for the purity of the internal standard.

PROCEDURES

Preparation of Stock Solutions

A 1.00 mg/mL primary stock solution was prepared by placing 0.1020 g (0.1000 g as active 

ingredient) of 4-tert-octylphenol in a 100-mL volumetric flask and bringing it to volume with 

acetonitrile. A 10.0 mg/L secondary stock solution 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 volumetric flask and bringing it to volume with 

acetonitrile. The 10.0 mg/L secondary stock solution was used to prepare the high-level 

recovery samples. Two sub-stock solutions (0.100 and 1.00 mg/L) were prepared by placing 

0.100 and 1.00 mL, respectively, of the 10.0 mg/L secondary 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- and mid-level recovery samples, 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-octylphenol in a 100-mL volumetric flask and bringing it to volume 

with acetonitrile. A 10.0 mg/L secondary stock solution was prepared by placing 0.500 mL of 

the 1.00 mg/mL primary stock solution into a 50.0-mL volumetric 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 and 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 solutions were stored in a refrigerator 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 prepared in 50:50 acetonitrile:purified reagent water by fortifying 

with the 1.00 mg/L sub-stock solution to yield concentrations of 0.500, 1.00, 5.00, 15.0, 20.0 and 

25.0 μg/L. Internal standard (IS) was also fortified 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 samples according to the table below. Each concentration level was 

produced in triplicate. In addition, three samples were left unfortified 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 NAa 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 standards were prepared in 45-mL glass vials with PTFE-lined caps. 

Each sample was fortified in the glass vials and 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 then transferred to disposable 

glass vials and reduced to incipient 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 vortexed 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 transferred 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 

Volume1

(mL) 

Secondary 

Volume 

(mL) 

Final 

Volume2 

(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:purified reagent water 

ANALYSIS

Instrumental Conditions

The LC/MS/MS analysis was conducted utilizing 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 second set immediately following the analysis of the 

recovery samples. Injection of recovery samples and calibration standards onto the 

chromatographic system was performed 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 manipulated to give equation 2. The concentration of the test 

substance within each recovery sample was determined using the regression coefficients from the 
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quadratic equation, the peak area of the recovery sample, and the dilution factor. Equations 2 

and 3 were then used to calculate measured 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 final sample volume divided by the 

original sample volume) 

A = concentration of the analyte in the original sample 

The limit of quantitation (LOQ) was calculated using the following equation: 

INST CTRL

INST

(5) LOQ LOQ x DF

(4) LOQ b b 4aC 2



   

where: 

 

AreaLS = mean detector response (peak area) of the low 

concentration calibration standard (two injections) 

a, b, c = regression constants 

C = regression constant ; C = (c - AreaLS) 

LOQINST = limit of quantitation on the instrument 

DFCTRL = dilution factor of the control samples (smallest dilution 

factor used) 

LOQ = limit of quantitation reported for the analysis 

  3 A DC x DF

2a

4aC 2 b b (2) DC (x)

bx c 2 (1) y ax



   

  
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RESULTS AND DISCUSSION 

The mean recovery was 103% with a standard deviation 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, while 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 from the analysis of a calibration 

standard, recovery sample and a control sample are presented in Figure 7A through Figure 7C, 

respectively. A typical regression analysis for 4-tert-octylphenol 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 < 0.0397a NAb

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 below the limit of quantitation (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 analytical (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 30.00

Detector Response

Concentration

Polynomial Regression Analysis

Polynomial Regression Analysis 

R2

 = 0.99990 

y = 1.8702E-04x2

 + 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 F1 DAY 0 LENGTH AND WEIGHT 

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

Smithers Viscient Study No. 13888.6111 Page 419 

Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 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 Contract 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 

F1 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 Contract 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 

F1 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 Contract 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 

F1 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 Contract 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 

F1 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 Contract 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 

F1 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 Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 425 

APPENDIX 9 - POST-PAIRING SURVIVAL DATA - 4-TERTOCTYLPHENOL 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

Control 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 19 20 5 20 20 6 20 20 7 20 20 8 20 20 9 19 1 20 

10 17 18 2 2 11 17 18 2 2 12 16 1 18 2 2 

13* 10 12 8 8 100 100 

14 9 11 9 9 100 100 15 9 11 9 9 100 100 16 9 11 9 9 100 100 17 9 11 9 9 100 100 18 9 11 9 9 100 100 19 9 11 9 9 100 100 20 9 11 9 9 100 100 21 9 11 9 9 100 100 22 9 11 9 9 100 100 23 9 11 9 9 100 100 24 9 11 8 1 9 100 89 25 8 1 11 8 9 89 89 26 8 11 8 9 89 89 27 8 11 8 9 89 89 28 8 11 8 9 89 89 29 6 1 1 10 8 9 75 89 30 5 1 10 8 9 63 89 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

Control 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 19 1 20 5 19 20 6 19 20 7 19 20 8 19 20 9 17 2 20 

10 15 18 2 2 11 14 17 3 3 12 13 16 4 4 

13* 9 12 8 8 100 100 

14 9 12 8 8 100 100 15 9 12 8 8 100 100 16 9 12 8 8 100 100 17 9 12 8 8 100 100 18 9 12 8 8 100 100 19 9 12 8 8 100 100 20 8 1 12 8 8 89 100 21 8 12 8 8 89 100 22 8 12 8 8 89 100 23 8 12 8 8 89 100 24 7 1 12 8 8 78 100 25 7 12 7 1 8 78 88 26 7 12 7 8 78 88 27 7 12 7 8 78 88 28 7 12 6 1 7 78 86 29 7 12 6 7 78 86 30 7 12 6 7 78 86 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

Control 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 16 4 20 5 16 20 6 16 20 7 16 20 8 16 20 9 16 20 

10 14 18 2 2 11 14 18 2 2 12 14 18 2 2 

13* 7 10 10 10 100 100 

14 7 10 10 10 100 100 15 7 10 10 10 100 100 16 7 10 10 10 100 100 17 7 10 10 10 100 100 18 7 10 10 10 100 100 19 7 10 10 10 100 100 20 7 10 10 10 100 100 21 7 10 10 10 100 100 22 7 10 10 10 100 100 23 7 10 10 10 100 100 24 7 10 10 10 100 100 25 7 10 10 10 100 100 26 7 10 10 10 100 100 27 6 1 9 10 10 100 100 28 6 9 10 10 100 100 29 6 9 10 10 100 100 30 5 1 8 10 10 100 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

0.26 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 20 20 

10 17 17 3 3 11 16 16 4 4 12 15 1 16 4 4 

13* 11 12 8 8 100 100 

14 11 12 8 8 100 100 15 11 12 8 8 100 100 16 11 12 8 8 100 100 17 10 1 12 7 1 8 91 88 18 10 12 7 8 91 88 19 9 1 12 7 8 82 88 20 9 12 7 8 82 88 21 8 1 12 7 8 73 88 22 8 12 7 8 73 88 23 8 12 7 8 73 88 24 8 12 7 8 73 88 25 8 12 7 8 73 88 26 8 12 7 8 73 88 27 8 12 7 8 73 88 28 7 1 11 7 8 70 88 29 7 11 7 8 70 88 30 7 11 7 8 70 88 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

0.26 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 19 1 20 3 19 20 4 18 1 20 5 18 20 6 18 20 7 18 20 8 18 20 9 18 20 

10 16 18 2 2 11 15 17 3 3 12 15 17 3 3 

13* 10 1 13 7 7 100 100 

14 9 12 8 8 100 100 15 9 12 8 8 100 100 16 9 12 8 8 100 100 17 9 12 7 1 8 100 88 18 7 1 11 8 9 88 89 19 7 11 8 9 88 89 20 6 1 10 8 9 86 89 21 6 10 8 9 86 89 22 6 10 8 9 86 89 23 6 10 8 9 86 89 24 5 1 9 8 9 83 89 25 4 1 9 8 9 67 89 26 4 9 7 1 9 67 78 27 4 9 7 9 67 78 28 3 1 8 6 1 9 60 67 29 2 1 7 6 9 50 67 30 2 7 6 9 50 67 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

0.26 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 18 1 20 3 18 20 4 18 20 5 18 20 6 18 20 7 18 20 8 18 20 9 18 20 

10 18 19 1 1 11 17 18 2 2 12 13 14 6 6 

13* 10 11 9 9 100 100 

14 9 10 10 10 100 100 15 9 10 10 10 100 100 16 9 10 10 10 100 100 17 6 1 8 11 1 12 86 92 18 6 8 11 12 86 92 19 6 8 11 12 86 92 20 6 8 10 1 11 86 91 21 6 8 10 11 86 91 22 6 8 10 11 86 91 23 6 8 10 11 86 91 24 5 7 11 12 83 92 25 5 7 9 2 10 83 90 26 5 7 9 10 83 90 27 4 1 6 9 10 80 90 28 4 6 8 1 9 80 89 29 4 6 8 9 80 89 30 3 1 6 6 2 7 60 86 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

0.64 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 19 20 3 19 20 4 19 20 5 19 20 6 19 20 7 19 20 8 19 20 9 19 20 

10 16 17 3 3 11 16 17 3 3 12 15 16 4 4 

13* 8 9 11 11 100 100 

14 8 9 11 11 100 100 15 8 9 11 11 100 100 16 8 9 11 11 100 100 17 8 9 10 1 11 100 91 18 8 9 10 11 100 91 19 8 9 10 11 100 91 20 7 1 9 10 11 88 91 21 7 9 9 1 11 88 82 22 7 9 9 11 88 82 23 7 9 9 11 88 82 24 7 9 9 11 88 82 25 7 9 9 11 88 82 26 7 9 9 11 88 82 27 6 1 8 8 1 10 86 80 28 6 8 8 10 86 80 29 6 8 8 10 86 80 30 6 8 8 10 86 80 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

0.64 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 18 1 20 9 18 20 

10 18 20 11 16 18 2 2 12 16 17 3 3 

13* 9 10 10 10 100 100 

14 10 11 9 9 100 100 15 9 1 10 9 9 100 100 16 9 10 9 9 100 100 17 9 10 9 9 100 100 18 9 10 8 1 9 100 89 19 9 10 8 9 100 89 20 9 10 8 9 100 89 21 9 10 8 9 100 89 22 9 10 8 9 100 89 23 9 10 8 9 100 89 24 9 10 8 9 100 89 25 9 10 8 9 100 89 26 9 10 8 9 100 89 27 9 10 7 1 8 100 88 28 8 1 9 7 8 100 88 29 8 9 7 8 100 88 30 7 1 8 7 8 100 88 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

0.64 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 18 1 20 3 17 1 20 4 17 20 5 17 20 6 17 20 7 17 20 8 15 2 20 9 15 20 

10 12 1 18 2 2 11 11 1 18 2 2 12 11 18 2 2 

13* 7 10 10 10 100 100 

14 7 10 10 10 100 100 15 7 10 10 10 100 100 16 7 10 10 10 100 100 17 7 10 10 10 100 100 18 7 10 10 10 100 100 19 7 10 10 10 100 100 20 7 10 10 10 100 100 21 7 10 10 10 100 100 22 7 10 10 10 100 100 23 7 10 10 10 100 100 24 7 10 10 10 100 100 25 7 10 10 10 100 100 26 7 10 10 10 100 100 27 6 1 10 9 1 10 86 90 28 6 10 8 1 10 86 80 29 5 1 9 8 10 83 80 30 5 9 8 10 83 80 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

1.6 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 18 1 20 3 18 20 4 17 1 20 5 17 20 6 17 20 7 17 20 8 17 20 9 16 20 

10 15 18 2 2 11 15 18 2 2 12 14 17 3 3 

13* 8 11 9 9 100 100 

14 8 11 9 9 100 100 15 8 11 9 9 100 100 16 8 11 9 9 100 100 17 8 11 9 9 100 100 18 8 11 9 9 100 100 19 8 11 9 9 100 100 20 8 11 9 9 100 100 21 8 11 9 9 100 100 22 8 11 9 9 100 100 23 7 1 11 9 9 88 100 24 7 11 9 9 88 100 25 7 11 9 9 88 100 26 7 11 9 9 88 100 27 7 11 8 1 9 88 89 28 7 11 8 9 88 89 29 7 11 8 9 88 89 30 7 11 8 9 88 89 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

1.6 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 20 20 

10 18 18 2 2 11 18 18 2 2 12 16 16 4 4 

13* 12 12 8 8 100 100 

14 11 11 9 9 100 100 15 11 11 9 9 100 100 16 11 11 9 9 100 100 17 10 1 11 9 9 91 100 18 10 11 9 9 91 100 19 9 1 10 8 1 8 90 100 20 9 10 8 8 90 100 21 9 10 8 8 90 100 22 8 1 9 8 8 89 100 23 7 1 8 7 1 7 88 100 24 5 2 8 7 7 63 100 25 5 8 7 7 63 100 26 5 8 7 7 63 100 27 5 8 7 7 63 100 28 5 8 6 1 7 63 86 29 5 8 6 7 63 86 30 5 8 6 7 63 86 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

1.6 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 19 20 3 19 20 4 19 20 5 19 20 6 18 1 20 7 18 20 8 18 20 9 18 20 

10 18 20 11 16 18 2 2 12 15 17 3 3 

13* 7 9 11 11 100 100 

14 7 9 11 11 100 100 15 7 9 11 11 100 100 16 7 9 11 11 100 100 17 7 9 10 1 11 100 91 18 7 9 9 1 11 100 82 19 7 9 9 11 100 82 20 6 1 9 9 11 86 82 21 6 9 9 11 86 82 22 6 9 8 1 11 86 73 23 6 9 8 11 86 73 24 6 9 8 11 86 73 25 5 1 8 8 11 83 73 26 5 8 8 11 83 73 27 5 8 8 11 83 73 28 5 8 7 1 11 83 64 29 5 8 7 11 83 64 30 5 8 7 11 83 64 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

4.0 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 19 1 20 2 19 20 3 19 20 4 19 20 5 19 20 6 19 20 7 19 20 8 19 20 9 19 20 

10 16 1 18 2 2 11 15 17 3 3 12 13 2 17 3 3 

13* 8 12 8 8 100 100 

14 8 12 8 8 100 100 15 8 12 8 8 100 100 16 8 12 8 8 100 100 17 8 12 8 8 100 100 18 7 1 12 8 8 88 100 19 7 12 8 8 88 100 20 7 12 8 8 88 100 21 7 12 8 8 88 100 22 7 12 8 8 88 100 23 7 12 8 8 88 100 24 7 12 8 8 88 100 25 7 12 8 8 88 100 26 7 12 8 8 88 100 27 7 12 7 1 7 88 100 28 7 12 7 7 88 100 29 7 12 7 7 88 100 30 7 12 7 7 88 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

4.0 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 17 3 20 2 16 1 20 3 16 20 4 16 20 5 16 20 6 16 20 7 16 20 8 15 1 20 9 15 20 

10 11 16 4 4 11 13 18 2 2 12 11 16 4 4 

13* 7 1 13 7 7 100 100 

14 7 13 7 7 100 100 15 7 13 7 7 100 100 16 7 13 7 7 100 100 17 7 13 7 7 100 100 18 7 13 7 7 100 100 19 6 1 13 7 7 86 100 20 6 13 7 7 86 100 21 6 13 7 7 86 100 22 6 13 7 7 86 100 23 6 13 7 7 86 100 24 6 13 7 7 86 100 25 6 13 7 7 86 100 26 6 13 7 7 86 100 27 5 1 12 7 7 83 100 28 5 12 7 7 83 100 29 5 12 7 7 83 100 30 5 12 7 7 83 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

4.0 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 20 20 6 20 20 7 20 20 8 20 20 9 20 20 

10 17 17 3 3 11 18 18 2 2 12 15 1 16 4 4 

13* 9 9 11 11 100 100 

14 8 8 12 12 100 100 15 8 8 12 12 100 100 16 8 8 12 12 100 100 17 8 8 12 12 100 100 18 8 8 12 12 100 100 19 8 8 11 1 12 100 92 20 8 8 11 12 100 92 21 7 1 7 11 12 100 92 22 7 7 11 12 100 92 23 7 7 11 12 100 92 24 7 7 11 12 100 92 25 7 7 11 12 100 92 26 7 7 11 12 100 92 27 7 7 11 12 100 92 28 7 7 11 12 100 92 29 7 7 10 1 12 100 83 30 7 7 9 1 11 100 82 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

10 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 20 20 5 19 1 20 6 19 20 7 19 20 8 19 20 9 19 20 

10 16 1 18 2 2 11 16 18 2 2 12 16 18 2 2 

13* 10 12 8 8 100 100 

14 10 12 8 8 100 100 15 9 1 11 8 8 100 100 16 9 11 8 8 100 100 17 8 10 9 9 100 100 18 8 10 9 9 100 100 19 8 10 8 1 9 100 89 20 8 10 7 1 9 100 78 21 8 10 7 9 100 78 22 8 10 7 9 100 78 23 8 10 7 9 100 78 24 8 10 6 1 9 100 67 25 8 10 5 1 8 100 63 26 8 10 5 8 100 63 27 8 10 5 8 100 63 28 6 1 1 9 5 8 86 63 29 5 1 8 5 8 83 63 30 5 8 4 1 7 83 57 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

10 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 19 1 20 3 19 20 4 19 20 5 19 20 6 19 20 7 19 20 8 19 20 9 19 20 

10 16 1 18 2 2 11 16 18 2 2 12 14 1 17 3 3 

13* 10 13 7 7 100 100 

14 10 13 7 7 100 100 15 10 13 7 7 100 100 16 10 13 7 7 100 100 17 10 13 7 7 100 100 18 10 13 7 7 100 100 19 9 1 12 7 7 100 100 20 9 12 7 7 100 100 21 8 1 11 7 7 100 100 22 8 11 7 7 100 100 23 7 1 10 7 7 100 100 24 7 10 7 7 100 100 25 6 1 10 7 7 86 100 26 6 10 7 7 86 100 27 6 10 7 7 86 100 28 6 10 7 7 86 100 29 6 10 7 7 86 100 30 6 10 7 7 86 100 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F0 Reproduction Basket Survival 

10 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRMa ♂/Immature b ♀ Dead/Missing NTRMa ♀ b ♂/Immature ♀ 

0 20 20 1 20 20 2 20 20 3 20 20 4 19 1 20 5 17 2 20 6 17 20 7 17 20 8 17 20 9 17 20 

10 14 17 3 3 11 14 17 3 3 12 13 16 4 4 

13* 7 10 10 10 100 100 

14 7 10 10 10 100 100 15 7 10 10 10 100 100 16 7 10 10 10 100 100 17 7 10 10 10 100 100 18 6 1 10 9 1 10 86 90 19 6 10 8 1 9 86 89 20 6 10 8 9 86 89 21 6 10 7 1 9 86 78 22 6 10 7 9 86 78 23 5 1 9 7 9 83 78 24 4 1 9 7 9 67 78 25 4 9 7 9 67 78 26 4 9 6 1 8 67 75 27 4 9 6 8 67 75 28 4 9 6 8 67 75 29 4 9 6 8 67 75 30 4 9 6 8 67 75 

a NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) b Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. e Day 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-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111 

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

Control 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 14 1 15 3 14 15 4 14 15 5 14 15 6 14 15 7 14 15 8 14 15 9 12 13 2 2 

10c 11 12 3 3 

11ab 11 12 3 3 100 100 

12 8 1 10 5 5 89 100 13 8 10 5 5 89 100 14 8 10 4 1 5 89 80 15 8 10 4 5 89 80 16 8 10 4 5 89 80 17 8 10 4 5 89 80 18 8 10 3 1 5 89 60 19 6 2 8 3 5 86 60 20 6 8 3 5 86 60 21 6 8 3 5 86 60 22 6 8 3 5 86 60 23 6 8 3 5 86 60 24 4 2 6 3 5 80 60 25 3 1 5 3 5 75 60 26 3 5 3 5 75 60 27 3 5 3 5 75 60 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

Control 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 11 11 4 4 

10c 8 8 7 7 

11ab 8 8 7 7 100 100 

12 5 5 10 10 100 100 13 5 5 10 10 100 100 14 5 5 10 10 100 100 15 5 5 9 1 9 100 100 16 5 5 9 9 100 100 17 5 5 9 9 100 100 18 5 5 9 9 100 100 19 5 5 9 9 100 100 20 5 5 9 9 100 100 21 5 5 9 9 100 100 22 5 5 7 1 1 8 100 88 23 5 5 7 8 100 88 24 5 5 7 8 100 88 25 5 5 7 8 100 88 26 5 5 7 8 100 88 27 5 5 7 8 100 88 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

Control 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 14 1 15 7 14 15 8 14 15 9 9 10 5 5 

10c 9 10 4 1 5 100 100 

11ab 8 1 9 7 8 100 100 

12 8 9 7 8 100 100 13 8 9 7 8 100 100 14 8 9 7 8 100 100 15 7 1 9 7 8 88 100 16 7 9 7 8 88 100 17 7 9 7 8 88 100 18 6 1 8 7 8 86 100 19 6 8 7 8 86 100 20 5 1 7 7 8 83 100 21 5 7 6 1 7 83 100 22 5 7 6 7 83 100 23 5 7 6 7 83 100 24 5 7 6 7 83 100 25 5 7 6 7 83 100 26 5 7 6 7 83 100 27 5 7 6 7 83 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

0.26 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 14 1 15 2 14 15 3 14 15 4 14 15 5 14 15 6 14 15 

7* 14 15 

8 14 15 9 14 15 

10 9 10 5 5 11 9 10 5 5 

12abc 8 9 6 6 100 100 

13 7 8 7 7 100 100 14 7 8 7 7 100 100 15 7 8 7 7 100 100 16 7 8 7 7 100 100 17 7 8 7 7 100 100 18 7 8 7 7 100 100 19 7 8 7 7 100 100 20 7 8 6 1 7 100 86 21 7 8 5 1 6 100 83 22 6 1 7 5 6 100 83 23 6 7 5 6 100 83 24 6 7 5 6 100 83 25 6 7 5 6 100 83 26 6 7 5 6 100 83 27 6 7 5 6 100 83 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

0.26 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 

7* 14 15 

8 14 15 9 14 15 

10 14 15 11 14 15 

12abc 7 1 7 7 7 100 100 

13 7 7 7 7 100 100 14 7 7 7 7 100 100 15 7 7 7 7 100 100 16 7 7 7 7 100 100 17 7 7 7 7 100 100 18 7 7 7 7 100 100 19 7 7 7 7 100 100 20 7 7 7 7 100 100 21 7 7 7 7 100 100 22 7 7 6 1 6 100 100 23 7 7 5 1 5 100 100 24 6 1 6 4 1 4 100 100 25 5 1 5 4 4 100 100 26 5 5 3 1 3 100 100 27 5 5 2 1 2 100 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

0.26 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 

7* 15 15 

8 15 15 9 14 1 15 

10 9 10 5 5 11 9 10 5 5 

12abc 5 6 9 9 100 100 

13 5 6 9 9 100 100 14 5 6 9 9 100 100 15 5 6 9 9 100 100 16 5 6 9 9 100 100 17 5 6 9 9 100 100 18 5 6 9 9 100 100 19 5 6 9 9 100 100 20 5 6 9 9 100 100 21 4 1 6 9 9 80 100 22 4 6 9 9 80 100 23 4 6 9 9 80 100 24 4 6 9 9 80 100 25 4 6 9 9 80 100 26 4 6 9 9 80 100 27 3 1 5 8 1 8 75 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired 

b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

0.64 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 14 1 15 4 14 15 5 14 15 6 14 15 7 14 15 8 14 15 9 14 15 

10 14 15 

11c 14 15 12a 6 1 8 7 7 

13 6 8 7 7 

14b 6 8 7 7 100 100 

15 6 8 7 7 100 100 16 6 8 7 7 100 100 17 6 8 7 7 100 100 18 6 8 7 7 100 100 19 6 8 7 7 100 100 20 6 8 7 7 100 100 21 6 8 7 7 100 100 22 6 8 7 7 100 100 23 6 8 7 7 100 100 24 4 2 6 7 7 100 100 25 4 6 6 1 6 100 100 26 4 6 6 6 100 100 27 4 6 6 6 100 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

0.64 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 14 1 15 3 14 15 4 14 15 5 12 2 15 6 12 15 7 12 15 8 12 15 9 12 15 

10 12 15 

11c 12 15 12a 12 15 

13 12 15 

14b 5 8 7 7 100 100 

15 5 8 7 7 100 100 16 5 8 7 7 100 100 17 5 8 7 7 100 100 18 4 1 7 7 7 100 100 19 4 7 7 7 100 100 20 4 7 7 7 100 100 21 4 7 7 7 100 100 22 4 7 7 7 100 100 23 3 1 6 6 1 6 100 100 24 3 6 6 6 100 100 25 3 6 6 6 100 100 26 3 6 6 6 100 100 27 2 1 5 6 6 100 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

0.64 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 14 1 15 3 14 15 4 14 15 5 12 2 15 6 12 15 7 12 15 8 12 15 9 12 15 

10 8 11 4 4 

11c 5 8 7 7 100 100 12a 8 11 7 7 100 100 

13 7 1 10 7 7 100 100 

14b 7 10 7 7 100 100 

15 7 10 7 7 100 100 16 7 10 7 7 100 100 17 7 10 5 1 1 6 100 83 18 7 10 5 6 100 83 19 7 10 5 6 100 83 20 7 10 5 6 100 83 21 6 1 9 5 6 100 83 22 6 9 4 1 5 100 80 23 5 1 8 4 5 100 80 24 5 8 4 5 100 80 25 5 8 4 5 100 80 26 5 8 4 5 100 80 27 5 8 4 5 100 80 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

1.6 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 14 1 15 4 14 15 5 14 15 6 14 15 7 14 15 8 14 15 9 14 15 

10 10 1 12 3 3 

11c 8 10 5 5 

12ab 6 8 7 7 100 100 

13 5 7 8 8 100 100 14 5 7 8 8 100 100 15 5 7 8 8 100 100 16 5 7 8 8 100 100 17 5 7 8 8 100 100 18 5 7 8 8 100 100 19 5 7 8 8 100 100 20 5 7 8 8 100 100 21 5 7 8 8 100 100 22 4 1 7 8 8 80 100 23 4 7 8 8 80 100 24 4 7 8 8 80 100 25 4 7 8 8 80 100 26 4 7 8 8 80 100 27 3 1 6 8 8 75 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

1.6 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 14 1 15 2 14 15 3 14 15 4 13 1 15 5 12 1 15 6 12 15 7 12 15 8 12 15 9 12 15 

10 9 12 3 3 

11c 6 9 6 6 

12ab 3 1 7 8 8 100 100 

13 5 9 8 8 100 100 14 5 9 8 8 100 100 15 4 1 9 8 8 80 100 16 4 9 8 8 80 100 17 4 9 8 8 80 100 18 4 9 8 8 80 100 19 4 9 8 8 80 100 20 4 9 8 8 80 100 21 4 9 8 8 80 100 22 4 9 8 8 80 100 23 4 9 8 8 80 100 24 4 9 8 8 80 100 25 4 9 8 8 80 100 26 4 9 8 8 80 100 27 4 9 8 8 80 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

1.6 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 14 1 15 3 14 15 4 14 15 5 14 15 6 14 15 7 14 15 8 14 15 9 13 1 15 

10 10 12 3 3 

11c 6 8 7 7 100 100 

12ab 7 9 8 8 100 100 

13 6 1 9 8 8 86 100 14 6 9 8 8 86 100 15 6 9 8 8 86 100 16 6 9 8 8 86 100 17 6 9 8 8 86 100 18 6 9 8 8 86 100 19 6 9 8 8 86 100 20 5 1 9 8 8 71 100 21 3 1 1 8 7 1 7 50 100 22 3 8 7 7 50 100 23 3 8 7 7 50 100 24 2 1 7 7 7 40 100 25 2 7 7 7 40 100 26 2 7 5 2 5 40 100 27 2 7 4 1 4 40 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

4.0 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 14 1 15 4 14 15 5 14 15 6 14 15 7 14 15 8 14 15 9 14 15 

10 11 12 3 3 

11c 6 7 8 8 

12 6 7 8 8 13 6 7 8 8 

14ab 6 7 8 8 100 100 

15 6 7 8 8 100 100 16 6 7 8 8 100 100 17 6 7 8 8 100 100 18 6 7 8 8 100 100 19 6 7 8 8 100 100 20 6 7 8 8 100 100 21 6 7 8 8 100 100 22 6 7 8 8 100 100 23 5 1 7 8 8 83 100 24 5 7 8 8 83 100 25 5 7 8 8 83 100 26 5 7 8 8 83 100 27 5 7 8 8 83 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

4.0 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10 11 11 4 4 

11c 8 1 9 6 6 

12 8 9 6 6 13 8 9 6 6 

14ab 7 8 7 7 100 100 

15 7 8 7 7 100 100 16 6 1 7 7 7 100 100 17 5 1 6 7 7 100 100 18 5 6 7 7 100 100 19 5 6 6 1 6 100 100 20 5 6 6 6 100 100 21 5 6 6 6 100 100 22 5 6 6 6 100 100 23 5 6 6 6 100 100 24 5 6 6 6 100 100 25 5 6 6 6 100 100 26 5 6 6 6 100 100 27 5 6 6 6 100 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet. 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

4.0 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 14 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 14 1 15 8 14 15 9 11 12 3 3 

10 10 11 4 4 

11c 7 8 7 7 100 100 

12 7 8 7 7 100 100 13 7 8 7 7 100 100 

14ab 7 8 7 7 100 100 

15 7 8 7 7 100 100 16 7 8 7 7 100 100 17 7 8 6 1 6 100 100 18 7 8 6 6 100 100 19 7 8 6 6 100 100 20 7 8 6 6 100 100 21 7 8 6 6 100 100 22 7 8 6 6 100 100 23 7 8 6 6 100 100 24 7 8 6 6 100 100 25 7 8 5 1 6 100 83 26 7 8 5 6 100 83 27 7 8 4 1 5 100 80 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

10 μg/L 

Replicate A 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 12 12 3 3 9 12 12 3 3 

10a 9 9 6 6 100 100 11c 9 9 6 6 100 100 

12b 9 9 6 6 100 100 

13 9 9 6 6 100 100 14 9 9 6 6 100 100 15 9 9 6 6 100 100 16 9 9 6 6 100 100 17 9 9 6 6 100 100 18 8 1 8 6 6 100 100 19 8 8 6 6 100 100 20 7 1 7 6 6 100 100 21 7 7 6 6 100 100 22 7 7 6 6 100 100 23 7 7 5 1 5 100 100 24 7 7 5 5 100 100 25 6 1 6 5 5 100 100 26 6 6 4 1 4 100 100 27 5 1 6 4 4 83 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

10 μg/L 

Replicate B 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 15 15 

10a 15 15 11c 11 11 4 4 

12b 5 5 10 10 100 100 

13 5 5 10 10 100 100 14 5 5 10 10 100 100 15 5 5 10 10 100 100 16 5 5 10 10 100 100 17 5 5 10 10 100 100 18 5 5 10 10 100 100 19 5 5 10 10 100 100 20 5 5 10 10 100 100 21 5 5 10 10 100 100 22 5 5 10 10 100 100 23 5 5 10 10 100 100 24 5 5 10 10 100 100 25 5 5 10 10 100 100 26 4 1 4 10 10 100 100 27 4 4 10 10 100 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Study Number: 13888.6111

Mysid Two Generation Exposure with 4-tert-octylphenol 

F1 Reproduction Basket Survival 

10 μg/L 

Replicate C 

Test # Live # of ♂ ♂ Total # # Live # of ♀ ♀ Total # % Survival % Survival 

Day ♂/Immature Dead/Missing NTRM ♂/Immature ^ ♀ Dead/Missing NTRM ♀ ^ ♂/Immature ♀ 

0 15 15 1 15 15 2 15 15 3 15 15 4 15 15 5 15 15 6 15 15 7 15 15 8 15 15 9 14 14 1 1 

10a 11 11 4 4 11c 9 9 6 6 100 100 

12b 9 9 6 6 100 100 

13 8 1 9 6 6 89 100 14 8 9 6 6 89 100 15 8 9 5 1 5 89 100 16 8 9 5 5 89 100 17 8 9 5 5 89 100 18 8 9 5 5 89 100 19 8 9 5 5 89 100 20 8 9 5 5 89 100 21 7 1 8 5 5 88 100 22 6 1 7 3 2 3 86 100 23 6 7 3 3 86 100 24 6 7 3 3 86 100 25 6 7 3 3 86 100 26 6 7 3 3 86 100 27 5 1 6 3 3 83 100 

NTRM = non-toxicant related mortalities (e.g. impingement, inadvertent kills during cleaning, etc.) ^ Survival adjusted for non-toxicant related mortalities (i.e. impingement, etc.) d Following test initiation until mysid pairing, counts are considered to be estimations. a Day Rep A was paired b Day Rep B was paired c 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. Missinspreadsheet.
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 462 

APPENDIX 10 - BRINE SHRIMP NAUPLII ANALYSIS 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

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

Smithers Viscient Study No. 13888.6111 Page 464 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 465 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 466 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 467 
RTI Project No. 0210114.030 EPA Contract No. EP-W-06-026 

Smithers Viscient Study No. 13888.6111 Page 468