DRAFT FINAL REPORT on COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE DISRUPTING CHEMICALS EPA CONTRACT NUMBER 68 W 01 023 WORK ASSIGNMENTS 2 18 and 3 8 July 2003 Prepared for LES TOUART, PH. D. WORK ASSIGNMENT MANAGER U. S. ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, DC BATTELLE 505 King Avenue Columbus, Ohio 43201 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) iii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 CONTENTS 1.0 INTRODUCTION.............................................................................................................................. 1 1.1 Purpose..................................................................................................................................... 2 2.0 MATERIALS AND METHODS....................................................................................................... 3 2.1 Test Material and Exposure Regime ........................................................................................ 3 2.2 Test Material ............................................................................................................................ 3 2.3 Preparation and Sampling of Chemical Exposure Water......................................................... 3 2.4 Analytical Procedures .............................................................................................................. 6 2.4.1 Methoxychlor .............................................................................................................. 6 2.4.2 Trenbolone .................................................................................................................. 6 2.4.3 Flutamide..................................................................................................................... 6 2.4.4 Fadrozole..................................................................................................................... 7 2.5 Animals and Husbandry........................................................................................................... 7 2.6 Study Schedule and Design...................................................................................................... 8 2.7 Description of Study Protocols ................................................................................................ 9 2.7.1 Summary of the 14 day Non spawning Method ......................................................... 9 2.7.2 Summary of the 14 day EPA Method ......................................................................... 9 2.7.3 Summary of 21 day EPA Method............................................................................. 10 2.7.4 Summary of Assay Endpoints ................................................................................... 11 2.8 Statistical Analyses ................................................................................................................ 18 2.9 Quality Assurance.................................................................................................................. 18 2.9.1 Technical Systems Audits ......................................................................................... 18 2.9.2 Audits of Data Quality .............................................................................................. 19 2.10 Storage of Records and Data Management ............................................................... 20 3.0 RESULTS: METHOXYCHLOR.................................................................................................... 21 3.1 EPA 14 Day Assay for Methoxychlor ................................................................................... 21 3.1.1 Survival ..................................................................................................................... 21 3.1.2 Vitellogenin............................................................................................................... 21 3.1.3 Appearance / Secondary Sex Characteristics ............................................................ 23 3.1.4 Gonadosomatic Index................................................................................................ 24 3.1.5 Female Gonad Histology........................................................................................... 26 3.1.6 Male Gonad Histology .............................................................................................. 29 3.1.7 Plasma Steroid Concentrations.................................................................................. 31 3.1.8 Body Weight ............................................................................................................. 37 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) iv Preliminary Data: May be subject to change following QA and Management review July 30, 2003 3.1.9 Fecundity................................................................................................................... 37 3.1.10 Fertilization Success.................................................................................................. 40 3.1.11 Hatchability and Larval Development....................................................................... 41 3.2 EPA 21 Day Assay for Methoxychlor ................................................................................... 43 3.2.1 Survival ..................................................................................................................... 43 3.2.2 Vitellogenin............................................................................................................... 44 3.2.3 Appearance / Secondary Sex Characteristics ............................................................ 46 3.2.4 Gonadosomatic Index................................................................................................ 46 3.2.5 Female Gonad Histology........................................................................................... 48 3.2.6 Male Gonad Histology .............................................................................................. 51 3.2.7 Plasma Steroid Concentrations.................................................................................. 53 3.2.8 Body Weight ............................................................................................................. 59 3.2.9 Fecundity................................................................................................................... 60 3.2.10 Fertilization Success.................................................................................................. 63 3.2.11 Hatchability and Larval Development....................................................................... 65 3.3 Non spawning Adult 14 day Assay for Methoxychlor.......................................................... 67 3.3.1 Survival ..................................................................................................................... 68 3.3.2 Vitellogenin............................................................................................................... 68 3.3.3 Appearance / Secondary Sex Characteristics ............................................................ 70 3.3.4 Gonadosomatic Index................................................................................................ 71 3.3.5 Female Gonad Histology........................................................................................... 73 3.3.6 Male Gonad Histology .............................................................................................. 77 3.3.7 Plasma Steroid Concentrations.................................................................................. 80 3.3.8 Body Weight and Length .......................................................................................... 85 4.0 RESULTS: TRENBOLONE........................................................................................................... 90 4.1 EPA 14 Day Assay for Trenbolone ....................................................................................... 90 4.1.1 Survival ..................................................................................................................... 90 4.1.2 Vitellogenin............................................................................................................... 90 4.1.3 Appearance / Secondary Sex Characteristics ............................................................ 92 4.1.4 Gonadosomatic Index................................................................................................ 92 4.1.5 Female Gonad Histology........................................................................................... 94 4.1.6 Male Gonad Histology .............................................................................................. 97 4.1.7 Plasma Steroid Concentrations.................................................................................. 99 4.1.8 Fecundity................................................................................................................. 104 4.1.9 Fertilization Success................................................................................................ 106 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) v Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.1.10 Hatchability and Larval Development..................................................................... 108 4.1.11 Body Weight ........................................................................................................... 110 4.2 EPA 21 Day Assay for Trenbolone ..................................................................................... 110 4.2.2 Vitellogenin............................................................................................................. 110 4.2.3 Appearance / Secondary Sex Characteristics .......................................................... 112 4.2.4 Gonadosomatic Index.............................................................................................. 112 4.2.5 Female Gonad Histology......................................................................................... 114 4.2.6 Male Gonad Histology ............................................................................................ 117 4.2.7 Plasma Steroid Concentrations................................................................................ 119 4.2.8 Fecundity................................................................................................................. 124 4.2.9 Fertilization Success................................................................................................ 128 4.2.10 Hatchability and Larval Development..................................................................... 129 4.2.11 Body Weight ........................................................................................................... 132 4.3 Non spawning Adult 14 Day Assay for Trenbolone ........................................................... 132 4.3.1 Survival ................................................................................................................... 132 4.3.2 Vitellogenin............................................................................................................. 132 4.3.3 Appearance / Secondary Sex Characteristics .......................................................... 134 4.3.4 Gonadosomatic Index.............................................................................................. 135 4.3.5 Female Gonad Histology......................................................................................... 137 4.3.6 Male Gonad Histology ............................................................................................ 140 4.3.7 Plasma Steroid Concentrations................................................................................ 142 4.3.8 Body Weight and Length ........................................................................................ 147 5.0 RESULTS: FLUTAMIDE ............................................................................................................ 152 5.1 EPA 14 Day Assay for Flutamide ....................................................................................... 152 5.1.1 Survival ................................................................................................................... 152 5.1.2 Vitellogenin............................................................................................................. 152 5.1.3 Appearance / Secondary Sex Characteristics .......................................................... 154 5.1.4 Gonadosomatic Index.............................................................................................. 154 5.1.5 Female Gonad Histology......................................................................................... 156 5.1.6 Male Gonad Histology ............................................................................................ 159 5.1.7 Plasma Steroid Concentrations................................................................................ 161 5.1.8 Fecundity................................................................................................................. 166 5.1.9 Fertilization Success................................................................................................ 169 5.1.10 Hatchability and Larval Development..................................................................... 170 5.1.11 Body Weight ........................................................................................................... 172 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) vi Preliminary Data: May be subject to change following QA and Management review July 30, 2003 5.2 EPA 21 Day Assay for Flutamide ....................................................................................... 172 5.2.1 Survival ................................................................................................................... 172 5.2.2 Vitellogenin............................................................................................................. 172 5.2.3 Appearance / Secondary Sex Characteristics .......................................................... 174 5.2.4 Gonadosomatic Index.............................................................................................. 175 5.2.5 Female Gonad Histology......................................................................................... 176 5.2.6 Male Gonad Histology ............................................................................................ 179 5.2.7 Plasma Steroid Concentrations................................................................................ 181 5.2.8 Fecundity................................................................................................................. 186 5.2.9 Fertilization Success................................................................................................ 190 5.2.10 Hatchability and Larval Development..................................................................... 191 5.2.11 Body Weight ........................................................................................................... 193 5.3 Non spawning Adult 14 Day Assay for Flutamide ............................................................. 194 5.3.1 Survival ................................................................................................................... 194 5.3.2 Vitellogenin............................................................................................................. 194 5.3.3 Appearance / Secondary Sex Characteristics .......................................................... 196 5.3.4 Gonadosomatic Index.............................................................................................. 197 5.3.5 Female Gonad Histology......................................................................................... 199 5.3.6 Male Gonad Histology ............................................................................................ 202 5.3.7 Plasma Steroid Concentrations................................................................................ 205 5.3.8 Body Weight and Length ........................................................................................ 211 6.0 RESULTS: FADROZOLE............................................................................................................ 216 6.1 EPA 14 Day Assay for Fadrozole........................................................................................ 216 6.1.1 Survival ................................................................................................................... 216 6.1.2 Vitellogenin............................................................................................................. 216 6.1.3 Appearance / Secondary Sex Characteristics .......................................................... 218 6.1.4 Gonadosomatic Index.............................................................................................. 218 6.1.5 Female Gonad Histology......................................................................................... 220 6.1.6 Male Gonad Histology ............................................................................................ 223 6.1.7 Plasma Steroid Concentrations................................................................................ 226 6.1.8 Fecundity................................................................................................................. 230 6.1.9 Fertilization Success................................................................................................ 233 6.1.10 Hatchability and Larval Development..................................................................... 234 6.1.11 Body Weight ........................................................................................................... 236 6.2 EPA 21 Day Assay for Fadrozole........................................................................................ 236 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) vii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 6.2.1 Survival ................................................................................................................... 236 6.2.2 Vitellogenin............................................................................................................. 236 6.2.3 Appearance / Secondary Sex Characteristics .......................................................... 238 6.2.4 Gonadosomatic Index.............................................................................................. 238 6.2.5 Female Gonad Histology......................................................................................... 240 6.2.6 Male Gonad Histology ............................................................................................ 244 6.2.7 Plasma Steroid Concentrations................................................................................ 247 6.2.8 Fecundity................................................................................................................. 252 6.2.9 Fertilization Success................................................................................................ 256 6.2.10 Hatchability and Larval Development..................................................................... 257 6.2.11 Body Weight ........................................................................................................... 259 6.3 Non spawning Adult 14 day Assay for Fadrozole............................................................... 260 6.3.1 Survival ................................................................................................................... 260 6.3.2 Vitellogenin............................................................................................................. 260 6.3.3 Appearance / Secondary Sex Characteristics .......................................................... 262 6.3.4 Gonadosomatic Index.............................................................................................. 263 6.3.5 Female Gonad Histology......................................................................................... 265 6.3.6 Male Gonad Histology ............................................................................................ 268 6.3.7 Plasma Steroid Concentrations................................................................................ 272 6.3.8 Body Weight and Length ........................................................................................ 277 7.0 DISCUSSION ................................................................................................................................ 282 7.1 Fecundity.............................................................................................................................. 282 7.2 Histology and Gonadosomatic index ( GSI) ......................................................................... 283 7.3 Vitellogenin and Plasma Steroid Levels .............................................................................. 284 7.3.1 Test Results: Transferability and Inter Protocol Comparison................................. 286 7.3.2 Methoxychlor ( mode of action: weak estrogen)...................................................... 287 7.3.3 Trenbolone ( anabolic steroid) ................................................................................. 288 7.3.4 Flutamide ( anti androgen)....................................................................................... 289 7.3.5 Fadrozole ( aromatase inhibitor, indirect anti estrogen) .......................................... 290 7.4 Conclusion ........................................................................................................................... 291 7.5 Protocol Selection ................................................................................................................ 292 7.6 Transferability...................................................................................................................... 292 8.0 REFERENCES ........................................................................................................................... 294 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) viii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 FIGURES Figure 2.1. Saturator column apparatus...................................................................................................... 4 Figure 2.2. Continuous flow proportional diluter system......................................................................... 5 Figure 2.3. Terracotta dish with nytex screen......................................................................................... 10 Figure 2.4. Staff members count eggs in support of fecundity measurements ....................................... 11 Figure 2.5. Flow through embryo incubation chambers ........................................................................ 11 Figure 2.6. Collection of the liver and gonads ....................................................................................... 13 Figure 3. 1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Methoxychlor assay...................................................................................................... 22 Figure 3.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Methoxychlor assay ............................................................................................................. 23 Figure 3.3. Boxplot of female GSI by treatment for the EPA 14 Day Methoxychlor assay .................. 24 Figure 3.4. Boxplot of male GSI by treatment for the EPA 14 Day Methoxychlor assay ..................... 25 Figure 3.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Methoxychlor assay ..................................................... 27 Figure 3.6. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 14 Day Methoxychlor assay ........................................................................................ 28 Figure 3.7. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 14 Day Methoxychlor assay................................................................................................. 28 Figure 3.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Methoxychlor assay ..................................................... 29 Figure 3.9. Boxplot of seminiferous tubule diameter ( mm) by treatment for the EPA 14 Day Methoxychlor assay ............................................................................................................. 30 Figure 3.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Methoxychlor assay ............................................................................................................. 32 Figure 3. 11. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Methoxychlor assay ............................................................................................................. 33 Figure 3.12. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Methoxychlor assay...................................................................................................... 34 Figure 3.13. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Methoxychlor assay ............................................................................................................. 35 Figure 3.14. Boxplot of female 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Methoxychlor assay. ....................................................................................... 36 Figure 3.15. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Methoxychlor assay................................................................................................. 37 Figure 3.16. Total Egg Production by Replicate per Treatment for the EPA 14 Day Methoxychlor Assay.................................................................................................................................... 38 Figure 3.17. Boxplot of the Number of Eggs Produced per Female Reproductive Day by Treatment for the EPA 14 Day Methoxychlor Assay .......................................................... 39 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) ix Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 3.18. Boxplot of the Proportion of Eggs Fertilized by Treatment for the EPA 14 Day Methoxychlor Assay ............................................................................................................ 40 Figure 3.19. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Methoxychlor assay...................................................................................................... 42 Figure 3.20. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Methoxychlor assay ............................................................................................................. 43 Figure 3.21. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Methoxychlor assay...................................................................................................... 44 Figure 3.22. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Methoxychlor assay ............................................................................................................. 45 Figure 3.23. Boxplot of female GSI by treatment for the EPA 21 Day Methoxychlor assay ................. 46 Figure 3.24. Boxplot of male GSI by treatment for the EPA 21 Day Methoxychlor assay .................... 47 Figure 3.25. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Methoxychlor assay ..................................................... 49 Figure 3.26. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Methoxychlor assay ........................................................................................ 50 Figure 3.27. Boxplot of the proportion corpora lutea per 300 follicles by treatment for the EPA 21 Day Methoxychlor assay................................................................................................. 50 Figure 3.28. Frequency Histogram Showing the Quantitative Developmental Staging of Testes for Each Treatment of the EPA 21 Day Methoxychlor Assay .................................................. 52 Figure 3.29. Boxplot of male seminiferous tubule diameter ( mm) by treatment for the EPA 21 Day Methoxychlor assay...................................................................................................... 53 Figure 3.30. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Methoxychlor assay ............................................................................................................. 54 Figure 3.31. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Methoxychlor assay ............................................................................................................. 55 Figure 3.32. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Methoxychlor assay...................................................................................................... 56 Figure 3.33. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Methoxychlor assay ............................................................................................................. 57 Figure 3.34. Boxplot of female 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Methoxychlor assay ........................................................................................ 58 Figure 3.35. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Methoxychlor assay................................................................................................. 59 Figure 3.36. Total egg production per treatment during the pre exposure and exposure periods of the EPA 21 Day Methoxychlor assay .................................................................................. 60 Figure 3.37. Total egg production by replicate per treatment during the EPA 21 Day Methoxychlor assay. .................................................................................................................................... 61 Figure 3.38. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Methoxychlor assay .................................................................................. 63 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) x Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 3.39. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Methoxychlor assay ............................................................................................................. 64 Figure 3.40. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Methoxychlor assay...................................................................................................... 66 Figure 3.41. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Methoxychlor assay ............................................................................................................. 67 Figure 3.42. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Methoxychlor assay ...................................................................... 69 Figure 3.43. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Methoxychlor assay ...................................................................... 70 Figure 3.44. Secondary sex characteristics of males used during the methoxychlor Non spawning Adult 14 Day assay. ............................................................................................................. 71 Figure 3.45. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Methoxychlor assay ............................................................................................................. 72 Figure 3.46. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Methoxychlor assay ..................................................................................................................................... 73 Figure 3.47. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Methoxychlor assay ........................... 75 Figure 3.48. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the Non spawning Adult 14 Day Methoxychlor assay .............................................................. 76 Figure 3.49. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the Nonspawning Adult 14 Day Methoxychlor assay. ..................................................................... 77 Figure 3.50. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Methoxychlor assay ........................... 78 Figure 3.51. Boxplot of seminiferous tubule diameter ( mm) by treatment for the Non spawning Adult 14 Day Methoxychlor assay ...................................................................................... 79 Figure 3.52. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Methoxychlor assay ...................................................................................... 80 Figure 3.53. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Methoxychlor assay ...................................................................................... 81 Figure 3.54. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Methoxychlor assay ...................................................................... 82 Figure 3.55. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Methoxychlor assay ...................................................................... 83 Figure 3.56. Boxplot of female 11 ketotesosterone concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Methoxychlor assay .............................................................. 84 Figure 3.57. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Methoxychlor assay. ..................................................................... 85 Figure 3.58. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Methoxychlor assay ............................................................................................................. 86 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xi Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 3.59. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Methoxychlor assay...................................................................................................... 87 Figure 3.60. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Methoxychlor assay ............................................................................................................. 88 Figure 3.61. Boxplot of male body length ( mm) by treatment for the Non spawning Adult 14 Day Methoxychlor assay ............................................................................................................. 89 Figure 4.1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Trenbolone assay. ....................................................................................................... 91 Figure 4.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Trenbolone assay. ............................................................................................................... 92 Figure 4.3. Boxplot of female GSI by treatment for the EPA 14 Day Trenbolone assay ...................... 93 Figure 4.4. Boxplot of male GSI by treatment for the EPA 14 Day Trenbolone assay ......................... 94 Figure 4.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Trenbolone assay.......................................................... 95 Figure 4.6. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 14 Day Trenbolone assay ............................................................................................ 96 Figure 4.7. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 14 Day Trenbolone assay..................................................................................................... 97 Figure 4.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Trenbolone assay.......................................................... 98 Figure 4.9. Boxplot of male seminiferous tubule diameter ( mm) by treatment for the EPA 14 Day Trenbolone assay.................................................................................................................. 99 Figure 4.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay................................................................................................................ 100 Figure 4.11. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay................................................................................................................ 101 Figure 4. 12. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay ........................................................................................................ 102 Figure 4.13. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay................................................................................................................ 103 Figure 4.14. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay................................................................................................... 104 Figure 4.15. Total egg production by replicate per treatment for the EPA 14 Day Trenbolone assay. .................................................................................................................................. 105 Figure 4.16. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 14 Day Trenbolone assay .................................................................................... 106 Figure 4.17. Boxplot of the proportion of eggs fertilized by treatment for the EPA 14 Day Trenbolone assay................................................................................................................ 107 Figure 4.18. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Trenbolone assayDay Trenbolone assay .................................................................... 108 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 4.19. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Trenbolone assay................................................................................................................ 109 Figure 4.20. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Trenbolone assay ........................................................................................................ 111 Figure 4.21. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 112 Figure 4.22. Boxplot of female GSI by treatment for the EPA 21 Day Trenbolone assay .................... 113 Figure 4.23. Boxplot of male GSI by treatment for the EPA 21 Day Trenbolone assay ....................... 114 Figure 4.24. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Trenbolone assay........................................................ 115 Figure 4.25. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Trenbolone assay .......................................................................................... 116 Figure 4.26. Boxplot of proportion of corpora lutea per 300 follicles by treatment for the EPA 21 Day Trenbolone assay ........................................................................................................ 116 Figure 4.27. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 21 Day Trenbolone assay........................................................ 118 Figure 4.28. Boxplot of male seminiferous tubule diameter ( mm) by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 119 Figure 4.29. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 120 Figure 4.30. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 121 Figure 4.31. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay ........................................................................................................ 122 Figure 4.32. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 123 Figure 4.33. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay................................................................................................... 124 Figure 4.34. Total egg production per treatment for the EPA 21 Day Trenbolone assay. The vertical line at Day 0 denotes the start of the exposure period........................................... 125 Figure 4.35. Total egg production by replicate per treatment for the EPA 21 Day Trenbolone assay. .................................................................................................................................. 126 Figure 4.36. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Trenbolone assay .................................................................................... 127 Figure 4.37. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 129 Figure 4.38. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Trenbolone assay ........................................................................................................ 130 Figure 4.39. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Trenbolone assay................................................................................................................ 131 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xiii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 4.40. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 133 Figure 4.41. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 134 Figure 4.42. Secondary sex characteristics of males used during the flutamide Non spawning Adult 14 Day assay. ........................................................................................................... 135 Figure 4.43. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Trenbolone assay ................................................................................................................................... 136 Figure 4.44. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Trenbolone assay ................................................................................................................................... 137 Figure 4.45. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Trenbolone assay.............................. 138 Figure 4.46. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the Non spawning Adult 14 Day Trenbolone assay ................................................................ 139 Figure 4.47. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 139 Figure 4. 48. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Trenbolone assay.............................. 141 Figure 4.49. Boxplot of male seminiferous tubule diameter ( mm) by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 142 Figure 4.50. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone assay......................................................................................... 143 Figure 4.51. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone assay......................................................................................... 144 Figure 4.52. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 145 Figure 4.53. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 146 Figure 4.54. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Trenbolone assay ........................................................................ 147 Figure 4.55. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Trenbolone assay................................................................................................................ 148 Figure 4.56. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Trenbolone assay ........................................................................................................ 149 Figure 4.57. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Trenbolone assay................................................................................................................ 150 Figure 4.58. Boxplot of male body length ( mm) by treatment for the Non spawning Adult 14 Day Trenbolone assay................................................................................................................ 151 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xiv Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 5.1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Flutamide assay. ......................................................................................................... 153 Figure 5.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 154 Figure 5.3. Boxplot of female GSI by treatment for the EPA 14 Day Flutamide assay. ..................... 155 Figure 5.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Flutamide assay.......................................................... 157 Figure 5.6. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 14 Day Flutamide assay ............................................................................................ 158 Figure 5.7. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 14 Day Flutamide assay ..................................................................................................... 159 Figure 5.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Flutamide assay.......................................................... 160 Figure 5.9. Boxplot of seminiferous tubule diameter (  m) by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 161 Figure 5.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 162 Figure 5.11. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 163 Figure 5.12. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay .......................................................................................................... 164 Figure 5.13. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 165 Figure 5.14. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay ..................................................................................................... 166 Figure 5.15. Total egg production by replicate per treatment for the EPA 14 Day Flutamide assay..... 167 Figure 5.16. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 14 Day Flutamide assay....................................................................................... 168 Figure 5.17. Boxplot of the proportion of eggs fertilized by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 169 Figure 5.18. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Flutamide assay .......................................................................................................... 171 Figure 5.19. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Flutamide assay.................................................................................................................. 172 Figure 5.20. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Flutamide assay .......................................................................................................... 173 Figure 5.21. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 174 Figure 5.22. Boxplot of female GSI by treatment for the EPA 21 Day Flutamide assay ...................... 175 Figure 5.23. Boxplot of male GSI by treatment for the EPA 21 Day Flutamide assay.......................... 176 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xv Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 5.24. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Flutamide assay.......................................................... 177 Figure 5.25. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Flutamide assay ............................................................................................ 178 Figure 5.26. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 21 Day Flutamide assay ..................................................................................................... 179 Figure 5.27. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 21 Day Flutamide assay.......................................................... 180 Figure 5.28. Boxplot of male seminiferous tubule diameter (  m) by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 181 Figure 5.29. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 182 Figure 5.30. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 183 Figure 5.31. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay .......................................................................................................... 184 Figure 5.32. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 185 Figure 5.33. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay. .................................................................................................... 186 Figure 5.34. Total egg production per treatment for the EPA 21 Day Flutamide assay. The vertical line denotes the initiation of the exposure assay. ............................................................... 187 Figure 5.35. Total egg production by replicate per treatment for the EPA 21 Day Flutamide assay..... 188 Figure 5.36. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Flutamide assay....................................................................................... 189 Figure 5.37. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 191 Figure 5.38. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Flutamide assay. ......................................................................................................... 192 Figure 5.39. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Flutamide assay.................................................................................................................. 193 Figure 5.40. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Flutamide assay........................................................................... 195 Figure 5.41. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Flutamide assay........................................................................... 196 Figure 5.42. Secondary sex characteristics of males used during the EPA Non spawning Adult 14 Day Flutamide assay. ......................................................................................................... 197 Figure 5.43. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Flutamide assay. .................................................................................................................................. 198 Figure 5.44. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Flutamide assay ................................................................................................................................... 199 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xvi Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 5.45. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Flutamide assay................................ 200 Figure 5.46. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the Non spawning Adult 14 Day Flutamide assay .................................................................. 201 Figure 5.47. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the Nonspawning Adult 14 Day Flutamide assay........................................................................... 202 Figure 5.48. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Flutamide assay................................ 203 Figure 5.49. Boxplot of seminiferous tubule diameter (  m) by treatment for the Non spawning Adult 14 Day Flutamide assay........................................................................................... 204 Figure 5.50. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay.......................................................................... 206 Figure 5.51. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay........................................................................................... 207 Figure 5.52. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay.......................................................................... 208 Figure 5.53. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay.......................................................................... 209 Figure 5.54. Boxplot of female 11 ketotesosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay.................................................................. 210 Figure 5.55. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay.......................................................................... 211 Figure 5.56. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Flutamide assay.................................................................................................................. 212 Figure 5.57. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Flutamide assay .......................................................................................................... 213 Figure 5.58. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Flutamide assay.................................................................................................................. 214 Figure 5.59. Boxplot of male body length ( mm) by treatment for the Non spawning Adult 14 Day Flutamide assay.................................................................................................................. 215 Figure 6.1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Fadrozole assay .......................................................................................................... 217 Figure 6.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Fadrozole assay .................................................................................................................. 218 Figure 6.3. Boxplot of female GSI by treatment for the EPA 14 Day Fadrozole assay....................... 219 Figure 6.4. Boxplot of male GSI by treatment for the EPA 14 Day Fadrozole assay.......................... 220 Figure 6.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Fadrozole assay .......................................................... 221 Figure 6.6. Boxplot of atretic follicles per 300 follicles by treatment for the EPA 14 Day Fadrozole assay .................................................................................................................. 222 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xvii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 6.7. Boxplot of corpora lutea per 300 follicles by treatment for the EPA 14 Day Fadrozole assay ................................................................................................................................... 223 Figure 6.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Fadrozole assay .......................................................... 224 Figure 6.9. Boxplot of seminiferous tubule diameter (  m) by treatment for the EPA 14 Day Fadrozole assay. ................................................................................................................. 225 Figure 6.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole assay .................................................................................................................. 226 Figure 6.11. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole assay. ......................................................................................................... 227 Figure 6.12. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole assay .................................................................................................................. 228 Figure 6.13. Boxplot of female 11 ketotestosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole assay............................................................................................. 229 Figure 6.14. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole assay ..................................................................................................... 230 Figure 6.15. Total egg production by replicate per treatment for the EPA 14 Day fadrozole assay...... 231 Figure 6.16. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 14 Day Fadrozole assay....................................................................................... 232 Figure 6.17. Boxplot of the proportion of eggs fertilized by treatment for the EPA 14 Day Fadrozole assay .................................................................................................................. 233 Figure 6.18. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Fadrozole assay .......................................................................................................... 235 Figure 6.19. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Fadrozole assay. ................................................................................................................. 236 Figure 6.20. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Fadrozole assay .......................................................................................................... 237 Figure 6.21. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Fadrozole assay. ................................................................................................................. 238 Figure 6.22. Boxplot of female GSI by treatment for the EPA 21 Day Fadrozole assay....................... 239 Figure 6.23. Boxplot of male GSI by treatment for the EPA 21 Day Fadrozole assay.......................... 240 Figure 6.24. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Fadrozole assay .......................................................... 242 Figure 6.25. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Fadrozole assay............................................................................................. 243 Figure 6.26. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 21 Day Fadrozole assay ..................................................................................................... 244 Figure 6.27. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 21 Day Fadrozole assay .......................................................... 245 Figure 6.28. Boxplot of male seminiferous tubule diameter (  m) by treatment for the EPA 21 Day Fadrozole assay .................................................................................................................. 246 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xviii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 6.29. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole assay .................................................................................................................. 247 Figure 6.30. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole assay .......................................................................................................... 249 Table 6.28. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay. ................................................................................ 249 Figure 6.31. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole assay .................................................................................................................. 250 Figure 6.32. Boxplot of female 11 ketotestosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole assay............................................................................................. 251 Figure 6.33. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole assay ..................................................................................................... 252 Figure 6.34. Total egg production per treatment for the 15 Day pre exposure assay. .......................... 253 Figure 6.35. Total egg production by replicate per treatment for the EPA 21 Day fadrozole assay...... 254 Figure 6.36. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Fadrozole assay....................................................................................... 255 Figure 6.37. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Fadrozole assay .................................................................................................................. 257 Figure 6.38. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Fadrozole assay .......................................................................................................... 258 Figure 6.39. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Fadrozole assay .................................................................................................................. 259 Figure 6.40. Boxplot of the body weights of males by treatment for the EPA 21 Day Fadrozole assay ................................................................................................................................... 260 Figure 6.41. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole assay........................................................................... 261 Figure 6.42. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole assay........................................................................... 262 Figure 6.43. Secondary sex characteristics of males used during the Non spawning Adult 14 Day Fadrozole assay. ................................................................................................................. 263 Figure 6.44. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Fadrozole assay ................................................................................................................................... 264 Figure 6.45. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Fadrozole assay ................................................................................................................................... 265 Figure 6.46. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Fadrozole assay ................................ 266 Figure 6.47. Boxplot of atretic follicles per 300 follicles by treatment for the Non spawning Adult 14 Day Fadrozole assay ..................................................................................................... 267 Figure 6.48. Boxplot of corpora lutea per 300 follicles by treatment for the Non spawning Adult 14 Day Fadrozole assay ..................................................................................................... 267 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xix Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 6.49. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Fadrozole assay ................................ 269 Figure 6.50. Boxplot of seminiferous tubule diameter (  m) by treatment for the Non spawning Adult 14 Day Fadrozole assay ........................................................................................... 270 Figure 6.51. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole assay ........................................................................................... 272 Figure 6.52. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole assay........................................................................... 274 Figure 6.53. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole assay........................................................................... 275 Figure 6.54. Boxplot of female 11 ketotestosterone concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole assay................................................................... 276 Figure 6.55. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole assay........................................................................... 277 Figure 6.56. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Fadrozole assay. ................................................................................................................. 278 Figure 6.57. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Fadrozole assay .......................................................................................................... 279 Figure 6.58. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Fadrozole assay .................................................................................................................. 280 Figure 6.59. Boxplot of male body length ( mm) by treatment for the Non spawning Adult 14 Day Fadrozole assay .................................................................................................................. 281 TABLES Table 2.1. Chemical concentrations used in the WA 2 18 program.......................................................... 3 Table 2.2. Recommended ranges of water quality characteristics for testing fathead minnows............... 8 Table 2.3. Testing schedule for P. promelas using four chemicals and three assays ................................ 8 Table 2.4. Histological stages of fathead minnow ovarian development. ............................................... 14 Table 2.5. Histological stages of fathead minnow testicular development. ............................................ 15 Table 2.6. Summary of the testing conditions for each assay ................................................................. 15 Table 3.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Methoxychlor assay............................................................................... 22 Table 3.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Methoxychlor assay............................................................................... 23 Table 3.3. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Methoxychlor assay .......................................................................................... 25 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xx Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 3.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Methoxychlor assay .......................................................................................... 26 Table 3.5. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 14 Day Methoxychlor assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments ........................................................................................ 27 Table 3.6. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 14 Day Methoxychlor assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments ........................................................................................ 30 Table 3.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Methoxychlor assay .................................................................................... 31 Table 3.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Methoxychlor assay............................................................................... 32 Table 3.9. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 14 Day Methoxychlor assay. ................................................................................... 33 Table 3.10. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Methoxychlor assay............................................................................... 34 Table 3.11. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Methoxychlor assay............................................................................... 35 Table 3.12. Summary statistics and power estimates for female 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Methoxychlor assay. ............................................................... 36 Table 3.13. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Methoxychlor assay. ............................................................... 37 Table 3.14. Summary Statistics and Power Estimates for Fecundity Data for the EPA 14 Day Methoxychlor Assay............................................................................................................... 38 Table 3.15. Summary Statistics and Power Estimates for Fecundity per Female Reproductive Day for the EPA 14 Day Methoxychlor Assay.............................................................................. 39 Table 3.16. Summary Statistics and Power Estimates for the Proportion of Eggs Fertilized for the EPA 14 Day Methoxychlor Assay ......................................................................................... 41 Table 3.17. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Methoxychlor assay............................................................................... 42 Table 3.18. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Methoxychlor assay .......................................................................................... 43 Table 3.19. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Methoxychlor assay............................................................................... 44 Table 3.20. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Methoxychlor assay............................................................................... 45 Table 3.21. Summary Statistics and Power Estimates for Female Gonadosomatic Index Data for the EPA 21 Day Methoxychlor Assay ......................................................................................... 47 Table 3.22. Summary Statistics and Power Estimates for Male Gonadosomatic Index Data for the EPA 21 Day Methoxychlor Assay ......................................................................................... 48 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxi Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 3.23 Descriptive Statistics of the Proportion of Ovarian Cells in each Developmental Stage for Females from the EPA 21 Day Methoxychlor Assay and Results of the Kruskal Wallis Test ( df = 2) Comparing Treatments........................................................................... 49 Table 3.24. Descriptive Statistics of the Proportion of Testes Cells in Each Developmental Stage for Males from the EPA 21 Day Methoxychlor Assay and Results of the Kruskal Wallis Test ( df = 2) Comparing Treatments........................................................................... 51 Table 3.25. Summary Statistics and Power Estimates for Male Seminiferous Tubule Diameter Data for the EPA 21 Day Methoxychlor Assay.............................................................................. 53 Table 3.26 Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Methoxychlor assay............................................................................... 54 Table 3.27. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Methoxychlor assay. ................................................................................... 54 Table 3.28. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Methoxychlor assay with all females having a measured testosterone value included. ................................................................................................... 56 Table 3.29. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Methoxychlor assay............................................................................... 57 Table 3.30. Summary statistics and power estimates for female 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Methoxychlor assay. ............................................................... 58 Table 3.31. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Methoxychlor assay. ............................................................... 59 Table 3.32. Summary Statistics and Power Estimates for Total Fecundity Data for the EPA 21 Day Methoxychlor Assay............................................................................................................... 61 Table 3.33. Summary Statistics and Power Estimates for Fecundity per Female Reproductive Day for the EPA 21 Day Methoxychlor Assay.............................................................................. 62 Table 3.34. Summary Statistics and Power Estimates for the Proportion of Eggs Fertilized for the EPA 21 Day Methoxychlor Assay ......................................................................................... 64 Table 3.35. Summary Statistics and Power Estimates for the Proportion of Fertile Eggs that Hatched for the EPA 21 Day Methoxychlor Assay ............................................................... 66 Table 3.36. Summary Statistics and Power Estimates for the Proportion of Normal Larvae for the EPA 21 Day Methoxychlor Assay ......................................................................................... 67 Table 3.37. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Methoxychlor assay..................................................... 68 Table 3.38. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Methoxychlor assay..................................................... 69 Table 3.39. Summary statistics and power estimates for female gonadosomatic index data for the Non spawning Adult 14 Day Methoxychlor assay ................................................................ 72 Table 3.40. Summary statistics and power estimates for male gonadosomatic index data for the Non spawning Adult 14 Day Methoxychlor assay ................................................................ 73 Table 3.41. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the Non spawning Adult 14 Day Methoxychlor assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................... 75 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 3.42. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the Non spawning Adult 14 Day Methoxychlor assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................... 78 Table 3.43. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Methoxychlor assay .......................................................... 79 Table 3.44. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Methoxychlor assay..................................................... 80 Table 3.45. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Methoxychlor assay. ......................................................... 81 Table 3.46. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Methoxychlor assay..................................................... 82 Table 3.47. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Methoxychlor assay..................................................... 83 Table 3.48. Summary statistics and power estimates for female 11 ketotesosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Methoxychlor assay. ..................................... 84 Table 3.49. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Methoxychlor assay. ..................................... 85 Table 3.50. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Methoxychlor assay ........................................................................ 86 Table 3.51. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Methoxychlor assay ........................................................................ 87 Table 3.52. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Methoxychlor assay ........................................................................ 88 Table 3.53. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Methoxychlor assay ........................................................................ 89 Table 4.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Trenbolone assay................................................................................... 90 Table 4.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Trenbolone assay................................................................................... 91 Table 4.3. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Trenbolone assay. ............................................................................................. 93 Table 4.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Trenbolone assay. ............................................................................................. 94 Table 4.5. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................... 95 Table 4.6. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................... 98 Table 4.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Trenbolone assay......................................................................................... 99 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxiii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay................................................................................. 100 Table 4.9. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay....................................................................................... 101 Table 4.10. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay................................................................................. 102 Table 4.11. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay................................................................................. 103 Table 4.12. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay................................................................... 104 Table 4.13. Summary statistics and power estimates for fecundity data for the EPA 14 Day Trenbolone assay.................................................................................................................. 105 Table 4.14. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 14 Day Trenbolone assay....................................................................................... 106 Table 4.15. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 14 Day Trenbolone assay. ........................................................................................... 107 Table 4.16. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Trenbolone assay................................................................................. 108 Table 4.17. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Trenbolone assay. ........................................................................................... 109 Table 4.18. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Trenbolone assay................................................................................. 110 Table 4.19. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Trenbolone assay................................................................................. 111 Table 4.20. Summary statistics and power estimates for female gonadosomatic index data for the EPA 21 Day Trenbolone assay ............................................................................................ 113 Table 4.21. Summary statistics and power estimates for male gonadosomatic index data for the EPA 21 Day Trenbolone assay ............................................................................................ 114 Table 4.22. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 21 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments .............................................................................................. 115 Table 4.23. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 21 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................. 117 Table 4.24. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 21 Day Trenbolone assay....................................................................................... 118 Table 4.25. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay................................................................................. 119 Table 4.26. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay....................................................................................... 120 Table 4.27. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay................................................................................. 121 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxiv Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.28. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay................................................................................. 122 Table 4.29. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay................................................................... 123 Table 4.30. Summary statistics and power estimates for fecundity data for the EPA 21 Day Trenbolone assay.................................................................................................................. 125 Table 4.31. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 21 Day Trenbolone assay....................................................................................... 127 Table 4.32. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 21 Day Trenbolone assay ............................................................................................ 128 Table 4.33. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 21 Day Trenbolone assay................................................................................. 130 Table 4.34. Summary statistics and power estimates for the proportion of normal larvae for the EPA 21 Day Trenbolone assay ............................................................................................ 131 Table 4.35. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Trenbolone assay....................................................... 132 Table 4.36. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Trenbolone assay....................................................... 133 Table 4.37. Summary statistics and power estimates for female gonadosomatic index data for the Non spawning Adult 14 Day Trenbolone assay. ................................................................. 135 Table 4.38. Summary statistics and power estimates for male gonadosomatic index data for the Non spawning Adult 14 Day Trenbolone assay. ................................................................. 136 Table 4.39. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the Non spawning Adult 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................. 138 Table 4.40. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the Non spawning Adult 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................. 140 Table 4.41. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Trenbolone assay............................................................. 141 Table 4.42. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay....................................................... 143 Table 4.43. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay............................................................. 144 Table 4.44. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay....................................................... 145 Table 4.45. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay....................................................... 146 Table 4.46. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay......................................... 147 Table 4.47. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Trenbolone assay........................................................................... 148 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxv Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.48. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Trenbolone assay........................................................................... 148 Table 4.49. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Trenbolone assay........................................................................... 149 Table 4.50. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Trenbolone assay........................................................................... 150 Table 5.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Flutamide assay. .................................................................................. 152 Table 5.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Flutamide assay. .................................................................................. 153 Table 5.3. Table E14FL GSI 1. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Flutamide assay. ....................................... 155 Table 5.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Flutamide assay............................................................................................... 156 Figure 5.4. Boxplot of male GSI by treatment for the EPA 14 Day Flutamide assay............................ 156 Table 5.5. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 14 Day Flutamide assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................. 157 Table 5.6. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 14 Day Flutamide assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.................................................................................................... 160 Table 5.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Flutamide assay......................................................................................... 161 Table 5.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay. .................................................................................. 162 Table 5.9. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay......................................................................................... 163 Table 5.10. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay. .................................................................................. 164 Table 5.11. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay. .................................................................................. 165 Table 5.12. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay..................................................................... 166 Table 5.13. Summary statistics and power estimates for fecundity data for the EPA 14 Day Flutamide assay. ................................................................................................................... 167 Table 5.14. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 14 Day Flutamide assay......................................................................................... 168 Table 5.15. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 14 Day Flutamide assay............................................................................................... 169 Table 5.16. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Flutamide assay. .................................................................................. 170 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxvi Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.17. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Flutamide assay............................................................................................... 171 Table 5.18. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Flutamide assay. .................................................................................. 173 Table 5.19. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Flutamide assay. .................................................................................. 174 Table 5.20. Summary statistics and power estimates for female gonadosomatic index data for the EPA 21 Day Flutamide assay............................................................................................... 175 Table 5.21. Summary statistics and power estimates for male gonadosomatic index data for the EPA 21 Day Flutamide assay............................................................................................... 176 Table 5.22. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 21 Day Flutamide assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................. 177 Table 5.23. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 21 Day Flutamide assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.................................................................................................... 180 Table 5.24. Summary statistics and power estimates for male seminiferous tubule diameter (  m) data for the EPA 21 Day Flutamide assay. .......................................................................... 181 Table 5.25. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay. .................................................................................. 182 Table 5.26. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay......................................................................................... 183 Table 5.27. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay. .................................................................................. 184 Table 5.28. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay. .................................................................................. 185 Table 5.29. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay..................................................................... 186 Table 5.30. Summary statistics and power estimates for total fecundity data for the EPA 21 Day Flutamide assay. ................................................................................................................... 187 Table 5.31. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 21 Day Flutamide assay......................................................................................... 189 Table 5.32. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 21 Day Flutamide assay............................................................................................... 190 Table 5.33. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 21 Day Flutamide assay. .................................................................................. 192 Table 5.34. Summary statistics and power estimates for the proportion of normal larvae for the EPA 21 Day Flutamide assay............................................................................................... 193 Table 5.35. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Flutamide assay. ........................................................ 194 Table 5.36. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Flutamide assay. ........................................................ 195 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxvii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.37. Summary statistics and power estimates for female gonadosomatic index data for the Non spawning Adult 14 Day Flutamide assay..................................................................... 197 Table 5.38. Summary statistics and power estimates for male gonadosomatic index data for the Non spawning Adult 14 Day Flutamide assay..................................................................... 198 Table 5.39. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the Non spawning Adult 14 Day Flutamide assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................. 200 Table 5.40. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the Non spawning Adult 14 Day Flutamide assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................. 203 Table 5.41. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Flutamide assay............................................................... 204 Table 5.42. Histological observations for males exposed to concentrations of flutamide during the Non spawning Adult 14 D assay. ........................................................................................ 205 Table 5.43. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay. ....................................................... 206 Table 5.44. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay. ............................................................. 207 Table 5.45. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay. ....................................................... 208 Table 5.46. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay. ....................................................... 209 Table 5.47. Summary statistics and power estimates for female 11 ketotesosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay. ......................................... 210 Table 5.48. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay. ......................................... 211 Table 5.49. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Flutamide assay............................................................................. 212 Table 5.50. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Flutamide assay............................................................................. 213 Table 5.51. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Flutamide assay............................................................................. 214 Table 5.52. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Flutamide assay............................................................................. 214 Table 6.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Fadrozole assay. .................................................................................. 216 Table 6.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Fadrozole assay. .................................................................................. 217 Table 6.3. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Fadrozole assay............................................................................................... 219 Table 6.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Fadrozole assay............................................................................................... 220 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxviii Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.5. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................. 221 Table 6.6. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.................................................................................................... 224 Table 6.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Fadrozole assay. ........................................................................................ 225 Table 6.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay. .................................................................................. 226 Table 6.9. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay. .................................................................................. 227 Table 6.10. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay. .................................................................................. 228 Table 6.11. Summary statistics and power estimates for female 11 ketotestosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay. .................................................................... 229 Table 6.12. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay. .................................................................... 230 Table 6.13. Summary statistics and power estimates for fecundity data for the EPA 14 Day Fadrozole assay. ................................................................................................................... 231 Table 6.14. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 14 Day Fadrozole assay. ........................................................................................ 232 Table 6.15. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 14 Day Fadrozole assay............................................................................................... 233 Table 6.16. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Fadrozole assay. .................................................................................. 234 Table 6.17. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Fadrozole assay............................................................................................... 235 Table 6.18. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Fadrozole assay. .................................................................................. 237 Table 6.19. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Fadrozole assay. .................................................................................. 238 Table 6.20. Summary statistics and power estimates for female gonadosomatic index data for the EPA 21 Day Fadrozole assay............................................................................................... 239 Table 6.21. Summary statistics and power estimates for male gonadosomatic index data for the EPA 21 Day Fadrozole assay............................................................................................... 240 Table 6.22. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 21 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments. ............................................................................................. 241 Table 6.23. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 21 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.................................................................................................... 245 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxix Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.24. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 21 Day Fadrozole assay. ........................................................................................ 246 Table 6.25. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay. .................................................................................. 247 Table 6.26. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay. ........................................................................................ 248 Table 6.27. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay. .................................................................................. 248 Table 6.29. Summary statistics and power estimates for female 11 ketotestosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay. .................................................................... 250 Table 6.30. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay. .................................................................... 251 Table 6.31. Summary statistics and power estimates for total fecundity data for the EPA 21 Day Fadrozole assay. ................................................................................................................... 253 Table 6.32. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 21 Day Fadrozole assay. ........................................................................................ 255 Table 6.33. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 21 Day Fadrozole assay............................................................................................... 256 Table 6.34. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 21 Day Fadrozole assay. .................................................................................. 258 Table 6.35. Summary statistics and power estimates for the proportion of normal larvae for the EPA 21 Day Fadrozole assay............................................................................................... 259 Table 6.36. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Fadrozole assay. ........................................................ 261 Table 6.37. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Fadrozole assay. ........................................................ 262 Table 6.38. Summary statistics and power estimates for female gonadosomatic index data for the Non spawning Adult 14 Day Fadrozole assay..................................................................... 263 Table 6.39. Summary statistics and power estimates for male gonadosomatic index data for the Non spawning Adult 14 Day Fadrozole assay..................................................................... 264 Table 6.40. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the Non spawning Adult 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................. 266 Table 6.41. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the Non spawning Adult 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments............................................................. 268 Table 6.42. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Fadrozole assay. .............................................................. 269 Table 6.43. Histological observations for males exposed to concentrations of fadrozole during the Non spawning Adult 14 D assay. ........................................................................................ 270 Table 6.44. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay. ........................................................ 272 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) xxx Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.45. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay. .............................................................. 273 Table 6.46. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay. ........................................................ 273 Table 6.47. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay. ........................................................ 274 Table 6.48. Summary statistics and power estimates for female 11 ketotestosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay. .......................................... 275 Table 6.49. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay. .......................................... 276 Table 6.50. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Fadrozole assay. ............................................................................ 277 Table 6.51. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Fadrozole assay. ............................................................................ 278 Table 6.52. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Fadrozole assay. ............................................................................ 279 Table 6.53. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Fadrozole assay. ............................................................................ 280 Table 7.1. Average Daily Fecundity in Control Fathead Minnows ( mean, mean ± SD) ......................... 283 Table 7.2. Average Plasma Markers in Control Female Fathead Minnows ( mean or mean ± SD)......... 285 Table 7.3. Average Plasma Markers in Control Male Fathead Minnows ( mean or mean ± SD) ............ 285 Table 7.4. Methoxychlor Summary ........................................................................................................ 287 Table 7.5. Trenbolone Summary............................................................................................................. 289 Table 7.6. Flutamide Summary............................................................................................................... 290 Table 7.7. Fadrozole Summary............................................................................................................... 291 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 1 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 1.0 INTRODUCTION The EPA has implemented an Endocrine Disruptor Screening Program ( EDSP). In 1996, the Food Quality Protection Act and the Safe Drinking Water Act were enacted by Congress to authorize the EPA to implement a screening program to evaluate whether pesticides and other chemicals found in food or water could affect endocrine systems in humans. In this program, comprehensive toxicological and ecotoxicological screens and assays are being developed to identify and characterize the endocrine effects of environmental contaminants, industrial chemicals, and pesticides. A two tiered approach is being utilized: Tier 1 employs a combination of in vivo and in vitro screens, and Tier 2 involves in vivo testing using two generation reproductive studies. Validation of the individual screens and assays is required, and the Endocrine Disruptor Methods Validation Subcommittee ( EDMVS) will provide advice and counsel on the validation assays.