DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 152 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 5.0 RESULTS: FLUTAMIDE 5.1 EPA 14 Day Assay for Flutamide The EPA 14 Day Flutamide exposure assay was conducted from February 18, 2003, to February 25, 2003 ( pre exposure assay), and from February 25, 2003, to March 11, 2003 ( exposure assay).

5.1.1 Survival All males and females in all treatments survived the EPA 14 Day Flutamide assay.

5.1.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the EPA 14 Day Flutamide assay ranged from 533,500 ng/ mL to 6,960,500 ng/ mL ( Figure 5.1). Among females exposed to the two flutamide concentrations, vitellogenin concentrations ranged from 1,781,000 ng/ mL to 8,388,000 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 5.1) were detected ( Kruskal Wallis, H = 3.49, p = 0.175, df = 2). The achieved power for this endpoint was 41%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 35 ( Table 5.1).

Table 5.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 15 3,077,733 1,532,872 50% 41% 35 low 16 3,489,000 1,686,463 48% high 15 4,188,333 1,706,450 41% 1 Calculated from natural log transformed data; with sample size = 15. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 153 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 8000000 6000000 4000000 2000000 0 treatment Flut VTG Figure 5.1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

Vitellogenin concentrations in Control treatment males used during the EPA 14 Day Flutamide assay ranged from 0 ng/ mL ( not detected) to 12,700 ng/ mL ( Figure 5.2). Among most males exposed to the two flutamide concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 15,212 ng/ mL. One male exposed to the High flutamide concentration had a vitellogenin concentration of 353,600 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 5.2) were detected ( Kruskal Wallis, H = 0.32, p = 0.850, df = 2). The achieved power for this endpoint was 5%, and the sample size required to detect a significant difference from the Control treatment at 80% power was > 1,000 ( Table 5.2).

Table 5.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 3,365 4,499 134% 5% > 1,000 low 8 4,497 5,469 122% high 8 44,563 124,870 280% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 154 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 300000 200000 100000 0 treatment Flut VTG Figure 5.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value and the asterisk represents a probable outlier.

5.1.3 Appearance / Secondary Sex Characteristics All of the females used during the EPA 14 Day Flutamide assay exhibited typical female morphology ( no fat pad, no tubercles, ovipositor present) with two exceptions. One female from the Control treatment lacked an ovipositor. One female from the High concentration that showed the dark vertical banding typically found in males.

Most males used during the EPA 14 Day Flutamide assay had typical male morphological features. One male from the High concentration lacked tubercles and a dorsal fat pad. Tubercles were present in all other males. Fat pads were present or pronounced in all other males from all treatments. Vertical banding was present or pronounced in all males.

5.1.4 Gonadosomatic Index The range of GSI values calculated for females in the Control treatments varied from 1.0 to 19.7 ( Figure 5.3), whereas the GSI values for the other treatments varied from 3.6 to 21.5 for females from the High concentration and from 8.1 to 16.3 for females from the Low concentration. The overall variability within each treatment was low to moderate ( CVs = 17%   40%; Table 5.3). There were no significant differences in mean GSI values ( Table 5.3) among treatments ( Kruskal Wallis, H = 0.14, p = 0.933, df = 2). The achieved power for this endpoint was 8%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 442 ( Table 5.3). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 155 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.3. Table E14FL GSI 1. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 11.4 4.4 38% 8% 442 low 16 11.3 1.9 17% high 16 12.2 4.9 40% 1 Calculated from arcsine square root transformed data; with sample size = 16. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 20 10 0 treatment GSI Figure 5.3. Boxplot of female GSI by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

The range of most GSI values calculated for males during the EPA 14 Day Flutamide assay, was small, ranging from 0.4 to 2.1 ( Figure 5.4), which approximates the typical range for reproductively active male fathead minnows. The highest and lowest male GSI values were 2.1 ( one fish in the High concentration) and 0.4 ( two fish in the Control treatment), respectively. Variability was moderate ( CVs = 25%   48%). There were no significant differences in mean GSI values ( Table 5.4) among treatments ( Kruskal Wallis, H = 3.44, p = 0.180, df = 2). The achieved power for this endpoint was 12%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 84 ( Table 5.4). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 156 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 1.1 0.5 48% 12% 84 low 8 0.8 0.2 25% high 8 1.2 0.5 40% 1 Calculated from arcsine square root transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 2.0 1.5 1.0 0.5 treatment GSI Figure 5.4. Boxplot of male GSI by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

5.1.5 Female Gonad Histology Histological analyses were conducted on the ovaries of 47 females exposed to flutamide during the EPA 14 Day Assay.

General Ovary Staging   Statistical analysis of the mean ovarian staging from 12 microscope fields per female from the EPA 14 Day Flutamide assay revealed no significant differences among treatments ( Kruskal Wallis, H = 4.73, p = 0.094, df = 2).

Quantitative Ovarian Staging   One hundred cells in each of three sections per female were examined to quantitatively determine the developmental stage of the ovaries. Ova from females from the Control treatment and High concentration ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages), whereas ovaries in females from the Low concentration treatment showed Stage 1a to Stage 4 development ( Figure 5.5). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 303% ( Table 5.5). Statistical analyses showed that there were no significant DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 157 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 difference among treatments in the proportion of cells in each of the five developmental stages ( Table 5.5). Therefore, there was no effect on ovarian developmental stage associated with flutamide dose.

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.

Control ( n = 15) Low ( n = 16) High ( n = 16) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1a 0.079 0.030 38% 0.094 0.037 39% 0.084 0.031 37% 0.95 0.621 1b 0.332 0.082 25% 0.305 0.040 13% 0.328 0.127 39% 2.39 0.302 2 0.174 0.039 23% 0.178 0.040 23% 0.158 0.071 45% 3.12 0.210 3 0.190 0.057 30% 0.188 0.044 23% 0.158 0.074 47% 2.19 0.335 4 0.193 0.090 47% 0.223 0.085 38% 0.215 0.116 54% 1.04 0.594 5 0.022 0.064 284% 0 0   0.033 0.100 303% 3.14 0.208 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 1a 1b 2 3 4 5 Ovarian Stage Proportion Control Low High Figure 5.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Flutamide assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation.

Atretic Follicles   The mean proportion of atretic follicles per 300 follicles ( counted per fish) ranged from 0.0004 follicles for females in the Control treatment to 0.015 follicles for females in the High concentration ( Figure 5.6). There was a significant difference in the mean proportions of atretic follicles among treatments ( Kruskal Wallis, H = 12.88, p = 0.002, df = 2). The proportion of atretic follicles was DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 158 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 greater for females from the High concentration than from the Control treatment or the Low concentration.

Low High Control 0.10 0.05 0.00 Treatment atretic_ follicles ( y )

Figure 5.6. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

Corpora Lutea   The mean proportion of corpora lutea per 300 follicles ( counted per fish) ranged from 0.009 for females in the High concentration to 0.010 for females in the Low concentration ( Figure 5.7). There were no significant differences in the mean proportion of corpora lutea among treatments ( Kruskal Wallis, H = 0.56, p = 0.757, df = 2). The value for the High concentration was significantly lower than those of the other two treatments. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 159 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 Treatment corpora_ lutea Figure 5.7. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

Observations   One fish from the Control treatment had only eight ova present in the three ovarian section examined. This fish was not included in the data analyses. Another fish in the Control treatment was observed to have some cortical alveolus stage ova ( Stage 2) that appeared abnormal because they had a very large yolk body next to the nucleus. Three fish exposed to the High flutamide concentration had very few ova or very few advanced stage ova in the ovaries.

5.1.6 Male Gonad Histology General Testes Staging   Testes from 24 males exposed to flutamide during the EPA 14 Day Flutamide assay were examined to determine the general developmental condition. Males in all treatments had welldeveloped testes with all showing Stage 4 and Stage 5 development ( see Methods for description of developmental stages). All of the 98 microscopic fields examined in the 8 Control treatment males showed Stage 4 ( 86 fields) or Stage 5 ( 12 fields) development. All of the 96 microscopic fields examined in the 8 Low concentration treatment males showed Stage 4 ( 90 fields) or Stage 5 ( 6 fields) development. All of the 96 microscopic fields examined in the 8 High concentration treatment males showed Stage 4 ( 81 fields) or Stage 5 ( 15 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed no significant differences among treatments ( Kruskal Wallis, H = 0.70, p = 0.703, df = 2).

Quantitative Testicular Staging   One hundred cells in each of three section per male were examined to quantitatively determine the developmental condition of the testes. The developmental stage all treatment testes ranged from Stage 2a to Stage 5 ( Figure 5.8). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 170% ( Table 5.6). Although statistical analyses showed that there were no significant differences among treatments in the proportion of cells in any developmental Stage, the probability value calculated for Stage 2a was slightly above the critical limit of 0.050 ( Table 5.6). Therefore, there was no effect on testicular developmental stage associated with flutamide dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 160 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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.

Control ( n = 8) Low ( n = 8) High ( n = 8) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1 0 0   0 0   0 0       2a 0.002 0.004 170% 0.006 0.005 83% 0.008 0.006 74% 5.82 0.054 2b 0.025 0.026 105% 0.015 0.013 85% 0.017 0.014 83% 0.43 0.805 3a 0.113 0.056 50% 0.135 0.058 43% 0.152 0.077 51% 1.86 0.394 3b 0.276 0.123 45% 0.287 0.076 26% 0.251 0.102 40% 0.59 0.746 4 0.174 0.079 45% 0.170 0.029 17% 0.124 0.068 55% 3.39 0.184 5 0.410 0.179 44% 0.386 0.085 22% 0.448 0.216 48% 0.23 0.893 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion of Cells Control Low High Figure 5.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Flutamide assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation.

Tubule Diameter   The diameter of the seminiferous tubules of males from the Control treatment ranged from 66.1 µ m to 161.9 µ m ( Figure 5.9). Tubule diameters of males from the two test concentrations ranged from 92.2 µ m to 164.2 µ m. No significant differences in the mean tubule diameter per treatment ( Table 5.7) were detected ( Kruskal Wallis, H = 2.42, p = 0.297, df = 2). The achieved power for this endpoint was 9%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 152 ( Table 5.7). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 161 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 118.6 29.0 24% 9% 152 low 8 108.4 10.8 10% high 8 125.1 22.2 18% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 160 110 60 Treatment diameter Figure 5.9. Boxplot of seminiferous tubule diameter ( µ m) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

Observations   No Sertoli cell proliferation was observed. One male from the Control treatment was observed to have Leydig cell proliferation. The same male was also noted to have disorganized development of the seminiferous tubules with early developmental stages shed prematurely into tubule lumina. No testicular atrophy was recorded and no ovatestes were observed for any treatment.

5.1.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the EPA 14 Day Flutamide assay ranged from 0 pg/ mL ( not detected) to 4,453 pg/ mL ( Figure 5.10). Among females exposed to the two flutamide concentrations, estradiol concentrations ranged from 282 pg/ mL to 5,837 pg/ mL. No significant differences in the mean estradiol concentration per treatment ( Table 5.8) were detected ( Kruskal Wallis, H = 2.02, p = 0.365, df = 2). The achieved power for this endpoint was 13%, and the DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 162 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 sample size required to detect a significant difference from the Control treatment at 80% power was 115 ( Table 5.8).

Table 5.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 12 2,241 1,338 60% 13% 115 low 15 2,351 1,294 55% high 15 1,693 1,221 72% 1 Calculated from natural log transformed data; with sample size = 12. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 6000 5000 4000 3000 2000 1000 0 treatment Estradiol Figure 5.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

Estradiol concentrations in Control treatment males used during the EPA 14 Day Flutamide assay ranged from 210 pg/ mL to 540 pg/ mL ( Figure 5.11). Among males exposed to the two flutamide concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 992 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 5.9) was detected ( Kruskal Wallis, H = 8.80, p = 0.012, df = 2). The mean concentration of estradiol in males from the High concentration was less than that in males from the Low concentration. The achieved power for this endpoint was 22%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 31 ( Table 5.9). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 163 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.9. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 7 302 110 36% 22% 31 low 8 400 245 61% high 7 200 97 48% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 1000 500 0 treatment Estradiol Figure 5.11. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisks represent probable outliers.

Testosterone   Testosterone concentrations in Control treatment females used during the EPA 14 Day Flutamide assay ranged from 0 pg/ mL ( not detected) to 2,989 pg/ mL ( Figure 5.12). Among females exposed to the two flutamide concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 4,874 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 5.10) were detected ( Kruskal Wallis, H = 2.59, p = 0.274, df = 2). The achieved power for this endpoint was 7%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 271 ( Table 5.10). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 164 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.10. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 11 1,227 851 69% 7% 271 low 9 1,298 791 61% high 10 2,613 1,884 72% 1 Calculated from natural log transformed data; with sample size = 9. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 5.12. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Testosterone concentrations in Control treatment males used during the EPA 14 Day Flutamide assay ranged from 949 pg/ mL to 6,258 pg/ mL ( Figure 5.13). Among males exposed to the two flutamide concentrations, testosterone concentrations ranged from 1,161 pg/ mL to 5,529 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 5.11) were detected ( Kruskal Wallis, H = 0.25, p = 0.883, df = 2). The achieved power for this endpoint was 6%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 760 ( Table 5.11). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 165 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.11. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 7 2,853 2,159 76% 6% 760 low 8 2,552 1,173 46% high 7 2,715 1,357 50% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 6000 5000 4000 3000 2000 1000 treatment Testosterone Figure 5.13. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

11 ketotestosterone   11 ketotesosterone was not detected in females from the Control treatment ( five individuals), the Low concentration ( six individuals), and the High concentration ( six individuals) exposed during the EPA 14 Day Flutamide assay.

11 ketotesosterone concentrations in Control treatment males used during the EPA 14 Day Flutamide assay ranged from 5,287 pg/ mL to 63,011 pg/ mL ( Figure 5.14). Among males exposed to the two flutamide concentrations, 11 ketotesosterone concentrations ranged from 3,942 pg/ mL to 48,001 pg/ mL. No significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 5.12) were detected ( Kruskal Wallis, H = 0.37, p = 0.830, df = 2). The achieved power for this endpoint was 6%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 356 ( Table 5.12). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 166 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.12. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 7 24,269 24,145 99% 6% 356 low 8 23,583 12,787 54% high 7 21,815 13,712 63% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 60000 50000 40000 30000 20000 10000 0 treatment 11 keto Figure 5.14. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

5.1.8 Fecundity Total Fecundity   Variability among treatments in the total number of eggs produced per replicate during the EPA 14 Day Flutamide assay was very high ( Figure 5.15). Total counts in the Control treatment ranged from 2,758 eggs to 4,346 eggs. Total counts for the High flutamide concentration treatment ranged from 1,004 eggs to 2,687 eggs. No eggs were laid after Day 10 in one High concentration replicate or after Day 12 in another replicate. The fecundity among replicates in the Low flutamide concentration ranged from 3,448 eggs to 4,876 eggs. A significant difference in the mean number of eggs ( square root transformed ) produced per treatment ( Table 5.13) was detected ( Kruskal Wallis, H = 8.77, p = 0.012, df = 2). The total fecundity of females in the High concentration treatment was significantly less than it was for females in the Low concentration or the Control treatment. The achieved power for this assay was 79%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 5 ( Table 5.13). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 167 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.13. Summary statistics and power estimates for fecundity data for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 3539 684 19% 79% 5 low 4 4501 702 16% high 4 1690 820 48% 1 Calculated from square root transformed data; with sample size = 4. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on square root transformed data.

0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 0 2 4 6 8 10 12 14 Day Cumulative Number of Eggs Control Low High Figure 5.15. Total egg production by replicate per treatment for the EPA 14 Day Flutamide assay.

Fecundity per Female Reproductive Day   During the EPA 14 Day Flutamide assay, the maximum number of female reproductive days was achieved for all treatments as no fish died during the assay ( Table 5.14). The number of eggs produced per female reproductive day in the Control treatment varied from 49.3 eggs to 77.6 eggs and from 61.6 eggs to 87.1 eggs in the Low concentration ( Figure 5.16). For the High concentration, the number of eggs produced per female reproductive day ranged from 17.9 eggs to 48.0 eggs, with fish in two of the replicates producing fewer than 20 eggs per day ( 17.9, 18.5). Because no fish died during the assay, the statistical results reported here are the same as those reported for total fecundity. A significant difference in the mean number of eggs laid per day per treatment ( Table 5.14) occurred. The number of eggs laid per day in the High concentration treatment was significantly less than it was for females in the Low concentration or the Control treatment. The achieved power for this assay was 77%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 5 ( Table 5.14). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 168 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.14. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 14 Day Flutamide assay.

Level Mean Number of Reproductive Days 1 N Mean Stdev CV Achieved Power 2 Sample Size Required 3 control 56 4 63.2 12.2 19% 77% 5 low 56 4 80.4 12.5 16% high 56 4 30.2 14.6 48% 1 Maximum number = 56. 2 Calculated from natural log transformed data; with sample size = 4. 3 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 90 80 70 60 50 40 30 20 treatment eggs/ ReproDay Figure 5.16. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Eggs on Tiles/ Dishes   The mean number of eggs laid on the tiles among the treatments during the EPA 14 Day Flutamide assay varied from 1,518 eggs for the High concentration to 4,148 eggs for the Low concentration ( Appendix E, Table 3.3). The number of eggs on dishes ranged from 172 eggs for the High concentration to 353 eggs for the Low concentration. Because of the variability in the total number of eggs laid per treatment, the proportional difference in the number of eggs on dishes versus those on tiles [ 1 (# eggs on dishes ÷ # eggs on tiles)] was calculated. The proportional difference ranged from 0.78 ( one High concentration replicate) to 0.96 ( one Control treatment replicate) ( Appendix E, Figure 3.2). There were no significant differences in this mean proportional difference among treatments ( Kruskal Wallis, H = 1.04, p = 0.595, df = 2). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 169 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 5.1.9 Fertilization Success Total Fertilization   The total ( tiles + dishes) fertilization success rates for all treatment replicates during the EPA 14 Day Flutamide assay were high, ranging from 0.993 ( one High concentration replicate) to 1.00 ( two High concentration replicates, three Control treatment replicates) ( Figure 5.17). No significant differences in mean fertilization success rates ( Table 5.15) among treatments were detected ( Kruskal Wallis, H = 1.53, p = 0.465, df = 2). The achieved power for this assay was 17%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 22 ( Table 5.15).

Table 5.15. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 1.000 0.001 0.1% 17% 22 low 4 1.000 0.000 0.0% high 4 0.997 0.003 0.3% 1 Calculated from arcsine square root transformed data; with sample size = 4. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.000 0.999 0.998 0.997 0.996 0.995 0.994 0.993 treatment Total Prop fert Figure 5.17. Boxplot of the proportion of eggs fertilized by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Fertilization of Eggs on Tiles and Dishes   The fertilization success rates for most treatment replicates for eggs laid on tiles during the EPA 14 Day Flutamide assay were high, ranging from 0.992 ( one High concentration replicate) to 1.00 ( two High concentration replicates, three Control treatment replicates) ( Appendix E, Figure 3.3). No significant differences in mean fertilization success rates ( Appendix E, Table 3.4) among treatments were detected ( Kruskal Wallis, H = 1.53, p = 0.465, df = 2). The fertilization success rates for all treatment replicates for eggs laid on dishes during the assay were very DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 170 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 high, ranging from 0.997 ( one Control treatment replicate) to 1.00 ( all remaining replicates from all treatments) ( Appendix E, Figure 3.4). No significant differences in mean fertilization success rates ( Appendix E, Table 3.4) among treatments were detected ( Kruskal Wallis, H = 2.00, p = 0.368, df = 2).

5.1.10 Hatchability and Larval Development Eggs were collected during the 14 day pre exposure period for the evaluation of hatchability. The mean proportion of fertilized eggs that hatched in the Control treatment was 0.96 ( sd = 0.05), 0.94 ( sd = 0.03) in the Low concentration, and 0.98 ( sd = 0.02) in the High concentration. There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 2.20, p = 0.333, df = 2).

Eggs were collected during the EPA 14 Day Flutamide assay for the evaluation of hatchability. The proportion of fertilized eggs that hatched ranged from 0.94 to 1.00 in the Control treatment and from 0.80 to 1.00 for the two test concentrations ( Figure 5.18). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 2.99, p = 0.224, df = 2). The achieved power for this endpoint was 20%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 18 ( Table 5.16).

Table 5.16. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 0.98 0.03 3% 20% 18 low 5 0.97 0.04 4% high 5 0.91 0.08 9% 1 Calculated from arcsine square root transformed data; with sample size = 4. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.0 0.9 0.8 treatment Prop Hatch DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 171 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 5.18. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Eggs were collected during the pre exposure period for the evaluation of larval development. The mean proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) in the Control treatment was 1.00 ( sd = 0.01). The mean proportion of normal larvae in the remaining treatments was 0.97 ( sd = 0.02) in the Low concentration and 0.98 ( sd = 0.02) in the High concentration. There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 3.46, p = 0.178, df = 2).

Eggs were collected during the EPA 14 Day Flutamide assay for the evaluation of larval development. The proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) ranged from 0.96 to 1.00 for the Control treatment and ranged from 0.95 to 1.00 for the two test concentrations ( Figure 5.19). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 3.38, p = 0.185, df = 2). The achieved power for this endpoint was 12%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 34 ( Table 5.17).

Table 5.17. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 0.98 0.02 2% 12% 34 low 5 0.99 0.01 1% high 5 0.97 0.02 2% 1 Calculated from arcsine square root transformed data; with sample size = 4. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.00 0.99 0.98 0.97 0.96 0.95 treatment Prop Norm DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 172 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Figure 5.19. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

5.1.11 Body Weight The body weight of females used in the EPA 14 Day Flutamide assay ranged from 0.9 g to 3.1 g. There were no significant differences in mean body weight among treatments, although the probability was only slighter greater than the critical limit of 0.05 ( Kruskal Wallis, H = 5.72, p = 0.057, df = 2). Females from the Control treatment tended to be larger than those form the other two treatments. The body weight of males used in the EPA 14 Day Flutamide assay ranged from 2.8 g to 5.6 g. There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 2.72, p = 0.257, df = 2).

5.2 EPA 21 Day Assay for Flutamide The EPA 21 Day Flutamide assay was conducted from February 11, 2003, to February 25, 2003 ( preexposure assay), and from February 25, 2003, to March 18, 2003 ( exposure assay).

5.2.1 Survival All males and females in all treatments survived the EPA 21 Day Flutamide assay.

5.2.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the EPA 21 Day Flutamide assay ranged from 252,800 ng/ mL to 9,071,500 ng/ mL ( Figure 5.20). Among females exposed to the two flutamide concentrations, vitellogenin concentrations ranged from 3,006,000 ng/ mL to 9,830,000 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 5.18) were detected ( Kruskal Wallis, H = 5.08, p = 0.079, df = 2). The achieved power for this endpoint was 44%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 31 ( Table 5.18).

Table 5.18. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 4,917,456 2,135,773 43% 44% 31 low 15 6,182,160 1,860,122 30% high 14 6,483,000 1,538,230 24% 1 Calculated from natural log transformed data; with sample size = 14. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 173 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 10000000 5000000 0 treatment Flut VTG ( y )

Figure 5.20. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Vitellogenin concentrations in Control treatment males used during the EPA 21 Day Flutamide assay ranged from 51 ng/ mL to 13,485 ng/ mL ( Figure 5.21). Among males exposed to the two flutamide concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 35,355 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 5.19) were detected ( Kruskal Wallis, H = 2.95, p = 0.229, df = 2). The achieved power for this endpoint was 13%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 70 ( Table 5.19).

Table 5.19. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 2,418 4,547 188% 13% 70 low 8 9,681 13,513 140% high 8 12,497 14,887 119% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 174 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 30000 20000 10000 0 treatment Flut VTG Figure 5.21. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, ant the asterisk represents a probable outlier.

5.2.3 Appearance / Secondary Sex Characteristics All of the females used during the EPA 21 Day Flutamide assay exhibited typical female morphology ( no fat pad, no tubercles, ovipositor present) except that one female from the High concentration that showed the dark vertical banding typically found in males.

Most males during the EPA 21 Day Flutamide assay had typical male morphological features. One male from the Low concentration lacked tubercles and a dorsal fat pad. Tubercles were present in all other males. Fat pads were present or pronounced in all other males from all treatments. Vertical banding was present or pronounced in all males.

5.2.4 Gonadosomatic Index The range of GSI values calculated for females in the Control treatment varied from 2.1 to 14.3 ( Figure 5.22), whereas the GSI values for the other treatments varied from 8.2 to 15.7. The overall variability within the treatment was moderate ( CVs = 18%   35%; Table 5.20). No significant differences in the mean GSI value per treatment ( Table 5.20) were detected ( Kruskal Wallis, H = 3.00, p = 0.223, df = 2). The achieved power for this endpoint was 40%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 39 ( Table 5.20).

Table 5.20. Summary statistics and power estimates for female gonadosomatic index data for the EPA 21 Day Flutamide assay. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 175 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 10.0 3.5 35% 40% 39 low 16 11.8 2.2 19% high 16 10.7 2.0 18% 1 Calculated from arcsine square root transformed data; with sample size = 16. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 15 10 5 treatment GSI Figure 5.22. Boxplot of female GSI by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

The range of most GSI values calculated for males during the EPA 21 Day Flutamide assay, was small, ranging from 0.6 to 1.7 ( Figure 5.23), which approximates the typical range for reproductively active male fathead minnows. There were no significant differences in mean GSI values ( Table 5.21) among treatments ( Kruskal Wallis, H = 1.06, p = 0.590, df = 2). The achieved power for this endpoint was 17%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 48 ( Table 5.21).

Table 5.21. Summary statistics and power estimates for male gonadosomatic index data for the EPA 21 Day Flutamide assay. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 176 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 1.0 0.3 29% 17% 48 low 8 1.1 0.3 31% high 8 1.2 0.3 26% 1 Calculated from arcsine square root transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.5 1.0 0.5 treatment GSI Figure 5.23. Boxplot of male GSI by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

5.2.5 Female Gonad Histology Histological analyses were conducted on the ovaries of 48 females exposed to flutamide during the EPA 21 Day Assay.

General Ovary Staging   Statistical analysis of the mean ovarian staging from 12 microscopic fields per female in the EPA 21 Day Flutamide assay revealed no significant differences among treatments ( Kruskal Wallis, H = 2.10, p = 0.350, df = 2).

Quantitative Ovarian Staging   One hundred cells in each of three sections per female were examined to quantitatively determine the developmental stage of the ovaries. Ova from fish in all treatments ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages ( Figure 5.24). Variability within treatments for each stage, particularly for Stage 5, was very high as indicated by CVs that ranged as high as 400% ( Table 5.22). Statistical analyses showed that within each of the five developmental stages there were no significant differences among treatments in the proportion of cells occurring in the stage ( Table 5.22). Therefore, flutamide dose had no effect on ovarian developmental stage. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 177 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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.

Control ( n = 16) Low ( n = 16) High ( n = 16) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1a 0.079 0.028 35% 0.082 0.040 49% 0.080 0.035 44% 0.21 0.900 1b 0.303 0.083 27% 0.270 0.078 29% 0.270 0.057 21% 1.70 0.427 2 0.182 0.027 15% 0.204 0.051 25% 0.193 0.046 24% 0.68 0.714 3 0.193 0.037 19% 0.204 0.053 26% 0.184 0.075 40% 1.88 0.391 4 0.197 0.105 53% 0.218 0.119 55% 0.206 0.110 53% 0.65 0.722 5 0.015 0.043 294% 0.010 0.038 400% 0.014 0.034 246% 1.89 0.389 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 1a 1b 2 3 4 5 Ovarian Stage Proportion Control Low High Figure 5.24. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Flutamide assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation.

Atretic Follicles   The mean proportion of atretic follicles per 300 follicles ( counted per fish) ranged from 0.002 follicles for females in the Low concentration to 0.019 follicles for females in the Control treatment ( Figure 5.25). One fish from the Control treatment had a very high proportion of atretic follicles ( 0.27). A significant difference in the proportions of atretic follicles among treatments was detected ( Kruskal Wallis, H = 9.92, p = 0.007, df = 2). The majority of the observations from the High concentration ranked higher than those of the remaining treatments. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 178 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.3 0.2 0.1 0.0 Treatment atretic_ follicles Figure 5.25. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

Corpora Lutea   The mean proportion of corpora lutea per 300 follicles ( counted per fish) ranged from 0.011 for females in the Low concentration to 0.040 for females in the High concentration ( Figure 5.26). Variability within each treatment was very high, ranging from 150% for the High concentration to 236% for the Low concentration. There were no significant differences in the mean proportion of corpora lutea among treatments ( Kruskal Wallis, H = 3.90, p = 0.142, df = 2). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 179 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.2 0.1 0.0 Treatment corpora_ lutea Figure 5.26. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

Observations   Two fish from the Control treatment were observed to have very few eggs in the ovaries. Two fish from the Low concentration were observed to have very few eggs in the ovaries. One of these two fish was also noted to have very small yolk particles in some cortical alveoli cells. One fish from the High concentration were observed to have very few eggs in the ovaries. One other fish from the High concentration had extensive epithelial and ovarian debris in the ovary and one had debris from disintegrated vitellogenic ova in the ovary.

5.2.6 Male Gonad Histology General Testes Staging   Testes from 24 males exposed to flutamide during the EPA 21 Day Flutamide assay were examined to determine the general developmental condition. Males in all treatments had welldeveloped testes with most showing Stage 4 and Stage 5 development ( see Methods for description of developmental stages). All of the 96 microscopic fields examined in the 8 Control treatment males showed Stage 4 ( 76 fields) or Stage 5 ( 20 fields) development. Ninety four of the 96 microscopic fields examined in the 8 Low concentration treatment males showed Stage 4 ( 87 fields) or Stage 5 ( 7 fields) development. Two microscopic fields showed Stage 3 development. All of the 96 microscopic fields examined in the 8 High concentration treatment males showed Stage 4 ( 68 fields) or Stage 5 ( 28 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed no significant differences among treatments ( Kruskal Wallis, H = 4.21, p = 0.122, df = 2).

Quantitative Testicular Staging   One hundred cells in each of three sections per male were examined to quantitatively determine the developmental condition of the testes. The developmental stage all treatment testes ranged from Stage 2a to Stage 5 ( Figure 5.27). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 129% ( Table 5.23). Although statistical analyses showed that there were no significant differences among treatments in the proportion of cells in any of the developmental stages, the probability value calculated for Stage 3b was slightly above the DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 180 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 critical limit of 0.050 ( Table 5.23). The largest difference in the mean proportion of cells in this developmental stage was between the Low and High concentration treatments. Therefore, there was no effect on testicular developmental stage associated with flutamide dose.

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.

Control ( n = 8) Low ( n = 8) High ( n = 8) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1 0 0   0 0   0 0       2a 0.003 0.004 129% 0.003 0.003 93% 0.006 0.005 79% 2.21 0.331 2b 0.009 0.009 101% 0.012 0.013 112% 0.010 0.009 96% 0.04 0.980 3a 0.123 0.096 78% 0.094 0.048 51% 0.128 0.081 64% 0.60 0.740 3b 0.205 0.118 57% 0.325 0.102 31% 0.211 0.064 30% 5.95 0.051 4 0.118 0.073 62% 0.152 0.086 56% 0.151 0.064 42% 0.60 0.740 5 0.543 0.271 50% 0.415 0.132 32% 0.495 0.168 34% 1.03 0.598 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion of Cells Control Low High Figure 5.27. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 21 Day Flutamide assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation.

Tubule Diameter   The average diameter of the seminiferous tubules of males from the Control treatment ranged from 112.5 µ m to 165.6 µ m ( Figure 5.28). Tubule diameters of males from the two test concentrations ranged from 96.7 µ m to 148.9 µ m. No significant differences in the mean tubule diameter DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 181 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 per treatment ( Table 5.24) were detected ( Kruskal Wallis, H = 5.57, p = 0.062, df = 2). The achieved power for this endpoint was 63%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 12 ( Table 5.24).

Table 5.24. Summary statistics and power estimates for male seminiferous tubule diameter ( µ m) data for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 137.4 18.3 13% 63% 12 low 8 116.0 16.5 14% high 8 132.0 11.7 9% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 170 160 150 140 130 120 110 100 90 Treatment diameter Figure 5.28. Boxplot of male seminiferous tubule diameter ( µ m) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value.

Observations   One male in the High concentration treatment showed multifocal proliferation of Sertoli cells and Leydig cells. One male from the Control treatment and two males from the Low concentration showed focal proliferation of Leydig cells. No testicular atrophy was recorded and no ovatestes were observed for any treatment.

5.2.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the EPA 21 Day Flutamide assay ranged from 0 pg/ mL ( not detected) to 3,854 pg/ mL ( Figure 5.29). Among females exposed to the two flutamide concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 182 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 12,367 pg/ mL. No significant differences in the mean estradiol concentration per treatment ( Table 5.25) were detected ( Kruskal Wallis, H = 3.12, p = 0.210, df = 2). The achieved power for this endpoint was 22%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 65 ( Table 5.25).

Table 5.25. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 15 1,225 1,260 103% 22% 65 low 14 1,570 1,172 75% high 16 2,477 3,074 124% 1 Calculated from natural log transformed data; with sample size = 14. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 10000 5000 0 treatment Estradiol Figure 5.29. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisks represent probable outliers.

Estradiol concentrations in Control treatment males used during the EPA 21 Day Flutamide assay ranged from 0 pg/ mL ( not detected) to 298 pg/ mL ( Figure 5.30). Among males exposed to the two flutamide concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 467 pg/ mL. Significant differences in the mean estradiol concentration per treatment ( Table 5.26) were detected ( Kruskal Wallis, H = 9.63, p = 0.008, df = 2). The mean concentration of estradiol in males from the High concentration was less than those of males from the Low concentration and the Control treatment. The achieved power for this endpoint was 79%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 8 ( Table 5.26). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 183 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.26. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 197 87 44% 79% 8 low 7 301 86 28% high 8 68 125 185% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 500 400 300 200 100 0 treatment Estradiol Figure 5.30. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisks represent probable outliers.

Testosterone   Testosterone concentrations in Control treatment females used during the EPA 21 Day Flutamide assay ranged from 0 pg/ mL ( not detected) to 3,475 pg/ mL ( Figure 5.31). Among females exposed to the two flutamide concentrations, testosterone concentrations ranged from 340 pg/ mL to 4,612 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 5.27) were detected ( Kruskal Wallis, H = 6.68, p = 0.035, df = 2). The mean testosterone concentration in females from the High concentration was greater than that of females from the Low concentration and the Control treatment. The achieved power for this endpoint was 45%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 21 ( Table 5.27). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 184 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.27. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 1,329 1,062 80% 45% 21 low 11 1,430 886 62% high 12 2,692 1,420 53% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 5.31. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Testosterone concentrations in Control treatment males used during the EPA 21 Day Flutamide assay ranged from 1,044 pg/ mL to 4,437 pg/ mL ( Figure 5.32). Among males exposed to the two flutamide concentrations, testosterone concentrations ranged from 390 pg/ mL to 6,131 pg/ mL. A significant difference in the mean testosterone concentration per treatment ( Table 5.28) was detected ( Kruskal Wallis, H = 7.26, p = 0.027, df = 2). The mean testosterone concentrations in males from the Low concentration and the Control treatment was less than that of males from the High concentration. The achieved power for this endpoint was 48%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 16 ( Table 5.28). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 185 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.28. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 2,218 1,168 53% 48% 16 low 8 2,278 1,751 77% high 8 4,344 1,220 28% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 6000 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 5.32. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

11 ketotestosterone   11 ketotesosterone was not detected in females from the Control treatment ( six individuals) or from the Low concentration ( one individual). No females from the High concentration were available for 11 ketotestosteron analyses exposed during the EPA 21 Day Flutamide assay.

11 ketotesosterone concentrations in Control treatment males used during the EPA 21 Day Flutamide assay ranged from 8,555 pg/ mL to 31,716 pg/ mL ( Figure 5.33). Among males exposed to the two flutamide concentrations, 11 ketotesosterone concentrations ranged from 1,814 pg/ mL to 95,944 pg/ mL. Significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 5.29) were detected ( Kruskal Wallis, H = 9.38, p = 0.009, df = 2). The mean 11 ketotesosterone concentration in males from the High concentration was greater than those of males from the Low concentration and the Control treatment. The achieved power for this endpoint was 58%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 13 ( Table 5.29).

Table 5.29. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Flutamide assay. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 186 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 16,888 8,477 50% 58% 13 low 8 23,603 28,698 122% high 8 53,209 24,556 46% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 100000 50000 0 treatment 11 keto Figure 5.33. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

5.2.8 Fecundity Total Fecundity   A 14 day pre exposure evaluation of total egg production was performed. Total 14 day counts among the tanks that were eventually used for the three treatments in the exposure assay ( individual tank values summed for each treatment) ranged from about 12,000 eggs to 13,000 eggs ( Figure 5.34). No significant differences in the mean 14 day egg production among the groups of replicates eventually used in the flutamide exposure assay were detected ( Kruskal Wallis, H = 0.15, p = 0.926, df = 2). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 187 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000  14  12  10  8  6  4  2 1 3 5 7 9 11 13 15 17 19 21 Day Cumulative Number of Eggs Control Low High 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.

During the EPA 21 Day Flutamide assay, total counts in the Control treatment were varied almost 1.5 fold among replicates, varying from 3,804 eggs to 5,391 eggs ( Figure 5.35). Variability in total egg production among Low concentration replicates was somewhat less, ranging from 4,003 eggs to 5,275 eggs. Total counts among the High concentration replicates varied about four fold, ranging from 874 eggs to 3,647 eggs. Statistical analysis of square root transformed egg counts showed significant amongtreatment differences ( Kruskal Wallis, H = 7.38, p = 0.025, df = 2) in mean total numbers of eggs produced ( Table 5.30). The total fecundity of females in the High concentration treatment was significantly less than it was for females in the Low concentration or the Control treatment. The achieved power for this assay was 83%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 4 ( Table 5.30).

Table 5.30. Summary statistics and power estimates for total fecundity data for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 4677 772 17% 83% 4 low 4 4777 552 12% high 4 2147 1192 56% 1 Calculated from square root transformed data; with sample size = 4. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on square root transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 188 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0 1,000 2,000 3,000 4,000 5,000 6,000 0 5 10 15 20 Day Cumulative Number of Eggs Control Low High Figure 5.35. Total egg production by replicate per treatment for the EPA 21 Day Flutamide assay.

Fecundity per Female Reproductive Day   During the 14 Day pre exposure evaluation, the mean number of eggs produced per female reproductive day ranged from 54.4 eggs/ day for the tanks that would be used for the Low concentration treatment to 58.8 eggs/ day for the tanks that would be used for the High concentration during the 21 day exposure assay. There were no significant differences among treatments in the numbers of eggs produced per reproductive day during the pre exposure period ( Kruskal Wallis, H = 0.11, p = 0.990, df = 2).

During the EPA 21 Day Flutamide assay, the maximum number of female reproductive days was achieved for all treatments ( Table 5.31). The number of eggs produced per female reproductive day varied from 45.3 eggs to 64.2 eggs in the Control treatment and from 47.7 to 62.8 in the Low concentration ( Figure 5.36). For the High concentration, the number of eggs produced per female reproductive day ranged from 10.4 eggs to 43.4 eggs, although fish in two of the replicates yielded fewer than 20 eggs per day ( 10.4, 19.1). Because no fish died during the assay, the statistical results reported here are the same as those reported for total fecundity. A significant difference in the mean number of eggs laid per day per treatment ( Table 5.31) occurred. The number of eggs laid per day in the Highconcentration treatment was significantly less than it was for females in the Low concentration or the Control treatment. The achieved power for this assay was 73%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 5 ( Table 5.31). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 189 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.31. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 21 Day Flutamide assay.

Level Mean Number of Reproductive Days 1 N Mean Stdev CV Achieved Power 2 Sample Size Required 3 control 84 4 55.7 9.2 17% 73% 5 low 84 4 56.9 6.6 12% high 84 4 25.6 14.2 56% 1 Maximum number = 84. 2 Calculated from natural log transformed data; with sample size = 4. 3 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 70 60 50 40 30 20 10 treatment eggs/ ReproDay Figure 5.36. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Eggs on Tiles/ Dishes   The mean number of eggs laid on the tiles during the 14 day pre exposure assay varied from 1,944 eggs for the tanks that would be used for the Low concentration to 2,355 eggs for the tanks that would be used for the Control treatment. The mean number of eggs on dishes ranged from 704 eggs for the Control treatment to 1,223 eggs for the High concentration. Because of the variability in the total number of eggs laid per treatment, the proportional difference in the number of eggs on dishes versus those on tiles [ 1 (# eggs on dishes ÷ # eggs on tiles)] was calculated. There were no significant differences in the mean proportional difference among treatments during the 14 day pre exposure assay ( Kruskal Wallis, H = 5.73, p = 0.126, df = 3).

The mean number of eggs laid on the tiles among the treatments during the EPA 21 Day Flutamide assay varied from 1,343 eggs for the High concentration to 3,463 eggs for the Low concentration treatment ( Appendix E, Table 3.7). The number of eggs on dishes ranged from 207 eggs for the Control treatment to 350 eggs for the High concentration. The proportional difference ranged from 0.16 ( one High DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 190 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 concentration replicate) to 0.87 ( one Control treatment replicate) ( Appendix E, Figure 3.6). There was a significant difference in the mean proportional difference among treatments ( Kruskal Wallis, H = 8.35, p = 0.015, df = 2). The mean proportional difference in the number of eggs on dishes versus those on tiles for the High concentration was significantly lower than those for the Low concentration and the Control treatment.

5.2.9 Fertilization Success Total Fertilization   Eggs were collected during the 14 day pre exposure period for the evaluation of fertilization success rate. The mean proportion of eggs fertilized in the Control treatment was 0.972 [ standard deviation ( sd) = 0.053], 0.999 ( sd = 0.004) in the Low concentration, and 0.999 ( sd = 0.005) in the High concentration. The mean proportion of eggs fertilized in the replicates that were not used in the 21 day validation assay was 0.997 ( sd = 0.004). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 5.47, p = 0.140, df = 3).

The total ( tiles + dishes) fertilization success rates for most treatment replicates during the EPA 21 Day Flutamide assay were high, ranging from 0.994 ( one Low concentration replicate) to 1.00 ( two Lowconcentration replicates) ( Figure 5.37). One High concentration replicate had a relatively low proportion of eggs fertilized ( 0.857%). No significant differences in mean fertilization success rates ( Table 5.32) among treatments were detected ( Kruskal Wallis, H = 4.32, p = 0.115, df = 2). The achieved power for this assay was 20%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 17 ( Table 5.32).

Table 5.32. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 0.999 0.001 0.1% 20% 17 low 4 0.998 0.003 0.3% high 4 0.961 0.070 7% 1 Calculated from arcsine square root transformed data; with sample size = 4. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 191 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 1.00 0.95 0.90 0.85 treatment Total Prop fert Figure 5.37. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Fertilization of Eggs on Tiles and Dishes   During the 14 Day pre validation assay, there were no significant differences in the fertilization success rates among treatments for eggs laid in tiles ( Kruskal Wallis, H = 7.44, p = 0.059, df = 3) or on dishes ( Kruskal Wallis, H = 4.85, p = 0.183, df = 3). The fertilization success rates for all treatment replicates for eggs laid on tiles during the EPA 21 Day Flutamide assay were high, ranging from 0.993 ( one Low concentration replicate) to 1.00 ( three Low and one High concentration replicates) ( Appendix E, Figure 3.7). No significant differences in mean fertilization success rates for eggs laid on tiles ( Appendix E, Table 3.8) among treatments were detected ( Kruskal Wallis, H = 1.33, p = 0.516, df = 2). The fertilization success rates for most treatment replicates for eggs laid on dishes during the assay were high, ranging from 0.972 ( one High concentration replicate) to 1.00 ( several replicates; including all treatments) ( Appendix E, Figure 3.8). One High concentration replicate had a relatively low proportion of eggs ( laid on tiles) that were fertilized ( 0.693%). No significant differences in mean fertilization success rates ( Appendix E, Table 3.8) among treatments were detected ( Kruskal Wallis, H = 4.91, p = 0.086, df = 2).

5.2.10 Hatchability and Larval Development Eggs were collected during the 14 day pre exposure period for the evaluation of hatchability. The mean proportion of fertilized eggs that hatched in the Control treatment was 0.98 [ standard deviation ( sd) = 0.03], 1.00 ( sd = 0.01) in the Low concentration, and 1.00 ( sd = 0.01) in the High concentration. There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 1.04, p = 0.594, df = 2).

Eggs were collected during the EPA 21 Day Flutamide assay for the evaluation of hatchability. The proportion of fertilized eggs that hatched ranged from 0.92 to 1.00 in the Control treatment and from 0.33 to 1.00 for the two test concentrations ( Figure 5.38). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 1.15, p = 0.563, df = 2). The achieved power for this endpoint was 18%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 44 ( Table 5.33). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 192 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.33. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 9 0.98 0.03 3% 18% 44 low 9 0.97 0.04 4% high 8 0.89 0.23 26% 1 Calculated from arcsine square root transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 treatment Prop Hatch Figure 5.38. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

Eggs were collected during the pre exposure period for the evaluation of larval development. The mean proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) in the Control treatment was 0.99 ( sd = 0.01). The mean proportion of normal larvae in the remaining treatments was 0.98 ( sd = 0.03) in the Low concentration and 0.98 ( sd = 0.02) in the High concentration. There were no significant differences among treatments in the proportion of normal larvae ( Kruskal Wallis, H = 0.71, p = 0.703, df = 2).

Eggs were collected during the EPA 21 Day Flutamide assay for the evaluation of larval development. The proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) ranged from 0.84 to 1.00 in the Control treatment and from 0.70 to 1.00 for the two test concentrations ( Figure 5.39). There was a significant difference among treatments in the proportion of normal larvae ( Kruskal Wallis, H = 8.17, p = 0.017, df = 2). The Mean proportion of larvae that developed normally DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 193 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 was significantly less in the High concentration than it was in the Control treatment. The achieved power for this endpoint was 75%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 9 ( Table 5.34).

Table 5.34. Summary statistics and power estimates for the proportion of normal larvae for the EPA 21 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 9 0.97 0.05 5% 75% 9 low 9 0.96 0.04 4% high 8 0.89 0.11 12% 1 Calculated from arcsine square root transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.0 0.9 0.8 0.7 treatment Prop Norm Figure 5.39. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

5.2.11 Body Weight The body weight of females used in the EPA 21 Day Flutamide assay ranged from 1.1 g to 2.8 g. There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 5.19, p = 0.075, df = 2). The body weight of males used in the EPA 21 Day Flutamide assay ranged from 3.1 g to 5.6 g. There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 3.80, p = 0.150, df = 2). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 194 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 5.3 Non spawning Adult 14 Day Assay for Flutamide The Non spawning Adult 14 Day Flutamide assay was conducted from March 10, 2003 to March 24, 2003 ( exposure assay).

5.3.1 Survival All males and females in all treatments survived the Non spawning Adult 14 Day Flutamide assay.

5.3.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the Non spawning Adult 14 Day Flutamide assay ranged from 1,252,000 ng/ mL to 5,983,000 ng/ mL ( Figure 5.40). Among females exposed to the two flutamide concentrations, vitellogenin concentrations ranged from 3,325,000 ng/ mL to 10,905,000 ng/ mL. Significant differences in the mean vitellogenin concentration among treatments ( Table 5.35) were detected ( Kruskal Wallis, H = 10.88, p = 0.012, df = 2). Mean vitellogenin concentrations among females exposed to the High and Medium flutamide concentrations were greater than the mean concentration for Control treatment females. The achieved power for this endpoint was 82%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 10 ( Table 5.35).

Table 5.35. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 4,189,350 1,516,107 36% 82% 10 low 10 6,147,250 2,219,643 36% Medium 10 7,342,100 2,699,666 37% High 10 7,546,150 2,137,789 28% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 195 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 10000000 5000000 0 treatment Flut VTG Figure 5.40. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Vitellogenin concentrations in Control treatment males used during the Non spawning Adult 14 Day Flutamide assay ranged from 0 ng/ mL ( not detected) to 1,523 ng/ mL ( Figure 5.41). Among most males exposed to the two flutamide concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 3,285 ng/ mL. One male exposed to the Low flutamide concentration had a vitellogenin concentration of 10,575 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 5.36) were detected ( Kruskal Wallis, H = 3.66, p = 0.300, df = 2). The achieved power for this endpoint was 12%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 94 ( Table 5.36).

Table 5.36. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 243 461 190% 12% 94 low 10 1,337 3,342 250% Medium 10 631 1,325 210% High 10 76 89 117% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 196 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 10000 5000 0 treatment Flut VTG Figure 5.41. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

5.3.3 Appearance / Secondary Sex Characteristics All females used during the Non spawning Adult 14 Day Flutamide assay showed normal female morphology.

Morphological development among males used during the flutamide Non spawning Adult 14 day assay varied among treatments ( Figure 5.42). One male exposed to the Medium concentration exhibited a female body shape. Fourteen of the 40 males used during the assay lacked tubercles. Most of these males also lacked a fat pad and vertical banding. There was no consistent dose related pattern to these variations in morphology. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 197 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Tubercles Abs Tubercles Prs Fat Pad NP Fat Pad P Fat Pad Pro Vert Banding NP Vert Banding P Vert Banding Pro Proportion Control Low Mid High Figure 5.42. Secondary sex characteristics of males used during the EPA Non spawning Adult 14 Day Flutamide assay.

5.3.4 Gonadosomatic Index The range of GSI values calculated for females in the Control treatments varied from 5.5 to 24.8 ( Figure 5.43), whereas the GSI values for the other treatments varied from 3.8 to 19.7. The overall withintreatment variability was moderate ( CVs = 29%   43%; Table 5.37). There were no significant differences in mean GSI values ( Table 5.37) among treatments ( Kruskal Wallis, H = 2.78, p = 0.426, df = 3). The achieved power for this endpoint was 18%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 55 ( Table 5.37).

Table 5.37. Summary statistics and power estimates for female gonadosomatic index data for the Nonspawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 13.7 5.6 41% 18% 55 low 10 11.7 4.5 38% medium 10 13.0 5.6 43% high 10 10.4 3.0 29% 1 Calculated from arcsine square root transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 198 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 25 15 5 treatment GSI Figure 5.43. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

The range of most GSI values calculated for most males during the Non spawning Adult 14 Day Flutamide assay, was large, varying from 0.3 to 2.7 ( Figure 5.44), which approximates the typical range for reproductively active male fathead minnows. One male from the Low concentration had an exceptionally high GSI value of 21.3. This male was small ( 3.2 g) and had a hypertrophied gonad ( 0.68 g) that was much larger than the average gonad weight ( 0.068 g, sd = 0.031) for the other males used during this test. This male was excluded from all analyses. Overall within treatment variability was moderate to high ( CVs = 31%   50%; Table 5.38). The highest, excluding the male mentioned above, and lowest male GSI values were 2.6   2.7 ( for two fish in the High concentration) and 0.3   0.4 ( three fish, one from each treatment except the Medium concentration), respectively. However, there were no significant differences in mean GSI values ( Table 5.38) among treatments ( Kruskal Wallis, H = 1.23, p = 0.746, df = 3). The achieved power for this endpoint was 10%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 108 ( Table 5.38).

Table 5.38. Summary statistics and power estimates for male gonadosomatic index data for the Nonspawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 1.3 0.5 39% 10% 108 low 9 1.4 0.7 50% medium 10 1.3 0.4 31% high 10 1.6 0.8 47% 1 Calculated from arcsine square root transformed data; with sample size = 9. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 199 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 3 2 1 0 treatment GSI Figure 5.44. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

5.3.5 Female Gonad Histology Histological analyses were conducted on the ovaries of 40 females exposed to flutamide during the Non Spawning Adult 14 Day Assay.

General Ovary Staging   Statistical analysis of the mean ovarian staging from 12 microscope fields per fish in the Non spawning Adult 14 Day Flutamide assay revealed no significant differences among treatments ( Kruskal Wallis, H = 1.08, p = 0.782, df = 3).

Quantitative Ovarian Staging   One hundred cells in each of three sections per female were examined to quantitatively determine the developmental condition of the ovaries. Ova from fish in the three exposure treatments ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages), whereas ova from females from the Control treatment showed Stage 1a to Stage 4 development ( Figure 5.45). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 316% ( Table 5.39). Although statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stages 1b and 3, there were no significant differences among treatments in the proportion of cells in the developmental Stages 1a, 2, 4, and 5 ( Table 5.39). The proportion of cells in developmental Stages 1b and 3 in the High concentration were significantly greater than those in the Low concentration treatments. There was no consistent pattern of significant difference associated with flutamide dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 200 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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.

Control ( n = 10) Low ( n = 10) Medium ( n = 10) High ( n = 10) Kruskal Wallis Stag e Mean Stdev CV Mean Stdev CV Mean Stdev CV Mean Stdev CV H p 1a 0.087 0.028 32% 0.072 0.030 42% 0.062 0.022 36% 0.091 0.021 23% 6.79 0.079 1b 0.256 0.072 28% 0.229 0.075 33% 0.265 0.073 27% 0.326 0.058 18% 8.45 0.038 * 2 0.183 0.049 27% 0.195 0.055 28% 0.209 0.071 34% 0.213 0.049 23% 2.23 0.526 3 0.136 0.045 33% 0.171 0.054 31% 0.141 0.039 28% 0.103 0.046 45% 8.25 0.041 * 4 0.287 0.115 40% 0.226 0.082 36% 0.238 0.103 43% 0.168 0.071 42% 6.56 0.087 5 0 0   0.010 0.033 316% 0.001 0.003 316% 0.016 0.052 316% 1.06 0.787 * p < 0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 1a 1b 2 3 4 5 Ovarian Stage Proportion Control Low Mid High Figure 5.45. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Flutamide assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation.

Atretic Follicles   The mean proportion of atretic follicles per 300 follicles ( counted per fish) ranged from 0.012 follicles for females in the High concentration to 0.030 follicles for females in the Medium concentration treatment ( Figure 5.46). There were no significant differences in the mean proportion of atretic follicles among treatments ( Kruskal Wallis, H = 2.83, p = 0.419, df = 3). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 201 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 0.3 0.2 0.1 0.0 Treatment atretic_ follicles Figure 5.46. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the Nonspawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, asterisks represent probable outliers.

Corpora Lutea   The mean proportion of corpora lutea per 300 follicles ( counted per fish) ranged from 0.001 for females in the Medium concentration to 0.016 for females in the Low concentration ( Figure 5.47). There was a significant differences in the mean proportion of corpora lutea among treatments ( Kruskal Wallis, H = 10.81, p = 0.013, df = 3). The mean proportion of corpora lutea in ovaries from females in the High concentration was greater than those for females from the Medium and Low concentrations, but did not differ from that for females from the Control treatment. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 202 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 0.15 0.10 0.05 0.00 Treatment corpora_ lutea Figure 5.47. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the Nonspawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

Observations   One fish from the Control treatment was observed to have interstitial inflammation. The ovary of one fish exposed to the Low flutamide concentration had a zone of only Stage 5 eggs and another zone with developing ova, containing mix of all stages, including Stage 5. The Stage 5 eggs were abnormally sequestered in the ovary of this fish. One fish exposed to the High flutamide concentration had Stage 5 eggs segregated in the ovary.

5.3.6 Male Gonad Histology General Testes Staging   Testes from 39 males exposed to flutamide during the Non spawning Adult 14 Day Flutamide assay were examined to determine the general developmental condition. Males in all treatments had well developed testes with most showing Stage 4 and Stage 5 development ( see Methods for description of developmental stages). All except 1 of the 120 microscopic fields examined in the 10 Control treatment males showed Stage 4 ( 45 fields) or Stage 5 ( 74 fields) development. Most of the 120 microscopic fields examined in the 9 Low concentration treatment males showed Stage 4 ( 49 fields) or Stage 5 ( 47 fields) development. Twelve microscopic fields showed Stage 1 development. In the 10 Medium concentration males available for examination, 112 of the 120 microscopic fields showed Stage 4 ( 64 fields) or Stage 5 ( 48 fields) development. In the 10 High concentration males available for examination, 108 of the 120 microscopic fields showed Stage 4 ( 53 fields) or Stage 5 ( 55 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed that no significant differences among treatments ( Kruskal Wallis, H = 3.35, p = 0.341, df = 3). Quantitative Testicular Staging   One hundred cells in each of three sections per male were examined to quantitatively determine the developmental condition of the testes. The developmental stage all treatment testes ranged from Stage 2a to Stage 5 ( Figure 5.48). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 216% ( Table 5.40). There were no significant differences among treatments in the proportion of cells in any of the developmental stages ( Table 5.40). Therefore, there was no effect on testicular developmental stage associated with flutamide dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 203 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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.

Control ( n = 10) Low ( n = 8) Medium ( n = 10) High ( n = 10) Kruskal Wallis Stag e Mean Stdev CV Mean Stdev CV Mean Stdev CV Mean Stdev CV H p 1 0 0   0 0   0 0   0 0       2a 0.002 0.004 211% 0.003 0.004 142% 0.001 0.003 211% 0.001 0.002 216% 1.41 0.703 2b 0.008 0.010 131% 0.013 0.015 112% 0.034 0.048 140% 0.015 0.030 200% 5.83 0.120 3a 0.074 0.112 152% 0.043 0.030 72% 0.069 0.098 141% 0.107 0.203 190% 0.01 1.000 3b 0.104 0.134 129% 0.124 0.066 54% 0.110 0.117 106% 0.115 0.064 56% 3.01 0.390 4 0.057 0.068 120% 0.077 0.072 94% 0.099 0.099 100% 0.059 0.057 96% 1.54 0.673 5 0.755 0.250 33% 0.741 0.148 20% 0.686 0.280 41% 0.703 0.272 39% 1.34 0.720 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion of Cells Control Low Mid High Figure 5.48. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Flutamide assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation.

Tubule Diameter   The diameter of the seminiferous tubules of males from the Control treatment ranged from 92.2 µ m to 194.4 µ m ( Figure 5.49). Tubule diameters of males from the three test concentrations ranged from 52.5 µ m to 254.4 µ m. No significant differences in the mean tubule diameter per treatment ( Table 5.41) were detected ( Kruskal Wallis, H = 0.22, p = 0.974, df = 3). The achieved power for this endpoint was 5%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 1,361 ( Table 5.41). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 204 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.41. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 147.1 37.0 25% 5% 1,361 low 9 144.8 45.4 31% medium 10 146.6 37.6 26% high 10 153.1 51.3 34% 1 Calculated from natural log transformed data; with sample size = 9. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 250 150 50 Treatment diameter Figure 5.49. Boxplot of seminiferous tubule diameter ( µ m) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Observations   No Sertoli cell proliferation was observed for any treatment. One male exposed to the Medium flutamide concentration showed multifocal Leydig cell proliferation. Three males exposed to the High concentration were found to have focal or multifocal Leydig cell proliferation. One male from the Medium flutamide concentration had an egg embedded in the testes. Testicular atrophy was observed for one of the High concentration males ( Fish ID 218632 in Table 5.42) that showed multifocal Leydig cell proliferation. Several males exposed to flutamide had abnormal testicular conditions ( Table 5.42). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 205 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.42. Histological observations for males exposed to concentrations of flutamide during the Nonspawning Adult 14 D assay.

Fish ID Concentration Leydig Cell Proliferation Observations 218604 Low None Premature release of secondary spermatocytes ( stage 3b) in lumina. High concentration of spermatogonia in mature tubules.

218620 Medium None Premature release of secondary spermatocytes ( stage 3b) in lumina. Some released stage 3b cells necrotic. Lumina generally occluded.

218623 Medium None Multifocal necrotic primary and secondary spermatocytes ( 3a and 3b) in crypts of tubules 218624 Medium None Premature release of secondary spermatocytes ( stage 3b) in lumina. Multifocal necrosis of secondary spermatocytes ( stage 3b). 218625 Medium Multifocal Basophilic nodules in testicular duct.

218626 Medium None Focal area proliferation and sequestration of sperm cells. 218627 Medium None Nodules in spermatic ducts and some tubules.

218628 High Focal Multifocal but not extensive necrosis of secondary spermatocytes ( stage 3b).

218632 High Multifocal Multifocal tubules with encysted structures that were possibly abnormal oocytes. Multifocal but sparse necrotic spermatogonia. One ova in section, probably an artifact.

218635 High Multifocal Tubule lumina occluded. Multifocal necrosis of secondary spermatocytes ( stage 3b).

218636 High None Multifocal necrosis of secondary spermatocytes ( stage 3b).

5.3.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the Non Spawning Adult 14 Day Flutamide assay ranged from 105 pg/ mL to 2,990 pg/ mL ( Figure 5.50). Among females exposed to the three flutamide concentrations, estradiol concentrations ranged from 478 pg/ mL to 5,591 pg/ mL. No significant differences in the mean estradiol concentration per treatment ( Table 5.43) were detected ( Kruskal Wallis, H = 3.06, p = 0.383, df = 2). The achieved power for this endpoint was 8%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 196 ( Table 5.43). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 206 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.43. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 1,638 850 52% 8% 196 low 10 1,371 842 61% medium 10 1,909 1,423 75% high 10 1,180 400 34% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 6000 5000 4000 3000 2000 1000 0 treatment Estradiol Figure 5.50. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

Estradiol was not detected in the 10 Control treatment males used during the Non Spawning Adult 14 Day Flutamide assay ( Figure 5.51). Among males exposed to the three flutamide concentrations, estradiol was not detected in the 9 Low concentration males or the 10 Medium concentration males ( Table 5.44). Estradiol was detected in two of the eight males from the High concentration that were analyzed. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 207 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.44. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 1 control 10 0 0       low 9 0 0   medium 10 0 0   high 8 65 124 191% 1 Not calculated.

Mid Low High Control 300 200 100 0 treatment Estradiol Figure 5.51. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Testosterone   Testosterone concentrations in Control treatment females used during the Non Spawning Adult 14 Day Flutamide assay ranged from 771 pg/ mL to 3,053 pg/ mL ( Figure 5.52). Among females exposed to the three flutamide concentrations, testosterone concentrations ranged from 145 pg/ mL to 4,057 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 5.45) were detected ( Kruskal Wallis, H = 5.51, p = 0.138, df = 2). The achieved power for this endpoint was 23%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 32 ( Table 5.45). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 208 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.45. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 1,565 857 55% 23% 32 low 8 880 475 54% medium 9 2,340 1,478 63% high 10 1,235 816 66% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 4000 3000 2000 1000 0 treatment Testosterone Figure 5.52. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

Testosterone concentrations in Control treatment males used during the Non Spawning Adult 14 Day Flutamide assay ranged from 607 pg/ mL to 4,044 pg/ mL ( Figure 5.53). Among males exposed to the three flutamide concentrations, testosterone concentrations ranged from 513 pg/ mL to 6,506 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 5.46) were detected ( Kruskal Wallis, H = 5.00, p = 0.172, df = 2). The achieved power for this endpoint was 28%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 26 ( Table 5.46). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 209 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.46. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 2,096 1,180 56% 28% 26 low 10 1,737 1,787 103% medium 10 1,045 405 39% high 8 2,042 1,596 78% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 7000 6000 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 5.53. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisks represent probable outliers.

11 ketotestosterone   11 ketotesosterone was detected in one of seven Control treatment females and in one of five females from the Low concentration during the Non Spawning Adult 14 Day Flutamide assay ( Figure 5.54). 11 ketotesosterone was not detected in females from the Medium concentration ( six individuals) and the High concentration ( five individuals) exposed during the Non Spawning Adult 14 Day Flutamide assay. No significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 5.47) were detected ( Kruskal Wallis, H = 1.98, p = 0.577, df = 2). The achieved power for this endpoint was 8%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 91 ( Table 5.47). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 210 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.47. Summary statistics and power estimates for female 11 ketotesosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 7 70 184 265% 8% 91 low 5 86 192 223% medium 6 0 0   high 5 0 0   1 Calculated from natural log transformed data; with sample size = 5. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 500 400 300 200 100 0 treatment 11 keto Figure 5.54. Boxplot of female 11 ketotesosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier.

11 ketotesosterone concentrations in Control treatment males used during the Non Spawning Adult 14 Day Flutamide assay ranged from 2,467 pg/ mL to 29,425 pg/ mL ( Figure 5.55). Among males exposed to the three flutamide concentrations, 11 ketotesosterone concentrations ranged from 1,816 pg/ mL to 89,227 pg/ mL. No significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 5.48) were detected ( Kruskal Wallis, H = 0.67, p = 0.879, df = 2). The achieved power for this endpoint was 6%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 295 ( Table 5.48). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 211 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.48. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non Spawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 10,190 8,876 87% 6% 295 low 10 19,906 27,524 138% medium 10 6,502 2,949 45% high 7 13,341 12,900 97%

1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 100000 50000 0 treatment 11 keto Figure 5.55. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Non Spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and asterisk represent probable outliers.

5.3.8 Body Weight and Length The body weight of females used in the Non spawning Adult 14 Day Flutamide assay ranged from 1.9 g to 3.7 g ( Figure 5.56). There were no significant differences in mean body weight ( natural log transformed) among treatments ( Kruskal Wallis, H = 3.90, p = 0.272, df = 3). The achieved power for this endpoint was 36%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 26 ( Table 5.49).

The body ( fork) length of females used in the Non spawning Adult 14 Day Flutamide assay ranged from 46 mm to 61 mm ( Figure 5.57). There were no significant differences in mean body length ( natural log transformed) among treatments ( Kruskal Wallis, H = 1.74, p = 0.627, df = 3). The achieved power for DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 212 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 this endpoint was 10%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 123 ( Table 5.50).

Table 5.49. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 2.9 0.5 19% 36% 26 low 10 2.6 0.4 15% medium 10 2.5 0.3 13% high 10 2.5 0.4 15% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 3.5 3.0 2.5 2.0 treatment fish_ wgt_ whole Figure 5.56. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 213 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.50. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 53.6 5.3 10% 10% 123 low 10 53.4 3.3 6% medium 10 53.1 2.7 5% high 10 55.0 2.8 5% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 60 55 50 45 treatment fish_ length_ fork Figure 5.57. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

The body weight of males used in the Non spawning Adult 14 Day Flutamide assay ranged from 2.8 g to 6.9 g ( Figure 5.58). There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 7.08, p = 0.069, df = 3). The achieved power for this endpoint was 30%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 28 ( Table 5.51).

The body ( fork) length of males used in the Non spawning Adult 14 Day Flutamide assay ranged from 54 mm to 75 mm ( Figure 5.59). There were no significant differences in mean body length ( natural log transformed) among treatments ( Kruskal Wallis, H = 5.42, p = 0.143, df = 3). The achieved power for this endpoint was 17%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 52 ( Table 5.52). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 214 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 5.51. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 5.0 0.5 10% 30% 28 low 9 4.9 0.9 18% medium 10 5.3 0.8 16% high 10 4.3 0.7 16% 1 Calculated from natural log transformed data; with sample size = 9. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 7 6 5 4 3 treatment fish_ wgt_ whole Figure 5.58. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Table 5.52. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Flutamide assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 65.4 1.8 3% 17% 52 low 9 68.0 4.0 6% medium 10 68.2 4.6 7% high 10 64.2 5.0 8% 1 Calculated from natural log transformed data; with sample size = 9. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 215 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 75 65 55 treatment fish_ length_ fork Figure 5.59. Boxplot of male body length ( mm) by treatment for the Non spawning Adult 14 Day Flutamide assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.