DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 216 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 6.0 RESULTS: FADROZOLE 6.1 EPA 14 Day Assay for Fadrozole The EPA 14 Day Fadrozole assay was conducted from March 17, 2003, to March 27, 2003 ( pre exposure assay), and from March 27, 2003, to April 10, 2003 ( exposure assay).

6.1.1 Survival All males and females in all treatments survived the EPA 14 Day Fadrozole assay.

6.1.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the EPA 14 Day fadrozole assay ranged from 3,426,500 ng/ mL to 6,920,500 ng/ mL ( Figure 6.1). Among females exposed to the two fadrozole concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 2,798,000 ng/ mL. Significant differences in the mean vitellogenin concentration per treatment ( Table 6.1) were detected ( Kruskal Wallis, H = 41.80, p = < 0.001, df = 2). Vitellogenin concentrations in Controltreatment females were significantly greater than those in females exposed to the Low and High fadrozole concentrations. Additionally, vitellogenin concentrations in females from the Low concentration were greater than those in females from the High concentration. The achieved power for this endpoint was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 2 ( Table 6.1).

Table 6.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 5,364,000 1,059,237 20% 100% 2 low 16 1,148,553 703,249 61% high 16 1,693 1,986 117% 1 Calculated from natural log transformed data; with sample size = 16. 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) 217 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 6000000 4000000 2000000 0 treatment Fad VTG Figure 6.1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Fadrozole 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.

Vitellogenin concentrations in Control treatment males used during the EPA 14 Day fadrozole assay ranged from 0 ng/ mL ( not detected) to 7,123 ng/ mL ( Figure 6.2). Among most males exposed to the two fadrozole concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 5,604 ng/ mL. One male exposed to the Low fadrozole concentration treatment had a vitellogenin concentration of 15,191 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 6.2) were detected ( Kruskal Wallis, H = 0.67, p = 0.716, 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 528 ( Table 6.2).

Table 6.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 2,150 3,088 144% 6% 528 low 8 2,856 5,111 179% high 8 1,056 1,914 181% 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) 218 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 15000 10000 5000 0 treatment Fad VTG Figure 6.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Fadrozole 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.

6.1.3 Appearance / Secondary Sex Characteristics All of the females used during the EPA 14 Day Fadrozole assay exhibited typical female morphology ( no fat pad, no tubercles, no vertical banding, ovipositor present).

Most of the males used during the EPA 14 Day Fadrozole assay exhibited typical male morphology ( fat pads, tubercles, vertical banding, no ovipositor present). One male from the Control treatment lacked a fat pad and vertical banding.

6.1.4 Gonadosomatic Index The range of GSI values calculated for females in the all treatments varied about two fold ( Figure 6.3), and the overall variability within the treatment was moderate ( CVs = 22%   24%; Table E14FAD GSI 1). The highest female GSI values were about 21   23 ( two fish each in the Low and High concentrations). Significant differences in mean GSI values ( Table 6.3) among treatments were detected ( Kruskal Wallis, H = 13.54, p = 0.001, df = 2). The mean GSI values for females exposed to the Low and High concentrations were significantly greater than GSI values for females from the Control treatment. The achieved power for this endpoint was 92%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 12 ( Table 6.3). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 219 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.3. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 11.0 2.4 22% 92% 12 low 16 15.1 3.5 23% high 16 14.8 3.5 24% 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 23 18 13 8 treatment GSI Figure 6.3. Boxplot of female GSI by treatment for the EPA 14 Day Fadrozole 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 Fadrozole assay, was small, ranging from 0.7 to 1.6 ( Figure 6.4), which approximates the typical range for reproductively active male fathead minnows. However, five fish had GSI values   2.0 ( at least one fish from each treatment). The highest male GSI value was 3.6 for a small fish ( 2.5 g body weight) exposed to the Low fadrozole concentration that had a relatively large gonad ( 0.09 g). There were no significant differences in mean GSI values ( Table 6.4) among treatments ( Kruskal Wallis, H = 4.12, p = 0.128, df = 2). The achieved power for this endpoint was 24%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 32 ( Table 6.4). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 220 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 1.23 0.42 34% 24% 32 low 8 1.45 0.87 60% high 8 1.66 0.45 27% 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 3.5 2.5 1.5 0.5 treatment GSI Figure 6.4. Boxplot of male GSI by treatment for the EPA 14 Day Fadrozole 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.

6.1.5 Female Gonad Histology Histological analyses were conducted on the ovaries of 48 females exposed to fadrozole 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 fadrozole assay revealed significant differences among treatments ( Kruskal Wallis, H = 8.78, p = 0.012, df = 2). The mean ovarian stage of females from the High concentration was significantly less than those of females from the Control treatment and the Low concentration.

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 Low concentration ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages), whereas ovaries in females from the High concentration treatment showed Stage 1a to Stage 4 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 221 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 development ( Figure 6.5). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 400% ( Table 6.5). Statistical analyses revealed significant difference among treatments in the proportion of cells in three of the five developmental stages ( Table 6.5). The proportion of cells in developmental Stage 1a was greater in females from the Control treatment than in females from the High and Low concentrations. Also, the proportion of cells in developmental Stage 1b was greater in females from the Control treatment than in females from the High concentration. The proportion of cells in developmental Stage 3 was greater in females from the High concentration than in females from the Control treatment and the Low concentration. The proportion of cells in developmental Stage 4 was less in females from the High concentration than in females from the Control treatment and the Low concentration.

Table 6.5. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 16) Low ( n = 16) High ( n = 16) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1a 0.085 0.028 33% 0.058 0.019 32% 0.050 0.025 50% 14.19 0.001* 1b 0.277 0.067 24% 0.220 0.057 26% 0.205 0.067 33% 8.83 0.012** 2 0.200 0.048 24% 0.198 0.032 16% 0.189 0.073 39% 1.21 0.545 3 0.192 0.046 24% 0.228 0.060 26% 0.351 0.100 28% 20.70 < 0.001*** 4 0.206 0.107 52% 0.272 0.080 29% 0.103 0.065 63% 20.79 < 0.001*** 5 0.015 0.045 308% 0.000 0.002 400% 0 0   2.18 0.337 * p < 0.01 ** p < 0.05 *** p < 0.001 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 222 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0.00 0.10 0.20 0.30 0.40 0.50 0.60 1a 1b 2 3 4 5 Ovarian Stage Proportion Control Low High Figure 6.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Fadrozole 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 0.009 follicles for females in the Control treatment to 0.102 follicles for females in the High concentration ( Figure 6.6). There was a significant difference in the mean proportions of atretic follicles among treatments ( Kruskal Wallis, H = 14.26, p = 0.001, df = 2). The proportion of atretic follicles was greater for females from the High concentration than for those from the Control treatment or the Low concentration. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 223 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Treatment atretic_ follicles Figure 6.6. Boxplot of atretic follicles per 300 follicles by treatment for the EPA 14 Day Fadrozole 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 0.001 for females in the High concentration to 0.012 for females in the Control treatment ( Figure 6.7). There were significant differences in the mean proportion of corpora lutea among treatments ( Kruskal Wallis, H = 11.24, p = 0.004, df = 2). The value for the Control treatment was significantly greater than those of the other two treatments.

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 6.7. Boxplot of corpora lutea per 300 follicles by treatment for the EPA 14 Day Fadrozole 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 224 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Observations   One female from the Control treatment was observed to have abnormal appearing Stage 2 ova that had crenulated surfaces. Six females from the High concentration had vacuoles in the nuclei of some Stage 3 and/ or Stage 4 cells. One additional female from the High concentration had inflammatory cells in the ovary.

6.1.6 Male Gonad Histology General Testes Staging   Testes from 24 males exposed to fadrozole during the EPA 14 Day Fadrozole assay were examined to determine the general developmental condition. Males in all treatments had welldeveloped testes, 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 ( 49 fields) or Stage 5 ( 47 fields) development. All of the 96 microscopic fields examined in the 8 Low concentration treatment males showed Stage 4 ( 67 fields) or Stage 5 ( 29 fields) development. All of the 96 microscopic fields examined in the 8 High concentration treatment males showed Stage 4 ( 40 fields) or Stage 5 ( 56 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed no significant differences among treatments ( Kruskal Wallis, H = 3.15, p = 0.207, 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 6.8). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 197% ( Table 6.6). Statistical analyses showed that there were significant differences among treatments in the proportion of cells in developmental Stage 4. The proportion of cells showing Stage 4 development was greater in males from the Low concentration than in males from the Control treatment or the High concentration. The probability value calculated for Stage 5 was slightly above the critical limit of 0.050 ( Table E14FAD MHist 1). The greatest difference was between the mean proportion of cells showing Stage 5 development in males from the High concentration ( 0.68) and the Low concentration ( 0.40). Therefore, there was no effect on testicular developmental stage associated with fadrozole dose.

Table 6.6. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

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 0.007 0.010 148% 0.003 0.006 197% 0.003 0.003 95% 0.93 0.629 2b 0.022 0.020 91% 0.026 0.013 48% 0.014 0.012 88% 4.27 0.118 3a 0.120 0.114 95% 0.208 0.131 63% 0.115 0.108 94% 3.40 0.183 3b 0.160 0.108 68% 0.154 0.092 60% 0.085 0.067 78% 2.89 0.235 4 0.098 0.058 59% 0.207 0.093 45% 0.102 0.011 11% 8.22 0.016* 5 0.592 0.252 43% 0.403 0.198 49% 0.681 0.158 23% 5.81 0.055 * p < 0.05 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 225 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion Control Low High Figure 6.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Fadrozole 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 145.0 µ m to 248.3 µ m ( Figure 6.9). Tubule diameters of males from the two test concentrations ranged from 114.7 µ m to 240.3 µ m. No significant differences in the mean tubule diameter per treatment ( Table 6.7) were detected ( Kruskal Wallis, H = 4.07, p = 0.131, 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 67 ( Table 6.7).

Table 6.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 167.9 35.8 21% 13% 67 low 8 151.2 23.0 15% high 8 177.2 34.5 19% 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) 226 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 250 200 150 100 Treatment diameter Figure 6.9. Boxplot of seminiferous tubule diameter ( µ m) by treatment for the EPA 14 Day Fadrozole 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   One male from the Control treatment showed Sertoli cell proliferation with sperm phagocytosis. The same male also showed focal tubule disorganization, multifocal proliferation of secondary spermatogonia, and sperm clumping in the tubule lamina. One male from the Low concentration and three males from the High concentration were observed to have multifocal Leydig cell proliferation. One male from the Low concentration had an abnormal appearing arrangement of components of spermatogenesis. Another male from the Low concentration showed tubules in developmental Stage 5 that appeared to have a smaller diameter than normal and had a lower than normal density of sperm in tubule lumina. Ovatestes, containing three Stage 3 ova, were detected in one male from the High concentration. One male of the males from the High concentration that had multifocal Leydig cell proliferation also showed focal disruption of tubule developmental sequence, with the proliferation of developmental Stage 2b cells in mature tubules containing developmental Stage 5 cells and had occasional foci of two or three necrotic Stage 3a or Stage 3b cells. Another male from the High concentration had multifocal areas of three or four abnormal developmental Stage 3b and Stage 4 cells with the Stage 4 cell clumps appearing to be inside cell wall of the stage 3b cells. No testicular atrophy was recorded and no ovatestes were observed for any treatment.

6.1.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the EPA 14 Day fadrozole assay ranged from 1,164 pg/ mL to 5,523 pg/ mL ( Figure 6.10). Among females exposed to the two fadrozole concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 2,158 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 6.8) was detected ( Kruskal Wallis, H = 36.86, p < 0.001, df = 2). The mean estradiol concentration in females from the High concentration was less than that in females from the Control treatment and the Low concentration. The achieved power for this endpoint was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 3 ( Table 6.8). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 227 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 15 2,064 1,176 57% 100% 3 low 16 1,088 468 43% high 16 39 86 218% 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.

Low High Control 6000 5000 4000 3000 2000 1000 0 treatment Estradiol Figure 6.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole 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 was not detected in males from the Control treatment ( 8 individuals) or in those exposed to the Low and High fadrozole concentrations ( 8 individuals per treatment).

Testosterone   Testosterone concentrations in Control treatment females used during the EPA 14 Day fadrozole assay ranged from 221 pg/ mL to 2,644 pg/ mL ( Figure 6.11). Among females exposed to the two fadrozole concentrations, testosterone concentrations ranged from 732 pg/ mL to 4,992 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 6.9) were detected ( Kruskal Wallis, H = 14.91, p = 0.001, df = 2). The mean testosterone concentration in females from the Control treatment was less than those in females from the Low and High concentrations. The achieved power for this endpoint was 96%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 9 ( Table 6.9). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 228 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.9. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 13 934 629 67% 96% 9 low 14 2,356 1,268 54% high 16 2,355 1,175 50% 1 Calculated from natural log transformed data; with sample size = 13. 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 6.11. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole 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 EPA 14 Day fadrozole assay ranged from 198 pg/ mL to 3,569 pg/ mL ( Figure 6.12). Among males exposed to the two fadrozole concentrations, testosterone concentrations ranged from 1,182 pg/ mL to 7,124 pg/ mL. A significant difference in the mean testosterone concentration per treatment ( Table 6.10) was detected ( Kruskal Wallis, H = 7.12, p = 0.029, df = 2). The mean testosterone concentration in males from the Control treatment was less than those in males from the High concentration. The achieved power for this endpoint was 68%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 11 ( Table 6.10). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 229 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.10. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 1,790 997 56% 68% 11 low 8 3,169 833 26% high 8 4,431 2,294 52% 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 7000 6000 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 6.12. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole 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 detected in only 1 of 11 Control treatment females used during the EPA 14 Day fadrozole assay ( Figure 6.13). Among females exposed to the two fadrozole concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 1,528 pg/ mL. No significant differences in the mean 11 ketotestosterone concentration per treatment ( Table 6.11) were detected ( Kruskal Wallis, H = 5.69, p = 0.058, df = 2). However, the calculated probability was slightly greater than the selected critical limit ( 0.050) for the test. The greatest difference in mean 11 ketotestosterone concentration was between females from the Control treatment ( 60 pg/ mL) and those from the High concentration ( 426 pg/ mL). The mean value for Control females was also much less than that for females from the Low concentration ( 400 pg/ mL). The achieved power for this endpoint was 54%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 19 ( Table 6.11). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 230 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.11. Summary statistics and power estimates for female 11 ketotestosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 11 60 197 332% 54% 19 low 11 400 516 129% high 14 426 430 101% 1 Calculated from natural log transformed data; with sample size = 11. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 1500 1000 500 0 treatment 11 keto Figure 6.13. Boxplot of female 11 ketotestosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole 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 EPA 14 Day fadrozole assay ranged from 959pg/ mL to 41,357 pg/ mL ( Figure 6.14g). Among males exposed to the two fadrozole concentrations, 11 ketotesosterone concentrations ranged from 1,665 pg/ mL to 103,500 pg/ mL. A significant difference in the mean 11 ketotesosterone concentration per treatment ( Table 6.12) was detected ( Kruskal Wallis, H = 6.54, p = 0.038, df = 2). The mean 11 ketotestosterone concentration in males from the Control treatment was less than that in males from the Low concentration. The achieved power for this endpoint was 64%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 11 ( Table 6.12). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 231 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.12. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 14,376 13,061 91% 64% 11 low 8 56,077 31,055 55% high 8 41,690 36,973 89% 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 6.14. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Fadrozole 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.

6.1.8 Fecundity Total Fecundity   Variability among treatments in the total number of eggs produced during the EPA 14 Day Fadrozole assay was moderate to high ( Figure 6.15). Total counts in the Control treatment ranged from 3,063 eggs to 5,252 eggs and from 2,164 eggs to 3,408 eggs in the Low concentration. Total counts for three replicates of the High fadrozole concentration treatment were similar, ranging from 644 eggs to 1,041 eggs, whereas the total number of eggs produced in the fourth replicate was 32. Egg production by females in the High concentration stopped after Day 4 of the assay. Significant differences in the mean numbers of eggs ( square root transformed ) produced per treatment ( Table 6.13) were detected ( Kruskal Wallis, H = 9.27, p = 0.010, df = 2). The mean numbers of eggs produced by females in the Control treatment and the Low concentration were greater than the mean numbers of eggs laid by females in the High concentration. The achieved power for this assay was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 3 ( Table 6.13). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 232 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.13. Summary statistics and power estimates for fecundity data for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 4462 961 22% 100% 3 low 4 2517 602 24% high 4 609 422 69% 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 1,000 2,000 3,000 4,000 5,000 6,000 0 2 4 6 8 10 12 14 Day Cumulative Number of Eggs Control Low High Figure 6.15. Total egg production by replicate per treatment for the EPA 14 Day fadrozole assay.

Fecundity per Female Reproductive Day   During the EPA 14 Day Fadrozole assay, the maximum number of female reproductive days was achieved for all treatments ( Table 6.14). The number of eggs produced per female reproductive day in the Control treatment varied from 54.7 eggs to 93.8 eggs and from 38.3 eggs to 60.9 eggs in the Low concentration ( Figure6.16). For the High concentration, the number of eggs produced per female reproductive day ranged from 0.6 eggs to 18.6 eggs. Because no fish died during the assay, the statistical results reported here are the same as those reported for total fecundity. Females in the High concentration produced significantly fewer eggs per reproductive day than those in the Control treatment and the Low concentration ( Kruskal Wallis, H = 9.27, p = 0.010, df = 2). The achieved power for this assay was 82%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 4 ( Table 6.14). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 233 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.14. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 14 Day Fadrozole assay.

Level Mean Number of Reproductive Days 1 N Mean Stdev CV Achieved Power 2 Sample Size Required 3 control 56 4 79.7 17.2 22% 82% 4 low 56 4 45.0 10.8 24% high 56 4 10.9 7.5 69% 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 100 90 80 70 60 50 40 30 20 10 0 treatment eggs/ ReproDay Figure 6.16. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 14 Day Fadrozole 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 Fadrozole assay varied from 551 eggs for the High concentration to 4,015 eggs for the Control treatment ( Appendix E, Table 4.3). The number of eggs on dishes ranged from 59 eggs for the High concentration to 448 eggs for the Control treatment. 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 ( Appendix E, Figure 4.2). There were no significant differences in this mean proportional difference among treatments ( Kruskal Wallis, H = 0.81, p = 0.668, df = 2). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 234 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 6.1.9 Fertilization Success Total Fertilization   The total ( tiles + dishes) fertilization success rates for most treatment replicates during the EPA 14 Day Fadrozole assay were high, ranging from 0.997 ( Control treatment and Lowconcentration replicates) to 1.00 ( replicates from all treatments) ( Figure 6.17). The proportion of eggs fertilized for the High concentration was 1.00 for all replicates. No significant differences in mean fertilization success rates ( Table 6.15) among treatments were detected ( Kruskal Wallis, H = 5.93, p = 0.052, df = 2). The achieved power for this assay was 48%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 7 ( Table 6.15).

Table 6.15. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 0.998 0.001 0.1% 48% 7 low 4 0.999 0.002 0.2% high 4 1.000 0 0.0% 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 treatment Total Prop fert Figure 6.17. Boxplot of the proportion of eggs fertilized by treatment for the EPA 14 Day Fadrozole 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 all treatment replicates for eggs laid on tiles during the EPA 14 Day Fadrozole assay were high, ranging from 0.996 ( Low concentration replicate) to 1.00 ( High and Low concentration replicates) ( Appendix E, Figure 4.3). No significant differences in mean fertilization success rates ( Appendix E, Table 4.4) among treatments were detected ( Kruskal Wallis, H = 5.93, p = 0.052, df = 2). The fertilization success rates for all treatment replicates for eggs laid on dishes during the assay were high, ranging from 0.995 ( Control treatment DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 235 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 replicate) to 1.00 ( several replicates, including all treatments) ( Appendix E, Figure 4.4). No significant differences in mean fertilization success rates ( Appendix E, Table 4.4) among treatments were detected ( Kruskal Wallis, H = 2.00, p = 0.368, df = 2).

6.1.10 Hatchability and Larval Development Eggs were collected during the 14 Day pre exposure period for the evaluation of hatchability. The proportion of fertilized eggs that hatched ranged from 0.96 to 1.00 for tanks that would be used for the Control treatment, from 0.78 to 0.96 for tanks that would constitute the Low concentration, and from 0.98 to 1.00 for tanks that would be assigned to the High concentration during the 14 Day exposure assay. Among the tanks evaluated during the pre exposure period, but that were not used in the 14 Day assay, the mean proportion of fertilized eggs that hatched was 0.98. There was a significant difference detected among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 8.42, p = 0.038, df = 3). The proportion of eggs that hatched in tanks destined for the Low concentration treatment was significantly less than that for eggs laid in tanks that would be used for the High concentration.

Eggs were collected during the EPA 14 Day Fadrozole 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 0.84 to 1.00 for the Low concentration ( Figure 6.18). No eggs were available to evaluate hatchability for the High concentration because the evaluation was started on Day 12 of the assay and no eggs were laid in those tanks after Day 4. There were no significant differences between the Control treatment and the Low concentration in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.05, p = 0.821, 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 6.16).

Table 6.16. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 6 0.973 0.031 3% 5% > 1000 low 7 0.959 0.062 6% high 0       1 Calculated from arcsine square root transformed data; with sample size = 6. 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) 236 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 low control 1.00 0.95 0.90 0.85 treatment Prop Hatch Figure 6.18. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Fadrozole 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) was 0.95 ( sd = 0.05) for all tanks that would be used in the Control treatment. The mean proportion of normal larvae in the remaining treatments was 0.97 ( sd = 0.03) in the Low concentration and 0.98 ( sd = 0.02) in the High concentration. Among the tanks evaluated during the pre exposure period, but that were not used in the 21 Day assay, the mean proportion of normal larvae was 0.97 ( sd = 0.01). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 1.38, p = 0.710, df = 3).

Eggs were collected during the EPA 14 Day Fadrozole assay for the evaluation of larval development. The proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) ranged from 0.74 to 1.00 for the Control treatment and from 0.92 to 1.00 for the Low concentrations ( Figure 6.19). There were no larvae available form the High concentration for the evaluation of development. There were no significant differences between the Control treatment and the Low concentration in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.08, p = 0.772, df = 1). 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 402 ( Table 6.17).

Table 6.17. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 6 0.916 0.123 13% 6% 402 low 7 0.970 0.026 3% high 0       1 Calculated from arcsine square root transformed data; with sample size = 6. 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) 237 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 low control 1.0 0.9 0.8 treatment Prop Norm Figure 6.19. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Fadrozole 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.

6.1.11 Body Weight The body weight of females used in the EPA 14 Day Fadrozole assay ranged from 1.5 g to 3.6 g. A significant difference in mean body weight among treatments was detected ( Kruskal Wallis, H = 12.33, p = 0.002, df = 2). The mean body weight of females exposed to the High concentration was significantly greater than that of females in the Control treatment. The body weight of males used in the EPA 14 Day Fadrozole assay ranged from 2.5 g to 7.5 g. There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 2.90, p = 0.235, df = 2).

6.2 EPA 21 Day Assay for Fadrozole The EPA 21 Day Fadrozole assay was conducted from March 10, 2003, to March 25, 2003 ( pre exposure assay), and from March 25, 2003, to April 15, 2003 ( exposure assay).

6.2.1 Survival All males and females in all treatments survived the EPA 21 Day Fadrozole assay.

6.2.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the EPA 21 Day fadrozole assay ranged from 2,667,000 ng/ mL to 10,890,000 ng/ mL ( Figure 6.20). Among females exposed to the two fadrozole concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 2,768,500 ng/ mL. Significant differences in the mean vitellogenin concentration per treatment ( Table 6.18) were detected ( Kruskal Wallis, H = 39.41, p = < 0.001, df = 2). Vitellogenin concentrations in Control treatment females were significantly greater than those in females exposed to the Low and High fadrozole DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 238 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 concentrations. Additionally, vitellogenin concentrations in females from the Low concentration were greater than those in females from the High concentration. The achieved power for this endpoint was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 3 ( Table 6.18).

Table 6.18. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 5,949,000 2,252,237 38% 100% 3 low 16 1,197,869 754,539 63% high 16 4,914 10,679 217% 1 Calculated from natural log transformed data; with sample size = 16. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 10000000 5000000 0 treatment ( y )

Figure 6.20. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Fadrozole 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 fadrozole assay ranged from 168 ng/ mL to 8,288 ng/ mL ( Figure 6.21). Among males exposed to the two fadrozole concentrations, vitellogenin concentrations ranged from 14 ng/ mL to 15,097 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 6.19) were detected ( Kruskal Wallis, H = 0.85, p = 0.655, 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 67 ( Table 6.19). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 239 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.19. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 2,334 2,639 113% 13% 67 low 8 3,576 5,554 155% high 8 2,074 2,792 135% 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 15000 10000 5000 0 treatment Fad VTG Figure 6.21. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Fadrozole 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.

6.2.3 Appearance / Secondary Sex Characteristics All of the females used during the EPA 21 Day Fadrozole assay exhibited typical female morphology ( no fat pad, no tubercles, no vertical banding, ovipositor present).

All of the males used during the EPA 21 Day Fadrozole assay exhibited typical male morphology ( fat pads, tubercles, vertical banding, no ovipositor present).

6.2.4 Gonadosomatic Index The range of GSI values calculated for females in the all treatments varied from three to seven fold ( Figure 6.22), and the overall variability within the treatment was moderate to high ( CVs = 28%   31%; Table 6.20). The highest value ( GSI = 22.2) was obtained for a female exposed to the High fadrozole concentration. One female exposed to the Low fadrozole concentration had a GSI value of 21.3. A significant difference in the mean GSI value per treatment ( Table 6.20) was detected ( Kruskal Wallis, H = DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 240 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 10.50, p = 0.005, df = 2). The mean GSI value for females from the High concentration was greater than that for females from the Control treatment. The achieved power for this endpoint was 80%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 16 ( Table 6.20).

Table 6.20. Summary statistics and power estimates for female gonadosomatic index data for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 10.3 2.9 28% 80% 16 low 16 13.3 4.1 31% high 16 15.2 4.8 31% 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 22 12 2 treatment GSI Figure 6.22. Boxplot of female GSI by treatment for the EPA 21 Day Fadrozole 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 21 Day Fadrozole assay, was small, ranging from 0.7 to 1.8 ( Figure 6.23), which approximates the typical range for reproductively active male fathead minnows. One male in the Low concentration had a GSI value of 2.3, attributable to its relatively high gonad weight of 0.10 g and moderate body size ( 4.5 g). The three large males did not have abnormally high GSI values ( 0.8   1.6), which reflects their proportionally high gonad weights ( 0.07 g   0.13 g). There were significant differences in mean GSI values ( Table 6.21) among treatments ( Kruskal Wallis, H = 10.19, p = 0.006, df = 2). The mean GSI values for fish from the High and Low concentrations were greater than the mean value for fish from the Control treatment. The achieved power DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 241 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 for this endpoint was 87%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 7 ( Table 6.21).

Table 6.21. Summary statistics and power estimates for male gonadosomatic index data for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 1.00 0.20 20% 87% 7 low 8 1.37 0.45 33% high 8 1.52 0.18 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 2.5 2.0 1.5 1.0 treatment GSI ( y )

Figure 6.23. Boxplot of male GSI by treatment for the EPA 21 Day Fadrozole 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.

6.2.5 Female Gonad Histology Histological analyses were conducted on the ovaries of 48 females exposed to fadrozole 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 Fadrozole assay revealed significant differences among treatments ( Kruskal Wallis, H = 34.09, p = < 0.001, df = 2). The mean ovarian stage of females exposed to the High concentration was significantly less than that of females from the Control treatment or the Low concentration. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 242 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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 from the Control treatment and Low concentration ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages ( Figure 6.24). Ova from fish from the High concentration ranged from Stage 1a to Stage 4. Variability within treatments for each stage, particularly for Stage 5, was very high as indicated by CVs that ranged as high as 400% ( Table 6.22). Statistical analyses showed that within each of the five developmental stages, except Stage 5, there were significant differences among treatments in the proportion of cells occurring in the stage ( Table 6.22). The proportion of ova in Stage 1a was significantly greater in the Control treatment females than in High concentration females. The proportion of ova in Stage 1b was significantly greater in the Control treatment females than in High or Lowconcentration females. The proportion of ova in Stage 2 was significantly greater in the Controltreatment and High concentration females than in Low concentration females. The proportion of ova in Stage 3 was significantly greater in the High concentration females than in Control treatment or Lowconcentration females. The proportion of ova in Stage 4 was significantly less in the High concentration females than in Control treatment or Low concentration females.

Table 6.22. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 21 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 16) Low ( n = 16) High ( n = 16) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1a 0.064 0.020 31% 0.049 0.026 53% 0.037 0.020 54% 10.43 0.005* 1b 0.246 0.059 24% 0.180 0.067 37% 0.170 0.066 39% 9.75 0.008* 2 0.230 0.030 13% 0.180 0.034 19% 0.211 0.053 25% 13.55 0.001* 3 0.243 0.055 23% 0.305 0.105 34% 0.425 0.148 35% 14.94 0.001* 4 0.180 0.118 66% 0.240 0.109 46% 0.015 0.018 115% 28.14 < 0.001** 5 0.006 0.017 288% 0.000 0.001 400% 0 0  2.18 0.337 * p < 0.01 ** p < 0.001 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 243 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0.00 0.10 0.20 0.30 0.40 0.50 0.60 1a 1b 2 3 4 5 Ovarian Stage Proportion Control Low High Figure 6.24. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Fadrozole 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 0.008 follicles for females in the Control treatment to 0.126 follicles for females in the High concentration ( Figure 6.25). One fish from the High concentration 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 = 24.42, p < 0.001, df = 2). The proportion of atretic follicles per 300 follicles in fish from the High concentration was greater than those of fish from the remaining two treatments. Additionally, the proportion of atretic follicles per 300 follicles in fish from the Low concentration was greater than that in fish from the Control treatment. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 244 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 6.25. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Fadrozole 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 0.006 for females in the Low concentration to 0.024 for females in the Control treatment ( Figure 6.26). Variability within each treatment was very high, ranging from 132% for the Control treatment to 388% for the High concentration. Despite the high variability, there was a significant difference in the mean proportion of corpora lutea among treatments ( Kruskal Wallis, H = 7.65, p = 0.022, df = 2). The proportion of corpora lutea in fish from the Control treatment was significantly greater than that in fish from the High concentration. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 245 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.25 0.20 0.15 0.10 0.05 0.00 Treatment corpora_ lutea Figure 6.26. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 21 Day Fadrozole 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 Low concentration were observed to have most of the atretic follicles at the margins of the ovaries. One fish from the High concentration were observed to have inflammatory cells in the ovary. Five other fish from the High concentration had atretic follicles that were Stage 3 ( one fish) or Stage 4 ( four fish) cells.

6.2.6 Male Gonad Histology General Testes Staging   Testes from 24 males exposed to fadrozole during the EPA 21 Day Fadrozole 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 ( 88 fields) or Stage 5 ( 8 fields) development. Ninety four of the 96 microscopic fields examined in the 8 Low concentration treatment males showed Stage 4 ( 85 fields) or Stage 5 ( 9 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 ( 69 fields) or Stage 5 ( 27 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed no significant differences among treatments ( Kruskal Wallis, H = 3.92, p = 0.141, 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 6.27). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 198% ( Table 6.23). Statistical analyses showed that there were significant differences among treatments in the proportion of cells in any of the developmental stages ( Table 6.23). The mean proportion of cells in developmental Stage 3a was greater in fish from the Low concentration than in fish from the Control treatment. The observed proportions of cells in developmental Stage 3b ranked higher in fish from the Low concentration and the Control treatment than in fish from the High concentration. The mean proportion of cells in developmental Stage DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 246 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 5 was greater in fish from the Low concentration than in fish from the High concentration. Therefore, there did not appear to be an effect on testicular developmental stage associated with fadrozole dose.

Table 6.23. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 21 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

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 0.004 0.006 147% 0.002 0.003 151% 0.001 0.002 198% 1.30 0.523 2b 0.019 0.016 84% 0.014 0.010 68% 0.010 0.008 80% 1.26 0.532 3a 0.092 0.054 59% 0.204 0.079 39% 0.141 0.091 64% 6.52 0.038* 3b 0.292 0.110 38% 0.274 0.082 30% 0.176 0.079 45% 6.18 0.046* 4 0.235 0.093 40% 0.223 0.048 22% 0.162 0.076 47% 3.51 0.173 5 0.358 0.139 39% 0.283 0.135 48% 0.510 0.199 39% 6.50 0.039* * p < 0.05 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion Control Low High Figure 6.27. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 21 Day Fadrozole 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 104.2 µ m to 136.1 µ m ( Figure 6.28). Tubule diameters of males from the two test concentrations ranged from 101.1 µ m to 202.2 µ m. A significant difference in the mean tubule diameter per treatment ( Table 6.24) was detected ( Kruskal Wallis, H = 12.89, p = 0.002, df = 2). The mean tubule DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 247 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 diameter in males form the High concentration was greater than those in males from the Low concentration and the Control treatment. The achieved power for this endpoint was 97%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 5 ( Table 6.24).

Table 6.24. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 115.0 10.8 9% 97% 5 low 8 123.5 12.7 10% high 8 151.8 25.0 16% 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 200 150 100 Treatment diameter ( y )

Figure 6.28. Boxplot of male seminiferous tubule diameter ( µ m) by treatment for the EPA 21 Day Fadrozole 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. Four males from the High concentration were found to have focal proliferation of Leydig cells. One male from the High concentration had focal proliferation of secondary spermatogonia. Four males from the High concentration had multifocal proliferation of secondary spermatogonia. In two of these fish, the proliferation extended into the lumen and included Stages 3b, 4, and 5 cells. In one fish, the proliferation extended into mature tubules. No testicular atrophy was recorded and no ovatestes were observed for any treatment. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 248 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 6.2.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the EPA 21 Day fadrozole assay ranged from 372 pg/ mL to 5,847 pg/ mL ( Figure 6.29). Among females exposed to the Lowfadrozole concentrations, estradiol concentrations ranged from 401 pg/ mL to 2,587 pg/ mL. No females from the High concentration were available for estradiol determination. A significant difference in the mean estradiol concentration per treatment ( Table 6.25) was detected ( Kruskal Wallis, H = 5.16, p = 0.023, df = 2). The mean estradiol concentration in females from the Low concentration was less than that in females from the Control treatment. 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 17 ( Table 6.25).

Table 6.25. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 2,861 1,492 52% 40% 17 low 7 1,343 756 56% high 0       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 Control 6000 5000 4000 3000 2000 1000 0 treatment Estradiol Figure 6.29. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole 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.

Estradiol concentrations in Control treatment males used during the EPA 21 Day Fadrozole assay ranged from 0 pg/ mL ( not detected) to 193 pg/ mL ( Figure 6.29). Estradiol was not detected in males exposed to DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 249 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 the Low fadrozole concentrations ( 4 individuals). No males from the High concentration were available for estradiol determination ( Table 6.26).

Table 6.26. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 1 control 8 70 96 138%     low 4 0 0   high 0       1 Not calculated.

Testosterone   Testosterone concentrations in Control treatment females used during the EPA 21 Day Fadrozole assay ranged from 199 pg/ mL to 3,828 pg/ mL ( Figure 6.30). Among females exposed to the two fadrozole concentrations, testosterone concentrations ranged from 561 pg/ mL to 7,275 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 6.27) were detected ( Kruskal Wallis, H = 13.68, p = 0.001, df = 2). The mean testosterone concentration in females from the Control treatment was less than those in females from the Low and High concentrations. The achieved power for this endpoint was 93%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 10 ( Table 6.27).

Table 6.27. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 1,096 938 86% 93% 10 low 13 1,977 834 42% high 16 2,928 1,951 67% 1 Calculated from natural log transformed data; with sample size = 13. 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) 250 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 8000 7000 6000 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 6.30. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole 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 concentrations in Control treatment males used during the EPA 21 Day Fadrozole assay ranged from 953 pg/ mL to 3,926 pg/ mL ( Figure 6.31). Among males exposed to the two fadrozole concentrations, testosterone concentrations ranged from 3,282 pg/ mL to 15,262 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 6.28) were detected ( Kruskal Wallis, H = 14.92, p = 0.001, df = 2). The mean testosterone concentration in males from the Control treatment was less than those in males from the Low and High concentrations. The achieved power for this endpoint was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 4 ( Table 6.28).

Table 6.28. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 2,208 1,116 51% 100% 4 low 8 6,593 1,600 24% high 8 9,030 4,252 47% 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) 251 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 15000 10000 5000 0 treatment Testosterone Figure 6.31. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole 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 concentrations in Control treatment females used during the EPA 21 Day Fadrozole assay ranged from 0 pg/ mL ( not detected) to 594 pg/ mL ( Figure 6.32). However, 11 ketotestosterone was only detected in 2 of the 16 females from the Control treatment that were analyzed. Among females exposed to the two fadrozole concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 1,966 pg/ mL. A significant difference in the mean 11 ketotestosterone concentration per treatment ( Table 6.29) was detected ( Kruskal Wallis, H = 9.05, p = 0.011, df = 2). The mean 11 ketotestosterone concentration in females from the Control treatment was less than those in females from the High concentration. However, the achieved power for this endpoint was only 48%, because of the high variability in the Control response. The sample size required to detect a significant difference from the Control treatment at 80% power was 22 ( Table 6.29).

Table 6.29. Summary statistics and power estimates for female 11 ketotestosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 16 71 193 274% 48% 22 low 11 369 377 102% high 16 515 593 115% 1 Calculated from natural log transformed data; with sample size = 13. 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) 252 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 2000 1000 0 treatment 11 keto Figure 6.32. Boxplot of female 11 ketotestosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole 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.

11 ketotesosterone concentrations in Control treatment males used during the EPA 21 Day Fadrozole assay ranged from 4,697 pg/ mL to 43,298 pg/ mL ( Figure 6.33). Among males exposed to the two fadrozole concentrations, 11 ketotesosterone concentrations ranged from 14,292 pg/ mL to 192,900 pg/ mL. Significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 6.30) were detected ( Kruskal Wallis, H = 14.89, p = 0.001, df = 2). The mean 11 ketotestosterone concentration in males from the Control treatment was less than those in males from the Low and High concentrations. The achieved power for this endpoint was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 4 ( Table 6.30).

Table 6.30. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 8 19,770 15,109 76% 100% 4 low 8 130,695 47,538 36% high 8 77,822 54,034 69% 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) 253 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 200000 100000 0 treatment 11 keto Figure 6.33. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Fadrozole 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.

6.2.8 Fecundity Total Fecundity   A 15 day pre exposure evaluation of total egg production was performed. Total 15 day counts among the three treatments in the exposure assay ( individual tank values summed for each treatment) ranged from about 17,000 eggs to 22,000 eggs ( Figure 6.34). A significant difference in the mean 15 day egg production among the groups of replicates evaluated during the pre exposure assay was detected ( Kruskal Wallis, H = 8.74, p = 0.033, df = 3). However, the only significant difference was that the numbers of eggs laid in the tanks that were not subsequently used during the 21 Day fadrozole assay were lower than the number for the tanks that would eventually constitute the High concentration during the assay. There were no differences in the numbers of eggs laid among the tanks that would eventually be used for the three treatments during the 21 Day assay. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 254 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 40,000 45,000  14  12  10  8  6  4  2 0 2 4 6 8 10 12 14 16 18 20 Day Cumulative Number of Eggs Control Low High Figure 6.34. Total egg production per treatment for the 15 Day pre exposure assay.

During the EPA 21 Day Fadrozole assay, total counts in the Control treatment were reasonably consistent among replicates, varying from 5,193 eggs to 6,131 eggs ( Figure 6.35). Variability in total egg production among Low concentration replicates was somewhat greater, ranging from 3,224 eggs to 6,356 eggs. Total counts among the High concentration replicates varied from 413 eggs to 922 eggs. Statistical analysis of square root transformed egg counts showed significant among treatment differences ( Kruskal Wallis, H = 8.00, p = 0.018, df = 2) in mean total numbers of eggs produced ( Table 6.31). The mean total number of eggs produced by females in the High concentration was significantly less than the mean total numbers of eggs produced by females in the Control treatment and the Low concentration. The achieved power for this assay was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 2 ( Table 6.31).

Table 6.31. Summary statistics and power estimates for total fecundity data for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 5548 421 8% 100% 2 low 4 4587 1305 28% high 4 654 210 32% 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) 255 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 7,000 0 5 10 15 20 Day Cumulative Number of Eggs Control Low High Figure 6.35. Total egg production by replicate per treatment for the EPA 21 Day fadrozole assay.

Fecundity per Female Reproductive Day   During the 15 Day pre exposure evaluation, the mean number of eggs produced per female reproductive day ranged from 61.1 eggs/ day for the tanks that would be used for the Control treatment to 104.4 eggs/ day for the tanks that would be used for the High concentration during the 21 day exposure assay. The mean number of eggs produced per female reproductive day for the tanks that would not be used during the 21 day exposure assay was 60.6 eggs/ day. There were no significant differences among treatments in the mean numbers of eggs produced per reproductive day during the pre exposure period ( Kruskal Wallis, H = 7.65, p = 0.054, df = 2). The largest difference in eggs per female reproductive day was between the tanks that would constitute the High concentration and those that were not subsequently used during the exposure assay.

During the EPA 21 Day Fadrozole assay, the maximum number of female reproductive days was achieved for all treatments ( Table 6.32). The number of eggs produced per female reproductive day varied from 61.8 eggs to 73.0 eggs in the Control treatment and from 38.4 to 75.7 in the Low concentration ( Figure 6.36). For the High concentration, the number of eggs produced per female reproductive day ranged from 4.9 eggs to 11.0 eggs. Because no fish died during the assay, the statistical results reported here are the same as those reported for total fecundity. The number of eggs produced per day by females in the High concentration was significantly less than the numbers produced by females in the Control treatment and the Low concentration ( Kruskal Wallis, H = 8.00, p = 0.018, df = 2). The achieved power for this assay was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 2 ( Table 6.32). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 256 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.32. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 21 Day Fadrozole assay.

Level Mean Number of Reproductive Days 1 N Mean Stdev CV Achieved Power 2 Sample Size Required 3 control 84 4 66.0 5.0 8% 100% 2 low 84 4 54.6 15.5 28% high 84 4 7.8 2.5 32% 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 80 70 60 50 40 30 20 10 0 treatment eggs/ ReproDay Figure 6.36. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Fadrozole 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 15 day pre exposure assay varied from 2,859 eggs for the tanks that would be used for the Control treatment to 3,791 eggs for the tanks that would be used for the High concentration. The mean number of eggs on dishes ranged from 1,464 eggs for the Control treatment to 1,781 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 15 day pre exposure assay ( Kruskal Wallis, H = 7.26, p = 0.064, df = 3).

The mean number of eggs laid on the tiles among the treatments during the EPA 21 Day Fadrozole assay varied from 534 eggs for the High concentration to 4,938 eggs for the Control treatment ( Appendix E; DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 257 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.7). The number of eggs on dishes ranged from 120 eggs for the High concentration to 610 eggs for the Control treatment. 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 ( Appendix E; Figure 4.6). A significant difference in this mean proportional difference among treatments was detected ( Kruskal Wallis, H = 6.96, p = 0.031, df = 2). Proportionally more eggs occurred on tiles in the Control treatment than in the High concentration treatment.

6.2.9 Fertilization Success Total Fertilization   Eggs were collected during the 15 day pre exposure period for the evaluation of fertilization success rate. The mean proportion of eggs fertilized in the Control treatment was 0.997 [ standard deviation ( sd) = 0.006], 1.000 ( sd = 0.0003) in the Low concentration, and 1.000 ( sd = 0.0001) in the High concentration. The mean proportion of eggs fertilized in the replicates that were not used in the 21 day exposure assay was 1.000 ( sd = 0.0005). There were no significant differences among treatments in the proportion of eggs that were fertilized ( Kruskal Wallis, H = 1.17, p = 0.760, df = 3).

The total ( tiles + dishes) fertilization success rates for all treatment replicates during the EPA 21 Day Fadrozole assay were high, ranging from 0.995 ( High concentration replicate) to 1.00 ( Highconcentration and Control treatment replicates) ( Figure 6.37). No significant differences in mean fertilization success rates ( Table 6.33) among treatments were detected ( Kruskal Wallis, H = 1.56, p = 0.457, df = 2). The achieved power for this assay was 6%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 201 ( Table 6.33).

Table 6.33. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 4 0.999 0.001 0.1% 6% 201 low 4 0.999 0.001 0.1% high 4 0.999 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 258 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 1.000 0.999 0.998 0.997 0.996 0.995 treatment Total Prop fert Figure 6.37. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Fadrozole 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 15 Day pre validation assay, there were no significant differences in the fertilization success rates among treatments for eggs laid on tiles ( Kruskal Wallis, H = 1.17, p = 0.760, df = 3) or on dishes as all eggs that occurred on dishes were fertilized. The fertilization success rates for all treatment replicates for eggs laid on tiles during the EPA 21 Day Fadrozole assay were high, ranging from 0.992 ( High concentration replicate) to 1.00 ( Highconcentration and Control treatment replicates) ( Appendix E, Figure 4.7). No significant differences in mean fertilization success rates ( Appendix E, Table 4.8) among treatments were detected ( Kruskal Wallis, H = 1.56, p = 0.457, df = 2). The fertilization success rates for all treatment replicates for eggs laid on dishes during the assay were high, ranging from 0.998 ( Control treatment replicate) to 1.00 ( all High and Low concentration replicates) ( Appendix E, Figure 4.8). No significant differences in mean fertilization success rates ( Appendix E, Table 4.8) among treatments were detected ( Kruskal Wallis, H = 2.00, p = 0.368, df = 2).

6.2.10 Hatchability and Larval Development Eggs were collected during the 15 day pre exposure period for the evaluation of hatchability. The mean proportion of fertilized eggs that hatched in the Control treatment was 0.93 [ standard deviation ( sd) = 0.14], 0.99 ( sd = 0.02) in the Low concentration, and 0.97 ( sd = 0.06) in the High concentration. There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.50, p = 0.779, df = 2).

Eggs were collected during the EPA 21 Day Fadrozole assay for the evaluation of hatchability. The proportion of fertilized eggs that hatched ranged from 0.88 to 1.00 in the Control treatment and from 0.83 to 1.00 for the Low concentration ( Figure 6.38). No eggs were available to evaluate hatchability for the High concentration because the evaluation was started on Day 12 of the assay and no eggs were laid in those tanks after Day 9. There were no significant differences between the Low concentration and the Control treatment in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.14, p = 0.708, df = 1). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 259 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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 767 ( Table 6.34).

Table 6.34. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 11 0.962 0.045 5% 6% 767 low 11 0.942 0.071 8% high 0       1 Calculated from arcsine square root transformed data; with sample size = 11. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

low control 1.00 0.95 0.90 0.85 treatment Prop Hatch Figure 6.38. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Fadrozole 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 0.97 ( sd = 0.02). The mean proportion of normal larvae in the remaining treatments was 0.99 ( sd = 0.01) in the Low concentration and 0.96 ( sd = 0.03) in the High concentration. There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 3.77, p = 0.152, df = 2).

Eggs were collected during the EPA 21 Day Fadrozole 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 Low concentration ( Figure 6.39). Again, no larvae were available in the High concentration to evaluate development. There DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 260 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 were no significant differences between the Low concentration and the Control treatment in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.76, p = 0.382, df = 1). 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 102 ( Table 6.35).

Table 6.35. Summary statistics and power estimates for the proportion of normal larvae for the EPA 21 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 11 0.955 0.059 6% 13% 102 low 11 0.917 0.107 12% high 0       1 Calculated from arcsine square root transformed data; with sample size = 11. 2 To detect a significant difference from control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

low control 1.0 0.9 0.8 0.7 treatment Prop Norm ( y )

Figure 6.39. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Fadrozole 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.

6.2.11 Body Weight The body weight of females used in the EPA 21 Day Fadrozole assay ranged from 1.5 g to 4.4 g. There were significant differences in mean body weight among all treatments ( Kruskal Wallis, H = 24.62, p = < 0.001, df = 2). The mean body weight of females in the High concentration was greater than that of females in the other two treatments and mean body weight of females in the Low concentration was greater than that of females in the Control treatment. The body weight of males used in the EPA 21 Day Fadrozole assay ranged from 3.8 g to 9.2 g. Three males were unusually large ( Figure 6.40). Two that DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 261 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 were exposed to the High concentration weighed 9.2 g and 6.6 g. One male from the Control treatment weighed 8.3 g. However, there were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 1.94, p = 0.380, df = 2).

Low High Control 9 8 7 6 5 4 treatment fish_ wgt_ whole Figure 6.40. Boxplot of the body weights of males by treatment for the EPA 21 Day Fadrozole 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.

6.3 Non spawning Adult 14 day Assay for Fadrozole The Non spawning Adult 14 Day Fadrozole assay was conducted from April 1, 2003 to April 15, 2003 ( exposure assay).

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

6.3.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the Non spawning Adult 14 Day fadrozole assay ranged from 2,245,000 ng/ mL to 11,020,000 ng/ mL ( Figure 6.41). Among females exposed to the three fadrozole concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 1,612,000 ng/ mL. Significant differences in the mean vitellogenin concentration among treatments ( Table 6.36) were detected ( Kruskal Wallis, H = 32.71, p = < 0.001, df = 2). Mean vitellogenin concentrations among Control treatment females were significantly greater than the mean concentrations for females exposed to the High, and Medium fadrozole concentrations. Additionally, the mean concentration of vitellogenin from Low concentration females was significantly greater than those from females in the Medium and High fadrozole concentrations. The achieved power for this endpoint was 100%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 3 ( Table 6.36). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 262 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.36. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 4,934,800 2,619,357 53% 100% 3 low 10 793,075 447,921 56% medium 10 18,431 30,153 164% high 9 8,962 17,857 199% 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 10000000 5000000 0 treatment Fad VTG Figure 6.41. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole 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.

Vitellogenin concentrations in Control treatment males used during the Non spawning Adult 14 Day fadrozole assay ranged from 0 ng/ mL ( not detected) to 3,734 ng/ mL ( Figure 6.42). Among most males exposed to the three fadrozole concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 829 ng/ mL. One male exposed to the High fadrozole concentration had a vitellogenin concentration of 6,377 ng/ mL and a second had a concentration of 5,498 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 6.37) were detected ( Kruskal Wallis, H = 1.67, p = 0.644, 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 174 ( Table 6.37). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 263 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.37. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 675 1,261 187% 9% 174 low 10 74 82 111% 10 192 236 123% high 10 1,395 2,422 174% 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 7000 6000 5000 4000 3000 2000 1000 0 treatment Fad VTG Figure 6.42. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole 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.

6.3.3 Appearance / Secondary Sex Characteristics Most of the females used during the Non spawning Adult 14 Day Fadrozole assay showed normal female morphology. One female from the High concentration had the fat pad typical of males.

Morphological development among males used during the Non spawning Adult 14 Day Fadrozole assay varied among treatments ( Figure 6.43). Three males, one exposed to the Medium concentration and two exposed to the High concentration, lacked tubercles. Two males exposed to the Medium concentration also lacked fat pads. Sixteen of the 40 males used during the assay lacked 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) 264 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 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 6.43. Secondary sex characteristics of males used during the Non spawning Adult 14 Day Fadrozole assay.

6.3.4 Gonadosomatic Index The range of GSI values calculated for females in the all treatments varied from about two to eight fold ( Figure 6.44), and the overall within treatment variability was moderate ( CVs = 2%   51%; Table 6.38). The highest female GSI value was 21.7 ( one fish in the Low concentration), but several fish had GSI values ~ 19   20. A significant difference in mean GSI values ( Table 6.38) among treatments was detected ( Kruskal Wallis, H = 9.21, p = 0.027, df = 3). The mean GSI value for females exposed to the High fadrozole concentration was significantly less than the mean GSI for females from the Control treatment. The achieved power for this endpoint was 59%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 15 ( Table 6.38).

Table 6.38. Summary statistics and power estimates for female gonadosomatic index data for the Nonspawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 15.3 3.1 20% 59% 15 low 10 13.5 3.9 29% medium 10 10.5 3.9 38% high 10 10.2 5.2 51% 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) 265 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 20 10 0 treatment GSI Figure 6.44. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Fadrozole 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.

The range of most GSI values calculated for males during the Non spawning Adult 14 Day Fadrozole assay, was large, ranging from 0.5 to 2.1 ( Figure 6.45), which approximates the typical range for reproductively active male fathead minnows. Overall within treatment variability was moderate to high ( CVs = 30%   63%; Table 6.39). The highest and lowest male GSI values were 2.4 and 2.6 ( for one fish from the High concentration and one from the Control treatment) and 0.1 and 0.4 ( two fish from the Control treatment), respectively. However, there were no significant differences in mean GSI values ( Table OECD14FAD GSI 2) among treatments ( Kruskal Wallis, H = 1.29, p = 0.731, df = 3). The achieved power for this endpoint was 15%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 68 ( Table 6.39).

Table 6.39. Summary statistics and power estimates for male gonadosomatic index data for the Nonspawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 1.19 0.75 63% 15% 68 low 10 1.27 0.57 45% medium 10 1.24 0.37 30% high 10 1.45 0.52 36% 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) 266 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 2.5 2.0 1.5 1.0 0.5 0.0 treatment GSI Figure 6.45. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Fadrozole 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.

6.3.5 Female Gonad Histology Histological analyses were conducted on the ovaries of 40 females exposed to fadrozole 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 Fadrozole assay revealed no significant differences among treatments ( Kruskal Wallis, H = 3.93, p = 0.269, 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 Medium and High concentrations ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages), whereas ova from females from the Control treatment and Low concentration showed Stage 1a to Stage 4 development ( Figure 6.46). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 316% ( Table 6.40). Statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stages 1a, Stage 3, and Stage 4, but the were no significant differences among treatments in the proportion of cells in the developmental Stages 1b, 2, and 5 ( Table 6.40). The proportion of cells in developmental Stage 1a in the Low and Medium concentrations were significantly greater than those in the High concentration. The proportion of cells in developmental Stage 3 in the High concentration was significantly greater than that in the Control treatment. The proportion of cells in developmental Stage 4 in the Medium concentration was significantly less than that in the Control treatment. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 267 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 6.40. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the Non spawning Adult 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 10) Low ( n = 10) Medium ( n = 10) High ( n = 10) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Mean Stdev CV Mean Stdev CV H p 1a 0.050 0.022 45% 0.064 0.030 47% 0.075 0.028 37% 0.037 0.013 34% 13.34 0.004** 1b 0.284 0.071 25% 0.277 0.117 42% 0.277 0.075 27% 0.197 0.085 43% 7.41 0.060 2 0.159 0.057 36% 0.141 0.066 47% 0.203 0.070 34% 0.175 0.054 31% 4.55 0.208 3 0.143 0.054 38% 0.195 0.065 33% 0.226 0.088 39% 0.266 0.103 39% 9.88 0.020* 4 0.281 0.123 44% 0.247 0.156 63% 0.134 0.095 71% 0.157 0.109 69% 8.23 0.041* 5 0.000 0.000   0.000 0.000   0.000 0.001 316% 0.001 0.002 316% 2.05 0.561 0.00 0.10 0.20 0.30 0.40 0.50 0.60 1a 1b 2 3 4 5 Ovarian Stage Proportion Control Low Mid High Figure 6.46. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Fadrozole 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 0.060 follicles for females in the Medium concentration to 0.166 follicles for females in the High concentration treatment ( Figure 6.47). There were significant differences in the mean proportion of atretic follicles among treatments ( Kruskal Wallis, H = 11.34, p = 0.010, df = 3). The proportion of atretic follicles in females from the High concentration was greater than those in females from the other three treatments. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 268 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 0.5 0.4 0.3 0.2 0.1 0.0 Treatment atretic_ follicles Figure 6.47. Boxplot of atretic follicles per 300 follicles by treatment for the Non spawning Adult 14 Day Fadrozole 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 0.0003 for females in the Low concentration and Control treatment to 0.025 for females in the Medium concentration ( Figure 6.48). There were no significant differences in the mean proportion of corpora lutea among treatments ( Kruskal Wallis, H = 5.90, p = 0.116, df = 3).

Mid Low High Control 0.25 0.20 0.15 0.10 0.05 0.00 Treatment corpora_ lutea Figure 6.48. Boxplot of corpora lutea per 300 follicles by treatment for the Non spawning Adult 14 Day Fadrozole 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 269 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Observations   One female from the Control treatment was observed to have many Stage 4 cells didn't develop and became atretic. Six females from the High concentration had atretic follicles that were Stage 4 cells. One of these females also had atretic follicles that were Stage 3 cells.

6.3.6 Male Gonad Histology General Testes Staging   Testes from 40 males exposed to fadrozole during the Non spawning Adult 14 Day Fadrozole assay were examined to determine the general developmental condition. However, the cassette containing a gonad sample for one male from the Control treatment contained too little tissue to permit a definitive evaluation and this male was excluded from the analyses. 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 8 of the 108 microscopic fields examined in the 9 Control treatment males showed Stage 4 ( 36 fields) or Stage 5 ( 64 fields) development. Most of the 120 microscopic fields examined in the 10 Low concentration treatment males showed Stage 4 ( 58 fields) or Stage 5 ( 56 fields) development. Six microscopic fields showed Stage 3 development. In the 10 Medium concentration males available for examination, all of the 120 microscopic fields showed Stage 4 ( 64 fields) or Stage 5 ( 56 fields) development. In the 10 High concentration males available for examination, 115 of the 120 microscopic fields showed Stage 4 ( 62 fields) or Stage 5 ( 53 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed that no significant differences among treatments ( Kruskal Wallis, H = 0.74, p = 0.863, 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 6.49). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 175% ( Table 6.41). There were no significant differences among treatments in the proportion of cells in any of the developmental stages ( Table 6.41). Therefore, there was no effect on testicular developmental stage associated with fadrozole dose.

Table 6.41. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the Non spawning Adult 14 Day Fadrozole assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 9) 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 1 0 0   0 0   0 0   0 0       2a 0.004 0.005 119% 0.003 0.005 152% 0.004 0.005 115% 0.001 0.002 175% 2.54 0.467 2b 0.021 0.032 149% 0.054 0.083 154% 0.027 0.029 108% 0.040 0.055 136% 1.01 0.799 3a 0.080 0.108 135% 0.092 0.085 92% 0.049 0.048 98% 0.108 0.110 102% 1.74 0.628 3b 0.134 0.101 75% 0.120 0.078 65% 0.097 0.090 93% 0.111 0.092 83% 0.86 0.835 4 0.116 0.067 58% 0.072 0.059 81% 0.115 0.077 67% 0.116 0.051 44% 3.93 0.269 5 0.644 0.269 42% 0.659 0.238 36% 0.708 0.191 27% 0.624 0.228 37% 0.40 0.940 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 270 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion Control Low Mid High Figure 6.49. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Fadrozole 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 121.7 µ m to 267.5 µ m ( Figure 6.50). Tubule diameters of males from the three test concentrations ranged from 82.2 µ m to 294.7 µ m. No significant differences in the mean tubule diameter per treatment ( Table 6.42) were detected ( Kruskal Wallis, H = 4.03, p = 0.258, df = 3). 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 36 ( Table 6.42).

Table 6.42. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 9 193.8 55.4 29% 23% 36 low 10 189.2 58.4 31% medium 10 149.0 42.1 28% high 10 168.2 63.7 38% 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) 271 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 300 200 100 Treatment diameter Figure 6.50. Boxplot of seminiferous tubule diameter ( µ m) by treatment for the Non spawning Adult 14 Day Fadrozole 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   One male from the High concentration showed Sertoli cell proliferation. One male exposed to the Low concentration and one male from the Medium concentration showed multifocal Leydig cell proliferation. Three males exposed to the High concentration were found to have focal or multifocal Leydig cell proliferation. No testicular atrophy and no ovatestes were observed in males from any treatment. Several males were observed to have abnormal testicular development ( Table 6.43).

Table 6.43. Histological observations for males exposed to concentrations of fadrozole during the Nonspawning Adult 14 D assay.

Fish ID Concentration Leydig Cell Proliferation Observations 218733 Control None Multifocal basophilic cystic structures with concentric laminations inside tubules and ducts, up to 45 µ m diameter 218736 Control None Transition from stage 3b to stage 4 results in foci of nuclear fragments and spermatids and mixed stage nuclear fragments in tubule lumina.

Large numbers of Stage 2 b cells in tubules 218737 Control None Multifocal proliferation of secondary spermatogonia 218738 Control None Stage 4 ova in section   probably an artifact 218750 Low None Tubules in stage 5 appeared to have smaller diameter than normal Multifocal partial divisions of Stage 3b and Stage 4 cells in tubule lumina Abnormal developmental sequence   a high concentration Stage 2b cells and very few Stage 4 cells DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 272 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Fish ID Concentration Leydig Cell Proliferation Observations Tubule lumina occluded in some cases 218756 Low Multifocal Multifocal proliferation of secondary spermatogonia 218762 Medium None Tubules in stage 5 appear to have smaller diameter than normal Proliferation of state 2b and 3a cells along tubules filled with mature sperm 218763 Medium None Large numbers of stage 2b cells in tubules 218764 Medium None Tubules in stage 5 appeared to have smaller diameter than normal 218765 Medium None Cystic structures ( concretions) in some tubule lumina   irregular to 20 µ m diameter 218767 Medium None Stage 3 ova in section   probably an artifact 218782 High multifocal Most tubules normal, but some with multifocal areas of Sertoli cell proliferation and Leydig cell proliferation Also, possible secondary spermatocyte proliferation.

218783 High None Multifocal proliferation of secondary spermatogonia A few syncytia of Stage 5 cells A few clumps of Stage 3b and Stage 4 cells in tubule lumina 218784 High None Stage 3 ova in section   probably an artifact 218787 High None Necrotic cells in tubule lumina associated with dense clusters of sperm Multifocal proliferation of secondary spermatogonia 218788 High None Multifocal proliferation of secondary spermatogonia, filled with Stage 5 cells Occasional incomplete division of Stage 3 cells , which appear necrotic Abnormal appearing arrangement of components of spermatogenesis Areas where tubule lumina occluded with apparent Stage 2b cells and some necrotic cells in tubule lumina 218789 High None Clumps of Stage 5 cells in tubule lumina DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 273 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 6.3.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the Non spawning Adult 14 Day Fadrozole assay ranged from 0 pg/ mL ( not detected) to 3,502 pg/ mL ( Figure 6.51). Among females exposed to the three fadrozole concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 1,956 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 6.44) was detected ( Kruskal Wallis, H = 18.33, p < 0.001, df = 2). The mean estradiol concentration in females from the High concentration was less than that in females from the Control treatment. The achieved power for this endpoint was 54%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 15 ( Table 6.44).

Table 6.44. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 1,519 1,132 75% 53% 15 low 10 727 524 72% medium 9 320 457 143% high 10 142 129 90% 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 3000 2000 1000 0 treatment Estradiol Figure 6.51. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 274 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Estradiol was detected only in one male exposed to the Medium concentration during the Non spawning Adult 14 Day Fadrozole assay ( 1,814 pg/ mL). Estradiol was not detected in males from the Control treatment ( 9 individuals) or in those exposed to the Low and High concentrations ( 10 individuals per treatment) ( Table 6.45).

Table 6.45. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 1 control 9 0 0       low 10 0 0   medium 10 181 574 316% high 10 0 0   1 Not calculated.

Testosterone   Testosterone concentrations in Control treatment females used during the Non spawning Adult 14 Day Fadrozole assay ranged from 146 pg/ mL to 2,053 pg/ mL ( Figure 6.52). Among females exposed to the three fadrozole concentrations, testosterone concentrations ranged from 394 pg/ mL to 7,314 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 6.46) were detected ( Kruskal Wallis, H = 4.61, p = 0.203, df = 2. The achieved power for this endpoint was 29%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 26 ( Table 6.46).

Table 6.46. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 9 1,095 712 65% 29% 26 low 10 1,730 822 48% medium 8 986 407 41% high 9 2,067 2,084 101% 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) 275 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 8000 7000 6000 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 6.52. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole 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 Fadrozole assay ranged from 1,005 pg/ mL to 2,603 pg/ mL ( Figure 6.53). Among males exposed to the three fadrozole concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 13,606 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 6.47) were detected ( Kruskal Wallis, H = 3.28, p = 0.350, df = 2). The achieved power for this endpoint was 21%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 41 ( Table 6.47).

Table 6.47. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 9 1,889 493 26% 21% 41 low 10 1,658 805 49% medium 10 1,413 813 58% high 10 3,256 3,860 119% 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) 276 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 14000 12000 10000 8000 6000 4000 2000 0 treatment Testosterone Figure 6.53. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Fadrozole 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 Control treatment females, nor in females exposed to the Low fadrozole concentration, during the Non spawning Adult 14 Day Fadrozole assay ( Figure 6.54). 11 ketotesosterone was detected in one of seven females exposed to the Medium concentration ( 519 pg/ mL). Among the 10 females from the High fadrozole concentrations, 11 ketotestosterone was detected in 6, and the concentrations for those ranged from 329 pg/ mL to 897 pg/ mL. Significant differences in the mean 11 ketotestosterone concentration per treatment ( Table 6.48) were detected ( Kruskal Wallis, H = 11.23, p = 0.011, df = 2). 11 ketotestosterone was primarily detected in females from the High concentration, with one female from the Medium concentration also having a detectable level of the compound. The achieved power for this endpoint was 59%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 8 ( Table 6.48).

Table 6.48. Summary statistics and power estimates for female 11 ketotestosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 7 0 0 59% 8 low 5 0 0 medium 7 74 196 264% high 10 397 378 95% 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 277 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 900 800 700 600 500 400 300 200 100 0 treatment 11 keto Figure 6.54. Boxplot of female 11 ketotestosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole 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 Fadrozole assay ranged from 0 pg/ mL ( not detected) to 19,106 pg/ mL ( Figure 6.55). Among males exposed to the three fadrozole concentrations, 11 ketotesosterone concentrations ranged from 0 pg/ mL ( not detected) to 57,374 pg/ mL. No significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 6.49) were detected ( Kruskal Wallis, H = 1.32, p = 0.724, 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 170 ( Table 6.49).

Table 6.49. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 7,585 6,499 86% 9% 170 low 10 7,422 8,541 115% medium 10 6,101 7,505 123% high 10 12,535 17,396 139% 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) 278 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 60000 50000 40000 30000 20000 10000 0 treatment 11 keto Figure 6.55. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Fadrozole 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.

6.3.8 Body Weight and Length The body weight of females used in the Non spawning Adult 14 Day Fadrozole assay ranged from 1.7 g to 4.8 g ( Figure 6.56). There were no significant differences in mean body weight ( natural log transformed) among treatments ( Kruskal Wallis, H = 1.05, p = 0.790, df = 3). 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 186 ( Table 6.50).

The body ( fork) length of females used in the Non spawning Adult 14 Day Fadrozole assay ranged from 46 mm to 62 mm ( Figure 6.57). There were no significant differences in mean body weight ( natural log transformed) among treatments ( Kruskal Wallis, H = 1.96, p = 0.581, 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 53 ( Table 6.51).

Table 6.50. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 2.9 0.8 27% 8% 186 low 10 2.7 0.6 23% medium 10 2.9 0.7 24% high 10 2.9 0.5 18% 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) 279 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 5 4 3 2 treatment fish_ wgt_ whole Figure 6.56. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Fadrozole 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.

Table 6.51. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 54.2 3.4 6% 18% 53 low 10 54.3 5.4 10% medium 10 55.4 4.5 8% high 10 56.9 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 280 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 60 55 50 45 treatment fish_ length_ fork Figure 6.57. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Fadrozole 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 Fadrozole assay ranged from 3.9 g to 8.1 g ( Figure 6.58). There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 3.33, p = 0.343, df = 3). The achieved power for this endpoint was 21%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 46 ( Table 6.52).

The body ( fork) length of males used in the Non spawning Adult 14 Day Fadrozole assay ranged from 57 mm to 80 mm ( Figure 6.59). There were no significant differences in mean body weight ( natural log transformed) among treatments ( Kruskal Wallis, H = 5.89, p = 0.117, 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 32 ( Table 6.53).

Table 6.52. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Fadrozole assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 control 10 5.8 1.3 23% 21% 46 low 10 5.5 1.0 19% medium 10 5.1 0.7 14% high 10 5.6 0.8 13% 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) 281 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 8 7 6 5 4 treatment fish_ wgt_ whole Figure 6.58. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Fadrozole 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.

Table 6.53. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Fadrozole assay.