DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 90 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.0 RESULTS: TRENBOLONE 4.1 EPA 14 Day Assay for Trenbolone The EPA 14 Day Trenbolone assay was conducted from February 4, 2003 to February 11, 2003 ( prevalidation test) and from February 11, 2003 to February 25, 2003 ( validation test).

4.1.1 Survival All males in all treatments and all females in the Control and High concentration treatments survived the EPA 14 Day Trenbolone assay. Two females in the Low concentration treatment died during the assay ( 88% survival).

4.1.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the EPA 14 Day Trenbolone assay ranged from 1,385,500 ng/ mL to 3,722,500 ng/ mL ( Figure 4.1). Among females exposed to the two trenbolone concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 5,847,500 ng/ mL. Significant differences in the mean vitellogenin concentration per treatment ( Table 4.1) were detected ( Kruskal Wallis, H = 31.72, p = < 0.001, df = 2). Vitellogenin concentrations in Controltreatment females and Low concentration females were significantly greater than those in females exposed to the High trenbolone 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 4.1).

Table 4.1. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 16 2,608,969 592,805 23% 100% 4 Low 14 2,237,993 1,798,862 80% High 16 40,910 79,868 195% 1 Calculated from natural log transformed data; with sample size = 14. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 91 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 6000000 5000000 4000000 3000000 2000000 1000000 0 treatment Trenb VTG Figure 4.1. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Trenbolone 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, the asterisk represents a probable outlier.

Vitellogenin concentrations in Control treatment males used during the EPA 14 Day Trenbolone assay ranged from 0 ng/ mL ( not detected) to 8,813 ng/ mL ( Figure 4.2). Among males exposed to the two trenbolone concentrations, vitellogenin concentrations ranged from 0 ng/ mL to 2,333 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 4.2) were detected ( Kruskal Wallis, H = 1.26, p = 0.532, df = 2). The achieved power for this endpoint was 14%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 66 ( Table 4.2).

Table 4.2. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 1,811 3,061 169% 14% 66 Low 8 774 1,033 133% High 8 447 226 51% 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) 92 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 treatment Trenb VTG Figure 4.2. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 14 Day Trenbolone 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, the asterisk represents a probable outlier.

4.1.3 Appearance / Secondary Sex Characteristics All of the Control treatment females used during the trenbolone EPA 14 Day Trenbolone assay exhibited typical female morphology ( no fat pad, no tubercles, no vertical banding, ovipositor present). Five of the 15 females exposed to the Low concentration had vertical banding. Among the 16 females exposed to the High concentration, 13 had tubercles, 1 had a dorsal fat pad, and 12 had vertical banding. Thus, trenbolone appeared to have a dose related effect on female morphology.

All of the males used during the EPA 14 Day Trenbolone assay exhibited typical male morphology ( fat pads, tubercles, vertical banding, no ovipositor present).

4.1.4 Gonadosomatic Index The range of GSI values calculated for females in the all treatments varied from two to three fold ( Figure 4.3), and the overall variability within the treatment was moderate ( CVs = 23%   27%; Table 4.3). GIS values for fish in the Control treatment ranged from 6.4 to 14.3. The highest female GSI value was about 24 ( one fish in the High concentration). No other fish had a GSI value greater than 18.6. A significant difference in the mean GSI value per treatment ( Table 4.3) was detected ( Kruskal Wallis, H = 9.84, p = 0.007, df = 2). The test indicated that the mean GSI for females in the Control treatment was less than those for the other two treatments. However, the mean GSI values calculated for the Low and High trenbolone concentrations were not statistically different. The achieved power for this endpoint was 78%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 15 ( Table 4.3). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 93 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.3. Summary statistics and power estimates for female gonadosomatic index data for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 16 10.5 2.6 25% 78% 15 Low 14 13.7 3.1 23% High 16 14.3 3.8 27% 1 Calculated from arcsine square root transformed data; with sample size = 14. 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 25 15 5 treatment GSI Figure 4.3. Boxplot of female GSI by treatment for the EPA 14 Day Trenbolone 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 Trenbolone assay, was small, ranging from 0.7 to 1.8 ( Figure 4.4), which approximates the typical range for reproductively active male fathead minnows. The highest and lowest male GSI values were 2.2 ( one fish the High concentration) and 0.7 ( two fish in the Control treatment), respectively. A significant difference in the mean GSI value per treatment ( Table 4.4) was detected ( Kruskal Wallis, H = 7.66, p = 0.022, df = 2). The test indicated that the mean GSI for males in the High concentration was greater than those for the other two treatments. The achieved power for this endpoint was 71%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 10 ( Table 4.4). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 94 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.4. Summary statistics and power estimates for male gonadosomatic index data for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 1.04 0.28 27% 71% 10 Low 8 1.02 0.33 33% High 8 1.56 0.39 25% 1 Calculated from arcsine square root transformed data; with sample size = 8. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 2.0 1.5 1.0 treatment GSI ( y )

Figure 4.4. Boxplot of male GSI by treatment for the EPA 14 Day Trenbolone 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.

4.1.5 Female Gonad Histology General Ovary Staging   Statistical analysis of the mean ovarian staging from 12 microscopic fields per female in the EPA 14 Day Trenbolone assay revealed no significant difference among treatments ( Kruskal Wallis, H = 2.21, p = 0.331, df = 2).

Quantitative Ovarian Staging   One hundred cells in each of three sections per female were examined to quantitatively determine the developmental stage of the ovaries. Ova from fish in all treatments ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages ( Figure 4.5). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 400% ( Table 4.5). Although statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stage 3 and Stage 4, there were no significant differences among treatments in the proportion of cells in the developmental Stages 1a, 1b, 2, and 5 ( Table 4.5). The proportion of cells in developmental Stage 3 in the High concentration was significantly greater than that DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 95 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 in the Control treatment and the Low concentration. The proportion of cells in developmental Stage 4 in the High concentration was significantly lower than that in the Control treatment and the Low concentration. Thus, it can be concluded that the trenbolone High concentration inhibited the development of ova from the early to late vitellogenic stages ( i. e. from stage 3 to stage 4).

Table 4.5. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 16) Low ( n = 15) High ( n = 16) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1a 0.104 0.037 36% 0.096 0.033 35% 0.082 0.023 28% 2.31 0.315 1b 0.321 0.064 20% 0.304 0.057 19% 0.294 0.059 20% 1.56 0.459 2 0.194 0.050 26% 0.174 0.044 25% 0.161 0.042 26% 5.77 0.056 3 0.138 0.037 27% 0.158 0.051 32% 0.291 0.056 19% 29.47 < 0.001* 4 0.215 0.091 42% 0.241 0.079 33% 0.130 0.068 52% 14.39 0.001** 5 0.003 0.011 339% 0.001 0.003 300% 0.001 0.003 400% 0.42 0.809 * p < 0.001 ** p < 0.01 0.0 0.1 0.2 0.3 0.4 0.5 1a 1b 2 3 4 5 Ovarian Stage Proportion of Cells Control Low High Figure 4.5. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 14 Day Trenbolone assay. For each treatment, the columns represent the grand mean proportion of cells in each stage and the bars represent the standard deviation. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 96 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Atretic Follicles   The mean proportion of atretic follicles per 300 follicles ( counted per fish) ranged from 0.002 for females in the Control treatment to 0.03 follicles for females in the High concentration ( Figure 4.6). There was a significant difference in the proportions of atretic follicles among treatments ( Kruskal Wallis, H = 23.99, p = < 0.001, df = 2). The proportion of atretic follicles in females from the High concentration was significantly greater than that in Control treatment and Low concentration females. This appears to be related to the inhibition of development of ova from stage 3 to stage 4 in the High concentration treatment.

Low High Control 0.2 0.1 0.0 treatment atretic_ follicles Figure 4.6. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 14 Day Trenbolone 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 0.01 for females in the High concentration to 0.02 for females in the Control treatment and the Low concentration ( Figure 4.7). There was a significant difference in the proportions of corpora lutea among treatments ( Kruskal Wallis, H = 7.04, p = 0.030, df = 2). The value for the High concentration was significantly lower than that of the Low concentration treatment. This may be related to the inhibition of ovarian development in the High concentration, so that over time, while ovarian development tends to be stalled between stages 3 and 4, the corpora lutea in the High dose females are resorbed.

Observations   One female in the Control treatment had abnormal Stage 3 ova that were more typical of Stage 2, but had vitellogenin granules in the cytoplasm. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 97 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 treatment corpora_ lutea ( y )

Figure 4.7. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the EPA 14 Day Trenbolone 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.

4.1.6 Male Gonad Histology Testes Staging by Microscopic Field   Testes from males exposed to trenbolone during the EPA 14 Day Trenbolone assay were examined to determine the general developmental condition. Males in all treatments had well developed testes with most showing Stage 4 and Stage 5 development ( see Methods for description of developmental stages). All of the 96 microscopic fields examined in the 8 Control treatment males showed Stage 4 ( 91 fields) or Stage 5 ( 5 fields) development. All of the 96 fields examined in the 8 Low concentration treatment males showed Stage 4 ( 90 fields) or Stage 5 ( 6 fields) development. All of the 96 microscopic fields examined in the 8 High concentration treatment males showed Stage 4 ( 58 fields) or Stage 5 ( 38fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed a significant difference among treatments ( Kruskal Wallis, H = 12.36, p = 0.002, df = 2). The mean testes stage for males in the High concentration ( 4.40, sd = 0.29) was significantly greater than that in the Control treatment ( 4.05, sd = 0.09) and Low concentration treatment( 4.06, sd = 0.10).

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 of the testes from fish in all treatments ranged from Stage 2a to Stage 5 ( Figure 4.8). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 129% ( Table 4.6). Although statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stage 3a, there were no significant differences among treatments in the proportion of cells in developmental Stages 2a, 2b, 3b, 4, and 5 ( Table 4.6). Therefore, there was no consistent pattern of significant difference associated with trenbolone dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 98 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.6. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

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 0.006 0.005 72% 0.003 0.004 129% 0.003 0.003 113% 3.49 0.175 2b 0.018 0.008 41% 0.010 0.006 62% 0.013 0.012 92% 3.24 0.198 3a 0.167 0.060 36% 0.186 0.062 33% 0.045 0.037 80% 12.32 0.002* 3b 0.250 0.084 34% 0.204 0.095 47% 0.181 0.089 49% 2.22 0.329 4 0.173 0.056 33% 0.156 0.044 29% 0.127 0.111 88% 2.27 0.322 5 0.386 0.178 46% 0.441 0.184 42% 0.632 0.237 38% 4.75 0.093 * p < 0.01 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion of Cells Control Low High Figure 4.8. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 14 Day Trenbolone 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 103.6 µ m to 136.4 µ m ( Figure 4.9). Tubule diameters of males from the two test concentrations ranged from 90.8 µ m to 172.8 µ m. A significant difference in the mean tubule diameter per treatment ( Table 4.7) was detected ( Kruskal Wallis, H = 6.86, p = 0.032, df = 2). However, the pattern of significance ( Low < Control < High) was not consistent with the trenbolone dose. 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 33 ( Table 4.7). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 99 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.7. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 125.4 13.6 11% 24% 33 Low 8 112.1 20.7 18% High 8 138.6 23.4 17% 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 180 170 160 150 140 130 120 110 100 90 treatment diameter p y Figure 4.9. Boxplot of male seminiferous tubule diameter ( µ m) by treatment for the EPA 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the asterisk represents a probable outlier.

Observations   No interstitial Sertoli cell proliferation and no Leydig cell proliferation was observed for any treatment. No testicular atrophy was recorded and no ovatestes were observed for any treatment.

4.1.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the EPA 14 Day Trenbolone assay ranged from 1,434 pg/ mL to 4,553 pg/ mL ( Figure 4.10). Among females exposed to the two trenbolone concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 5,109 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 4.8) was detected ( Kruskal Wallis, H = 24.55, p < 0.001, df = 2). The mean estradiol concentration in females from the High concentration was less than that in females from the Low concentration and the Control treatment. The achieved power for this endpoint was 98%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 6 ( Table 4.8). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 100 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.8. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 15 2,907 972 33% 98% 6 Low 10 2,512 1,740 69% High 15 388 620 160% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 5000 4000 3000 2000 1000 0 treatment Estradiol ( y )

Figure 4.10. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisks represent probable outliers.

Estradiol concentrations in Control treatment males used during the EPA 14 Day Trenbolone assay ranged from 188 pg/ mL to 698 pg/ mL ( Figure 4.11). Among males exposed to the two trenbolone concentrations, estradiol concentrations ranged from 184 pg/ mL to 513 pg/ mL. No significant differences in the mean estradiol concentration per treatment ( Table 4.9) were detected ( Kruskal Wallis, H = 1.55, p = 0.460, df = 2). The achieved power for this endpoint was 7%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 99 ( Table 4.9). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 101 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.9. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 322 166 52% 7% 99 Low 4 331 143 43% High 6 352 62 18% 1 Calculated from natural log transformed data; with sample size = 4. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 700 600 500 400 300 200 treatment Estradiol ( y )

Figure 4.11. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone 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   Testosterone concentrations in Control treatment females used during the EPA 14 Day Trenbolone assay ranged from 492 pg/ mL to 3,047 pg/ mL ( Figure 4.12). Among females exposed to the two trenbolone concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 691 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 4.10) were detected ( Kruskal Wallis, H = 19.78, p < 0.001, df = 2). The mean testosterone concentration in females from the High concentration was less than that of females from the Low concentration and the Control treatment. The achieved power for this endpoint was 70%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 4 ( Table 4.10). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 102 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.10. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 1,515 911 60% 70% 4 Low 3 484 184 38% High 12 93 121 129% 1 Calculated from natural log transformed data; with sample size = 3. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 3000 2000 1000 0 treatment Testosterone ( means are indicated by solid circles)

Figure 4. 12. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone 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 Trenbolone assay ranged from 1,765 pg/ mL to 4,349 pg/ mL ( Figure 4.13). Among males exposed to the two trenbolone concentrations, testosterone concentrations ranged from 1,038 pg/ mL to 5,824 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 4.11) were detected ( Kruskal Wallis, H = 0.70, p = 0.703, df = 2). The achieved power for this endpoint was 8%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 113 ( Table 4.11). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 103 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.11. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 7 2,899 809 28% 8% 113 Low 6 3,227 1,668 52% High 7 2,787 2,002 72% 1 Calculated from natural log transformed data; with sample size = 6. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 6000 5000 4000 3000 2000 1000 treatment Testosterone Figure 4.13. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

11 ketotestosterone   11 ketotesosterone was not detected in females from the Control treatment ( 4 individuals), from the Low concentration ( 4 individuals), or from the High concentration ( 11 individuals).

11 ketotesosterone concentrations in Control treatment males used during the EPA 14 Day Trenbolone assay ranged from 14,810 pg/ mL to 33,093 pg/ mL ( Figure 4.14). Among males exposed to the two trenbolone concentrations, 11 ketotesosterone concentrations ranged from 2,090 pg/ mL to 120,800 pg/ mL. No significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 4.12) were detected ( Kruskal Wallis, H = 4.97, p = 0.083, df = 2). The achieved power for this endpoint was 31%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 22 ( Table 4.12). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 104 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.12. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 24,652 5,927 24% 31% 22 Low 7 34,349 19,619 57% High 8 23,835 40,136 168% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Low High Control 100000 50000 0 treatment 11 keto ( y )

Figure 4.14. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 14 Day Trenbolone 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.

4.1.8 Fecundity Total Fecundity   Variability among treatments in the total number of eggs produced during the EPA 14 Day Trenbolone assay was very high ( Figure 4.15). Total counts in the Control treatment ranged from 1,951 eggs to 3,227 eggs. Total counts for three replicates of the High trenbolone concentration treatment were similar, ranging from 280 eggs to 346 eggs, whereas the total number of eggs produced in the fourth replicate was 666. It is also notable that the primary egg production in three High trenbolone concentration replicates occurred only on Day 1 ( one replicate yielded 30 eggs on Day 12). Egg production in the fourth High trenbolone concentration occurred only on Days 2 and 3. The minimum and maximum production among replicates in the Low trenbolone concentration ranged from 2339 eggs to 3140 eggs. Statistical analysis of square root transformed egg counts showed significant amongtreatment differences in the mean cumulative number of eggs produced per treatment ( Table 4.13) ( 1 way ANOVA, F = 63.99, p < 0.001, df = 2, 9). Dunnett's comparison identified significant differences in mean egg production between the High trenbolone concentration and the Control treatment, and also between the High and Low concentrations. The Low concentration and the Control treatment fecundity DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 105 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 values were not statistically different. 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 4.13).

Table 4.13. Summary statistics and power estimates for fecundity data for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 2654 600 23% 100% 2 Low 4 2689 348 13% High 4 400 180 45% 1 Calculated from square root transformed data; with sample size = 4. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on square root transformed data.

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 12 14 16 Day Cumulative Number of Eggs Control Low High Figure 4.15. Total egg production by replicate per treatment for the EPA 14 Day Trenbolone assay.

Fecundity per Female Reproductive Day   During the EPA 14 Day Trenbolone assay, the maximum number of female reproductive days was achieved for the Control treatment and the High concentration, whereas 55 female reproductive days were achieved in the Low concentration ( Table 4.14). The number of eggs produced per female reproductive day in the Control treatment varied from 34.8 eggs to 57.6 eggs and from 41.8 eggs to 58.1 eggs in the Low concentration ( Figure 4.16). For the High concentration, the number of eggs produced per female reproductive day ranged from 5.0 eggs to 11.9 eggs, with fish in three of the replicates producing fewer than 7 eggs per day ( 5.0, 5.5, 6.2). A significant difference in the mean number of eggs produced per female reproductive day among treatments was detected ( Kruskal Wallis, H = 7.42, p = 0.024, df = 2). Fewer eggs were produced per female reproductive day in the High concentration than in either the Control treatment or the Low concentration. There was no difference in the mean number of eggs laid per female reproductive day between 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 4.14). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 106 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.14. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 14 Day Trenbolone assay.

Level Mean Number of Reproductive Days 1 N Mean Stdev CV Achieved Power 2 Sample Size Required 3 Control 56.0 4 47.4 10.7 23% 100% 2 Low 55.0 4 48.5 7.1 15% High 56.0 4 7.2 3.2 45% 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 60 50 40 30 20 10 0 treatment eggs/ ReproDay Figure 4.16. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 14 Day Trenbolone 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 Trenbolone assay varied from 350 eggs for the High concentration to 2526 eggs for the Low concentration ( Appendix E, Table 2.3). The number of eggs on dishes ranged from 50 eggs for the High concentration to 222 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. The proportional difference ranged from 0.78 ( one High concentration replicate) to 0.96 ( one Low concentration replicate) ( Appendix E, Figure 2.3). There were no significant differences in this mean proportional difference among treatments ( Kruskal Wallis, H = 3.23, p = 0.199, df = 2).

4.1.9 Fertilization Success Total Fertilization   The total ( tiles + dishes) fertilization success rates for most treatment replicates during the EPA 14 Day Trenbolone assay were high, ranging from 0.981 ( one High concentration DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 107 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 replicate) to 1.00 ( one Control treatment replicate) ( Figure 4.17). A significant difference in mean fertilization success rates ( Table 4.15) among treatments was detected ( Kruskal Wallis, H = 7.65, p = 0.022, df = 2). The fertilization success rate was lower in the High concentration than in the Control treatment or the Low concentration. There was no difference in the fertilization success rate between the Control treatment and the Low concentration. The achieved power for this assay was 89%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 4 ( Table 4.15).

Table 4.15. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 0.999 0.001 0% 89% 4 Low 4 0.998 0.002 0% High 4 0.990 0.007 1% 1 Calculated from arcsine square root transformed data; with sample size = 4. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.00 0.99 0.98 treatment Total Prop fert ( y )

Figure 4.17. Boxplot of the proportion of eggs fertilized by treatment for the EPA 14 Day Trenbolone 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 Trenbolone assay were high, ranging from 0.978 ( one High concentration replicate) to 1.00 ( two Control treatment replicates, one High and one Low concentration replicate) ( Appendix E, Figure 2.3). No significant differences in mean fertilization success rates for eggs laid on tiles ( Appendix E, Table 2.4) among treatments were detected ( Kruskal Wallis, H = 3.16, p = 0.206, df = 2). The fertilization success rates for all treatment replicates for eggs laid on dishes during the assay were high, ranging from 0.933 ( one High concentration replicate) to 1.00 ( several replicates; all treatments) ( Appendix E, Figure 2.4). No significant differences in mean fertilization success rates ( Appendix E, Table 2.4) among treatments were detected ( Kruskal Wallis, H = 0.04, p = 0.981, df = 2). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 108 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.1.10 Hatchability and Larval Development Eggs were collected during the 14 day pre exposure period for the evaluation of hatchability. The mean proportion of fertilized eggs that hatched was 0.98 ( sd = 0.03) for Control treatment, 0.94 ( sd = 0.03) for the Low concentration, and 0.97 ( sd = 0.03) for the High concentration. 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 0.75 ( sd = 0.23), which was significantly lower than those for the other treatments ( Kruskal Wallis, H = 9.59, p = 0.022, df = 3).

Eggs were collected during the EPA 14 Day Trenbolone assay for hatchability analysis. Fish in the High concentration did not produce eggs during that period, therefore, the High concentration was not included in the analysis. The proportion of fertilized eggs that hatched ranged from 0.96 to 1.00 in the Control treatment and was 1.00 for all replicates of the Low concentration ( Figure 4.18). There were no significant differences between the two treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 2.29, p = 0.131, df = 2). The achieved power for this endpoint was 11%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 13 ( Table 4.16).

Table 4.16. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 0.98 0.02 2% 11% 13 Low 4 1.00 0 0% High 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 Control 1.00 0.99 0.98 0.97 0.96 treatment Prop Hatch ( y )

Figure 4.18. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 14 Day Trenbolone 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 109 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 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 0.98 ( sd = 0.02) for Control treatment. The mean proportion of normal larvae in the remaining treatments was 0.98 ( sd = 0.02) in the Low concentration and 0.99 ( sd = 0.01) 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 0.98 ( sd = 0.03). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.84, p = 0.840, df = 3).

Eggs were collected on Days 7 through 10 during the EPA 14 Day Trenbolone assay for the evaluation of larval development. Fish in the High concentration did not produce eggs during that period, therefore, the High concentration was not included in the analysis. The proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) ranged from 0.96 to 1.00 in the Control treatment and from 0.78 to 1.00 for the Low concentration ( Figure 4.19). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.79, p = 0.375, df = 2). The achieved power for this endpoint was 8%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 29 ( Table 4.17).

Table 4.17. Summary statistics and power estimates for the proportion of normal larvae for the EPA 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 0.98 0.02 2% 8% 29 Low 4 0.92 0.10 11% High 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 Control 1.0 0.9 0.8 treatment Prop Norm Figure 4.19. Boxplot of the proportion of normal larvae by treatment for the EPA 14 Day Trenbolone 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 110 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.1.11 Body Weight The body weight of females used in the EPA 14 Day Trenbolone assay ranged from 1.0 g to 2.9 g. A significant difference in the mean female body weight per treatment was detected ( Kruskal Wallis, H = 10.77, p = 0.005, df = 2). The test indicated that the mean female body weight for females in the Lowconcentration treatment was less than those for the other two treatments. The body weight of males used in the EPA 14 Day Trenbolone assay ranged from 2.4 g to 4.7 g. There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 3.72, p = 0.156, df = 2).

4.2 EPA 21 Day Assay for Trenbolone The EPA 21 Day Trenbolone assay was conducted from January 20, 2003, to February 3, 2003 ( preexposure test), and from February 3, 2003, to February 24, 2003 ( exposure test).

4.2.1 Survival All males in all treatments and all females in the Control treatment survived the EPA 21 Day Trenbolone assay. One female in the High concentration and one female in the Low concentration treatments died during the 21 day exposure ( 94% survival in each case).

4.2.2 Vitellogenin Vitellogenin concentrations in most Control treatment females used during the EPA 21 Day Trenbolone assay ranged from 329,100 ng/ mL to 1,434,000 ng/ mL ( Figure 4.20). Two Control treatment females had vitellogenin concentrations of 3,443,500 ng/ mL and 4,162,000 ng/ mL. Among females exposed to the two trenbolone concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 3,184,500 ng/ mL. Significant differences in the mean vitellogenin concentration per treatment ( Table 4.18) were detected ( Kruskal Wallis, H = 21.37, p = < 0.001, df = 2). Vitellogenin concentrations in Control treatment females and Low concentration females were significantly greater than those in females exposed to the High trenbolone concentrations. The achieved power for this endpoint was 99%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 7 ( Table 4.18).

Table 4.18. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 13 1,289,831 1,164,016 90% 99% 7 Low 14 1,221,589 1,093,574 90% High 15 113,444 270,367 238% 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) 111 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 4000000 3000000 2000000 1000000 0 treatment Trenb VTG Figure 4.20. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Trenbolone 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 21 Day Trenbolone assay ranged from 0 ng/ mL ( not detected) to 8,368 ng/ mL ( Figure 4.21). Among males exposed to the two trenbolone concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 185,700 ng/ mL. No significant differences in the mean vitellogenin concentration per treatment ( Table 4.19) were detected ( Kruskal Wallis, H = 0.65, p = 0.722, df = 2). The achieved power for this endpoint was 8%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 140 ( Table 4.19).

Table 4.19. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 2,919 3,607 124% 8% 140 Low 8 43,439 76,533 176% High 7 6,983 11,964 171% 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 112 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 200000 100000 0 treatment Trenb VTG Figure 4.21. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the EPA 21 Day Trenbolone 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.

4.2.3 Appearance / Secondary Sex Characteristics All of the Control treatment females used during the EPA 21 Day Trenbolone assay exhibited typical female morphology ( no fat pad, no tubercles, no vertical banding, ovipositor present). One of the 15 females exposed to the Low concentration had a dorsal fat pad. Five had vertical banding. Among the 16 females exposed to the High concentration, 6 had tubercles, 8 had dorsal fat pads, 7 had vertical banding, and 3 lacked ovipositors. Thus, trenbolone appeared to have a dose related effect on female morphology.

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

4.2.4 Gonadosomatic Index The range of GSI values calculated for females in the all treatments varied from 4 to 22 fold ( Figure 4.22), and the overall within treatment variability, as indicated by CVs, ranged from 34%   52%; Table 4.20). Several fish had relatively high GSI values. The highest value was obtained for a female exposed to the Low trenbolone concentration had a GSI value of 29.8. The GSI values for several other fish exceeded 21.0, which is much greater than the typical upper limit for reproductively active female fathead minnows ( GSI ~ 13.0). The total body weight of these females were well within the normal range for the test animals and it appears the high GSI value resulted from each fish having unusually high gonad weights. The gonad weights of these fish ranged from 0.41 g to 0.76 g ( average = 0.59 g), greater than the average values for the remaining fish ( 0.2 g). A significant difference in the mean GSI value per treatment ( Table 4.20) was detected ( Kruskal Wallis, H = 8.45, p = 0. 015, df = 2). The test indicated that the mean GSI for females in the Control treatment was less than those for the other two treatments. However, the mean GSI values calculated for the Low and High trenbolone concentrations were not statistically different. The achieved power for this endpoint was 58%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 24 ( Table 4.20). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 113 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.20. Summary statistics and power estimates for female gonadosomatic index data for the EPA 21 Day Trenbolone assay Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 16 10.4 5.4 52% 58% 24 Low 15 15.9 5.4 34% High 15 15.9 7.7 48% 1 Calculated from arcsine square root transformed data; with sample size = 15. 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 30 20 10 0 treatment GSI ( y )

Figure 4.22. Boxplot of female GSI by treatment for the EPA 21 Day Trenbolone 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.

The range of most GSI values calculated for males during the EPA 21 Day Trenbolone assay, was small, ranging from 0.4 to 1.7 ( Figure 4.23). Most estimates were between about 1.0 and 1.7, which approximates the typical range for reproductively active male fathead minnows. There were no significant differences in mean GSI values ( Table 4.21) among treatments ( Kruskal Wallis, H = 0.97, p = 0.617, df = 2). The achieved power for this endpoint was 19%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 42 ( Table 4.21). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 114 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.21. Summary statistics and power estimates for male gonadosomatic index data for the EPA 21 Day Trenbolone assay Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 1.12 0.47 42% 19% 42 Low 8 1.20 0.30 25% High 8 1.35 0.27 20% 1 Calculated from arcsine square root transformed data; with sample size = 8. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.5 1.0 0.5 treatment GSI ( y )

Figure 4.23. Boxplot of male GSI by treatment for the EPA 21 Day Trenbolone 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.

4.2.5 Female Gonad Histology General Ovary Staging   Statistical analysis of the mean ovarian staging from 12 microscopic fields per female in the EPA 21 Day Trenbolone assay revealed no significant differences among treatments ( Kruskal Wallis, H = 4.71, p = 0.095, df = 2).

Quantitative Ovarian Staging   One hundred cells in each of three sections per female were examined to quantitatively determine the developmental stage of the ovaries. Ova from fish in all treatments ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages) ( Figure 4.24). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 412% ( Table 4.22. Although statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stages 1a, 1b, 2, and 3, there were no significant differences among treatments in the proportion of cells in the developmental Stages 4, and 5 ( Table 4.22). There was no consistent pattern of significant difference associated with trenbolone dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 115 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.22. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the EPA 21 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments Control ( n = 16) Low ( n = 13) High ( n = 17) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Value Stdev CV H p 1a 0.079 0.034 44% 0.099 0.021 22% 0.077 0.019 25% 6.94 0.031* 1b 0.365 0.065 18% 0.327 0.067 20% 0.265 0.089 34% 8.36 0.015* 2 0.202 0.057 28% 0.178 0.029 16% 0.250 0.096 38% 8.03 0.018* 3 0.102 0.052 51% 0.122 0.031 26% 0.192 0.096 50% 14.09 0.001** 4 0.215 0.096 45% 0.240 0.068 28% 0.156 0.106 68% 5.47 0.065 5 0.003 0.008 278% 0.001 0.003 361% 0.002 0.006 412% 2.76 0.252 * p < 0.05 ** p < 0.01 0.0 0.1 0.2 0.3 0.4 0.5 1a 1b 2 3 4 5 Ovarian Stage Proportion of Cells Control Low High Figure 4.24. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the EPA 21 Day Trenbolone 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 0.007 for females in the Control treatment to 0.05 for females in the High concentration ( Figure 4.25). There were significant differences in the proportions of atretic follicles among treatments ( Kruskal Wallis, H = 14.88, p = 0.001, df = 2). The proportions of atretic follicles in the High concentration were greater than those in the Control treatment, but not greater than those in the Lowconcentration treatment. Therefore, there was no consistent pattern of significant difference associated with trenbolone dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 116 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 0.2 0.1 0.0 treatment atretic_ follicles ( y )

Figure 4.25. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the EPA 21 Day Trenbolone 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 0.01 for females in the High concentration to 0.025 for females in the Low concentration ( Figure 4.26). There were significant differences in the proportions of corpora lutea among treatments ( Kruskal Wallis, H = 11.43, p = 0.003, df = 2). The mean proportion of corpora lutea in the High concentration was less than those in the Control treatment and the Low concentration.

Low High Control 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 treatment corpora_ lutea ( y )

Figure 4.26. Boxplot of proportion of corpora lutea per 300 follicles by treatment for the EPA 21 Day Trenbolone 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) 117 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.2.6 Male Gonad Histology Testes Staging by Microscopic Field   Testes from males exposed to trenbolone during the EPA 21 Day Trenbolone assay were examined to determine the general developmental condition. Males in all treatments had well developed testes with most showing Stage 4 and Stage 5 development ( see Methods for description of developmental stages). All of the 96 microscopic fields examined in the 8 Control treatment males showed Stage 4 ( 79 fields) or Stage 5 ( 17 fields) development. All of the 120 microscopic fields examined in the 10 Low concentration treatment males showed Stage 4 ( 99 fields) or Stage 5 ( 21 fields) development. All of the 72 microscopic fields examined in the 6 High concentration treatment males showed Stage 4 ( 48 fields) or Stage 5 ( 24 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed no significant differences in testicular development among treatments ( Kruskal Wallis, H = 3.59, p = 0.166, 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 of the Control treatment testes ranged from Stage 1 to Stage 5, whereas testes in males from the Low and Highconcentration treatments showed Stage 2a to Stage 5 development ( Figure 4.27). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 283% ( Table 4.23). Although statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stage 2a and Stage 3b, there were no significant differences among treatments in the proportion of cells in developmental Stages 1, 2b, 3a, 4, and 5 ( Table 4.23). Therefore, there was no consistent pattern of significant difference associated with trenbolone dose.

Table 4.23. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the EPA 21 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 8) Low ( n = 10) High ( n = 6) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Mean Stdev CV H p 1 0.0004 0.001 283% 0 0   0 0   2.00 0.368 2a 0.007 0.003 38% 0.004 0.005 125% 0.002 0.004 182% 6.05 0.049* 2b 0.016 0.009 54% 0.016 0.014 88% 0.014 0.016 114% 0.19 0.910 3a 0.117 0.068 58% 0.156 0.107 69% 0.102 0.077 76% 1.19 0.550 3b 0.280 0.063 22% 0.202 0.112 55% 0.114 0.065 57% 9.44 0.009** 4 0.213 0.081 38% 0.147 0.097 66% 0.115 0.053 46% 4.38 0.112 5 0.367 0.170 46% 0.475 0.252 53% 0.652 0.191 29% 4.84 0.089 * p < 0.05 ** p < 0.01 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 118 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion of Cells Control Low High Figure 4.27. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the EPA 21 Day Trenbolone 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 78.6 µ m to 160.3 µ m ( Figure 4.28). Tubule diameters of males from the two test concentrations ranged from 88.1 µ m to 215.8 µ m. No significant differences in the mean tubule diameter per treatment ( Table 4.24) were detected ( Kruskal Wallis, H = 3.33, p = 0.190, df = 2). The achieved power for this endpoint was 33%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 17 ( Table 4.24).

Table 4.24. Summary statistics and power estimates for male seminiferous tubule diameter data for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 114.4 28.0 24% 33% 17 Low 10 121.4 28.1 23% High 6 147.9 37.0 25% 1 Calculated from natural log transformed data; with sample size = 6. 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) 119 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 200 150 100 treatment diameter Figure 4.28. Boxplot of male seminiferous tubule diameter ( µ m) by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the asterisk represents a probable outlier.

Observations   One male in the Control treatment showed early stage cells in the lumen of some tubules. No interstitial Sertoli cell proliferation and no Leydig cell proliferation was observed for any treatment. No testicular atrophy was recorded and no ovatestes were observed for any treatment.

4.2.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the EPA 21 Day Trenbolone assay ranged from 357 pg/ mL to 5,112 pg/ mL ( Figure 4.29). Among females exposed to the two trenbolone concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 4,567 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 4.25) was detected ( Kruskal Wallis, H = 11.62, p = 0.003, df = 2). The mean estradiol concentration in females from the High concentration was less than that in females from the Low concentration. The achieved power for this endpoint was 39%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 22 ( Table 4.25).

Table 4.25. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 12 1,361 1,380 101% 39% 22 Low 9 1,696 1,553 92% High 15 396 393 99% 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) 120 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 5000 4000 3000 2000 1000 0 treatment Estradiol ( y )

Figure 4.29. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisks represent probable outliers.

Estradiol concentrations in Control treatment males used during the EPA 21 Day Trenbolone assay ranged from 0 pg/ mL ( not detected) to 645 pg/ mL ( Figure 4.30). Among males exposed to the two trenbolone concentrations, estradiol concentrations ranged from 153 pg/ mL to 732 pg/ mL. No significant differences in the mean estradiol concentration per treatment ( Table 4.26) were detected ( Kruskal Wallis, H = 4.50, p = 0.105, df = 2). The achieved power for this endpoint was 22%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 16 ( Table 4.26).

Table 4.26. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 6 323 266 82% 22% 16 Low 7 299 121 40% High 4 548 188 34% 1 Calculated from natural log transformed data; with sample size = 4. 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) 121 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 800 700 600 500 400 300 200 100 0 treatment Estradiol ( y )

Figure 4.30. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Testosterone   Testosterone concentrations in Control treatment females used during the EPA 21 Day Trenbolone assay ranged from 464 pg/ mL to 4,534 pg/ mL ( Figure 4.31). Among females exposed to the two trenbolone concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 667 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 4.27) were detected ( Kruskal Wallis, H = 15.24, p < 0.001, df = 2). The mean testosterone concentration in females from the Low concentration was less than that of females from the High concentration and the Control treatment. The achieved power for this endpoint was 60%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 8 ( Table 4.27).

Table 4.27. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 1,332 1,330 100% 60% 8 Low 5 180 126 70% High 12 475 137 29% 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) 122 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 5000 4000 3000 2000 1000 0 treatment Testosterone ( y )

Figure 4.31. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone 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 21 Day Trenbolone assay ranged from 893 pg/ mL to 2,592 pg/ mL ( Figure 4.32). Among males exposed to the two trenbolone concentrations, testosterone concentrations ranged from 481 pg/ mL to 4,709 pg/ mL. No significant differences in the mean testosterone concentration per treatment ( Table 4.28) were detected ( Kruskal Wallis, H = 2.89, p = 0.236, df = 2). The achieved power for this endpoint was 12%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 44 ( Table 4.28).

Table 4.28. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 1,414 600 42% 12% 44 Low 8 2,656 1,459 55% High 5 2,528 1,766 70% 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) 123 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 5000 4000 3000 2000 1000 0 treatment Testosterone ( y )

Figure 4.32. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

11 ketotestosterone   11 ketotesosterone was not detected in females from the Control treatment ( 2 individuals) or from the Low concentration ( three individuals). No females from the High concentration were available for 11 ketotestosteron analyses.

11 ketotesosterone concentrations in most Control treatment males used during the EPA 21 Day Trenbolone assay ranged from 0 pg/ mL ( not detected) to 13,938 pg/ mL ( Figure 4.33). One male from the Control treatment had a 11 ketotestosterone concentration of 56,704 pg/ mL. Among males exposed to the two trenbolone concentrations, 11 ketotesosterone concentrations ranged from 481 pg/ mL to 40,953 pg/ mL. No significant differences in the mean 11 ketotesosterone concentration per treatment ( Table 4.29) were detected ( Kruskal Wallis, H = 4.25, p = 0.120, df = 2). The achieved power for this endpoint was 26%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 26 ( Table 4.29).

Table 4.29. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the EPA 21 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 12,925 18,174 141% 26% 26 Low 8 26,128 14,008 54% High 7 14,128 11,455 81% 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. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 124 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Low High Control 60000 50000 40000 30000 20000 10000 0 treatment 11 keto ( y )

Figure 4.33. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the EPA 21 Day Trenbolone 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.

4.2.8 Fecundity Total Fecundity   Two pre exposure evaluations of total egg production were performed. One examined the total egg production at 14 days, which is within the protocol guidelines; the other evaluated egg production at day 7 of the pre exposure assay to determine whether or not the shorter pre exposure period would be sufficient to evaluate the potential reproductive success of the test fish. Total 14 day counts among the tanks that were eventually used for the three treatments in the exposure assay ( individual tank values summed for each treatment) ranged from about 12,000 eggs to 14,000 eggs ( Figure 4.34). No significant differences in the mean 14 day egg production among the groups of replicates that would be used in the trenbolone exposure assay were detected ( 1 way ANOVA, F = 0.11, p = 0.953, df = 3, 13). Production at Day 7 of the pre exposure test among the 12 replicates that were eventually used in the exposure assay ranged from 503 eggs to 1,923 eggs. No significant differences in the mean 7 day egg production among the groups of replicates that would be used in the trenbolone exposure assay were detected ( 1 way ANOVA, F = 0.11, p = 0.955, df = 3, 13). No significant differences were detected in the mean number of eggs laid per day per replicate group at 7 days versus 14 days ( Two Sample t Test, t =   0.85, p = 0.401, df = 25). However, daily within treatment variation ( indicated by fluctuating coefficient of variation values) was high through Day 9. Therefore, variability during a 7 day preexposure assay is likely to be much greater than that during longer pre exposure assay, which reduces the likelihood of reliably choosing replicates with successful reproduction histories for use in the exposure assay. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 125 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  14  12  10  8  6  4  2 1 3 5 7 9 11 13 15 17 19 21 Day Cumulative Number of Eggs Control Low High Figure 4.34. Total egg production per treatment for the EPA 21 Day Trenbolone assay. The vertical line at Day 0 denotes the start of the exposure period.

During the EPA 21 Day Trenbolone assay, total counts in the Control treatment were reasonably consistent among replicates, varying from 2,741 eggs to 4,334 eggs ( Figure 4.35). Variability in total egg production among Low concentration replicates was somewhat greater, ranging from 1,873 eggs to 4,017 eggs. Total counts among the High concentration replicates varied about five fold, ranging from 215 eggs to 1,095 eggs. Statistical analysis of square root transformed egg counts showed significant amongtreatment differences ( 1 way ANOVA, F = 23.14, p < 0.001, df = 2, 10) in mean cumulative numbers of eggs produced ( Table 4.30). Tukey's pairwise comparison identified significant differences in mean egg production between the High trenbolone concentration and the Control treatment, and also between the High and Low concentrations. The Low concentration and the Control treatment fecundity values were not statistically different. Daily within treatment variation ( indicated by fluctuating coefficient of variation values) was much reduced after Days 8   10, except for the High concentration. Egg production in the other four High concentration replicates was sporadic. Egg production stopped after Day 3 in two replicates, after Day 7 in a third replicate, and occurred only on Day 9 in the fourth replicate. 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 4.30).

Table 4.30. Summary statistics and power estimates for fecundity data for the EPA 21 Day Trenbolone assay Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 3672 795 22% 100% 3 Low 4 2818 914 32% High 4 590 426.5 72% 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) 126 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 0 2 4 6 8 10 12 14 16 18 20 22 Day Cumulative Number of Eggs Control Low High Figure 4.35. Total egg production by replicate per treatment for the EPA 21 Day Trenbolone assay.

Fecundity per Female Reproductive Day   During the 14 Day pre exposure evaluation, the mean number of eggs produced per female reproductive day ranged from 55.6 eggs/ day for the tanks that would be used for the Control treatment to 58.5 eggs/ day for the tanks that would be used for the High concentration during the 21 day exposure assay. There were no significant differences among treatments in the numbers of eggs produced per reproductive day during the pre exposure period ( Kruskal Wallis, H = 0.99, p = 0.804, df = 3).

During the EPA 21 Day Trenbolone assay, the maximum number of female reproductive days was achieved for the Control treatment, whereas 80 and 79.3 female reproductive days were achieved in the Low concentration and High concentration, respectively ( Table 4.31). The number of eggs produced per female reproductive day varied from 32.6 eggs to 51.6 eggs in the Control treatment and from 27.5 to 47.8 in the Low concentration ( Figure 4.36). For the High concentration, the number of eggs produced per female reproductive day ranged from 2.6 eggs to 13.0 eggs. A significant difference in the mean number of eggs produced per female reproductive day among treatments was detected ( Kruskal Wallis, H = 8.38, p = 0.015, df = 2). The mean number of eggs produced per female reproductive day was significantly smaller in the High concentration than in either the Control treatment or the Low concentration. The mean number of eggs produced in the Low concentration did not differ from that produced in the Control treatment. 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 4.31). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 127 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.31. Summary statistics and power estimates for fecundity per female reproductive day for the EPA 21 Day Trenbolone assay Level Mean Number of Reproductive Days 1 N Mean Stdev CV Achieved Power 2 Sample Size Required 3 Control 84.0 4 43.7 9.5 22% 100% 3 Low 80.0 4 34.8 9.3 27% High 79.3 4 7.2 4.8 67% 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 50 40 30 20 10 0 treatment eggs/ ReproDay Figure 4.36. Boxplot of the number of eggs produced per female reproductive day by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Eggs on Tiles/ Dishes   The mean number of eggs laid on the tiles during the 14 day pre exposure assay varied from 2,101 eggs for the tanks that would be used for the Control treatment to 2,561 eggs for the tanks that would be used for the High concentration treatment. The mean number of eggs laid on the dishes during the 14 day pre exposure assay varied from 733 eggs for the tanks that would be used for the High concentration treatment to 1,005 eggs for the tanks that would be used 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. There were no significant differences in the mean proportional difference among treatments during the 14 day pre exposure assay ( Kruskal Wallis, H = 1.39, p = 0.707, df = 3).

The mean number of eggs laid on the tiles among the treatments during the EPA 21 Day Trenbolone assay varied from 500 eggs for the High concentration to 3,384 eggs for the Control treatment ( Appendix E, Table 2.7). The number of eggs on dishes ranged from 90 eggs for the High concentration to 288 eggs DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 128 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 for the Control treatment. The proportional difference ranged from   0.10 ( one High concentration replicate had more eggs on the dish than on the tile) to 0.96 ( one Control treatment replicate) ( Appendix E, Figure 2.6). There were no significant differences in this mean proportional difference among treatments ( Kruskal Wallis, H = 2.46, p = 0.292, df = 2).

4.2.9 Fertilization Success Total Fertilization   Eggs were collected during the 14 day pre exposure period for the evaluation of fertilization success rate. The mean proportion of eggs fertilized in the Control treatment was 0.981 [ standard deviation ( sd) = 0.028], 0.997 ( sd = 0.002) in the Low concentration, and 0.985 ( sd = 0.023) in the High concentration. The mean proportion of eggs fertilized in the replicates that were not used in the 21 day validation assay was 0.977 ( sd = 0.041). There were no significant differences among treatments in the proportion of eggs that were fertilized ( Kruskal Wallis, H = 2.42, p = 0.490, df = 3).

The total ( tiles + dishes) fertilization success rates for all treatment replicates during the EPA 21 Day Trenbolone assay were high, ranging from 0.991 ( one Low concentration replicate) to 1.00 ( one Highconcentration replicate) ( Figure 4.37). No significant differences in mean fertilization success rates ( Table 4.32) among treatments were detected ( Kruskal Wallis, H = 0.04, p = 0.981, df = 2). The achieved power for this assay was 5%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 764 ( Table 4.32).

Table 4.32. Summary statistics and power estimates for the proportion of eggs fertilized for the EPA 21 Day Trenbolone assay Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 0.997 0.001 0.1% 5% 764 Low 4 0.996 0.004 0.4% High 4 0.997 0.004 0.4% 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) 129 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 0.994 0.993 0.992 0.991 treatment Total Prop fert Figure 4.37. Boxplot of the proportion of eggs fertilized by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Fertilization of Eggs on Tiles and Dishes   During the 14 Day pre validation assay, there were no significant differences in the fertilization success rates among treatments for eggs laid in tiles ( Kruskal Wallis, H = 4.51, p = 0.211, df = 3) or on dishes ( Kruskal Wallis, H = 2.67, p = 0.446, df = 3).

The fertilization success rates for all treatment replicates for eggs laid on tiles during the EPA 21 Day Trenbolone assay were high, ranging from 0.990 ( one Low concentration replicate) to 1.00 ( two Highconcentration replicates) ( Appendix E, Figure 2.7). No significant differences in mean fertilizationsuccess rates ( Appendix E, Table 2.8) among treatments were detected ( Kruskal Wallis, H = 0.35, p = 0.841, df = 2). The fertilization success rates for all treatment replicates for eggs laid on dishes during the assay were high, ranging from 0.958 ( one Control treatment replicate) to 1.00 ( several replicates; including all treatments) ( Appendix E, Figure 2.8). No significant differences in mean fertilizationsuccess rates ( Appendix E, Table 2.8) among treatments were detected ( Kruskal Wallis, H = 0.62, p = 0.733, df = 2).

4.2.10 Hatchability and Larval Development Eggs were collected during the pre exposure period for the evaluation of hatchability. The mean proportion of fertilized eggs that hatched was 0.97 ( sd = 0.02) for Control treatment, 0.98 ( sd = 0.02) for the Low concentration, and 0.94 ( sd = 0.06) for 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 fertilized eggs that hatched was 0.92 ( sd = 0.03). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 6.91, p = 0.075, df = 3).

Eggs were collected during the EPA 21 Day Trenbolone assay for the evaluation of hatchability. The proportion of fertilized eggs that hatched ranged from 0.26 to 1.00 in the Control treatment and from 0.82 to 1.00 for the two test concentrations ( Figure 4.38). There were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.07, p = 0.964, 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 185 ( Table 4.33). One of the Control treatment replicates had a very low proportion of the eggs hatch ( 0.26) during the second week of the hatch evaluation. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 130 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Because the data sheet for that replicate indicated that the eggs may have been abnormal at the initiation of the evaluation, the analyses were rerun excluding those data. The resulting mean proportion hatch for the Control treatment was 0.95 ( sd = 0.08), however there still were no significant differences among treatments in the proportion of eggs that hatched ( Kruskal Wallis, H = 0.59, p = 0.746, df = 2).

Table 4.33. Summary statistics and power estimates for the proportion of fertile eggs that hatched for the EPA 21 Day Trenbolone assay Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 0.86 0.26 30% 5% 185 Low 8 0.94 0.06 7% High 2 0.94 0.08 9% 1 Calculated from arcsine square root transformed data; with sample size = 2. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 treatment Prop Hatch ( y )

Figure 4.38. Boxplot of the proportion of fertile eggs that hatched by treatment for the EPA 21 Day Trenbolone 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.

Eggs were collected during the pre exposure period for the evaluation of larval development. The proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) was 1.00 for all replicates of the Control treatment. The mean proportion of normal larvae in the remaining treatments was 0.96 ( sd = 0.01) in the Low concentration and 0.95 ( sd = 0.07) 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 0.93 ( sd = 0.05). There were no significant differences among treatments in the proportion of normal larvae ( Kruskal Wallis, H = 6.26, p = 0.099, df = 3). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 131 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Eggs were collected on Days 7   10, 14   16, and 21 during the EPA 21 Day Trenbolone assay for the evaluation of larval development. The proportion of larvae that developed normally ( i. e., that showed no morphological abnormalities) ranged from 0.08 to 1.00 in the Control treatment and from 0.98 to 1.00 for the two test concentrations ( Figure 4.39). There were no significant differences among treatments in the proportion of normal larvae ( Kruskal Wallis, H = 2.77, p = 0.250, 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 16 ( Table 4.34). As for the hatchability analyses, the larval development analysis was rerun excluding the one Control treatment replicate that may have had abnormal eggs at the initiation of the evaluation. The resulting mean proportion of larvae that developed normally for the Control treatment was 0.98 ( sd = 0.22), however there were no significant differences among treatments in the proportion of normal larvae ( Kruskal Wallis, H = 1.89, p = 0.390, df = 2).

Table 4.34. Summary statistics and power estimates for the proportion of normal larvae for the EPA 21 Day Trenbolone assay Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 0.78 0.35 45% 9% 16 Low 8 0.997 0.01 1% High 2 0.99 0.02 2% 1 Calculated from arcsine square root transformed data; with sample size = 2. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on arcsine square root transformed data.

Low High Control 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 treatment Prop Norm Figure 4.39. Boxplot of the proportion of normal larvae by treatment for the EPA 21 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisk represents a probable outlier. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 132 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.2.11 Body Weight The body weight of females used in the EPA 21 Day Trenbolone assay ranged from 1.1 g to 3.7 g. A significant difference in the mean female body weight per treatment was detected ( Kruskal Wallis, H = 16.34, p < 0.001, df = 2). The test indicated that the mean female body weight for females in the Highconcentration treatment was greater than those for the other two treatments. The body weight of males used in the EPA 21 Day Trenbolone assay ranged from 2.6 g to 6.0 g. There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 0.97, p = 0.616, df = 2).

4.3 Non spawning Adult 14 Day Assay for Trenbolone The Non spawning Adult 14 Day Trenbolone assay was conducted from February 10, 2003 to February 24, 2003.

4.3.1 Survival Total survival in the Control and all three test concentrations during the Non spawning Adult 14 Day Trenbolone assay was 100%.

4.3.2 Vitellogenin Vitellogenin concentrations in Control treatment females used during the Non spawning Adult 14 Day Trenbolone assay ranged from 1,409,500 ng/ mL to 4,440,000 ng/ mL ( Figure 4.40). Among most females exposed to the three trenbolone concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 915,750 ng/ mL. Two females exposed to the Low trenbolone concentration had vitellogenin concentrations of 1,549,500 ng/ mL and 3,821,000 ng/ mL. Significant differences in the mean vitellogenin concentration among treatments ( Table 4.35) were detected ( Kruskal Wallis, H = 23.23, p = < 0.001, df = 2). Mean vitellogenin concentrations among Control treatment females were significantly greater than the mean concentrations for females exposed to the all three trenbolone 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 6 ( Table 4.35).

Table 4.35. Summary statistics and power estimates for female vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 2,810,125 1,019,160 36% 96% 6 Low 10 907,905 1,123,918 124% Medium 9 125,801 241,178 192% High 9 112,255 145,135 129% 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) 133 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 4000000 3000000 2000000 1000000 0 treatment Trenb VTG Figure 4.40. Boxplot of female vitellogenin concentration ( ng/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone 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.

Vitellogenin concentrations in Control treatment males used during the Non spawning Adult 14 Day Trenbolone assay ranged from 0 ng/ mL ( not detected) to 7,672 ng/ mL ( Figure 4.41). Vitellogenin was not detected above the MDL for four of the eight males in the Control treatment. Among most males exposed to the three trenbolone concentrations, vitellogenin concentrations ranged from 0 ng/ mL ( not detected) to 1,020 ng/ mL. Vitellogenin concentrations were not detected above the MDL for 18 of the 20 males exposed to the Low and Medium trenbolone concentrations. One male exposed to the High trenbolone concentration had a vitellogenin concentration of 11,390 ng/ mL. Significant differences in the mean vitellogenin concentration per treatment ( Table 4.36) were detected ( Kruskal Wallis, H = 16.81, p = 0.001, df = 2). Vitellogenin concentrations in males form the Control treatment and the High concentration ranked higher than those from males in the Low and Medium concentrations. The achieved power for this endpoint was 41%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 20 ( Table 4.36).

Table 4.36. Summary statistics and power estimates for male vitellogenin concentrations ( ng/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 9 1,337 2,561 192% 41% 20 Low 10 102 323 317% Medium 10 30 96 320% High 10 1,449 3,500 242% 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) 134 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 10000 5000 0 treatment Trenb VTG ( y )

Figure 4.41. Boxplot of male vitellogenin concentration ( ng/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone 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.

4.3.3 Appearance / Secondary Sex Characteristics All of the Control treatment and Low concentration females used during the Non spawning Adult 14 Day Trenbolone assay showed normal female morphology. Six of 10 females from the High concentration had tubercles typical of males. One female each from the Medium and High concentrations had dorsal fat pads. Four females from the High concentration had vertical banding. Therefore trenbolone likely had a dose related effect on female morphology.

Morphological development among males used during the Non spawning Adult 14 Day Trenbolone assay varied among treatments ( Figure 4.42). Five males, three from the Control treatment and two from the Low concentration, had female body shapes. Three males for the same two treatments lacked tubercles. Two males from the Control treatment lacked fat pads. Twelve 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) 135 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 4.42. Secondary sex characteristics of males used during the flutamide Non spawning Adult 14 Day assay.

4.3.4 Gonadosomatic Index The range of GSI values calculated for females in the all treatments varied from two to ten fold ( Figure 4.43), and the overall within treatment variability was moderate ( CVs = 22%   50%; Table 4.37). The highest female GSI value was 24.9 ( one fish in the High concentration), but several fish had GSI values > 20. There were no significant differences in mean GSI values ( Table 4.37) among treatments ( Kruskal Wallis, H = 3.05, p = 0.384, df = 3). The achieved power for this endpoint was 18%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 55 ( Table 4.37).

Table 4.37. Summary statistics and power estimates for female gonadosomatic index data for the Nonspawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 13.2 5.0 38% 18% 55 Low 10 13.8 3.5 25% Medium 10 16.4 3.6 22% High 10 14.8 7.4 50% 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) 136 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 25 20 15 10 5 0 treatment GSI ( y )

Figure 4.43. Boxplot of female GSI by treatment for the Non spawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

The range of most GSI values calculated for males during the Non spawning Adult 14 Day Trenbolone assay, varying from 0.8 to 2.0 ( Figure 4.44), which approximates the typical range for reproductivelyactive male fathead minnows. Within treatment variability was moderate, as indicated by CVs that ranged from 24% to 59%; Table 4.38). The highest and lowest male GSI values were 2.2   2.3 ( for one fish each in the Low and High concentrations) and 0.2   0.3 ( one fish each in the Low and Medium concentrations), respectively. However, there were no significant differences among treatments in mean GSI values ( Table 4.38) among treatments ( Kruskal Wallis, H = 4.60, p = 0.203, df = 3). The achieved power for this endpoint was 16%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 64 ( Table 4.38).

Table 4.38. Summary statistics and power estimates for male gonadosomatic index data for the Nonspawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 1.21 0.41 34% 16% 64 Low 10 1.11 0.66 59% Medium 10 1.34 0.56 42% High 10 1.60 0.38 24% 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) 137 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 2 1 0 treatment GSI Figure 4.44. Boxplot of male GSI by treatment for the Non spawning Adult 14 Day Trenbolone 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.

4.3.5 Female Gonad Histology General Ovary Staging   Statistical analysis of the mean ovarian staging from 12 microscopic fields per female in the Non spawning Adult 14 Day Trenbolone assay revealed a significant difference among treatments ( Kruskal Wallis, H = 9.13, p = 0.028, df = 2). The mean ovarian stage of females in the Control treatment was greater than that of females in the other three treatments.

Quantitative Ovarian Staging   One hundred cells in each of three sections per female were examined to quantitatively determine the developmental stage of the ovaries. Ova from fish in the Control treatment, Medium concentration, and High concentration ranged from Stage 1a to Stage 5 ( see Methods for a description of the stages), whereas ova from females from the Low concentration treatment showed Stage 1a to Stage 4 development ( Figure 4.45). Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 316% ( Table 4.39). Although statistical analyses showed that there was a significant difference among treatments in the proportion of cells in developmental Stages 3 and 5, there were no significant differences among treatments in the proportion of cells in the developmental Stages 1a, 1b, 2, and 4 ( Table 4.39). Therefore, there was no consistent pattern of significant difference associated with trenbolone dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 138 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.39. Descriptive statistics of the proportion of ovarian cells in each developmental stage for females from the Non spawning Adult 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

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 0.107 0.034 32% 0.086 0.029 33% 0.082 0.041 51% 0.069 0.047 68% 5.65 0.130 1b 0.370 0.085 23% 0.325 0.080 25% 0.257 0.080 31% 0.291 0.128 44% 7.70 0.053 2 0.135 0.048 35% 0.137 0.046 33% 0.108 0.043 40% 0.151 0.052 34% 3.31 0.346 3 0.096 0.047 48% 0.202 0.093 46% 0.236 0.072 31% 0.238 0.088 37% 15.03 0.002* 4 0.211 0.154 73% 0.233 0.116 50% 0.251 0.131 52% 0.197 0.134 68% 1.41 0.704 5 0.034 0.052 151% 0.000 0.000   0.002 0.006 316% 0.002 0.004 179% 9.28 0.026** * p < 0.01 ** p < 0.05 0.0 0.1 0.2 0.3 0.4 0.5 1a 1b 2 3 4 5 Ovarian Stage Proportion of Cells Control Low Medium High Figure 4.45. Frequency histogram showing the quantitative developmental staging of ovaries for each treatment of the Non spawning Adult 14 Day Trenbolone 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 0.01 follicles for females in the Low concentration to 0.05 follicles for females in the High and Medium concentrations ( Figure 4.46). There were significant differences in the proportions of atretic follicles among treatments ( Kruskal Wallis, H = 10.32, p = 0.016, df = 2). The value for females in the Low concentration was lower than those for females in the other three treatments. However, there was no consistent pattern of significant difference associated with trenbolone dose. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 139 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 treatment atretic_ follicles Figure 4.46. Boxplot of the proportion of atretic follicles per 300 follicles by treatment for the Nonspawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the asterisk represents a probable outlier.

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 0.011 for females in the Control treatment ( Figure 4.47). There were significant differences in the proportions of corpora lutea among treatments ( Kruskal Wallis, H = 10.16, p = 0.017, df = 2). The mean value for fish from the High concentration was less than that for the Low concentration, but not than that for the Control treatment.

Mid Low High Control 0.04 0.03 0.02 0.01 0.00 treatment corpora_ lutea Figure 4.47. Boxplot of the proportion of corpora lutea per 300 follicles by treatment for the Nonspawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and asterisks represent probable outliers. DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 140 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 4.3.6 Male Gonad Histology Testes Staging by Microscopic Field   Testes from males exposed to trenbolone during the Nonspawning Adult 14 Day Trenbolone assay were examined to determine the general developmental condition. Males in all treatments had well developed testes with most showing Stage 4 and Stage 5 development ( see Methods for description of developmental stages). All of the 120 microscopic fields examined in the 10 Control treatment males showed Stage 4 ( 58 fields) or Stage 5 ( 62 fields) development. All of the 108 microscopic fields examined in the 9 Low concentration treatment males showed Stage 4 ( 74 fields) or Stage 5 ( 34 fields) development. All of the 120 microscopic fields examined in the 10 Medium concentration treatment males showed Stage 4 ( 50 fields) or Stage 5 ( 70 fields) development. All of the 120 microscopic fields examined in the 10 High concentration treatment males showed Stage 4 ( 76 fields) or Stage 5 ( 44 fields) development. Statistical analysis of the mean staging from 12 microscopic fields per fish revealed no significant differences among treatments ( Kruskal Wallis, H = 4.24, p = 0.236, 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 of the testes from fish in all treatments, except the Low concentration, ranged from Stage 2a to Stage 5 ( Figure 4.48). The developmental stage of the testes from fish in the Low concentration ranged from Stage 2b to Stage 5. Variability within treatments for each stage was very high as indicated by CVs that ranged as high as 211% ( Table 4.40). There were no significant differences among treatments in the proportion of cells in developmental Stages 2a, 2b, 3a, 3b, 4, and 5 ( Table 4.40). Therefore, there was no consistent pattern of significant difference associated with trenbolone dose.

Table 4.40. Descriptive statistics of the proportion of testes cells in each developmental stage for males from the Non spawning Adult 14 Day Trenbolone assay and results of the Kruskal Wallis Test ( df = 2) comparing treatments.

Control ( n = 10) Low ( n = 9) Medium ( n = 10) High ( n = 10) Kruskal Wallis Stage Mean Stdev CV Mean Stdev CV Mean Stdev CV Mean Stdev CV H p 1 0 0   0 0   0 0   0 0       2a 0.001 0.002 175% 0 0   0.002 0.003 194% 0.001 0.001 211% 3.44 0.329 2b 0.009 0.013 150% 0.010 0.012 121% 0.005 0.008 159% 0.007 0.008 125% 1.10 0.777 3a 0.027 0.032 119% 0.066 0.055 84% 0.046 0.076 165% 0.055 0.031 56% 5.69 0.128 3b 0.116 0.111 95% 0.149 0.082 55% 0.152 0.162 107% 0.117 0.093 79% 1.62 0.654 4 0.082 0.084 102% 0.090 0.061 67% 0.064 0.074 115% 0.087 0.066 76% 1.77 0.621 5 0.765 0.224 29% 0.685 0.166 24% 0.731 0.305 42% 0.700 0.170 24% 1.79 0.618 DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 141 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1 2a 2b 3a 3b 4 5 Testicular Stage Proportion of Cells Control Low Mid High Figure 4. 48. Frequency histogram showing the quantitative developmental staging of testes for each treatment of the Non spawning Adult 14 Day Trenbolone 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 85.3 µ m to 236.7 µ m ( Figure 4.49). Tubule diameters of males from the three test concentrations ranged from 87.2 µ m to 250.0 µ m. No significant differences in the mean tubule diameter per treatment ( Table 4.41) were detected ( Kruskal Wallis, H = 2.54, p = 0.468, 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 78 ( Table 4.41).

Table 4.41. Summary statistics and power estimates for male seminiferous tubule diameter data for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 159.3 54.8 10 13% 78 Low 9 133.3 41.8 9 Medium 10 162.2 42.4 10 High 10 160.3 39.5 10 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) 142 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 250 200 150 100 treatment diameter Figure 4.49. Boxplot of male seminiferous tubule diameter ( µ m) by treatment for the Non spawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the asterisk represents a probable outlier.

Observations   Two males in the Medium concentration treatment showed disorganized tubular components. No interstitial Sertoli cell proliferation and no Leydig cell proliferation was observed for any treatment. No testicular atrophy was recorded and no ovatestes were observed for any treatment.

4.3.7 Plasma Steroid Concentrations Estradiol   Estradiol concentrations in Control treatment females used during the Non spawning Adult 14 Day Trenbolone assay ranged from 234 pg/ mL to 3,382 pg/ mL ( Figure 4.50). Among females exposed to the three trenbolone concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 3,913 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 4.42) was detected ( Kruskal Wallis, H = 11.87, p = 0.008, df = 2). The mean estradiol concentration in females from the Medium concentration was less than that in females from the Low concentration and 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 11 ( Table 4.42). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 143 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.42. Summary statistics and power estimates for female estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 7 1,346 1,013 75% 59% 11 Low 10 1,730 1,272 74% Medium 10 378 461 122% High 9 475 430 91% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 4000 3000 2000 1000 0 treatment Estradiol Figure 4.50. Boxplot of female estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, the circle is the mean value, and the asterisks represent probable outliers.

Estradiol concentrations in Control treatment males used during the Non spawning Adult 14 Day Trenbolone assay ranged from 0 pg/ mL ( not detected) to 566 pg/ mL ( Figure 4.51). Among males exposed to the three trenbolone concentrations, estradiol concentrations ranged from 0 pg/ mL ( not detected) to 520 pg/ mL. A significant difference in the mean estradiol concentration per treatment ( Table 4.43) was detected ( Kruskal Wallis, H = 8.02, p = 0.046, df = 2). Estradiol concentrations in males from the Low concentration consistently ranked lower than those form the Medium concentration. The achieved power for this endpoint was 17%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 33 ( Table 4.43). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 144 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.43. Summary statistics and power estimates for male estradiol concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 6 209 210 101% 17% 33 Low 6 78 121 156% Medium 8 276 81 30% High 7 320 168 52% 1 Calculated from natural log transformed data; with sample size = 6. 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 600 500 400 300 200 100 0 treatment Estradiol Figure 4.51. Boxplot of male estradiol concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone 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   Testosterone concentrations in Control treatment females used during the Non spawning Adult 14 Day Trenbolone assay ranged from 790 pg/ mL to 1,145 pg/ mL ( Figure 4.52). Among females exposed to the three trenbolone concentrations, testosterone concentrations ranged from 0 pg/ mL ( not detected) to 526 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 4.44) were detected ( Kruskal Wallis, H = 9.56, p = 0.023, df = 2). The mean testosterone concentration in females from the High concentration was less than that of females from the Low and Medium concentrations and from the Control treatment. The achieved power for this endpoint was 10%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 14 ( Table 4.44). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 145 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.44. Summary statistics and power estimates for female testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 4 975 156 16% 10% 14 Low 8 217 245 113% Medium 2 138 195 141% High 4 134 156 116% 1 Calculated from natural log transformed data; with sample size = 2. 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 1000 500 0 treatment Testosterone Figure 4.52. Boxplot of female testosterone concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Testosterone concentrations in Control treatment males used during the Non spawning Adult 14 Day Trenbolone assay ranged from 1,125 pg/ mL to 6,094 pg/ mL ( Figure 4.53). Among males exposed to the three trenbolone concentrations, testosterone concentrations ranged from 226 pg/ mL to 4,796 pg/ mL. Significant differences in the mean testosterone concentration per treatment ( Table 4.45) were detected ( Kruskal Wallis, H = 11.14, p = 0.011, df = 2). The mean testosterone concentration in males from the Medium and Low concentrations were less than that in males from the Control treatment. The achieved power for this endpoint was 76%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 8 ( Table 4.45). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 146 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.45. Summary statistics and power estimates for male testosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 7 3,487 1,649 47% 76% 8 Low 8 1,018 955 94% Medium 9 1,283 1,075 84% High 9 1,818 1,514 83% 1 Calculated from natural log transformed data; with sample size = 7. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 6000 5000 4000 3000 2000 1000 0 treatment Testosterone Figure 4.53. Boxplot of male testosterone concentration ( pg/ mL) by treatment for the Non spawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

11 ketotestosterone   11 ketotesosterone was not detected in females from the Control treatment ( 3 individuals), the Low concentration ( 3 individuals), the Medium concentration ( 2 individuals), or the High concentration ( 6 individuals).

11 ketotesosterone concentrations in Control treatment males used during the Non spawning Adult 14 Day Trenbolone assay ranged from 8,913 pg/ mL to 64,045 pg/ mL ( Figure 4.54). Among males exposed to the three trenbolone concentrations, 11 ketotesosterone concentrations ranged from 0 pg/ mL ( not detected) to 57,115 pg/ mL. A significant difference in the mean 11 ketotesosterone concentration per treatment ( Table 4.46) was detected ( Kruskal Wallis, H = 8.04, p = 0.045, df = 2). The mean 11 ketotesosterone concentration in males from the Low concentration was less than that in males from the Control treatment. The achieved power for this endpoint was 53%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 14 ( Table 4.46). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 147 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.46. Summary statistics and power estimates for male 11 ketotesosterone concentrations ( pg/ mL) for the Non spawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 8 28,598 17,106 60% 53% 14 Low 9 7,926 11,034 139% Medium 10 13,943 16,314 117% High 10 16,500 22,096 134% 1 Calculated from natural log transformed data; with sample size = 8. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 70000 60000 50000 40000 30000 20000 10000 0 treatment 11 keto Figure 4.54. Boxplot of male 11 ketotesosterone concentration ( pg/ mL) by treatment for the Nonspawning Adult 14 Day Trenbolone 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 represent probably outliers.

4.3.8 Body Weight and Length The body weight of females used in the Non spawning Adult 14 Day Trenbolone assay ranged from 1.4 g to 3.3 g ( Figure 4.55). There were no significant differences in mean body weight ( natural log transformed) among treatments ( Kruskal Wallis, H = 4.05, p = 0.256, df = 3). The achieved power for this endpoint was 35%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 26 ( Table 4.47).

The body ( fork) length of females used in the Non spawning Adult 14 Day Trenbolone assay ranged from 47 mm to 63 mm ( Figure 4.56). There were no significant differences in mean body length ( natural log transformed) among treatments ( Kruskal Wallis, H = 5.90, p = 0.117, df = 3). The achieved power for this endpoint was 33%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 28 ( Table 4.48). DRAFT EPA WA 3 8 ( Report of WA 2 18 Study) 148 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Table 4.47. Summary statistics and power estimates for female body weight ( g) data for the Nonspawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 1.9 0.3 16% 35% 26 Low 10 2.1 0.4 19% Medium 10 2.3 0.5 22% High 10 2.1 0.5 23% 1 Calculated from natural log transformed data; with sample size = 10. 2 To detect a significant difference from Control treatment based on maximum achieved absolute difference; calculated on natural log transformed data.

Mid Low High Control 3.5 2.5 1.5 treatment fish_ wgt_ whole ( y )

Figure 4.55. Boxplot of female body weight ( g) by treatment for the Non spawning Adult 14 Day Trenbolone assay. The box represents the interquartile range, whiskers represent the data range, the horizontal line is the median value, and the circle is the mean value.

Table 4.48. Summary statistics and power estimates for female body length ( mm) data for the Nonspawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 51.8 3.9 7% 33% 28 Low 10 54.0 3.1 6% Medium 10 55.2 3.3 6% High 10 52.8 4.9 9% 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) 149 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 65 60 55 50 treatment fish_ length_ fork Figure 4.56. Boxplot of female body length ( mm) by treatment for the Non spawning Adult 14 Day Trenbolone 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 EPA 14 Day Trenbolone assay ranged from 2.4 g to 6.5 g ( Figure 4.57). There were no significant differences in mean body weight among treatments ( Kruskal Wallis, H = 5.34, p = 0.148, df = 3). The achieved power for this endpoint was 48%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 19 ( Table 4.49).

The body ( fork) length of males used in the Non spawning Adult 14 Day Trenbolone assay ranged from 51 mm to 79 mm ( Figure 4.58). There were no significant differences in mean body length ( natural log transformed) among treatments ( Kruskal Wallis, H = 6.72, p = 0.081, df = 3). The achieved power for this endpoint was 48%, and the sample size required to detect a significant difference from the Control treatment at 80% power was 19 ( Table 4.50).

Table 4.49. Summary statistics and power estimates for male body weight ( g) data for the Nonspawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 3.7 0.8 21% 48% 19 Low 10 4.0 0.8 21% Medium 10 4.7 1.1 23% High 10 4.4 0.9 21% 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) 150 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 6.5 5.5 4.5 3.5 2.5 treatment fish_ wgt_ whole ( y )

Figure 4.57. Boxplot of male body weight ( g) by treatment for the Non spawning Adult 14 Day Trenbolone 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 4.50. Summary statistics and power estimates for male body length ( mm) data for the Nonspawning Adult 14 Day Trenbolone assay.

Level N Mean Stdev CV Achieved Power 1 Sample Size Required 2 Control 10 59.9 5.4 9% 48% 19 Low 10 65.4 4.8 7% Medium 10 65.8 7.1 11% High 10 64.8 3.2 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) 151 Preliminary Data: May be subject to change following QA and Management review July 30, 2003 Mid Low High Control 80 70 60 50 treatment fish_ length_ fork ( y )

Figure 4.58. Boxplot of male body length ( mm) by treatment for the Non spawning Adult 14 Day Trenbolone 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.