1. COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE-DISRUPTING CHEMICALS Endocrine Disruptor Methods Validation Subcommittee August 2003 Presented by: Dr. Irv Schultz and Michael L. Blanton

2. 2 COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE-DISRUPTING CHEMICALS WORK PERFORMED BY On behalf of the United States Environmental Protection Agency EPA CONTRACT NUMBER 68-W-01-023

3. 3 BackgroundBackground EDSTAC (1998) recommends inclusion of fish (fathead minnow) recrudescence/reproduction assay as one of five Tier 1 screening tests for EDCs that could affect the HPG axis Evaluations conducted by EPA scientists at Duluth Mid- Continent Ecology Division (MED; 1998-1999) suggest that recrudescence assay would be problematic for routine testing due to logistic constraints Alternative short-term reproduction assay with fathead minnow described by Ankley et al. (2001; Environ. Toxicol. Chem. 20:1276) Detailed standard guaidance published by USEPA (2002; A Short-Term Test Method for Assessing the Reproductive Toxicity of Endocrine-Disrupting Chemicals using the Fathead Minnow (Pimephales promelas), EPA/600/R- 01/067)

4. 4 Background cont. Assay system evaluated/validated using materials reflective of MOA of concern: Estrogens (methoxychlor, Ankley et al. 2001; E2, Harries et al. 2000 1 ) Androgens (methyltestosterone, Ankley et al. 2001; 2003 2 ) Anti-androgens (vinclozolin, Makyenen et al. 2000 3 ; flutamide, Jensen et al. 4 ) Aromatase inhibitor (fadrozole, Ankley et al. 2002 5 ) 1 Environ. Sci. Technol. 34:3003 (conducted with paired- spawners) 2 Environ. Toxicol. Chem. 22:1350 3 Aquat. Toxicol. 48:461 4 Aquat. Toxicol. Submitted 5 Toxicol. Sci. 67:121

5. 5 Background cont. Organization for Economic Cooperation and Development (OECD) forms EDTA (Endocrine Disruptor Testing Advisory) oversight group with goal of international harmonization of EDC testing (1998) Validation Management-Ecology (VMG-eco) assembled as working group under EDTA (2001) to focus on EDC testing with non-mammalian vertebrates (and invertebrates) Fish Drafting Group (FDG) one of several formed to provide technical input to VMG-eco (2001) International ring testing of draft fish protocols initiated (2003)

6. 6 Background cont. Draft protocols provided for consideration to FDG for fish EDC testing: - Short-term (14 d) juvenile vitellogenin induction assay (fathead minnow) * - Short-term (21-d) reproduction assays with fathead minnow and medaka as described by Ankley et al. (2001) - Zebrafish (40-60 d) early developmental assay - Non-spawning (21-d ** ) assay with fathead minnow and medaka conducted under conditions similar to spawning assay * Emphasis decreased due to limitations in MOA detected (estrogens) ** Initially proposed for 14-d as opposed to 21-d

7. 7 Purpose of this Study Evaluate short-term screening assays designed to detect substances that interfere with the estrogen and androgen systems of fish evaluate the transferability and sensitivity of short-term reproduction assays with the fathead minnow to identify specific modes of action of endocrine disruptors using four model compounds conduct a side-by-side comparison of the 21-day fathead minnow short-term reproduction assay (EPA 2001) with two separate 14-day assays: one a shortened version of the 21- day assay with less intensive monitoring of reproductive performance and the other an assay using non-spawning fathead minnows (OECD Draft 31 December 2001)

8. 8 Model Chemicals used and Modes of Action Methoxychlor is considered a weak estrogen that is biotransformed into an estrogen like metabolite. Trenbolone is an anabolic steroid that mimics 11-KT and testosterone. This is expected to cause masculinization of females and perhaps enhance growth. Flutamide is an established mammalian anti-androgen. If this effect occurs in fish, then the normal effectiveness of testosterone and 11-KT would be reduced. Fadrozole inhibits aromatase, which is the key step in E2 synthesis.

9. MethodsMethods

10. 10 Experimental Test Concentrations and Chemical Analyses Methoxychlor: analyzed by gas chromatograph with an electron capture detector (GC-ECD) Trenbolone :control and low samples analyzed by GC with mass selective detection (MSD); mid- and high samples analyzed by high- performance liquid chromatograph (HPLC) analysis with a fluorescence detector Flutamide :analyzed by high performance liquid chromatography (HPLC) with a UV/VIS detector at the 220-nm wavelength Fadrozole : analyzed by HPLC with a UV/VIS detector Methoxychlor & flutamide prepared using a saturator column (Kahl et al. 1999) Chemical (ug/L) LowMid*High Methoxychlor1.02.55.0 Trenbolone0.10.51.0 Flutamide6.0350650 Fadrozole52550

11. 11 Proportional Diluter Continuous flow proportional diluter used for chemical delivery Adjusted to deliver three concentrations (including control) with four replicates per concentration for the EPA assays. Second diluter modified for the nonspawning assay to deliver four concentrations (including control) with two replicates per concentration. Chemical stock solution metered into the mixing cell of the diluter using a fluid metering pump. Diluter set to add chemical-laden water to the test chamber every 12 min, equal to six volume exchanges of water per day.

12. 12 Animal Husbandry 4-month-old P. promelas were obtained from EC&T for use in the methoxychlor experiments Based on methoxychlor results it was decided that younger P. promelas should be purchased and a in house culture established at Battelle 30- to 60-day old P. promelas were obtained from ABC Laboratories for use in the trenbolone, flutamide, and fadrozole experiments n Water conditions were maintained at 24  C to 26  C. A flow-through system provided adequate volume replacement while maintaining required constant temperature. Gentle aeration was provided to the tanks. P. promelas housed in 55 –gal tanks

13. 13 Summary of the 14-day Nonspawning Assay Protocol : OECD draft proposal–31 December 2001 (OECD 2001)  Healthy, sexually dimorphic nonspawning adults (males and females contained in separate chambers  Two replicate tanks (one for each gender) per treatment with four treatments: a dilution water control, and low, medium, and high concentrations  Gross morphological conditions, GSI determinations and histological analyses were performed  Plasma samples analyzed for sex steroids and VTG. Excising gonads

14. 14 Summary of the 14-day EPA Assay Pre-exposure (7days) four females/two males No quantitative measures of fecundity Larval hatching conducted once during pre- exposure and once during chemical exposure Successful breeding pairs transferred to the chemical exposure Four replicate containers, a dilution-water control, and a low and high concentration Behavior, fecundity and routine water chemistry measurements assessed Gross morphological conditions, GSI and histological analyses were performed Plasma samples analyzed for sex steroids and VTG. Terracotta tiles and screen used for egg collection Eggs on tile

15. 15 Summary of 21-day EPA Assay 21-day assay paralleled 14-day assay with some exceptions: Larval hatching once during pre- exposure phase and three times during chemical exposure (Days 7, 14 and 21) Pre-exposure conducted for 14-days; quantitative counts of fecundity Successful breeding pairs used for chemical exposure Test conducted using same stock solution, proportional diluter, and water table as the 14-day EPA assay a) View of pre-exposure tanks b) fecundity counts n Appearance, behavior, and fecundity assessed daily n Gross morphological conditions, GSI and histological analyses performed n Plasma analyzed for sex steroids and VTG.

16. HistologyHistology

17. 17 Female Histology Methods General Ovarian Staging General development scored from 1 to 5 (18 sections) Quantitative Staging Oogonia and oocytes were typed; 100 cells from each of three sections were rated according to developmental stage Atretic Follicles Proportion of atretic follicles/100 cells was determined Corpora Lutea Proportion of corpora lutea/100 cells was determined

18. 18 Male Histology Methods General Testes Staging General development scored from 1 to 5 (12 sections) Quantitative Testes Staging spermatic cells were typed; 100 cells were from each of the three slides were rated according to developmental stage. Diameter of the seminiferous tubule was measured Other changes were noted, including changes to the interstitial tissues proliferation of Sertoli or Leydig cells premature shedding of spermatocytes presence of any ovatestes or patterns of testicular atrophy foci of necrotic spermatocytes

19. Methoxychor

20. 20 Methoxychlor Cumulative Fecundity

21. 21 Methoxychlor Cumulative Fecundity

22. 22 Methoxychlor Male Vitellogenin (mg/mL) 14-Day21-DayNon-Spawning Adult nMeanSDnMeanSDnMeanSD Control 7 <0.001 7 10 <0.0010.001 Low 7 <0.001 7 0.001 10 0.0020.003 Medium – –– – –– 10 0.001 High 4 0.560.65 1 10.89– 9 0.160.22 Significant ? NS

23. 23 Methoxychlor Steroid Results MED Data: 5  g/L  E2 in Females; T/11KT in males. EPA 21-Day assay: no dose-related effects. EPA 14-Day assay: no dose-related effects; low dose reduced E2 in females and T in males. Non-spawning Adult assay: no dose-related effects; mid (2.0  g/L) dose increased T in females. High variability within treatments.

24. 24 Methoxychlor Histology Results EPA 21-Day assay: no noticeable effects on gonad histology. EPA 14-Day assay: no noticeable effects on gonad histology. Non-spawning Adult: Low (0.8  g/L) and mid (2.0  g/L) concentrations had noticeable effects on male gonad histology.

25. 25 Effects of Methoxychlor on Fathead Minnow Reproduction EPA–MED -15-10-50510152025 Time (d) 0 1000 2000 3000 4000 5000 6000 7000 8000 Eggs Spawned Cumulative Number of Methoxychlor (µg/L) Control 0.5 5 *

26. 26 Methoxychlor Effects on Male VTG EPA-MED Methoxychlor (µg/L) Control 0.55.0000.0025.0050 4 5 6 Vitellogenin (mg/ml) *

27. Trenbolone

28. 28 Trenbolone Cumulative Fecundity

29. 29 Trenbolone Cumulative Fecundity

30. 30 Trenbolone Female Vitellogenin (mg/mL) 14-Day21-Day Non-Spawning Adult nMeanSDnMeanSDnMeanSD Control 16 2.610.59 13 1.291.16 8 2.811.02 Low 14 2.241.80 14 1.221.09 10 0.911.12 Medium ––––––9 0.130.24 High 16 0.040.08 15 0.110.27 9 0.110.15 Significant ? Control > High Low > High Control > High Low > High Control > High; Medium Low > Medium *

31. 31 Trenbolone Steroid Results MED Data: 0.5  g/L  E2, T in females EPA 21-Day assay: low dose (0.041  g/L) reduced T in females; high dose (0.60  g/L) reduced E2 in females versus low dose. EPA 14-Day assay: high dose (0.78  g/L) reduced E2 and T in females. Non-spawning Adult assay: low dose (0.071  g/L) reduced E2, T, and KT in males, perhaps reduced T in females; mid dose (0.45  g/L) reduced E2 in females and T in males and perhaps females; high dose (0.86  g/L) reduced E2 and T in females. High variability within treatments.

32. 32 Trenbolone Histology Results EPA 21-Day assay: High dose (0.60  g/L) reduced the proportion of corpora lutea and increased the proportion of atretic follicles in the ovaries. EPA 14-Day assay: High dose (0.78  g/L) reduced the proportion of atretic follicles in the ovaries compared to the control and low dose; progression to late vitellogenic stage inhibited. Testes in males from the high dose showed a later general developmental stage than those from the control and low dose. Non-spawning Adult: Low (0.07  g/L), mid (0.45  g/L), high (0.86  g/L) doses less advanced ovarian staging; low reduced atretic follicles.

33. 33 Effects of Trenbolone on Fathead Minnow Reproduction EPA-MED 02468101214161820 Exposure (d) 0 800 1600 2400 3200 4000 Cumulative Number of Eggs Spawned Control 0.005 0.05 0.5 5.0 50 Trenbolone (µg/L) * * * *

34. 34 Control0.0050.050.55.050 0 5 10 15 20 25 Tubercle Score * * * * Trenbolone (µg/l) Effects of Trenbolone on Female Tubercles EPA-MED

35. 35 Control0.0050.050.5 5.0 50 0 6 12 18 24 30 Vitellogenin (mg/ml) a a b c c b,c Trenbolone (µg/l) * * * * Trenbolone Effects on Females EPA-MED

36. 36 Masculinization by Trenbolone EPA-MED Control male and female Female 0.05 μg/L

37. FlutamideFlutamide

38. 38 Flutamide Cumulative Fecundity

39. 39 Flutamide Cumulative Fecundity

40. 40 Flutamide Steroid Results MED Data: T  in Females. EPA 21-Day assay: high dose (510  g/L) reduced E2 and increased T and KT in males; increased T in females. EPA 14-Day assay: high dose (519  g/L) reduced E2 in males versus low dose (46  g/L). Non-spawning Adult assay: no dose-related effects. High variability within treatments.

41. 41 Flutamide Histology Results EPA 21-Day assay: high dose (510  g/L) increased the proportion of atretic follicles in the ovaries compared to the control and low dose. EPA 14-Day assay: high dose (519  g/L) increased the proportion of atretic follicles and decreased the proportion of corpora lutea in the ovaries. Males from high dose showed more advanced testicular staging. Non-spawning Adult: no noticeable dose-related effects on histology; several males in all three doses had abnormal testes histology.

42. 42 Summary of EPA Flutamide Results Dose of 650  g/L slightly increased VTG in males and females, and slightly increased plasma T in females. Dose of 650  g/L increased early-stage follicles and atretic oocytes relative to controls. Dose of 650  g/L caused abnormal gonadal histology in males, with increased incidence of pychnotic and degenerating spermatocytes among healthy cysts.

43. 43 Summary of Battelle Flutamide Results There was no apparent effect on plasma VTG in males or females from any of the three designs. The high dose (510-519  g/L) decreased plasma E2 in males, increased T in females (21-Day assay), and KT in males (21-Day assay). The high dose (510-519  g/L) increased the proportion of atretic follicles in the ovaries compared to the control and low dose. Non-spawning assay showed abnormal male histology that was not related to flutamide dose.

44. 44 0246810121416182022 Exposure (d) 0 1000 2000 3000 4000 5000 6000 Cumulative Number of Eggs Spawned Control 62.8 649 Flutamide (µg/L) Effects of Flutamide on Fathead Minnow Reproduction EPA-MED *

45. 45 Flutamide Cumulative Fecundity

46. FadrozoleFadrozole

47. 47 Fadrozole Cumulative Fecundity

48. 48 Fadrozole Cumulative Fecundity

49. 49 Fadrozole Female Estradiol (pg/mL) 14-Day21-Day Non-Spawning Adult nMeanSDnMeanSDnMeanSD Control 152,0641,176162,8611,492101,5191,132 Low 161,08846871,34375610727524 Medium ––––––9320457 High 1639860––10142129 Significant ? Control > High Low > High Control > Low Control > High *

50. 50 Fadrozole Female Vitellogenin (mg/mL) 14-Day21-Day Non-Spawning Adult nMeanSDnMeanSDnMeanSD Control 16 5.361.06 16 5.952.25 10 4.932.62 Low 16 1.150.70 16 1.200.75 10 0.790.45 Medium – –– – –– 10 0.020.03 High 16 0.002 16 0.0050.011 9 0.010.02 Significant ? Control > High; Low Low > High Control > High; Low Low > High Control > High; Medium Low > High; Medium

51. 51 Fadrozole Steroid Results MED Data:  E2 in Females; T, 11KT  in Males at 50  g/L. EPA 21-Day assay: low (5.2  g/L) and high (55.7  g/L) doses increased T in females and males and increased KT in males. High dose increased KT in females. Low dose decreased E2 in females. EPA 14-Day assay: low (5.5  g/L) and high (59.9  g/L) doses increased T in females and perhaps males and increased KT in males and perhaps females. High dose decreased E2 in females. Non-spawning Adult assay: High dose (57.0  g/L) perhaps increased KT and T in males, increased KT and reduced E2 in females. High variability within treatments.

52. 52 Fadrozole Histology Results EPA 21-Day assay: high (55.7  g/L) dose females showed less advanced staging; low (5.2  g/L) and high doses had increased proportions of atretic follicles in the ovaries; high decreased proportion of corpora lutea. Males from high dose had increased tubule diameter. Some males had abnormal testes histology. EPA 14-Day assay: low (5.5  g/L) and high (59.9  g/L) doses showed less advanced staging and decreased corpora lutea; high increased proportion of atretic follicles in the ovaries. Non-spawning Adult: mid (31.7  g/L) and high (57.0  g/L) doses showed less advanced quantitative staging; high increased proportion of atretic follicles. Several males in all three doses had abnormal testes histology.

53. 53 Fadrozole Effects on Fathead Minnow Reproduction EPA-MED -20-18-16-14-12-10-8-6-4-202468101214161820 Exposure (d) 0 2 4 6 8 10 (Thousands) Cumulative Number of Eggs Spawned Control 2 10 50 Fadrozole (ug/L) * * *

54. 54 8 0 2 4 6 E2 (ng/ml) * * 0 10 20 Vtg (mg/ml) * * * Control21050 Fadrozole effects on female Fathead Minnow EPA-MED Fadrozole (ug/l)

55. Spawning Tile and Egg Dish Comparison

56. 56 Proportional Difference of Eggs on Tiles Versus Eggs on Dishes MethoxychlorTrenboloneFlutamideFadrozole EPA 14-Day Control0.860.910.900.89 Low0.920.930.910.86 High0.750.86 0.65 CVs23%–115%15%–68%9%–54%20%–95% Significant ?NS EPA 21-Day Control0.900.920.940.87 Low0.870.880.910.87 High0.720.610.690.72 CVs6%–342%21%–83%14%–60%9%–41% Significant ?NS High < Low High < Control

57. 57 Major Conclusions Good agreement between EPA and Battelle for key apical and diagnostic data generated for four different chemicals using 21-d reproduction protocol for fathead minnow Methoxychlor Fecundity reductions comparable Effects on male VTG induction qualitatively similar Trenbolone Fecundity reductions comparable Changes in secondary sex characteristics comparable Decreases in female VTG similar Flutamide Fecundity reductions comparable Subtle VTG effects and histological responses not similar Fadrozole Fecundity reductions comparable VTG and E2 reductions in females similar

58. 58 Suggested Protocol 14-day spawning protocol shows promise for use as a fish endocrine-disruptor screening protocol based on: Consistency in overall response to the 21-day protocol Insufficient need for quantitative pre-exposure fecundity measurements Reduced cost compared with the 21-day protocol Lack of response at the low-exposure level and frequent negative responses at the high-exposure level in the non-spawning protocol. This observation suggests a higher risk of false-negative results, particularly with weak-acting endocrine disruptors.

59. 59 It is recommended that additional development of the abbreviated 14-day spawning protocol be performed to evaluate how well the assay performs in comparison to the full 21-day protocol with weaker acting and/or high log-P compounds ( agonists/antagonists spanning range of MOA (including mixed MOA), as well as “negative” chemicals) A Mutli-Chemical Evaluation is currently underway with the following chemicals to help provide additional information. AtrazineDDE Bisphenol A Perchlorate Dibutyl PhthalateCadmium Additional Research, Testing and Questions

60. 60 Work on endpoint measurement techniques to standardize/reduce variability Fecundity (capture of detached eggs?) Histopathology (quantification?) Steroids (RIA vs. ELISA?) Others? Additional Research, Testing and Questions cont.