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COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE-DISRUPTING CHEMICALS

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.

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COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE-DISRUPTING CHEMICALS

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  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. 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 2

  3. Background • 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) 3

  4. Background cont. • Assay system evaluated/validated using materials reflective of MOA of concern: • Estrogens (methoxychlor, Ankley et al. 2001; E2, Harries et al. 20001) •  Androgens (methyltestosterone, Ankley et al. 2001; 20032) •  Anti-androgens (vinclozolin, Makyenen et al. 20003; flutamide, Jensen et al.4) •  Aromatase inhibitor (fadrozole, Ankley et al. 20025) • 1Environ. Sci. Technol. 34:3003 (conducted with paired-spawners) • 2Environ. Toxicol. Chem. 22:1350 • 3Aquat. Toxicol. 48:461 • 4Aquat. Toxicol. Submitted • 5Toxicol. Sci. 67:121 4

  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) 5

  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 6

  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) 7

  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. 8

  9. Methods

  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) 10

  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. 11

  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 P. promelas housed in 55 –gal tanks • 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. 12

  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 Excising gonads • Gross morphological conditions, GSI determinations and histological analyses were performed • Plasma samples analyzed for sex steroids and VTG. 13

  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 14

  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 • Appearance, behavior, and fecundity assessed daily • Gross morphological conditions, GSI and histological analyses performed • Plasma analyzed for sex steroids and VTG. 15

  16. Histology

  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 17

  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 18

  19. Methoxychor

  20. Methoxychlor Cumulative Fecundity 20

  21. Methoxychlor Cumulative Fecundity 21

  22. Methoxychlor Male Vitellogenin(mg/mL) 22

  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. 23

  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. 24

  25. Effects of Methoxychlor on Fathead Minnow Reproduction EPA–MED 8000 7000 6000 Methoxychlor (µg/L) 5000 * Control Eggs Spawned 4000 0.5 Cumulative Number of 5 3000 2000 1000 0 -15 -10 -5 0 5 10 15 20 25 Time (d) 25

  26. Methoxychlor Effects on Male VTG EPA-MED * 6 5 4 .0050 Vitellogenin (mg/ml) .0025 .0000 0.5 5 Control Methoxychlor (µg/L) 26

  27. Trenbolone

  28. Trenbolone Cumulative Fecundity 28

  29. Trenbolone Cumulative Fecundity 29

  30. Trenbolone Female Vitellogenin(mg/mL) 30

  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. 31

  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. 32

  33. Effects of Trenbolone on Fathead Minnow Reproduction EPA-MED 4000 Trenbolone (µg/L) Control 3200 0.005 0.05 0.5 5.0 50 2400 Cumulative Number of Eggs Spawned 1600 * 800 * * * 0 0 2 4 6 8 10 12 14 16 18 20 Exposure (d) 33

  34. Effects of Trenbolone on Female Tubercles EPA-MED 25 * * 20 * 15 Tubercle Score 10 5 * 0 Control 0.005 0.05 0.5 5.0 50 Trenbolone (µg/l) 34

  35. Trenbolone Effects on Females EPA-MED 30 a a 24 18 Vitellogenin (mg/ml) b * 12 b,c 6 * c c * * 0 Control 0.005 0.05 0.5 50 5.0 Trenbolone (µg/l) 35

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

  37. Flutamide

  38. Flutamide Cumulative Fecundity 38

  39. Flutamide Cumulative Fecundity 39

  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. 40

  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. 41

  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. 42

  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. 43

  44. Effects of Flutamide on Fathead Minnow Reproduction EPA-MED 6000 Flutamide (µg/L) 5000 Control 62.8 4000 649 Cumulative Number of Eggs Spawned 3000 2000 * 1000 0 0 2 4 6 8 10 12 14 16 18 20 22 Exposure (d) 44

  45. Flutamide Cumulative Fecundity 45

  46. Fadrozole

  47. Fadrozole Cumulative Fecundity 47

  48. Fadrozole Cumulative Fecundity 48

  49. Fadrozole Female Estradiol(pg/mL) 49

  50. Fadrozole Female Vitellogenin(mg/mL) 50

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