COMPARATIVE EVALUATION OF COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR FATHEAD MINNOW ASSAYS FOR

DETECTING ENDOCRINE-DISRUPTING DETECTING ENDOCRINE-DISRUPTING CHEMICALSCHEMICALS

COMPARATIVE EVALUATION OF COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR FATHEAD MINNOW ASSAYS FOR

DETECTING ENDOCRINE-DISRUPTING DETECTING ENDOCRINE-DISRUPTING CHEMICALSCHEMICALS

Endocrine Disruptor Methods Validation SubcommitteeAugust 2003

Presented by: Dr. Irv Schultz and Michael L. Blanton

2

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

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

WORK PERFORMED BY

On behalf of the United States Environmental Protection AgencyEPA CONTRACT NUMBER 68-W-01-023

3

BackgroundBackgroundBackgroundBackground

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 axisEvaluations 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 constraintsAlternative 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

Background cont.Background cont.Background cont.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 3 Aquat. Toxicol. 48:461 4Aquat. Toxicol. Submitted 5Toxicol. Sci. 67:121

5

Background cont.Background cont.Background cont.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

Background cont.Background cont.Background cont.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

Purpose of this StudyPurpose of this StudyPurpose of this StudyPurpose 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

Model Chemicals used and Model Chemicals used and Modes of ActionModes of Action

Model Chemicals used and Model Chemicals used and Modes of ActionModes 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.

MethodsMethodsMethodsMethods

10

Experimental Test Concentrations and Experimental Test Concentrations and Chemical AnalysesChemical Analyses

Experimental Test Concentrations and Experimental Test Concentrations and Chemical AnalysesChemical 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)

Low Mid* High

Methoxychlor 1.0 2.5 5.0

Trenbolone 0.1 0.5 1.0

Flutamide 6.0 350 650

Fadrozole 5 25 50

11

Proportional DiluterProportional DiluterProportional DiluterProportional Diluter

Continuous flow proportional diluter used for chemical deliveryAdjusted 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

Animal HusbandryAnimal HusbandryAnimal HusbandryAnimal 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 Battelle30- to 60-day old P. promelas were obtained from ABC Laboratories for use in the trenbolone, flutamide, and fadrozole experiments

Water conditions were maintained at 24C to 26C. 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

Summary of the 14-day Nonspawning AssaySummary of the 14-day Nonspawning AssaySummary of the 14-day Nonspawning AssaySummary 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

Summary of the 14-day EPA AssaySummary of the 14-day EPA AssaySummary of the 14-day EPA AssaySummary 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

Summary of 21-day EPA AssaySummary of 21-day EPA AssaySummary of 21-day EPA AssaySummary 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 exposureTest 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.

HistologyHistologyHistologyHistology

17

Female Histology MethodsFemale Histology MethodsFemale Histology MethodsFemale 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

Male Histology MethodsMale Histology MethodsMale Histology MethodsMale 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 measuredOther 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

MethoxychorMethoxychor

20

Methoxychlor Cumulative FecundityMethoxychlor Cumulative FecundityMethoxychlor Cumulative FecundityMethoxychlor Cumulative FecundityEPA 21-Day Assay

0

5,000

10,000

15,000

20,000

25,000

-15 -10 -5 0 5 10 15 20

Day

Cu

mu

lati

ve

Nu

mb

er

of

Eg

gs

Control Low High

21

Methoxychlor Cumulative FecundityMethoxychlor Cumulative FecundityMethoxychlor Cumulative FecundityMethoxychlor Cumulative Fecundity

EPA14 Day Assay

0

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Day

Cu

mu

lati

ve N

um

ber

of

Eg

gs

Control Low High

22

Methoxychlor Male VitellogeninMethoxychlor Male Vitellogenin(mg/mL)(mg/mL)

Methoxychlor Male VitellogeninMethoxychlor Male Vitellogenin(mg/mL)(mg/mL)

14-Day 21-Day Non-Spawning Adult

n Mean SD n Mean SD n Mean SD

Control 7 <0.001 <0.001 7 <0.001 <0.001 10 <0.001 0.001

Low 7 <0.001 <0.001 7 0.001 0.001 10 0.002 0.003

Medium – – – – – – 10 0.001 0.001

High 4 0.56 0.65 1 10.89 – 9 0.16 0.22

Significant ?

NS NS NS

23

Methoxychlor Steroid ResultsMethoxychlor Steroid ResultsMethoxychlor Steroid ResultsMethoxychlor 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

Methoxychlor Histology ResultsMethoxychlor Histology ResultsMethoxychlor Histology ResultsMethoxychlor 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

Effects of Methoxychlor on Fathead Effects of Methoxychlor on Fathead Minnow Reproduction EPA–MED Minnow Reproduction EPA–MED

-15 -10 -5 0 5 10 15 20 25

Time (d)

0

1000

2000

3000

4000

5000

6000

7000

8000

Egg

s S

paw

ned

Cum

ulat

ive

Num

ber

of

Methoxychlor (µg/L)

Control

0.5

5

*

26

Methoxychlor Effects on Male VTG EPA-MED

Methoxychlor (µg/L)

Control 0.5 5.0000

.0025

.00504

5

6

Vite

llog

enin

(m

g /m

l)

*

TrenboloneTrenbolone

28

Trenbolone Cumulative FecundityTrenbolone Cumulative FecundityTrenbolone Cumulative FecundityTrenbolone Cumulative Fecundity

EPA21-Day Assay

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

Cu

mu

lati

ve

Nu

mb

er

of

Eg

gs

Control Low High

29

Trenbolone Cumulative FecundityTrenbolone Cumulative FecundityTrenbolone Cumulative FecundityTrenbolone Cumulative FecundityEPA 14-Day Assay

0

2,000

4,000

6,000

8,000

10,000

12,000

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Day

Cu

mu

lati

ve N

um

ber

of

Eg

gs

Control Low High

30

Trenbolone Female VitellogeninTrenbolone Female Vitellogenin(mg/mL)(mg/mL)

Trenbolone Female VitellogeninTrenbolone Female Vitellogenin(mg/mL)(mg/mL)

14-Day 21-Day Non-Spawning Adult

n Mean SD n Mean SD n Mean SD

Control 16 2.61 0.59 13 1.29 1.16 8 2.81 1.02

Low 14 2.24 1.80 14 1.22 1.09 10 0.91 1.12

Medium – – – – – – 9 0.13 0.24

High 16 0.04 0.08 15 0.11 0.27 9 0.11 0.15

Significant ?

Control > HighLow > High

Control > HighLow > High

Control > High; MediumLow > Medium *

31

Trenbolone Steroid ResultsTrenbolone Steroid ResultsTrenbolone Steroid ResultsTrenbolone 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

Trenbolone Histology ResultsTrenbolone Histology ResultsTrenbolone Histology ResultsTrenbolone 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 (0.45g/L), high (0.86 g/L) doses less advanced ovarian staging; low reduced atretic follicles.

33

Effects of Trenbolone on Fathead Minnow Effects of Trenbolone on Fathead Minnow Reproduction EPA-MEDReproduction EPA-MED

0 2 4 6 8 10 12 14 16 18 20

Exposure (d)

0

800

1600

2400

3200

4000

Cum

ula

tive N

um

ber

of

Eg

gs

Spaw

ned

Control0.0050.050.55.050

Trenbolone (µg/L)

*

***

34

Control 0.005 0.05 0.5 5.0 500

5

10

15

20

25

Tu

berc

le S

core

*

*

* *

Trenbolone (µg/l)

Effects of Trenbolone on Female Tubercles EPA-MED

35

Control 0.005 0.05 0.5 5.0 500

6

12

18

24

30

Vite

llog

enin

(m

g/m

l)

aa

b

c c

b,c

Trenbolone (µg/l)

*

* *

*

Trenbolone Effects on Females EPA-MED

36

Masculinization by Trenbolone EPA-MED

Control male and female

Female 0.05 μg/L

FlutamideFlutamideFlutamideFlutamide

38

Flutamide Cumulative FecundityFlutamide Cumulative FecundityFlutamide Cumulative FecundityFlutamide Cumulative FecundityEPA 21-Day Assay

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

-14 -12 -10 -8 -6 -4 -2 1 3 5 7 9 11 13 15 17 19 21

Day

Cu

mu

lati

ve

Nu

mb

er

of

Eg

gs

Control Low High

39

Flutamide Cumulative FecundityFlutamide Cumulative FecundityFlutamide Cumulative FecundityFlutamide Cumulative FecundityEPA 14-Day Assay

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

20,000

0 2 4 6 8 10 12 14

Day

Cu

mu

lati

ve N

um

ber

of

Eg

gs

Control Low High

40

Flutamide Steroid ResultsFlutamide Steroid ResultsFlutamide Steroid ResultsFlutamide 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

Flutamide Histology ResultsFlutamide Histology ResultsFlutamide Histology ResultsFlutamide 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

Summary of EPA Flutamide ResultsSummary of EPA Flutamide ResultsSummary of EPA Flutamide ResultsSummary 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

Summary of Battelle Flutamide ResultsSummary of Battelle Flutamide ResultsSummary of Battelle Flutamide ResultsSummary 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

0 2 4 6 8 10 12 14 16 18 20 22

Exposure (d)

0

1000

2000

3000

4000

5000

6000C

um

ula

tive

Nu

mb

er

of

Eg

gs

Sp

aw

ne

d

Control

62.8

649

Flutamide (µg/L)

Effects of Flutamide on Fathead Minnow Reproduction EPA-MED

*

45

Flutamide Cumulative FecundityFlutamide Cumulative FecundityFlutamide Cumulative FecundityFlutamide Cumulative FecundityEPA 14-Day Assay

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

20,000

0 2 4 6 8 10 12 14

Day

Cu

mu

lati

ve

Nu

mb

er

of

Eg

gs

Control Low High

FadrozoleFadrozoleFadrozoleFadrozole

47

Fadrozole Cumulative FecundityFadrozole Cumulative FecundityFadrozole Cumulative FecundityFadrozole Cumulative FecundityEPA 21-Day Assay

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20

Day

Cu

mu

lati

ve N

um

ber

of

Eg

gs

Control Low High

48

Fadrozole Cumulative FecundityFadrozole Cumulative FecundityFadrozole Cumulative FecundityFadrozole Cumulative FecundityEPA 14-Day Assay

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

20,000

0 2 4 6 8 10 12 14

Day

Cu

mu

lati

ve

Nu

mb

er

of

Eg

gs

Control Low High

49

Fadrozole Female EstradiolFadrozole Female Estradiol (pg/mL)(pg/mL)

Fadrozole Female EstradiolFadrozole Female Estradiol (pg/mL)(pg/mL)

14-Day 21-DayNon-Spawning Adult

n Mean SD n Mean SD n Mean SD

Control 15 2,064 1,176 16 2,861 1,492 10 1,519 1,132

Low 16 1,088 468 7 1,343 756 10 727 524

Medium – – – – – – 9 320 457

High 16 39 86 0 – – 10 142 129

Significant ?

Control > HighLow > High

Control > Low Control > High *

50

Fadrozole Female VitellogeninFadrozole Female Vitellogenin(mg/mL)(mg/mL)

Fadrozole Female VitellogeninFadrozole Female Vitellogenin(mg/mL)(mg/mL)

14-Day 21-DayNon-Spawning Adult

n Mean SD n Mean SD n Mean SD

Control 16 5.36 1.06 16 5.95 2.25 10 4.93 2.62

Low 16 1.15 0.70 16 1.20 0.75 10 0.79 0.45

Medium – – – – – – 10 0.02 0.03

High 16 0.002 0.002 16 0.005 0.011 9 0.01 0.02

Significant ?

Control > High; LowLow > High

Control > High; LowLow > High

Control > High; MediumLow > High; Medium

51

Fadrozole Steroid ResultsFadrozole Steroid ResultsFadrozole Steroid ResultsFadrozole 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

Fadrozole Histology ResultsFadrozole Histology ResultsFadrozole Histology ResultsFadrozole 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

Fadrozole Effects on Fathead Minnow Reproduction EPA-MED

-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20

Exposure (d)

0

2

4

6

8

10

(Th

ou

san

ds)

Cu

mu

lativ

e N

um

be

r o

f E

gg

s S

pa

wn

ed

Control

2

10

50

Fadrozole (ug/L)

*

**

54

8

0

2

4

6

E2

(ng/

ml)

*

*

0

10

20

Vtg

(m

g/m

l)

*

**

Control 2 10 50

Fadrozole effects on female Fathead Minnow EPA-MED

Fadrozole (ug/l)

Spawning Tile and Spawning Tile and Egg Dish ComparisonEgg Dish Comparison

Spawning Tile and Spawning Tile and Egg Dish ComparisonEgg Dish Comparison

56

Proportional Difference of Eggs on Tiles Versus Proportional Difference of Eggs on Tiles Versus Eggs on DishesEggs on Dishes

Proportional Difference of Eggs on Tiles Versus Proportional Difference of Eggs on Tiles Versus Eggs on DishesEggs on Dishes

Methoxychlor Trenbolone Flutamide Fadrozole

EPA 14-Day

Control 0.86 0.91 0.90 0.89

Low 0.92 0.93 0.91 0.86

High 0.75 0.86 0.86 0.65

CVs 23%–115% 15%–68% 9%–54% 20%–95%

Significant ? NS NS NS NS

EPA 21-Day

Control 0.90 0.92 0.94 0.87

Low 0.87 0.88 0.91 0.87

High 0.72 0.61 0.69 0.72

CVs 6%–342% 21%–83% 14%–60% 9%–41%

Significant ? NS NS High < LowHigh < Control

High < Control

57

Major ConclusionsMajor ConclusionsMajor ConclusionsMajor ConclusionsGood 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

Suggested ProtocolSuggested ProtocolSuggested ProtocolSuggested 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

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.•Atrazine •DDE

•Bisphenol A •Perchlorate

•Dibutyl Phthalate •Cadmium

Additional Research, Testing and Additional Research, Testing and QuestionsQuestions

Additional Research, Testing and Additional Research, Testing and QuestionsQuestions

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 Additional Research, Testing and Questions cont.Questions cont.

Additional Research, Testing and Additional Research, Testing and Questions cont.Questions cont.