COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE-DISRUPTING CHEMICALS...
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Transcript of COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE-DISRUPTING CHEMICALS...
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.
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.
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
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)
*
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
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
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.