# Presentation: 081203

## 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

## 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

## 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)

## 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)
- 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

## 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)

## 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

## 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)

## 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.

## Methods

## 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) | 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 |

## 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.

## 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

- 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

## 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

## 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

## 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.

## Histology

## 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

**Notes:**
Histological stages of fathead minnow ovarian development.
Stage Characteristics
1. Primary Growth: Oogonia and primary oocytes
	1a. Oocytes in nests; small cytoplasmic volume
	1b. Oocytes larger, out of nests, surrounded by follicle cells; many 	pleiomorphic nucleoli bordering the nuclear envelope
2. Cortical Alveolus: Appearance of cortical alveoli and possibly small lipid droplets
3. Early Vitellogenic: Appearance of yolk bodies; initially few and small, ultimately many and variably-sized; centrally located germinal vesicle is round to oval with several peripheral nucleoli
4. Late Vitellogenic: Germinal vesicle loses nucleoli, moves towards the periphery and breaks down; yolk bodies frequently fill the entire center of the oocyte and a germinal vesicle may not be evident 
5. Mature/Spawning Oocytes: Germinal vesicle breakdown complete; yolk bodies fuse and may become larger than cortical alveoli

## 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

**Notes:**
Histological stages of fathead minnow testicular development.
Stage Characteristics
1. Resting Germ Cells:  No development
2. Spermatogonia:
	 2a. Primary Spermatogonia: Large cells near edges of tubule; have a lightly staining 	nucleus with a prominent nucleolus
	2b. Secondary Spermatogonia: Clusters of medium-sized cells with a round, lightly 	basophilic nucleus; cluster or cyst is the result of several mitotic divisions of primary 	spermatocyte
3. Spermatocytes: 
	3a. Primary Spermatocytes: Smaller cells with smaller, more basophilic nuclei than 	Spermatogonia; will undergo meiosis I to produce secondary spermatocytes
	3b. Secondary Spermatocytes: Small cells with smaller, more basophilic nuclei than 	primary spermatocytes; will undergo meiosis II to produce spermatids
4. Spermatids and some spermatozoa in lumen of seminiferous tubules; small tubule lumen:
	Spermatids have a small, intensely basophilic nucleus; mature into spermatozoa
5. Abundant sperm in an expanded lumen:

## Methoxychor

## Methoxychlor Cumulative Fecundity

**Notes:**
Note: There were  no significant differences in mean total number of eggs at Day 0 among treatments (i.e., at end of pre-exposure assay) (1-way ANOVA, F = 2.39, p = 0.124, df = 3, 11) .

Statistical analysis of square-root transformed egg counts showed significant among-treatment differences (1-way ANOVA, F = 7.29, p = 0.013, df = 2, 9) in mean total numbers of eggs produced.  Tukey’s pairwise comparison identified significant differences in mean egg production between the high-methoxychlor concentration and the control treatment, but NOT between the high and low concentrations or between the low concentration and the control treatment. 

## Methoxychlor Cumulative Fecundity

**Notes:**
No significant differences in the mean number of eggs (square-root transformed ) produced per treatment were detected (1-way ANOVA, F = 3.32, p = 0.083, df = 2, 9). 

Probably related to high within-treatment variability as shown by CVs ranging up to 109% (high treatment).

## Methoxychlor Male Vitellogenin(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 |  |

## 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.

## 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.

**Notes:**
Non-spawning Adult: Statistical analysis of the mean testicular staging from 12 microscopic fields per fish revealed that there was a significant difference among treatments (Kruskal-Wallis, H = 10.54, p = 0.014, df = 3).  The mean testes stage for males in the Control treatment (4.81, sd = 0.21) was significantly greater than that in the medium-concentration (4.40, sd = 0.37) and Low-concentration treatment (4.49, sd = 0.18), but not greater than that in the High-concentration treatment. 

That is, testes were “less” developed in low and mid dose males than in control males.

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

- -15

- -10

- -5

- 0

- 5

- 10

- 15

- 20

- 25

- Time (d)

- 0

- 1000

- 2000

- 3000

- 4000

- 5000

- 6000

- 7000

- 8000

- Eggs Spawned

- Cumulative Number of

- Methoxychlor (μg/L)

- Control

- 0.5

- 5

*****

**Notes:**
Dose response effect on fecundity

## Methoxychlor Effects on Male VTG EPA-MED

- Methoxychlor (μg/L)

- Control

- 0.5

- 5

- .0000

- .0025

- .0050

- 4

- 5

- 6

- Vitellogenin (mg/ml)

- *

**Notes:**
Note male vitellogenin induction

## Trenbolone

## Trenbolone Cumulative Fecundity

**Notes:**
Note:  At Day 0, 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).
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). 
Exposure Assay:
Statistical analysis of square-root transformed egg counts showed significant among-treatment differences (1-way ANOVA, F = 23.14, p < 0.001, df = 2, 10) in mean cumulative numbers of eggs produced (Table E21T Fec-1).  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.  
That is: High total eggs < Control and Low total eggs.

## Trenbolone Cumulative Fecundity

**Notes:**
Statistical analysis of square-root transformed egg counts showed significant among-treatment differences in the mean cumulative number of eggs produced per treatment (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. 

That is, High concentration total eggs < control treatment and low concentration total eggs.

The Low concentration and the Control treatment fecundity values were NOT statistically different. 

## Trenbolone Female Vitellogenin(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 > High
Low > High |  |  | Control > High
Low > High |  |  | Control > High; Medium
Low > Medium * |  |  |

**Notes:**
Non-Spawning Adult:
Significance test based on ranks. Low concentration values consistently ranked higher than Medium concentration ranks, but NOT consistently higher than High. Similarly, Control ranks did not differ from Low concentration ranks, thus shown as NS even though mean values appear quite different. See table below for median values and mean ranks.

## 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.

**Notes:**
Doses shown are means for individual assays.

Note: use of “perhaps” signifies that there was no statistically significant difference (probably because of high variability), but means were pretty different. Equals “~” used in report discussion.

## 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.

**Notes:**
EPA 21: The mean proportion of corpora lutea in the High concentration was less than those in the Control treatment and the Low concentration. 

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

- 0

- 2

- 4

- 6

- 8

- 10

- 12

- 14

- 16

- 18

- 20

- 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)

*****

*****

*****

*****

**Notes:**
Dose response reduction in fecundity

## *

- Control

- 0.005

- 0.05

- 0.5

- 5.0

- 50

- 0

- 5

- 10

- 15

- 20

- 25

- Tubercle Score

- Trenbolone (μg/l)

**Effects of Trenbolone on Female Tubercles EPA-MED**

**Notes:**
Scheme for quantifying tubercles

Ankley’ note above...we did not do this 

## *

- Control

- 0.005

- 0.05

- 0.5

- 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**

**Notes:**
Note reduction in all parameters (consistent and explainable)

## Masculinization by Trenbolone EPA-MED

**Masculinization by Trenbolone EPA-MED**

- Control male and female

- Female  0.05 μg/L

**Notes:**
Female tubercle induction 0.05  (less sensitive than other endpoints)

## Flutamide

## Flutamide Cumulative Fecundity

**Notes:**
Pre-exposure Period:
No significant differences in the mean 14-day egg production among the groups of replicates eventually used in the flutamide exposure assay were detected (Kruskal-Wallis, H = 0.15, p = 0.926, df = 2). 

Exposure Assay:  Statistical analysis of square-root transformed egg counts showed significant among-treatment differences (Kruskal-Wallis, H = 7.38, p = 0.025, df = 2) in mean total numbers of eggs produced (Table E21FL Fec-1).  

The total fecundity of females in the High-concentration treatment was significantly less than it was for females in the Low concentration or the Control treatment. 

That is: High total eggs < Control and Low total eggs.

## Flutamide Cumulative Fecundity

**Notes:**
Exposure Assay:
A significant difference in the mean number of eggs (square-root transformed ) produced per treatment (Table E14FL Fec-1) was detected (Kruskal-Wallis, H = 8.77, p = 0.012, df = 2).  
The total fecundity of females in the High-concentration treatment was significantly less than it was for females in the Low concentration or the Control treatment. 

That is, High concentration total eggs < control treatment and low concentration total eggs.
The Low concentration and the Control treatment fecundity values were NOT statistically different. 

## 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.

**Notes:**
Doses shown are means for individual assays.

## 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.

**Notes:**
Fish ID	Concentration	Leydig Cell Proliferation	Observations	
218604	Low	None	Premature release of secondary spermatocytes 			(stage 3b) in lumina. High concentration of 			spermatogonia in mature tubules.	
218620	Medium	None	Premature release of secondary spermatocytes 			(stage 3b) in lumina. Some released stage 3b 			cells necrotic. Lumina generally occluded.	
218623	Medium	None	Multifocal necrotic primary and secondary 			spermatocytes (3a and 3b) in crypts of tubules	
218624	Medium	None	Premature release of secondary spermatocytes 			(stage 3b) in lumina. Multifocal necrosis of 			secondary spermatocytes (stage 3b).	
218625	Medium	Multifocal	Basophilic nodules in testicular duct.	
218626	Medium	None	Focal area proliferation and sequestration of 			sperm cells.	
218627	Medium	None	Nodules in spermatic ducts and some tubules.	
218628	High	Focal	Multifocal but not extensive necrosis of secondary 			spermatocytes (stage 3b).	
218632	High	Multifocal	Multifocal tubules with encysted structures that 			were possibly abnormal oocytes. Multifocal but 			sparse necrotic spermatogonia. One ova in 			section, probably an artifact.	
218635	High	Multifocal	Tubule lumina occluded. Multifocal necrosis of 			secondary spermatocytes (stage 3b).	
218636	High	None	Multifocal necrosis of secondary spermatocytes 			(stage 3b).	

## 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.

## 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.

## Effects of Flutamide on Fathead Minnow Reproduction EPA-MED

- 0

- 2

- 4

- 6

- 8

- 10

- 12

- 14

- 16

- 18

- 20

- 22

- 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**

*****

**Notes:**
Note dose response

## Flutamide Cumulative Fecundity

**Notes:**
Exposure Assay:
Significant differences in the mean numbers of eggs (square-root transformed ) produced per treatment were detected (Kruskal-Wallis, H = 9.27, p = 0.010, df = 2).  
The mean numbers of eggs produced by females in the High concentration were less than the mean numbers of eggs laid by females in the Control treatment and the Low concentration. 
That is, High concentration total eggs < control treatment and low concentration total eggs.
Egg production by females in the High concentration stopped after Day 4 of the assay. 
The Low concentration and the Control treatment fecundity values were NOT statistically different. 

## Fadrozole

## Fadrozole Cumulative Fecundity

**Notes:**
Pre-exposure Period:
There were no significant differences in the numbers of eggs laid among the tanks that would eventually be used for the three treatments during the 21-Day assay. 

Exposure Assay:  Statistical analysis of square-root transformed egg counts showed significant among-treatment differences (Kruskal-Wallis, H = 8.00, p = 0.018, df = 2) in mean total numbers of eggs produced.  
The mean total number of eggs produced by females in the High concentration was significantly less than the mean total numbers of eggs produced by females in the Control treatment and the Low concentration. 

That is: High total eggs < Control and Low total eggs.

## Fadrozole Cumulative Fecundity

**Notes:**
Exposure Assay:
Significant differences in the mean numbers of eggs (square-root transformed ) produced per treatment were detected (Kruskal-Wallis, H = 9.27, p = 0.010, df = 2).  
The mean numbers of eggs produced by females in the High concentration were less than the mean numbers of eggs laid by females in the Control treatment and the Low concentration. 
That is, High concentration total eggs < control treatment and low concentration total eggs.
Egg production by females in the High concentration stopped after Day 4 of the assay. 
The Low concentration and the Control treatment fecundity values were NOT statistically different. 

## Fadrozole Female Estradiol (pg/mL)

|  | 14-Day |  |  | 21-Day |  |  | Non-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 > High
Low > High |  |  | Control > Low
 |  |  | Control > High *
 |  |  |

**Notes:**
Non-spawning Adult:

As for Trenbolone Female VTG, the statistical test is based on ranks. The only significant difference here is between the Control ranks and the High concentration ranks even though the means for other treatments seem quite different. Variability was very high for this test-endpoint with CVs ranging from 72% to 143%.

## Fadrozole Female Vitellogenin(mg/mL)

|  | 14-Day |  |  | 21-Day |  |  | Non-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; Low
Low > High |  |  | Control > High; Low
Low > High |  |  | Control > High; Medium
Low > High; Medium |  |  |

## 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.

**Notes:**
Doses shown are means for individual assays.

Note: again, “perhaps” means no statistically significant difference, but means well separated. High variability.

## 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.

**Notes:**
Fish ID	Concentration	Leydig Cell Proliferation	Observations	
218604	Low	None	Premature release of secondary spermatocytes 			(stage 3b) in lumina. High concentration of 			spermatogonia in mature tubules.	
218620	Medium	None	Premature release of secondary spermatocytes 			(stage 3b) in lumina. Some released stage 3b 			cells necrotic. Lumina generally occluded.	
218623	Medium	None	Multifocal necrotic primary and secondary 			spermatocytes (3a and 3b) in crypts of tubules	
218624	Medium	None	Premature release of secondary spermatocytes 			(stage 3b) in lumina. Multifocal necrosis of 			secondary spermatocytes (stage 3b).	
218625	Medium	Multifocal	Basophilic nodules in testicular duct.	
218626	Medium	None	Focal area proliferation and sequestration of 			sperm cells.	
218627	Medium	None	Nodules in spermatic ducts and some tubules.	
218628	High	Focal	Multifocal but not extensive necrosis of secondary 			spermatocytes (stage 3b).	
218632	High	Multifocal	Multifocal tubules with encysted structures that 			were possibly abnormal oocytes. Multifocal but 			sparse necrotic spermatogonia. One ova in 			section, probably an artifact.	
218635	High	Multifocal	Tubule lumina occluded. Multifocal necrosis of 			secondary spermatocytes (stage 3b).	
218636	High	None	Multifocal necrosis of secondary spermatocytes 			(stage 3b).	

## *

- -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

- (Thousands)

- Cumulative Number of Eggs Spawned

- Control

- 2

- 10

- 50

- Fadrozole (ug/L)

*****

*****

*****

**Notes:**
Note nearly immediate effects on fecundity 

## Fadrozole effects on female Fathead Minnow EPA-MED

**Fadrozole effects on female Fathead Minnow EPA-MED**

- Fadrozole (ug/l)

**Notes:**
Note decrease in E2 and VTG

## Spawning Tile and Egg Dish Comparison

## Proportional Difference of Eggs on Tiles Versus Eggs 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 < Low
High < Control | High < Control
 |

**Notes:**
Because of the variability in the total number of eggs laid per treatment, a direct comparison of the absolute numbers of eggs on tiles versus dishes is not possible.  Therefore, the proportional difference in the number of eggs on dishes versus those on tiles was calculated as
1[# eggs on dishes ÷ # eggs on tiles] 

Note that there seems to be a trend for decreasing eggs on tiles for High concentration treatments. Differences between High concentration and Control or Low concentration are not always statistically significant because of the high variability within treatments.  
This is particularly noticeable for methoxychlor 14-Day, trenbolone 21-Day, and fadrozole 14-D.

FERTILIZATION:
No separate slide for fertilization, which was ≥98% for all tests and chemicals except methoxychlor 14-D High (82%) and flutamide 21-D High-Dish (92%). There were no significant differences in % fertilization on dishes versus tiles among any treatment in any test or chemical.

## 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

## 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.

## 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

- 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 ****Questions**

## Work on endpoint measurement techniques to standardize/reduce variability
Fecundity (capture of detached eggs?)
Histopathology (quantification?)
Steroids (RIA vs. ELISA?)
Others?

- Fecundity (capture of detached eggs?)
- Histopathology (quantification?)
- Steroids (RIA vs. ELISA?)
- Others?

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