Fish Short-Term Reproduction Assay (FSTRA)

OCSPP 890.1350

Amy Blankinship, M.S.

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency

1
Overview

Background Information on Assay

Evaluation of Assays 

• Adherence to Performance Criteria

• Use of Incorrectly Sexed Fish in FSTRA

• Observation of Overt Toxicity

• General Observations of Measured Endpoints

Conclusions

2
Purpose of Assay

FSTRA is intended to serve in a screening capacity 

to provide information on reproductive fitness and 

potential endocrine interactions

FSTRA can screen for potential interactions with: 

• estrogen agonists or antagonists;

• androgen agonists or antagonists; 

• and/or chemicals that act on steriodogenesis

or at a higher level along the HPG axis.

Includes a suite of biochemical and histological 

endpoints that reflect changes associated with 

interactions to the HPG axis

3
Study Design

Aqueous Exposure

• 14-day Pre-exposure phase

• 21-day Exposure phase

Test species 

• fathead minnow (Pimephales promelas)

≥3 test concentrations plus control(s)

2 males and 4 females/replicate (newly mature adults)

4 reps/treatment

4
Summary Data in the Data Evaluation Record (DER)

5

Parameter Values to Report (by sex, if applicable)

Survival sample size, number and % survival

Body weight and length sample size, treatment mean, std. dev.

Fecundity treatment mean:

eggs/female/reproductive day/replicate

Fertilization success % based on embryos/eggs

Nuptial tubercle score sample size, treatment median

GSI sample size, treatment mean, std. dev.

Gonadal stage sample size, treatment median

Gonadal histopathology for each diagnostic observation: 

sample size and incidence, by severity grade 0–4

Plasma VTG, T, and E2 sample size, treatment mean, st. dev.

Other secondary sex characteristics 

and other clinical signs 

sample size and incidence by type, from daily 

observations throughout the study
Guideline Performance Criteria* for the FSTRA

6

 21 FSTRA studies with unknown endocrine potential evaluated

* = referred to as “test validity criteria” in Table 1 of guideline

Criterion Reference Value(s)

1 Measured Test Concentration 

Coefficient of Variance (CV) ≤20% 

2 Control Fertility ≥95% (of eggs produced)

3 Control Fecundity ≥15 eggs/female/day/replicate or 

spawning every 4 days (per replicate)

4 Control Survival ≥90%

5 Dissolved Oxygen ≥60% saturation (at 25°C)

6 Mean Water Temperature 25 ± 1°C
Summary of Studies Achieving Each Performance Criterion

21

15

20

15

17

12

0

6

1

6

4

9

0 5 10 15 20

Mean Temperature 

Dissolved Oxygen 

Control Survival 

Control Fecundity 

Control Fertility 

Test Concentration CV

# of Studies

met

didn't meet

7
Summary of Studies Achieving Performance Criteria

4

11

2

6

1

0

5

10

15

all 5 of 6 4 of 6 3 of 6 2 of 6

# of Studies

# of Met Criteria

8

 Test Validity Criterion Number of Studies

Measured Test Concentration CV 4

Control Fecundity 3

Dissolved Oxygen 2

Control Survival 1

Control Fertility 1

Single Unmet Criteria in the 11 Studies
Measured Test Concentration

Coefficient of Variance (CV)

9

CVs based on 21-day treatment mean or timeweighted average (TWA)

12 out of 21 studies met the criterion of CVs ≤ 20%

Range of Reported CVs # of Studies

≤ 20% 12

Up to 30% 4

Up to 40% 2

Up to 50% 2

Up to 80% 1
Control Fecundity

Fecundity measured daily in each replicate during 

both pre-exposure (min 14-days) and exposure 

phase (21-days)

Pre-exposure tanks assigned to treatment groups 

using a randomized block design based on 

fecundity

Recommended pre-exposure fecundity rate for 

potential use in exposure phase: 

• 2 spawns in 7 days preceding exposure

and > 15 eggs/female/reproductive day (E/F/R)

• 20 out of 21 studies met pre-exposure rate

10
Control Fecundity

Exposure phase criterion: 

1. ≥15 eggs/female/reproductive day/replicate (E/F/D/R)

 OR

2. spawning at least every 4 days per replicate

All replicates in 15 of the 21 studies met one of the two 

criteria above

Overall mean control fecundity >15 E/F/D in 17 studies

• Range in the four other studies: 6.5–13 E/F/D

11
Control Fertility and Survival

 Fertility assessed daily in each test replicate for 21 days

 Criterion —

• ≥95% fertility of the eggs produced in the control

• 17 of the 21 studies met criterion

• Fertility range in the 4 other studies was 90 – 94%

 Survival of adult males and females assessed daily

 Criterion —

• ≥90% control survival over 21 days

• 20 of the 21 studies met criterion

• Control survival in remaining study was 83%

12
Environmental Conditions

 Dissolved Oxygen (DO)

 Criterion —

• Maintain DO ≥ 60% saturation

• 15 of the 21 studies met criterion

• Other studies at least one measurement <60%

(minimum range 45–58% saturation)

• Laboratories added aeration when DO decreased

 Water Temperature

 All 21 studies were within the mean water temperature 

criterion of 25 ± 1°C

 Dilution water used in studies deemed adequate to support 

health, growth and reproduction of fathead minnows

13
Performance Criteria Conclusions

Control performance generally within or close to 

guideline criteria

4 studies met all criteria, 11 others met all but 

one (out of 21 studies) 

Laboratories responsive to decreases in DO 

and generally took measures to reduce 

variation in chemical exposure

14
Incorrectly Sexed Fish in FSTRA

15

 Fish distributed into tanks by 2 males and 4 females

 Guideline recognizes potential for misidentifying sex of fish and 

recommends adding additional tanks during pre-exposure

 30% of studies had at least one replicate with an incorrectly 

sexed fish (male mistaken for female)

3

1 1 1

1 Rep 2 Reps 3 Reps 5 Reps

0

1

2

3

# of Reps w/a single incorrectly sexed fish

# of Studies

 Fecundity adjusted to account for additional male

 For gender-specific endpoints (e.g., GSI), some labs excluded fish,

 others did not
Selection of Test Concentrations in the FSTRA

16

 Guideline suggests that highest concentration tested should not 

result in increased overt toxicity compared to control

 Guideline suggestions for setting the high concentration:

• 1⁄3 96-hr LC50;

• limit of solubility; or

• maximum concentration of 100 ppm (mg/L)

Method Used to Set High Concentration Number of Studies 

(out of 21)

1⁄3 LC50 (at least in part) 12

Solubility 2

Maximum necessary concentration (100 ppm) 2

Subchronic or chronic study results 2

Not reported 3
Observation of Overt Toxicity in FSTRA

17

 13 studies reported effects on survival and/or clinical signs of toxicity (including ↓ body wt)

 4 additional studies reported decreases in body weight with no other signs of overt toxicity

7

8 8

6

7

8

Survival Clinical Signs Dec. Body Wt

# of Studies

Observed effect

12

3

2

0

4

8

12

Only High High and Mid All Conc

# of Studies

Concentration(s) where overt toxicity observed
Evaluation of Measured Endpoints in FSTRA and Overt Toxicity

8 7 6

3 3 2 3

1 1

7

10

4

1

1

2 1

2

1

4

1

1

2

3

1

1

1

3

0

4

8

12

16

# of Studies (out of 21)

only in presence of overt tox

both w and w/o presence of overt tox

in absence of any overt tox

Response 

Observed:

* = sex steroids only 

measured in 3 studies

18
General Observations on Measured 

Endpoints in FSTRA

Control male and female GSI values within 

guideline reported range

Male nuptial tubercles present on control male 

fish and not on control female fish 

For VTG, potential outliers observed in several 

studies, primarily for male VTG

Sex steroids only measured in 3 of the 21 

studies

19
Male VTG Examples

20

11,700

Chemical L Chemical A

Potential 

outliers

Statistically 

significant p≤0.05
FSTRA Conclusions

 Generally, studies being conducted as expected in regards 

to control performance

• Majority of evaluated studies met all or most of the test performance criteria

(15 out of 21 studies)

 Prevalence of overt toxicity may warrant additional/revisiting 

guidance 

• Overt toxicity reported in majority of studies (17 out of 21), generally in highest 

treatment group which can allow for evaluation of potential endocrine interaction 

in lower treatments

• However, given the dose spacing, having three test concentrations 

available for evaluation of potential endocrine interaction would be optimal

 Recommend excluding incorrectly sexed fish from data 

analysis

 Although optional, the Agency emphasizes the importance in 

measuring sex steroids when sample is available

21