Data Evaluation Record on the Fish Short-Term Reproduction Assay with [TGAI or EP]

 EPA MRID Number [........]

Version: 22 September 2011

Page 1 f 33

Data Requirement: EPA DP Barcode [............]if applicable

OECD Data Point [............]if applicable

EPA MRID [............]if applicable

EPA Guideline 890.1350

Fish Short-Term Reproduction Assay

Test Material: [....................................................] Purity (%): [............]

Common Name [....................................................]

Chemical Name IUPAC [....................................................]

CAS Name [....................................................]

CAS No. [....................................................]

Synonyms [....................................................]

EPA PC Code [..................]

Primary Reviewer: [...........................................................] Date: [............] [EPA/OECD/PMRA]

Secondary Reviewer(s): [....................................................] Date: [............] [EPA/OECD/PMRA]

Date Evaluation Completed: [dd-mmm-yyyy]

CITATION: [Indicate: Author(s),Year, Study Title, Laboratory Name and Location, Laboratory Report Number, 

Sponsor, Full Study Date. If published, list the name of the journal, vol., pages, year.]

[Instructions, prompts, and example values for the individual(s) completing the DER are shown in the DER 

template in bracketed red text; these instructions and examples do not need to remain visible in the 

completed DER.]

Guideline recommendations are provided in italics; these recommendations should remain visible in the 

completed DER. 

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in 

preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test 

Guidelines, nor to provide any guidance on how the study should be conducted.
Data Evaluation Record on the Fish Short-Term Reproduction Assay with [TGAI or EP]

 EPA MRID Number [........]

Version: 22 September 2011

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EXECUTIVE SUMMARY

The 21-day short-term reproduction assay of [test chemical] with [common name and scientific name] was studied 

under [flow-through, specify if other] conditions. Adult fish [enter number of fish and age] were exposed to [control, 

solvent control (if applicable), and test chemical nominal/measured concentrations] of [x1, x2, x3, .... xn] mg a.i./L. The 

test system was maintained at [...] to [...]oC and a pH of [...] to [...]. 

[Modify as appropriate.] Spawning frequency and fecundity in controls, respectively, were every [...] days and [...]

eggs/female/day/replicate; fertilization success in controls was [...]%. [Test chemical] significantly [increased or 

decreased] fecundity at [list all relevant concentrations] mg a.i./L and [increased or decreased] fertilization success at 

[list all relevant concentrations] mg a.i./L. Plasma [vitellogenin, testosterone, and 17β-estradiol, if measured], 

respectively, were significantly [increased or decreased] in male fish at [list all relevant concentrations] mg a.i./L and 

were significantly [increased or decreased] in female fish at [list all relevant concentrations] mg a.i./L. Effects on 

gonadal histopathology were observed in male fish at [list all relevant concentrations] mg a.i./L and in female fish at [list 

all relevant concentrations] mg a.i./L. Histopathological effects included ......... [provide details of treatment-related 

effects, i.e., presence of testis-ova in males, increased oocyte atresia in females]. A significant [increase or decrease]

in gonado-somatic index (GSI) was observed in male fish at [list all relevant concentrations] mg a.i./L and in female fish 

at [list all relevant concentrations] mg a.i./L. Nuptial tubercle score was significantly [increased or decreased] in 

[female or males] at [list all relevant concentrations] mg a.i./L. [Test chemical] exposure was associated with an 

[increase or decrease] in [body weight and/or length] in [male or female] fish at [list all relevant concentrations] mg a.i./L. 

Other effects on secondary sex characteristics and clinical signs (i.e., behavioral and other sublethal effects) including 

[discoloration, changes in dorsal nape pad, lethargy, loss of equilibrium] were observed in [number of fish] at [each 

relevant concentration] mg a.i./L. Unless otherwise indicated, all effects are reported based on comparison to the 

negative (clean water) control.

This assay [does or does not] satisfy the Test Order requirement for a Fish Short-Term Reproduction Assay (OCSPP 

Guideline 890.1350). [If it does not satisfy the requirement, concisely list the major deficiencies.]
Data Evaluation Record on the Fish Short-Term Reproduction Assay with [TGAI or EP]

 EPA MRID Number [........]

Version: 22 September 2011

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Results Synopsis:

Test organism age at test initiation: [...] months

Mean body weight at test initiation (if measured): [...] g for males, [...] g for females

Mean length at test initiation (if measured): [...] mm for males, [...] mm for females

Test type: [flow-through, other]

Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with [test chemical].

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. 

M Male. NA Not applicable. T

Testosterone. VTG Vitellogenin.

1

 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.

2

 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be 

heavily weighted by the expert opinion of a board-certified pathologist.

Treatment

(mg a.i../L)

[measured]

Fecundity Fert. 

Success

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2

M F M F M F M F M F M F

Test conc. 1 No No Yes No Yes No Yes No No No No No No No

Test conc. 2

Test conc. 3

Test conc. n

Positive control, 

if used
Data Evaluation Record on the Fish Short-Term Reproduction Assay with [TGAI or EP]

 EPA MRID Number [........]

Version: 22 September 2011

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I. MATERIALS AND METHODS

Guideline Followed: [Specify the guideline(s) that were followed and any deviations from the guideline(s). 

State if the deviations affect the validity of the study.]

Compliance: [Indicate if signed and dated GLP, Quality Assurance and Data Confidentiality 

statements were provided.] 

A. Test Material [Complete this subsection using the information provided in the methodology 

section of the study report. Include the name of the test material and CAS 

number as cited in the study report.] 

Description: [eg. Chemical state of the test material]

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow, 

vapor pressure of test compound, expiration date.

Lot No./Batch No. : [....................]

Purity: [Indicate the % of active ingredient or purity of the test substance. If radiolabeled 

material was used, indicate the radiopurity and the location(s) of the label.]

Impurities: [Identify any impurities reported.]

Stability of Compound: [Briefly describe the stability of the test item and identify the source of information.]

Storage Conditions of 

Test Chemicals: [Indicate if the test material was frozen, refrigerated, maintained in the dark, and 

duration of storage.]
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 EPA MRID Number [........]

Version: 22 September 2011

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B. Test Organism

Table 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations

Species common name: [............................] EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: [............................]

Species strain (if stated): [............................]

Were fish obtained from a single 

laboratory stock?

[Yes/No] [Provide additional information about the 

source of animals, if available.]

EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same 

as definitive test?

[Yes/No] EPA recommends that fish be acclimated under 

water quality and illumination conditions that are 

similar to the definitive test.

Acclimation period: [...] days EPA recommends a minimum two-week 

acclimation period. Note that the acclimation 

period is different from the subsequent, in situ 

pre-exposure phase.

Details on health: [Describe the health of the stock: Were any 

behavioral abnormalities, deformities, other 

clinical signs, or mortality observed? ]

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be less 

than 5% of the culture population. If mortality 

during these 7 days is greater than 10%, EPA 

recommends that the fish be rejected. If 

mortality is between 5-10%, EPA recommends

that fish be held another 7 days. If mortalities 

greater than 5% occur during this extended 

acclimation period, EPA recommends that the 

fish not be used.

Type of food: [frozen brine shrimp, 

other]

EPA recommends that fish be fed frozen brine 

shrimp twice per day to promote active 

reproduction and maintain body condition. Source of food: [............................]

Frequency of feeding: [............................]

times/day

Details on feeding: [Describe any other relevant information 
Data Evaluation Record on the Fish Short-Term Reproduction Assay with [TGAI or EP]

 EPA MRID Number [........]

Version: 22 September 2011

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Parameter Value(s) Details or Remarks Guideline Recommendations

about the feeding regime, including the 

amount given. Provide additional 

information if feeding conditions differed 

between the acclimation, pre-exposure, and 

definitive test periods.]

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations

Age at test initiation: [...] months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months old.

Mean weight of males at test 

initiation (if determined):

[...] g [Indicate whether the mean is based on a 

subsample of male fish or based on all male 

fish selected for the test.]

EPA recommends that a subsample

of fish be weighed before the test to estimate the 

mean weight for each sex. It is recommended 

that the individual weight of each fish selected 

for the test be within ±20% of the estimated 

mean for each sex.

Range of individual weights 

(males) at test initiation (if 

determined):

[...] g : [...] g

Mean weight of females at test 

initiation (if determined):

[...] g [Indicate whether the mean is based on a 

subsample of female fish or based on all 

female fish selected for the test.]

Range of individual weights 

(females) at test initiation (if 

determined):

[...] g : [...] g

Mean length of males at test 

initiation (if determined):

[...] mm

Mean length of females at test 

initiation (if determined):

[...] mm

Duration of pre-exposure phase: [...] days EPA recommends a minimum of 14 days.

Were pre-exposure conditions 

identical to the definitive test?

[Yes/No] EPA recommends that pre-exposure conditions, 

including temperature, photoperiod, feeding, 

etc., be identical to definitive test conditions. 
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Version: 22 September 2011

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Parameter Value(s) Details or Remarks Guideline Recommendations

Number of pre-exposure tanks: [...] [Were extra replicates established?] EPA recommends that additional tanks set up at 

the beginning of pre-exposure will ensure that 

sufficient replicates with the correct sex ratio are 

available for the definitive test.

Number of males per tank: [...]

Number of females per tank: [...]

Pre-exposure fecundity: > [...] eggs/female/

reproductive day/

replicate

EPA recommends that pre-exposure fecundity 

in each replicate (tank) selected for use in the 

definitive test be at least 15 

eggs/female/reproductive day/replicate during 

the 7 days prior to the definitive test.

Number of spawns during 

pre-exposure:

> [...] times in 7 days EPA recommends that spawning occur at least 

twice in the 7 days prior to the definitive test.

Details on pre-exposure: [Were any pre-exposure replicates 

excluded due to mortality, incorrect sex 

ratio, or poor reproductive performance?]

C. Exposure System

Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.

Parameter Value(s) Details or Remarks Guideline Recommendations

Type of exposure: [flow-through, other] [Provide details if a different exposure system 

was used.]

EPA recommends the use of a flow-through 

system. As noted in the Corrections and 

Clarifications document1, the use of a static 

renewal system is not recommended for this 

assay.

 

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor 

Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. 

(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).
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Version: 22 September 2011

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Parameter Value(s) Details or Remarks Guideline Recommendations

Type of flow-through dilution 

system:

[intermittent flow 

proportional diluters, 

continuous flow serial 

diluters, other]

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: [...] mL/min Recommended flow-through rate is 45 

mL/min (2.7 L/hr), or at least 6 total volume 

exchanges per day.

Details on toxicant mixing for 

flow-through systems:

[Briefly summarize any relevant information 

about toxicant mixing, flow-splitting accuracy, 

and the performance of the flow-through 

system.]

Recommended toxicant mixing for 

flow-through systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration is 

not recommended for mixing; 3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is 

within 10%.

Aeration? [Yes/No] EPA recommends aeration if dissolved 

oxygen reaches <4.9 mg/L (< 60% 

saturation).

Source of dilution water: [natural water, 

reconstituted water, other]

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other possible 

contaminants. OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

Was dilution water analyzed for 

pesticides, heavy metals, and 

other contaminants?

[Yes/No]

Test vessel type/materials: [...................................] EPA and OECD recommend that 

water-contact portions of the system not 

 

2

Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.
Data Evaluation Record on the Fish Short-Term Reproduction Assay with [TGAI or EP]

 EPA MRID Number [........]

Version: 22 September 2011

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Parameter Value(s) Details or Remarks Guideline Recommendations

compromise the study (e.g., all glass vessels 

or glass vessels with stainless steel frames 

are acceptable examples).

Test vessel size: [report dimensions and/or 

total volume, specify units]

EPA recommends the use of 18 L test 

chambers (e.g., 40 x 20 x 20 cm).

Fill volume: [...] L EPA recommends 10 L solution per tank.

Spawning substrate material: [aged PVC, glass, 

stainless steel, other]

EPA recommends that each tank contain 

three semi-circular spawning substrates, 

e.g., aged PVC pipe, 10 - 20 cm in length, 

split lengthwise. Spawning substrate size: [...] cm

Additional details on exposure 

system:

[Briefly summarize other relevant details

regarding the test system as they relate to 

guideline recommendations.]

Table 5: Summary of Water Quality Characteristics in the Test System.

Parameter Minimum Maximum Mean Measurement 

Interval Guideline Recommendations

Temperature (°C)

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH EPA recommends pH 6.5 to 9.0.

Dissolved oxygen (mg/L) EPA recommends dissolved oxygen (DO) 

>4.9 mg/L (>60% air saturation)

Total alkalinity (mg/L as CaCO3)

EPA recommends total alkalinity > 20 mg/L 

as CaCO3.

Hardness [specify units]

Total organic carbon (mg/L) EPA recommends that total organic carbon 

in dilution water be < 2 mg/L.

Unionized ammonia ( g/L) EPA recommends that unionized ammonia 

in the dilution water be < 1 g/L.
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Parameter Minimum Maximum Mean Measurement 

Interval Guideline Recommendations

Residual chlorine ( g/L) EPA recommends that residual chlorine in 

dilution water be < 10 g/L.

Other [specify]

General recommendations for frequency of 

measurements: EPA recommends that 

temperature, pH, and dissolved oxygen be 

measured in all test tanks at least weekly and 

that hardness and alkalinity be measured in 

controls and in one tank at the highest test 

concentration at least weekly. In addition, 

continuous temperature monitoring of at 

least one tank is encouraged. 

Abbreviations: NA Not applicable.

D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations

Was a range-finder conducted? [Yes/No] EPA recommends conducting a range-finder if 

96-hour LC50 data for the fathead minnow are 

unavailable.

If yes, what was the method for 

determining the highest test 

concentration in the range-finder? 

[other reference study, 

solubility limit, 100 mg/L]

EPA recommends that the highest test 

concentration be selected based on toxicity data 

for other fish studies or species, if available. 

Otherwise, either the solubility limit of the test 

compound or 100 mg/L (whichever is lower) is 

appropriate.

Species: [scientific name]

Life stage: [...] EPA recommends that range-finding tests be 

performed with fish of similar age and size to 

those that would be utilized in the test.

Test duration: [ ...] days EPA recommends a 96-hour exposure.
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Additional details: [Briefly outline the range-finding test 

concentrations and other relevant 

conditions. Indicate the results, e.g., 

NOEC, LOEC, LC50 values if obtained, and 

note any relevant clinical observations.]

EPA recommends conducting a range-finder 

with five test concentrations plus a control (six 

total treatment levels), with four females and two 

males per exposure tank (36 fish total). The 

number of mortalities that occur may be used to 

develop a concentration-response curve. 

Based upon the results, the highest 

concentration that does not result in increased 

mortality or signs of overt morbidity compared to 

controls, or 1/3 the derived 96-hr LC50, may be 

selected as the highest exposure concentration 

in the 21-day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations

Test duration: [....] days EPA recommends that the duration of the 

definitive test be 21 days.

Method for selecting the highest 

test concentration in the definitive 

test:

[range-finder, other 

reference study, solubility 

limit, 100 mg/L, other]

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., 1/3 the 

96-hour LC50), whichever concentration is 

lowest.

Reference study citation (if 

applicable):

[MRID, if available, and 

additional citation 

information]

Separation of test concentrations: [...] EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or ten-fold) 

is scientifically acceptable1.

Number of test concentrations: [...] EPA recommends a minimum of 3 

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations 

adjusted for purity?

[Yes/No]
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Parameter Value(s) Details or Remarks Guideline Recommendations

Indicate the type of values 

presented for measured 

concentrations:

[geometric mean, 

time-weighted average, 

other]

Limit of quantification (LOQ): [...] mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): [...] mg a.i./L

Frequency of measurement: [...] days It is recommended that test item concentration 

be measured prior to the addition of fish in all 

tanks and at least weekly thereafter in two 

replicates per treatment level.

Was the randomized complete 

block design used?

[Yes/No] [Provide details if the test design differed 

from guideline recommendations.]

EPA recommends that all fish be randomly

assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure 

fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive 

test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls.

Number of replicates in control: [...] EPA recommends 4 replicates.

Number of replicates in solvent 

control (if applicable):

[...] EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is 

used. EPA recommends 4 replicates.

Number of replicates per test item 

treatment level:

[...] EPA recommends 4 replicates.

Number of male fish per replicate

at test initiation:

[...] EPA recommends 2 males per replicate.

Number of female fish per replicate 

at test initiation:

[...] EPA recommends 4 females per replicate.

Was a solvent used? [Yes/No]

Solvent type (if applicable): [e.g., DMF, acetone, 
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Parameter Value(s) Details or Remarks Guideline Recommendations

other]

Maximum solvent concentration (if 

applicable):

[...] mL/L EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any other 

adverse effects and that concentration not be 

greater than 0.1 ml/L4.

Was a positive control used? [Yes/No]

Positive control (if applicable): [name of chemical]

Positive control concentration(s) (if 

applicable):

[..................] mg a.i./L

Photoperiod: [... ] hrs light : 

[...] hrs dark

EPA recommends photoperiod 16:8 (light:dark).

Light intensity at water’s surface: [...] lux EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

Additional details: [Briefly summarize other relevant details 

regarding the study conditions for the 

definitive test, as they relate to guideline 

recommendations.] 

 

3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic

Toxicology, 76, pp.69–92.

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing 

and Assessment. No. 23. Paris, France.
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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with [test chemical].

Treatment ID

Nominal 

Concentration

(mg a.i./L)

Measured 

Concentration

(mg a.i./L)

Mean CV (%) Details or Remarks Guideline Recommendations

Control (dilution 

water only) 0.00 <LOQ N/A EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

≤20%. Solvent control (if 

applicable) 0.00 <LOQ N/A

Treatment 1

Treatment 2

Treatment 3

Treatment n

Abbreviations: CV Coefficient of variation.

E. Observations

Biological Endpoints: [List the parameters measured, including the specific clinical signs/sublethal effects that were considered. Include the measurement 

interval for each observation (e.g., daily, day 21, other).]

Were raw (individual) data provided? [Yes/No]

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At test 

termination (day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and plasma 

sex steroids (testosterone and 17β-estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the 

nearest 0.1 mg / body weight in mg x 100) at test termination.

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal behavior.

II. RESULTS AND DISCUSSION

A. Results

[Briefly summarize the results and complete the associated tables, modifying if needed to add test item concentrations. Example values are provided in red text 

in the tables.]
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[Describe any effects of the test substance on fish survival. Compare results of the test item treatments with the available reference toxicity endpoints. The 

guideline recommends that survival in controls be >90%.]

Table 9: Adult Fish Survival in [test organism].

Abbreviations: NA Not applicable.

[Briefly summarize any effects on growth, as indicated by body weight and length at test termination, respectively. Report all concentrations at which effects were 

observed and the direction of the effect, i.e., apparent increase or decrease when compared to control.] 

Treatment (mg a.i../L) [measured]

Males Females

n # Surviving % Survival n # Surviving % Survival

Control (dilution water only), if used 8 8 100 16 16 100

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used
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Table 10: Size at Test Termination in [test organism].

Treatment

(mg a.i./L)

[measured]

Body Weight Length

Males Females Males Females

n

Mean

(g)

±SD n

Mean

(g)

±SD n

Mean

(mm)

±SD n

Mean

(mm)

±SD

Control (dilution 

water only), if used

8 3.02 0.36 16 1.84 0.28 8 50.6 1.6 16 46.2 2.6

Solvent control, if 

used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if 

used

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
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[Briefly summarize any effects on fecundity and fertilization success. Report all concentrations at which 

effects were observed and the direction of the effect, i.e., apparent increase or decrease when compared to 

control.] 

Table 11: Fecundity and Fertilization Success in [test organism].

Abbreviations: NA Not applicable. 

ND Not determined. 

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2

Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100.

[Briefly summarize any effects on nuptial tubercle score. Report all concentrations at which effects were 

observed and the direction of the effect, i.e., apparent increase or decrease when compared to control.] 

Table 12: Nuptial Tubercle Score in [test organism].

Treatment (mg a.i../L) 

[measured]

Fecundity1Fertilization Success (%)2

Control (dilution water only), if 

used

15 95

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used

Treatment (mg a.i../L) 

[measured]

Males Females

n

Median Tubercle 

Score n

Median Tubercle 

Score

Control (dilution water only), if 

used

8 38 16 0

Solvent control, if used
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Abbreviations: NA Not applicable. 

ND Not determined. SD Standard deviation.

[Briefly summarize any effects on gonado-somatic index (GSI). Report all concentrations at which effects 

were observed and the direction of the effect, i.e., apparent increase or decrease when compared to control.] 

Table 13: Gonado-Somatic Index (GSI) in [test organism].

Abbreviations: NA Not applicable.

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.

[Briefly summarize any effects on gonadal stage. Report all concentrations at which effects were observed 

and the direction of the effect, i.e., apparent accelerated or inhibited development when compared to control.] 

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used

Treatment (mg a.i../L) 

[measured]

Males Females

n

Mean GSI1

(%) ±SD n

Mean GSI1

(%) ±SD

Control (dilution water only), if 

used

8 1.5 0.6 16 12.8 2.2

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used
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Table 14: Gonadal Staging in [test organism].

Abbreviations: J Juvenile. NA Not applicable. 

ND Not determined. UTS Unable to stage.

1

 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early 

spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2

 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early 

development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.

[Discuss the incidence of gonadal histopathology observations in male and female fish, respectively. 

Summarize qualitative (narrative) observations not included in the tables below, if provided. Identify any 

apparent treatment-related effects. Report whether the severity grades were assigned based on comparison 

to the negative control or based on comparison to normal histology as determined by the pathologist’s 

experience.] 

[Complete the following tables, specifying values for each severity grade listed. For example, if 8 control fish 

were examined for testis-ova and no testis-ova were observed, the incidence value for severity grade 0 (not 

remarkable) in the control would be 8, and the incidence values for severity grades 1-4 in the control would be 

0.] 

Table 15: Gonadal Histopathology in Male [test organism].

Treatment (mg 

a.i./L)

[measured]

Diagnostic Observations1

Severity

Increased 

Proportion of 

Spermatogonia

Presence of 

Testis-Ova

Increased 

Testicular 

Degeneration

Interstitial Cell 

Hypertrophy/ 

Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Control (dilution 0 8 8 8 8 8 8 8 8

1

Treatment (mg a.i../L) 

[measured]

Males Females

n Median Stage1n Median Stage2

Control (dilution water only), if 

used

8 3 16 5

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used
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Treatment (mg 

a.i./L)

[measured]

Diagnostic Observations1

Severity

Increased 

Proportion of 

Spermatogonia

Presence of 

Testis-Ova

Increased 

Testicular 

Degeneration

Interstitial Cell 

Hypertrophy/ 

Hyperplasia

n Incidence n Incidence n Incidence n Incidence

water only), if 

used

2

3

4

Solvent control, if 

used

0

1

2

3

4

Test 

concentration1

0

1

2

3

4

Test 

concentration 2

0

1

2

3

4

Test 

concentration 3

0

1

2

3

4

Test 

concentration n

0

1

2

3

4

Positive control, 

if used

0

1

2

3

4

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 
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3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.
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Table 16: Additional Gonadal Histopathology Observations in Male [test organism].

Treatment (mg a.i./L)

[measured]

Additional Diagnostic Observations1

Severity

Decreased 

Proportion of 

Spermatogonia

Increased 

Vascular or 

Interstitial 

Proteinaceous 

Fluid

Asynchronous 

Gonad 

Development

Altered Proportions 

of Spermatocytes or 

Spermatids

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence

Control (dilution water 

only), if used

0 8 8 8 8 8 8 8 8 8 8

1

2

3

4

Solvent control, if used 0

1

2

3

4

Test concentration1 0

1

2

3

4

Test concentration 2 0

1

2

3

4
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Treatment (mg a.i./L)

[measured]

Additional Diagnostic Observations1

Severity

Decreased 

Proportion of 

Spermatogonia

Increased 

Vascular or 

Interstitial 

Proteinaceous 

Fluid

Asynchronous 

Gonad 

Development

Altered Proportions 

of Spermatocytes or 

Spermatids

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence

Test concentration 3 0

1

2

3

4

Test concentration n 0

1

2

3

4

Positive control, if used 0

1

2

3

4

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test 

guideline for reference.
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Table 17: Gonadal Histopathology in Female [test organism].

Treatment (mg a.i./L)

[measured]

Diagnostic Observations1

Severity

Increased Oocyte 

Atresia

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy

Decreased Yolk 

Formation

n Incidence n Incidence n Incidence

Control (dilution water 

only), if used

0 16 16 16 16 16 16

1

2

3

4

Solvent control, if used 0

1

2

3

4

Test concentration1 0

1

2

3

4

Test concentration 2 0

1

2

3

4

Test concentration 3 0

1

2

3

4

Test concentration n 0

1

2

3

4

Positive control, if used 0

1

2
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Treatment (mg a.i./L)

[measured]

Diagnostic Observations1

Severity

Increased Oocyte 

Atresia

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy

Decreased Yolk 

Formation

n Incidence n Incidence n Incidence

3

4

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 

3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

Table 18: Additional Gonadal Histopathology Observations in Female [test organism].

Treatment 

(mg a.i./L)

[measured]

Additional Diagnostic Observations1

Severity

Interstitial 

Fibrosis

Egg Debris in 

Oviduct

Granulomatous 

Inflammation

Decreased 

Post-Ovulatory 

Follicles

n Incidence n Incidence n Incidence n Incidence

Control 

(dilution water 

only), if used

0 16 16 16 16 16 16 16 16

1

2

3

4

Solvent 

control, if used

0

1

2

3

4

Test 

concentration1

0

1

2

3

4

Test 

concentration 

2

0

1

2

3

4

Test 

concentration 

3

0

1

2

3
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Treatment 

(mg a.i./L)

[measured]

Additional Diagnostic Observations1

Severity

Interstitial 

Fibrosis

Egg Debris in 

Oviduct

Granulomatous 

Inflammation

Decreased 

Post-Ovulatory 

Follicles

n Incidence n Incidence n Incidence n Incidence

4

Test 

concentration 

n

0

1

2

3

4

Positive 

control, if used

0

1

2

3

4

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 

3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

[Briefly summarize any effects on biochemical endpoints, including plasma vitellogenin (VTG), 

testosterone (T), and 17β-estradiol, if measured. Report all test concentrations at which effects 

were observed and the direction of the effect, i.e., apparent increase or decrease when compared 

to control.] 
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Table 19: Plasma Vitellogenin in [test organism].

Treatment

(mg a.i./L)

[measured]

Plasma Vitellogenin (VTG)

Males Females

n

Mean

(ng/mL 

plasma)

±SD n

Mean

(ng/mL 

plasma)

±SD

Control (dilution water only), if 

used

8 528 39 16 1.2x1078.1x106

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
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Table 20: Plasma Sex Steroids in [test organism] (if measured).

Treatment

(mg a.i./L)

[measured]

Plasma Testosterone (T) Plasma 17β-estradiol (E2)

Males Females Males Females

n

Mean

(ng/mL 

plasma)

±SD n

Mean

(ng/mL 

plasma)

±SD n

Mean

(ng/mL 

plasma)

±SD n

Mean

(ng/mL 

plasma)

±SD

Control (dilution water 

only), if used

8 9.8 0.9 16 3.0 0.2 8 0.4 0.1 16 6.1 1.2

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
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[Discuss qualitative observations of secondary sex characteristics and the incidence of behavioral effects or other clinical signs. Identify when during the study 

the effects were observed and whether the effects are considered treatment-related. Complete the following table, using a separate line for each type of 

observation. Add rows as necessary.]

Table 21: Secondary Sex Characteristics and Clinical Signs in [test organism].

Treatment

(mg a.i./L)

[measured]

Secondary Sex Characteristics and Clinical Signs1

Males Females

Type n Incidence Type n Incidence

Control (dilution water 

only), if used

Light body color 8 1 Vertical bands 16 2

Solvent control, if used

Test concentration1

Test concentration 2

Test concentration 3

Test concentration n
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Treatment

(mg a.i./L)

[measured]

Secondary Sex Characteristics and Clinical Signs1

Males Females

Type n Incidence Type n Incidence

Positive control, if used 

1 Define any abbreviations used to identify secondary sex characteristics and clinical signs (i.e., sublethal effects, including behavioral effects) not specified elsewhere in the results. 

Examples include discoloration, changes in dorsal nape pad, abnormal swimming behavior, lethargy, loss of equilibrium, hemorrhage, malformations, lesions, etc. Add rows as 

necessary. Note that nuptial tubercle score is reported previously in Table 12.

B. Study Author’s Analysis and Conclusions

[List the parameters that were analyzed and the statistical tests that were performed by the study author. Indicate whether the statistical analyses were 

appropriate and whether they were consistent with the methods recommended in the guideline.]

[Briefly summarize the study author’s conclusions.]

C. Reviewer’s Analysis and Conclusions

[It is recommended that the reviewer confirm the accuracy of the statistical analyses by recalculating both summary statistics and pertinent statistical tests for 

each endpoint, as well as performing the tests including and excluding any outliers, as appropriate. Include percent differences from the negative control for each 

endpoint and the criteria for determining significance.] 

Statistical Methods: [Modify as appropriate.] The reviewer analyzed data for survival (mortality) using Fisher’s Exact Test [ToxStat 3.5 or CETIS v.xx]. Data 

for fecundity, normalized to the number of female reproductive days; fertility; gondo-somatic index (GSI), vitellogenin (VTG); sex steroids (if measured); weight; 

and length (respectively), ... 

[Choose the appropriate option below]

were consistent with a monotonic concentration-response and therefore were analyzed using the Jonckheere-Terpstra test [SAS v.xx or CETIS v.xx].

were not consistent with a monotonic concentration-response. The data were tested for normality using Shapiro-Wilks test and for homogeneity of 

variance using Levene’s test [ToxStat 3.5 or CETIS]. Data [specify] that met the assumptions of normality and homogeneity of variance were then 

analyzed using Dunnett’s test [ToxStat 3.5 or CETIS). Data [specify] that failed either of these assumptions [specify] were analyzed using the 

Mann-Whitney test with the Bonferroni-Holm adjustment [ToxStat 3.5 or CETIS].
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Replicate median values for nuptial tubercle score were analyzed using the Jonckheere-Terpstra test [SAS v.xx or CETIS v.xx]. Unless otherwise indicated, 

effects were considered statistically significant at [p<0.05]. [Describe any summary statistics presented for gonadal staging and histopathology.]

Conclusions:

[Briefly summarize the study findings and complete the following tables.]

Table 22: Reproductive and HPG Endpoints1,2 for Male [test organism] in the FSTRA with [test chemical].

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. TTestosterone. VTG

Vitellogenin.

1

 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

2

 Unless otherwise specified, effects are considered statistically significant at [p<0.05].

Treatment

(mg a.i../L)

[measured]

Tubercle Score GSI

Gonadal 

Staging and 

Histo.

Plasma VTG Plasma T Plasma E2

Median p % Diff. p

Effect? 

(Yes/No) % Diff. p % Diff. p % Diff. p

Control (dilution 

water only), if used

38 NA 0 NA NA 0 NA 0 NA 0 NA

Solvent control, if 

used

Test conc. 1

Test conc. 2

Test conc. 3

Test conc. n

Positive control, if 

used
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Table 23: Reproductive and HPG Endpoints1,2 for Female [test organism] in the FSTRA with [test chemical].

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. 

NA Not applicable. T

Testosterone. VTG Vitellogenin.

1

 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

2

 Unless otherwise specified, effects are considered statistically significant at [p<0.05].

Treatment

(mg a.i../L)

[measured]

Fecundity Fert. Success Tubercle Score GSI

Gonadal 

Staging and 

Histo.

Plasma VTG Plasma T Plasma E2

% 

Diff. p

% 

Diff. p Median p

% 

Diff. p

Effect? 

(Yes/No)

% 

Diff. p

% 

Diff. p % Diff. p

Control (dilution 

water only), if 

used

0 NA 0 NA 0 NA 0 NA NA 0 NA 0 NA 0 NA

Solvent control, 

if used

Test conc. 1

Test conc. 2

Test conc. 3

Test conc. n

Positive control, 

if used
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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with [test chemical].

Treatment

(mg a.i./L)

[measured]

Body Weight Length

Males Females Males Females

% Diff. p % Diff. p % Diff. p % Diff. p

Control (dilution water 

only), if used

0 NA 0 NA 0 NA 0 NA

Solvent control, if used

Test concentration 1

Test concentration 2

Test concentration 3

Test concentration n

Positive control, if used

Abbreviations: Diff. Difference. NA Not applicable. ND Not determined.

1

 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water) 

control.

2

 Unless otherwise specified, effects are considered statistically significant at [p<0.05].

E. Study Deficiencies

[Were validity and performance criteria met? Include a discussion of whether, to what extent, and in what 

way failure to meet the performance criteria had an impact on the quality or acceptability of the study e.g., 

major vs. minor deficiencies.]

F. Reviewer’s Comments

[Identify whether the reviewer’s interpretation is in agreement with that of the study author; discuss any 

differences and whether or not the differences substantively impact the analysis. Provide additional 

comments that do not appear under other sections of the template.]

III. REFERENCES

[Provide references that were cited in the study report, studies in the open literature, references to other study 

reports in the submission or other studies conducted by the sponsor. Do not include references to standard 

guidelines or methodologies.]