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

Page 2 f 33 EXECUTIVE SUMMARY

The 21

- day short

- term reproduction assay of [ test chemical ] with [ common name and scientific name ] was stud ied

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 [x 1 , x 2 , x 3

, .... x n ] mg a.i

. /L. The

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

.

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

eggs/female/day/replicate; fertili zation 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. Pl asma [vitellogenin, testosterone, and 17β

- estradiol , if measured]

, respectively, w ere 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 inclu ded ......... [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 co ncentrations] 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

Page 3 f 33 Results Synopsis :

Test o rganis m a ge 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 Effects 1,2 in the Fish Short

- Term Reproduction Assay (FSTRA) with [test chemical]

.

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β

- estradiol. F Female. Fert. Fertilization. GSI Gonad o

- 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. Con clusions regarding histopathology may be

heavil y 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 usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 4 f 33 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.]Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 5 f 33 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 rec ommends

that fish be held another 7 days. If mortalities

greater than 5% occur d uring 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 informationData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 6 f 33 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 t he 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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 7 f 33 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 document 1 , 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 Guideli nes for the Endocrine Disruptor

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

( htt p://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf ).Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 8 f 33 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 th e 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

contaminant

s . 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 Aqu atic Laboratory Studies.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 9 f 33 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±1 o C;

inter

- replicate and inter

- treatment

differentials should not exceed 1 o C.

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 CaCO 3 ) EPA recommends total alkalinity > 20 mg/L

as CaCO 3.

Hardness [specify units]

Total o rganic c arbon (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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 10 f 33 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 LC

50 data for the fathead minnow are

un available.

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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 11 f 33 Additional details: [Briefly outline the range

- finding test

concentrations and other relevant

conditions. Indicate the results, e.g

., NOEC, LOEC, LC 50 values if obtained, and

note any relevant clinical observations.] EPA recommends conducting a range

- finder

with five test co ncentrations 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 LC 50

, 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 LC

50 ), whichev

er 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 acceptable 1

.

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]Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 12 f 33 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 tr eatment,

including controls.

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

Number of replicates in solvent

control (if applicable): [...] EPA recommend s 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,Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 13 f 33 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/L 3 . 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/L 4

.

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 1 6 : 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 M J, 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. Envir onmental Health and Safety Publications. Series on Testing

and Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 14 f 33 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 mea surement

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. A t test

termination (day 2 1), 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). Gonad o

- 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 m g x 100) at test termination.

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

II. RESULTS AND DISCUSSION



### A. Results

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

in the tables.]Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 15 f 33 [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 > 9 0%

. ]

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 a ll 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] Male s Female s

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 usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 16 f 33 Table 1 0 : 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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 17 f 33 [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 1 1 : 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 1 2 : Nuptial Tubercle Score in [test organism]

. Treatment (mg a.i../L)

[measured] Fecundity 1 Fertilization 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 usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 18 f 33 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 1 3 : 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 ( m g) 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 GSI 1

(%) ±SD n Mean GSI 1

(%) ±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 usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 19 f 33 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 fat head 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 no rmal 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 value s for severity grades 1

- 4 in the control would be

0.]

Table 1 5 : Gonadal Histopathology in Male [test organism]

.

Treatment (mg

a.i./L)

[measured] Diagnostic Observations 1

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 Stage 1 n Median Stage 2

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 usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 20 f 33 Treatment (mg

a.i./L)

[measured] Diagnostic Observations 1

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 G onadal histopathology diagnostic observations are graded 0

– 4 based on severity: 0=Not remarkable, 1= Minimal, 2= Mild,Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 21 f 33 3 =Moderate, 4 =Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 22 f 33 Table 1 6 : Additional Gonadal Histopathology Observations in Male [test organism]

.

Treatment (mg a.i./L)

[measured] Additional Diagnostic Observations 1

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

4Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 23 f 33 Treatment (mg a.i./L)

[measured] Additional Diagnostic Observations 1

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 G onadal 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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 24 f 33 Table 17 : Gonadal Histopathology in Female [test organism]

.

Treatment (mg a.i./L)

[measured] Diagnostic Observations 1

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

2Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 25 f 33 Treatment (mg a.i./L)

[measured] Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation

n Incidence n Incidence n Incidence

3

4

1 G onadal 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 Observations 1

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

3Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 26 f 33 Treatment

(mg a.i./L)

[measured] Additional Diagnostic Observations 1

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.]Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 27 f 33 Table 1 9 : 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 1.2x10 7 8.1x10 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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 28 f 33 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.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 29 f 33 [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. Complet e 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 Signs 1

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 nData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 30 f 33 Treatment

(mg a.i./L)

[measured] Secondary Sex Characteristics and Clinical Signs 1

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 equi librium, hemorrhage

, malformations, lesions, etc. Add rows as

necessary. Note that nuptial tubercle score is reported previously in Table 1 2

.



### 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 statistic s 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 homogene ity 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]

.Data Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 31 f 33 Replica te 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 f or gonadal staging and histopathology.]

Conclusions:

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

Table 22 : Reproductive and HPG Endpoints 1,2 for Male [test organism] in the FSTRA with [test chemical]

.

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β

- estradiol. GSI Gonad o

- Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG

Vitellogenin.

1 Unless otherwise indicated, effects and percent (%) differences are repo rted 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 signif icant 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 3 8 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

usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011

Page 32 f 33 Table 23 : Reproductive and HPG Endpoints 1,2 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 usedData Evaluation Record on the Fish Short

- Term Reproduction Assay with [TGAI or EP]

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



### Version: 22 September 2011 Page 33 f 33 Table 24 : Growth Endpoints 1,2 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 additiona l

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 t he sponsor. Do not include references to standard

guidelines or methodologies.]