Data Evaluation Record on the Fish Short-Term Reproduction Assay with Atrazine

 EPA MRID Number 49694001

Version: 22 September 2011 

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Data Requirement: EPA DP Barcode Not Provided

OECD Data Point 

EPA MRID 49694001

EPA Guideline 890.1350 

Fish Short-Term Reproduction Assay 

Test Material: Atrazine Purity (%): 97.5% wt/wt 

Common Name Atrazine 

Chemical Name IUPAC Not Reported 

 CAS Name Not Reported 

CAS No. Not Reported 

Synonyms Not Reported

EPA PC Code 080803 

Primary Reviewer: John Marton, Ph.D., Environmental Scientist Date: 08/20/15 CDM Smith, Inc.

Secondary Reviewer(s): 

Teri S. Myers, Ph.D., Environmental Scientist Date: 08/25/15 CDM Smith, Inc.

Frank T. Farruggia, Ph.D., Biologist, USEPA/OCSPP/OPP/ERB3 Date: 08/25/15 USEPA 

Colleen Rossmeisl, Biologist, USEPA/OCSPP/OPP/ERB3 Date: 08/25/15 USEPA 

Date Evaluation Completed: 25-08-2015

COLLEEN 

ROSSMEISL

Digitally signed by COLLEEN ROSSMEISL 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=COLLEEN 

ROSSMEISL, dnQualifier=0000069649 

Date: 2016.04.12 11:55:37 -04'00'

Digitally signed by FRANK 

FARRUGGIA 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, 

cn=FRANK FARRUGGIA, 

dnQualifier=0000048865 

Date: 2016.04.12 12:35:26 

-04'00'
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CITATION: Schneider SC, Zhang L, Martin KH, Gallagher SP. 2015. Atrazine- Fish Short-Term Reproduction Assay with 

the Japanese Medaka (Oryzias latipes). Unpublished study performed by Wildlife International, Easton, Maryland. 

Laboratory report number 528A-275. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, North Carolina. 

Study completed July 31, 2015. 

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.
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EXECUTIVE SUMMARY

The 21-day short-term reproduction assay of atrazine with Japanese Medaka (Oryzias latipes) was studied under flowthrough conditions. Adult fish (40 per group, 4.5 months old) were exposed to nominal concentrations of 0 (negative 

control), 0.00049, 0.0049, and 0.049 mg a.i./L; mean-measured concentrations were <0.000125 (<LOQ; control), 

0.00059, 0.0054, and 0.052 mg a.i./L. The test system was maintained at 23.9 to 25.7°C and a pH of 7.9 to 8.4. 

Fecundity in the negative control was 40.9 eggs/female/day/replicate, with eggs produced daily; fertilization success in 

controls was 91.6%. Atrazine did not significantly alter fecundity or fertility at any treatment level. Plasma vitellogenin was

significantly increased (p = 0.0090) in male fish at the mean-measured 0.0054 mg a.i./L treatment level; no differences 

were detected for females. No remarkable effects on gonadal histopathology were observed in male fish at any treatment 

level. A small number of fish showed an increase in atretic oocytes, however, the overall score for oocyte atresia did not 

show a concentration related increase. Gonado-somatic index (GSI) was not assessed. Atrazine exposure was associated 

with an increase in female body length at the mean-measured 0.052 mg a.i./L treatment level. No other effects on 

secondary sex characteristics and clinical signs (i.e., behavioral and other sublethal effects) were observed. Unless 

otherwise indicated, all effects are reported based on comparison to the negative (clean water) control. Based on these 

findings, it is likely that atrazine does not interfere with the HPG axis of Japanese medaka. 

This assay was not conducted in response to an Endocrine Disruptor Screening Program (EDSP) Tier 1 Test Order; 

instead, it was submitted as a voluntary submission of consideration in re-registration review. The study is scientifically 

sound and its performance was generally consistent with the recommendations in the Fish Short-term Reproduction 

Assay guideline (OCSPP Guideline §890.1350). Deviations from the guideline are noted in Section IIA 8.1 of this data 

evaluation record (DER). Except for the deviation in test species and a few instances of pre-study fecundity not meeting 

guideline recommendations, the validity elements and performance criteria specified in §890.1350 were met (i.e., control 

survival and reproduction). 
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Results Synopsis:Test organism age at test initiation: 4.5 months 

Mean body weight at test initiation (if measured): 0.23 g for males, 0.23 g for females Mean length at test initiation (if measured): Not measured Test type: flow-through 

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

Treatment 

(mg a.i./L) 

Mean-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 

0.00059 No No -- -- -- -- No No No No -- -- -- -- 0.0054 No No -- -- -- -- No No Yes No -- -- -- -- 0.052 No No -- -- -- -- No No No No -- -- -- -- 
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Abbreviations: Conc. Concentration. Diff. Difference. E2 17 ȕ-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. 
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I. MATERIALS AND METHODS 

 Guideline Followed: This study was conducted following procedures outlined in the U.S. EPA Series 890- 

Endocrine Disruptor Screening Program Test Guidelines, OPPTS Number 

890.1350, Fish Short-Term Reproduction Assay; and the OECD Guidelines for 

Testing of Chemicals, Guideline 229, Fish Short Term Reproduction Assay. The 

following deviations from OCSPP 890.1350 were noted: 

1. Japanese medaka is not a recommended species in the OCSPP guidance. However, OECD guidelines 

allow the use of this species. 

2. All but three of the tanks selected for use in the definitive test met the minimum fecundity requirement 

during the pre-exposure phase (15 eggs/female/reproductive day). 

3. Unionized ammonia and residual chlorine concentrations of the dilution water were not reported. 

4. The gonado-somatic index was not assessed. Further, the count of anal fin papillae was used in 

replacement of the nuptial tubercle score. 

5. Fertilization success (91.6%) was below the recommended validity criterion outlined in OCSPP 890.1350 

(95%). 

6. Arsenic, copper, iron, and zinc were all detected in the periodic screening analysis of the dilution water. 

 Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements were 

provided. This study was conducted in compliance with Good Laboratory Practice 

Standards as published by the U.S. Environmental Protection Agency (40 CFR 

Parts 160 and 792); and OECD Principles of Good Laboratory Practice 

(ENV/MC/CHEM (98) 17), with the following exception: periodic analyses of water 

for potential contaminants were not performed according to Good Laboratory 

Practice Standards, but were performed using a certified laboratory and standard 

US EP analytical methods. 

A. Test Material Atrazine 

Description: Solid

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow, 
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Atrazine

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vapor pressure of test compound, expiration date.

Lot No./Batch No. : 726718 (Batch ID) 

Purity: 97.5% 

Impurities: None reported 

Stability of Compound: Considered stable until end of February 2017 when stored at <30°C (Certificate of 

Analysis).

Storage Conditions of 

Test Chemicals: Stored under ambient conditions 
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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: Japanese medaka EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Oryzias latipes

Species strain (if stated): Not applicable Were fish obtained from a single laboratory stock? 

Yes Fish were originally obtained from Osage 

Catfisheries, Inc, Osage Beach, Missouri. 

EPA recommends that fish be from a single laboratory stock.

Were acclimation conditions same as definitive test? 

Yes EPA recommends that fish be acclimated under 

water quality and illumination conditions that are 

similar to the definitive test. 

Acclimation period: At least 14 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: There was 1 mortality 

during the 7 days prior to pre-exposure, and no 

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be less 

than 5% of the culture population. If mortality 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

other fish showed any signs of disease or stress and therefore fish did not receive treatment for disease during the acclimation periods, the two-week pre-exposure 

period, or the exposure 

period. 

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: Brine shrimp nauplii 

(Artemia sp.)

EPA recommends that fish be fed frozen brine 

shrimp twice per day to promote active 

reproduction and maintain body condition. Source of food: INVE Aquaculture, Salt 

Lake City, Utah

Frequency of feeding: Two to three times/day Details on feeding: Fish were fed at a rate 

sufficient to promote active reproduction and maintain body condition. Fish were not fed for at 

Uneaten food and fecal material were removed from the test vessels daily by siphoning. 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

least 12 hours prior to test termination. Feeding rates in each replicate were adjusted weekly, if necessary, to account for mortality. 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 4.5 months EPA recommends reproductively mature 

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

Mean weight of males at test initiation (if determined): 

0.23 g EPA recommends thata 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): 

Not reported The range of individual weights at the start 

of the pre-exposure period was ±20% of the mean weight of each sex. 

Mean weight of females at test initiation (if determined): 

0.23 g 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Range of individual weights (females) at test initiation (if determined): 

Not reported The range of individual weights at the start 

of the pre-exposure period was ±20% of the mean weight of each sex. 

Mean length of males at test initiation (if determined): 

Not determined 

Mean length of females at test initiation (if determined): 

Not determined 

Duration of pre-exposure phase: 7 days EPA recommends a minimum of 14 days. Were pre-exposure conditions identical to the definitive test? 

Yes EPA recommends that pre-exposure conditions, 

including temperature, photoperiod, feeding, etc., 

be identical to definitive test conditions. 

Number of pre-exposure tanks: 32 Additional tanks were established so that 

breeding groups with a proven history of spawning could be selected for inclusion in the exposure phase of the test. 

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: 5 Number of females per tank: 5 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Pre-exposure fecundity: > 12.8 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 preexposure: > 2 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: Pre-exposure was 

conducted under conditions as similar as possible to those used during the exposure periods. 

A replicate was considered suitable for use in the test if there were at least 10 eggs per female per reproductive day, and spawning in that vessel occurred at least twice during the 7 days period. Any vessels with mortalities, incorrect sex ratio, or poor reproductive performance were excluded from use in the test. 

C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through 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.

Type of flow-through dilution 

system: 

Continuous-flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: 48.6 mL/min This was calculated based on a 10-L fill volume 

and a flow-rate sufficient to provide 7 volume additions per vessel per day. 

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: 

Fluid metering pumps were used to deliver volumes of a single test substance stock solution to mixing 

The proportion of the test and solution that was pumped into each replicate test vessel was checked prior to the test and approximately weekly during the test to ensure that flow rates 

Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration is not recommended for mixing; 3) A 

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). 

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

chambers randomly assigned to each treatment, where it was mixed with well water. The flow of dilution water was controlled using rotamaters. 

varied by no more than ±10% of the mean flow 

for the replicates. Delivery was initiated seven days prior to the introduction of test organisms. The general operation of the exposure system was checked visually at least two times during the test at least once on the last day of the test. 

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? None reported EPA recommends aeration if dissolved oxygen 

reaches <4.9 mg/L (< 60% saturation).

Source of dilution water: Water was obtained from 

an on-site well. Water was passed through a sand filter to remove particles greater than approximately 25 μm, pumped into a storage tank, and aerated. Water was then filtered to 0.45 μm and UV-sterilized. 

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.
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was dilution water analyzed for pesticides, heavy metals, and other contaminants? 

Yes. See Reviewer’s Comments section. 

Test vessel type/materials: Glass aquaria EPA and OECD recommend that watercontact portions of the system not 

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

or glass vessels with stainless steel frames 

are acceptable examples).

Test vessel size: 12 L EPA recommends the use of 18 L test 

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

Fill volume: 10 L Depth of test water was approximately 12.7 cm. EPA recommends 10 L solution per tank. Spawning substrate material: No spawning substrates 

were specified 

During observations for reproduction, egg clumps were gently removed from netted females and/or from the aquarium bottom each morning. 

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: N/A Additional details on exposure system: 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 23.9 25.7 25.1 

Measured in each 

vessel weekly, and 

continuously in one 

neg control replicate 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials 

should not exceed 1oC.

pH 7.9 8.4 8.1 

Measured in each 

vessel weekly 

EPA recommends pH 6.5 to 9.0.

Dissolved oxygen (mg/L) 7.4 8.2 8.0 

Measured in each 

vessel weekly 

EPA recommends dissolved oxygen (DO) 

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

Total alkalinity (mg/L as CaCO3) 140 180 166 

Measured in one 

replicate of the 

negative control and 

the highest 

concentration 

treatment group 

weekly during the test, 

with measurements 

typically alternating 

EPA recommends total alkalinity > 20 mg/L 

as CaCO3.
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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

between replicates. 

Hardness (mg/L as CaCO3) 124 140 133 

Measured in one 

replicate of the 

negative control and 

the highest 

concentration 

treatment group 

weekly during the test, 

with measurements 

typically alternating 

between replicates. 

Total organic carbon (mg/L) <1 4 weeks prior to test 

initiation 

EPA recommends that total organic carbon in 

dilution water be < 2 mg/L. 

Unionized ammonia (Pg/L) Not Reported 

EPA recommends that unionized ammonia in the dilution water be < 1 Pg/L. 

Residual chlorine (Pg/L) Not Reported 

EPA recommends that residual chlorine in 

dilution water be < 10 Pg/L. 

Conductivity (μS/cm) 275 452 346 Measured in one General recommendations for frequency of 
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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

replicate of the 

negative control and 

the highest 

concentration 

treatment group 

weekly during the test, 

with measurements 

typically alternating 

between replicates. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? No Non-GLP preliminary trials were conducted 

to determine the optimal male:female pairing. See Reviewer’s Comments. 

EPA recommends conducting a range-finder if 96-hour LC50 data for the fathead minnow are 

unavailable. 
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If yes, what was the method for determining the highest test concentration in the range-finder? 

N/A 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: N/A Life stage: N/A 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: N/A EPA recommends a 96-hour exposure. Additional details: N/A 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 
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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: 35 days EPA recommends that the duration of the 

definitive test be 21 days. 

Method for selecting the highest test concentration in the definitive test:

Nominal concentrations for the definitive study were determined from the findings in Papoulias et al. (2014).

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):

Papoulias DM, Tillitt DE, Talykina MG, Whyte JJ, Richter CA. 2014. Atrazine reduces reproduction 

in Japanese medaka (Oryzias latipes). Aquatic 

Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.05.022. 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Separation of test concentrations:

0.1 EPA suggests that a concentration 

separation of between 0.33 (or threefold) and 0.1 (or ten-fold) is 

scientifically acceptable1 . 

Number of test concentrations:

3 EPA recommends a minimum of 3 

concentrations and a control, plus 

solvent control if appropriate.

Are nominal concentrations adjusted for purity? 

No The reviewer corrected the nominal 

and mean-measured concentrations for the purity of the test material. The standard curve used in analyzing the analytical samples was prepared without correcting for the purity. Therefore, the calculated measured concentrations were not corrected for the purity. 

Indicate the type of values presented for measured concentrations: 

Mean-measured concentrations 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Limit of quantification (LOQ): 0.000125 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): Not Reported Frequency of measurement: Samples were collected for analysis on Days 0, 7, 

11, 21, 28, and 35. 

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 EPA recommends that all fish be 

randomly assigned to tanks during preexposure. 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. 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in control: 

4 EPA recommends 4 replicates.

Number of replicates in solvent control (if applicable): 

N/A; a solvent was not used 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: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

5 EPA recommends 2 males per 

replicate. 

Number of female fish per replicate at test initiation: 

5 EPA recommends 4 females per 

replicate. 

Was a solvent used? No Solvent type (if applicable): N/A Maximum solvent concentration (if applicable): 

N/A EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD 

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 
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Parameter Value(s) Details or Remarks Guideline Recommendations 

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? No Positive control (if applicable): 

N/A 

Positive control concentration(s) (if applicable): 

N/A 

Photoperiod: 16 hrs light : 

8 hrs dark 

A 30-minute transition period of low-light intensity was included. 

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

Light intensity at water’s surface: 

536-940 lux EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

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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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: None 

Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Atrazine. 

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.000125 N/A N/A EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 20%.

Treatment 1 0.00049 0.00059 4.58 N/A Treatment 2 0.0049 0.0054 4.81 N/A Treatment 3 0.049 0.052 2.42 N/A 

Abbreviations: CV Coefficient of variation. 

E. Observations

Biological Endpoints: Daily- survival, general observations (sub-lethal effects), fecundity, fertility 

 Test termination- sex-specific length and wet weight, liver VTG, necropsy and secondary sex characteristics, and histology and 

histopathology
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Were raw (individual) data provided? Yes, except for VTG. VTG data were expressed as a fold change, no further details were provided.

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 17 ȕ-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

After 35 days of exposure survival averaged 100% in the negative control and 100, 97.5, and 97.5% in the mean-measured 0.00059, 0.0054, and 0.052 mg 

a.i./L treatment groups, respectively. One female died in the mean-measured 0.0054 mg a.i./L treatment group, and one male died in the mean-measured 0.052 

mg a.i./L treatment group (Table 9). No significant differences were reported. 

Table 9: Adult Fish Survival in Oryzias latipes.

Treatment (mg a.i./L) 

Mean-Measured

Males Females 
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Abbreviations: NA Not applicable. 

There were no significant differences in body weight or length for either males or females at any treatment level relative to the negative control (Table 10; 

Dunnett’s, p  0.05). However, a significant increase in female length was detected at the top level relative to the negative control based on the JonckheereTerpstra Trend test (p  0.05). This increase was slight (~4%) and considered to be within the range of biological activity. 

n # Surviving % Survival n # Surviving % Survival 

Negative Control 20 20 100 20 20 100 0.00059 20 20 100 20 20 100 0.0054 20 20 100 20 19 95 0.052 20 19 95 20 20 100 
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Table 10: Size at Test Termination in 

Oryzias latipes

Treatment 

(mg a.i./L) 

Mean-measured

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Negative Control 4 0.46 0.02 4 0.62 0.04 4 29 0.7 4 31 0.8 0.00059 4 0.45 0.03 4 0.60 0.04 4 29 0.6 4 31 0.8 0.0054 4 0.49 0.05 4 0.61 0.03 4 30 0.8 4 31 0.6 0.052 4 0.46 0.04 4 0.63 0.05 4 29 0.6 4 32a 0.6 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. a Significantly increased relative to the negative control based on the Jonckheere-Terpstra Trend test (

p

 0.05). 
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Mean fecundity in the control and treatment groups ranged from 38.2 to 40.9 eggs per female per reproductive 

day, while mean fertility ranged from 90.5 to 93.0% (Table 11). No significant differences were detected at any 

treatment level relative to the control based on either the Dunnett’s or Jonckheere-Terpstra Trend test (p 

0.05). 

Table 11: Fecundity and Fertilization Success in Oryzias latipes.

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. 

The mean anal fin papillae count for males ranged from 162 to 194 in the control and treatment groups; no 

papillae were detected in females (Table 12). No significant differences were detected (Dunnett’s & 

Jonckheere-Terpstra Trend Tests, p  0.05). 

Treatment (mg a.i./L) Meanmeasured

Fecundity1 Fertilization Success (%)2

Negative Control 40.9±2.6 91.7±2.1 

0.00059 38.2±2.3 90.5±4.7 

0.0054 40.2±2.6 93.0±1.6 

0.052 39.2±2.3 92.9±1.8 
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Table 12: Anal Fin Papillae Count in Oryzias latipes.

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 

Gonado-somatic index was not assessed. 

Table 13: Gonado-Somatic Index (GSI) in Oryzias latipes.

Abbreviations: NA Not applicable. 

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

Treatment (mg a.i./L) Meanmeasured

Males Females 

n 

Mean Count 

±S.D. 

n 

Mean Count 

±S.D. 

Negative Control 4 162±32.6 4 0.0±0.00 

0.00059 4 179±15.2 4 0.0±0.00 

0.0054 4 172±34.8 4 0.0±0.00 

0.052 4 194±10.4 4 0.0±0.00 

Treatment (mg a.i./L) Meanmeasured

Males Females 

n Mean GSI1

(%) 

±SD n Mean GSI1

(%) 

±SD 

Negative Control 4 -- -- 4 -- -- 

0.00059 4 -- -- 4 -- -- 

0.0054 4 -- -- 4 -- -- 

0.052 4 -- -- 4 -- -- 
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Median gonad stage was 2 in the control and treatment groups for both males and females (Table 14). No 

differences were detected at any treatment level relative to the negative control (Jonckheere-Terpstra Trend 

Test, p > 0.05). 

Table 14: Gonadal Staging in Oryzias latipes.

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. 

The majority of testes in the control and exposure groups were normal in appearance (Table 15). The only 

abnormality observed in the testes was the sporadic occurrence of oogonia and early oocytes in a few testes 

(Tables 15 and 16). 

A minimal increase in atretic oocytes was observed in ovaries of seven of 79 females. Ovaries of one female in 

the negative control group showed a minimal severity of atretic oocytes, ovaries of two females in the middle 

dose showed a minimum severity, and ovaries of four females in the top dose showed minimal severity (Table 

17). The increase was significant at the top dose (Jonckheere-Terpstra Trend test, p = 0.038). However, the 

overall score for oocyte atresia (based on the sum of the number of atretic oocytes and post-atretic follicles) 

did not show a concentration related increase. There were no other remarkable or significant differences in 

Treatment (mg a.i./L) Meanmeasured

Males Females 

n Median Stage1 n Median Stage2

Negative Control 4 2 4 2 

0.00059 4 2 4 2 

0.0054 4 2 4 2 

0.052 4 2 4 2 
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primary or secondary guideline diagnoses.

Table 15: Gonadal Histopathology in Male Oryzias latipes.

Treatment (mg 

a.i./L) 

Mean-measured 

Diagnostic Observations1

Severity

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

(Oogonia/Early 

Oocytes) 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hypertrophy/ 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 N/A 20 18 20 N/A 20 N/A 

1 20 N/A 20 1 20 N/A 20 N/A 

2 20 N/A 20 0 20 N/A 20 N/A 

3 20 N/A 20 1 20 N/A 20 N/A 

4 20 N/A 20 0 20 N/A 20 N/A 

0.00059 0 20 N/A 20 20 20 N/A 20 N/A 

1 20 N/A 20 0 20 N/A 20 N/A 

2 20 N/A 20 0 20 N/A 20 N/A 

3 20 N/A 20 0 20 N/A 20 N/A 

4 20 N/A 20 0 20 N/A 20 N/A 

0.0054 0 20 N/A 20 19 20 N/A 20 N/A 

1 20 N/A 20 1 20 N/A 20 N/A 

2 20 N/A 20 0 20 N/A 20 N/A 

3 20 N/A 20 0 20 N/A 20 N/A 

4 20 N/A 20 0 20 N/A 20 N/A 

0.052 0 19 N/A 19 17 19 N/A 19 N/A 

1 19 N/A 19 1 19 N/A 19 N/A 

2 19 N/A 19 1 19 N/A 19 N/A 

3 19 N/A 19 0 19 N/A 19 N/A 

4 19 N/A 19 0 19 N/A 19 N/A 

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 16: Additional Gonadal Histopathology Observations in Male 

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

Mean-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 

Negative Control 0 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

1 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

2 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 3 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 4 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

0.00059 0 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

1 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

2 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 3 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 4 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

0.0054 0 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

1 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 

2 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 3 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 4 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A 
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Treatment (mg a.i./L) 

Mean-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 

0.052 0 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A 

1 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A 

2 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A 3 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A 4 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A 

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 Oryzias latipes.

Treatment (mg a.i./L) 

Mean-measured 

Diagnostic Observations1

Severity

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Increased Atretic 

Oocytes 

n Incidence n Incidence n Incidence 

Negative Control 0 20 17 20 N/A 20 19 

1 20 3 20 N/A 20 1 

2 20 0 20 N/A 20 0 

3 20 0 20 N/A 20 0 

4 20 0 20 N/A 20 0 

0.00059 0 20 20 20 N/A 20 20 

1 20 0 20 N/A 20 0 

2 20 0 20 N/A 20 0 

3 20 0 20 N/A 20 0 

4 20 0 20 N/A 20 0 

0.0054 0 19 17 19 N/A 19 17 

1 19 2 19 N/A 19 2 

2 19 0 19 N/A 19 0 

3 19 0 19 N/A 19 0 

4 19 0 19 N/A 19 0 

0.052 0 20 16 20 N/A 20 16 

1 20 4 20 N/A 20 4 

2 20 0 20 N/A 20 0 

3 20 0 20 N/A 20 0 

4 20 0 20 N/A 20 0 

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 18: Additional Gonadal Histopathology Observations in Female Oryzias latipes.

Treatment (mg 

a.i./L) 

Meanmeasured 

Additional Diagnostic Observations1

Severity

Increased Mature 

Oocytes 

Granulomatous 

Inflammation 

Increased PostAtretic Follicles Increased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

0 20 16 20 20 20 18 20 2 

1 20 4 20 0 20 2 20 8 

2 20 0 20 0 20 0 20 10 

3 20 0 20 0 20 0 20 0 

4 20 0 20 0 20 0 20 0 

0.00059 0 20 20 20 19 20 20 20 2 

1 20 0 20 1 20 0 20 9 

2 20 0 20 0 20 0 20 8 

3 20 0 20 0 20 0 20 1 

4 20 0 20 0 20 0 20 0 

0.0054 0 19 17 19 19 19 19 19 6 

1 19 1 19 0 19 0 19 7 

2 19 1 19 0 19 0 19 6 

3 19 0 19 0 19 0 19 0 

4 19 0 19 0 19 0 19 0 

0.052 0 20 19 20 20 20 19 20 0 

1 20 1 20 0 20 1 20 14 

2 20 0 20 0 20 0 20 5 

3 20 0 20 0 20 0 20 1 

4 20 0 20 0 20 0 20 0 

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. 

There were no significant effects on male or female hepatic vitellogenin gene expression (Table 19). 

There was a statistically significant increase (p < 0.05) in males at the middle dose group relative 

to the negative control, but this was not considered to be treatment related as there were no effects 
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seen on reproductive endpoints, secondary sex characteristics, or gonadal histopathology. 

Plasma sex steroids were not measured (Table 20). 

Table 19: Plasma Vitellogenin in Oryzias latipes.

Treatment 

(mg a.i./L) 

Mean-measured

VTG Gene Expression 

Males Females 

n Mean

(fold change) 

±SD n Mean

(fold change) 

±SD 

Negative Control 4 1.4 0.36 4 1.4 0.21 

0.00059 4 1.6 1.4 4 1.7 0.48 

0.0054 4 6.9* 3.8 4 0.75 0.20 

0.052 4 3.7 1.8 4 1.6 0.63 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 

* Significantly increased relative to the negative control (Dunnett’s, p < 0.05).
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Table 20: Plasma Sex Steroids in 

Oryzias latipes (Not Measured).

Treatment 

(mg a.i./L) 

[measured]

Plasma Testosterone (T) Plasma 17ȕ-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 

Negative Control 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A 0.00059 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A 0.0054 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A 0.052 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 
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Aside from the male anal fin papillae, no secondary sex characteristics were reported (Table 21). 

Table 21: Secondary Sex Characteristics and Clinical Signs in Oryzias latipes.

Treatment 

(mg a.i./L) 

Mean-measured 

Secondary Sex Characteristics and Clinical Signs1

Males Females 

Type n Incidence Type n Incidence 

Negative Control None 20 N/A None 20 N/A 0.00059 None 20 N/A None 20 N/A 0.0054 None 20 N/A None 19 N/A 0.052 None 19 N/A None 20 N/A 

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

The study authors analyzed survival, sex-specific growth (standard length and wet weight), fecundity, fertility, sex-specific hepatic VTG gene expression, occurrence 

and number of anal fin papillae, gonad development stage, and gonadal histology (incidence of severity of gonad abnormalities). Unless otherwise noted, the unit of statistical analysis was the replicate test vessel. If responses appeared to be monotonic a step-down Jonckheere-Terpstra trend test was used to evaluate possible 
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trends in the ranks of replicate means to determine concentration responsive trends. All endpoints, except survival, were analyzed by performing pair-wise comparisons 

using Dunnett’s multiple comparison test to determine which treatment groups differed statistically from the control. For survival, the most suitable unit of statistical analysis was the individual animal. Therefore, survival was analyzed using Fishers Exact test. Analyses were conducted at Į = 0.05 using SAS software. 

The study authors reported no significant treatment-related effects on survival, growth, reproduction, secondary sex characteristic, or gonad histopathology of Japanese medaka following 35 days of exposure to atrazine. These results would suggest that atrazine does not interfere with the HPG axis. 

 C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival, sex-specific growth and VTG fold change, fertility, and fecundity using CETIS statistical software version 

1.8.7.12 with database backend settings implemented by EFED on 3/25/14. Data were tested for normality using the Shaprio-Wilk’s test (Į = 0.01) and for homogeneity of variance using Levene’s test (Į = 0.01). Survival data failed to meet these assumptions and were therefore analyzed using the Mann-Whitney U 

Two-Sample test. All other endpoints met these assumptions and were analyzed using analysis of variance followed by the Dunnett Multiple comparison post-hoc 

test. Female body length data exhibited a suggestive monotonically increasing trend and were therefore also analyzed using the Jonckheere-Terpstra Trend test. Unless specified otherwise, all analyses were conducted at Į = 0.05 using the mean-measured concentrations. 

 Conclusions: 

The Jonckheere-Terpstra Trend test detected a significant increase in female body length at the highest treatment level (p = 0.0151). However, this increase was 

slight (3.44% increase). There was a significant increase in male VTG gene expression fold change at middle dose level (Dunnett, p = 0.0090), though this 

increase did not follow a dose-dependent response. Overall, it does not appear that atrazine interferes with the HPG axis of Japanese medaka. 
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Table 22: Reproductive and HPG Endpoints1,2 for Male Oryzias latipes in the FSTRA with Atrazine. 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17 ȕ-estradiol. 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) 

Mean-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 

Negative Control -- -- -- -- -- -- -- -- -- -- -- 0.00059 -- -- -- -- No 19.7 0.99 -- -- -- -- 0.0054 -- -- -- -- No 398 0.0090 -- -- -- -- 0.052 -- -- -- -- No 178 0.29 -- -- -- -- Statistical Test -- -- -- Dunnett -- -- 
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Table 23: Reproductive and HPG Endpoints1,2 for Female Oryzias latipes in the FSTRA with Atrazine. 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-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) 

Mean-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 

Negative Control -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 0.00059 -6.5 0.33 -1.3 0.89 -- -- -- -- No 20.5 0.66 -- -- -- -- 0.0054 -1.6 0.96 1.4 0.86 -- -- -- -- No -46.0 0.12 -- -- -- -- 0.052 -4.2 0.65 1.3 0.88 -- -- -- -- No 8.2 0.96 -- -- -- -- Statistical Test Dunnett Dunnett -- -- -- Dunnett -- -- 
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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Atrazine.

Treatment 

(mg a.i./L) 

Mean-measured

Body Weight Length 

Males Females Males Females 

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

Negative Control -- -- -- -- -- -- -- -- 

0.00059 -3.2 0.89 -2.4 0.93 0.52 0.98 0.66 0.28 

0.0054 7.0 0.47 -0.4 1.0 1.7 0.60 1.4 0.19 

0.052 -0.54 1.0 2.9 0.90 1.0 0.86 3.4 0.015 

Statistical Test Dunnett Dunnett Dunnett Jonckheere 

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 

Fertilization success averaged 91.6% in the negative control, which is lower than the recommended validity 

criterion specified in OCSPP 890.1350 (95%).

F. Reviewer’s Comments 

The reviewer’s results agreed with those reported by the study authors. 

A non-GLP reproduction trial was conducted prior to the definitive study to evaluate two different pairing designs. 

The trial was conducted to mimic the length of a definitive study (4 weeks) using groups consisting of either 1 

male and 4 females (1M:4F) as used in Papoulias et al. (2014) or 5 males and 5 females (5M:5F). Exposure 

was to dilution water only. Fecundity and fertility were monitored during the 4 week reproductive trial. Twelve 

replicates for each pairing type were initiated and the first 7 days of the trial mimicked the pre-exposure period. 

The top 8 performers were then carried on to mimic the last 21-day exposure period. Fecundity in the 1ML4F 
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and 5M:5F pairing designs averaged 30.7 and 39.6 eggs per female per reproductive day, respectively, and 

fertility averaged 82 and 91%, respectively. 

Results from the periodic screening analysis of the dilution water indicated the presence of the following analytes: 

arsenic (19.1 mg/kg), calcium (627 mg/kg), chloride (1800 mg/kg), copper (7.90 mg/kg), fluoride (34.2 

mg/kg), iron (538 mg/kg), magnesium (2280 mg/kg), manganese (7.95 mg/kg), mercury (0.125 mg/kg), 

potassium (7670 mg/kg), selenium (2.86 mg/kg), sodium (2120 mg/kg), sulfate (240 mg/kg), and zinc (82.9 

mg/kg). 

The in-life portion of the definitive toxicity test was conducted from December 12, 2014 to January 16, 2015. 

III. REFERENCES 

Papoulias DM, Tillitt DE, Talykina MG, Whyte JJ, Richter CA. 2014. Atrazine reduces reproduction in Japanese medaka 

(Oryzias latipes). Aquatic Toxicology (2014). http://dx.doi.org/10.1016/j.aquatox.2014.05.022. 

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc., Cary, North Carolina.