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

EPA MRID Number 49694001



# Version: 22 September 2011 Page 1 R f 46 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'

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

EPA MRID Number 49694001



# Version: 22 September 2011 Page 2 R f 46 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 Unpublished study performed by Wildlife International, Easton, Maryland.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.

D

i s

c

l a

i m

e

r : 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 Atrazine

EPA MRID Number 49694001



# Version: 22 September 2011 Page 3 R f 46 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).Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page4Rf46Results Synopsis:Test organism age at test initiation: 4.5 monthsMean body weight at test initiation (if measured): 0.23 g for males, 0.23 g for femalesMean length at test initiation (if measured): Not measuredTest type: flow-throughTable 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Atrazine.Treatment(mg a.i./L)Mean-measuredFecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.00059No No -- -- -- -- No No No No -- -- -- --0.0054No No -- -- -- -- No No Yes No -- -- -- --0.052 No No -- -- -- -- No No No No -- -- -- --Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page5Rf46Abbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.

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

EPA MRID Number 49694001



# Version: 22 September 2011 Page 6 R f 46 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

EPA MRID Number 49694001



# Version: 22 September 2011 Page 7 R f 46 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 conditionsData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page8Rf46B. Test OrganismTable 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: Japanese medaka 

# EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: 

# Oryzias latipesSpecies strain (if stated): Not applicableWere fish obtained from a singlelaboratory stock?Yes Fish were originally obtained from OsageCatfisheries, Inc, Osage Beach, Missouri. 

# EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameas 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 mortalityduring the 7 days prior topre-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 mortalityData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page9Rf46ParameterValue(s)Details or RemarksGuideline Recommendationsother fish showed anysigns of disease or stressand therefore fish did notreceive treatment fordisease during theacclimation periods, thetwo-week pre-exposureperiod, or the exposureperiod. 

# 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( 

# Artemiasp.) 

# 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, SaltLake City, UtahFrequency of feeding: Two to three times/dayDetails on feeding: Fish were fed at a ratesufficient to promoteactive reproduction andmaintain body condition.Fish were not fed for atUneaten food and fecal material wereremoved from the test vessels daily bysiphoning.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page10Rf46ParameterValue(s)Details or RemarksGuideline Recommendationsleast 12 hours prior totest termination. Feedingrates in each replicatewere adjusted weekly, ifnecessary, to account formortality.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: 4.5 months 

# EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):0.23 g 

# EPA recommends thata subsampleof fish be weighed before the test to estimatethe mean weight for each sex. It isrecommended that the individual weight ofeach fish selected for the test be within ±20%of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):Not reported The range of individual weights at the startof the pre-exposure period was±20% of themean weight of each sex.Mean weight of females at testinitiation (if determined):0.23 gData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page11Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsRange of individual weights(females) at test initiation (ifdetermined):Not reported The range of individual weights at the startof the pre-exposure period was±20% of themean weight of each sex.Mean length of males at testinitiation (if determined):Not determinedMean length of females at testinitiation (if determined):Not determinedDuration of pre-exposure phase: 7 days 

# EPA recommends a minimum of 14 days.Were pre-exposure conditionsidentical 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 thatbreeding groups with a proven history ofspawning could be selected for inclusion inthe 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: 5Number of females per tank: 5Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page12Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsPre-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 wasconducted underconditions as similar aspossible to those usedduring the exposureperiods.A replicate was considered suitable for usein the test if there were at least 10 eggs perfemale per reproductive day, and spawningin that vessel occurred at least twice duringthe 7 days period. Any vessels withmortalities, incorrect sex ratio, or poorreproductive performance were excludedfrom use in the test.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page13Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure: Flow-through 

# 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.Type of flow-through dilutionsystem:Continuous-flow diluter 

# Intermittent flow proportional diluters or continuous flow serial diluters are

recommended. 2Flow-through rate: 48.6 mL/minThis was calculated based on a 10-L fill volumeand a flow-rate sufficient to provide 7 volumeadditions 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 forflow-through systems:Fluid metering pumps wereused to deliver volumes ofa single test substancestock solution to mixingThe proportion of the test and solution that waspumped into each replicate test vessel waschecked prior to the test and approximatelyweekly 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) A1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram 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).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page14Rf46ParameterValue(s)Details or RemarksGuideline Recommendationschambers randomlyassigned to each treatment,where it was mixed withwell water. The flow ofdilution water wascontrolled using rotamaters.varied by no more than±10% of the mean flowfor the replicates. Delivery was initiated sevendays prior to the introduction of test organisms.The general operation of the exposure systemwas checked visually at least two times duringthe test at least once on the last day of thetest. 

# 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 froman on-site well. Water waspassed through a sand filterto remove particles greaterthan approximately 25μm,pumped into a storagetank, and aerated. Waterwas then filtered to 0.45μm 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page15Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes. See Reviewer’sComments 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 LDepth of test water was approximately 12.7 cm. 

# EPA recommends 10 L solution per tank.Spawning substrate material: No spawning substrateswere specifiedDuring observations for reproduction, eggclumps were gently removed from nettedfemales and/or from the aquarium bottom eachmorning. 

# 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/AAdditional details on exposuresystem:N/AData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page16Rf46Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C) 23.9 25.7 25.1Measured in eachvessel weekly, andcontinuously in oneneg control replicate 

# EPA recommends temperature 25±1 o

# C; interreplicate and inter-treatment differentials should not exceed 1 o 

# C.pH 7.9 8.4 8.1Measured in eachvessel weekly 

# EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L) 7.4 8.2 8.0Measured in eachvessel weekly 

# EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 140 180 166Measured in onereplicate of thenegative control andthe highestconcentrationtreatment groupweekly during the test,with measurementstypically alternating 

# EPA recommends total alkalinity > 20 mg/L

as CaCO 3.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page17Rf46ParameterMinimumMaximumMeanMeasurement IntervalGuideline Recommendationsbetween replicates.Hardness (mg/L as CaCO3) 124 140 133Measured in onereplicate of thenegative control andthe highestconcentrationtreatment groupweekly during the test,with measurementstypically alternatingbetween replicates.Total organic carbon (mg/L) <14 weeks prior to testinitiation 

# 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 P g/L.Residual chlorine (Pg/L) Not Reported 

# EPA recommends that residual chlorine in dilution water be < 10 P g/L.Conductivity (μS/cm) 275 452 346 Measured in one 

# General recommendations for frequency ofData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page18Rf46ParameterMinimumMaximumMeanMeasurement IntervalGuideline Recommendationsreplicate of thenegative control andthe highestconcentrationtreatment groupweekly during the test,with measurementstypically alternatingbetween 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:NANot applicable.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding StudyConditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?NoNon-GLP preliminary trials were conductedto determine the optimal male:female pairing.See Reviewer’s Comments. 

# EPA recommends conducting a range-finder if

96-hour LC 50 

# data for the fathead minnow are

unavailable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page19Rf46If yes, what was the method fordetermining the highest testconcentration 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/ALife 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, orData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page20Rf46 

# 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.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:35 days 

# EPA recommends that the duration of the definitive test be 21 days.Method for selecting thehighest test concentration inthe definitive test:Nominal concentrations for the definitive studywere determined from the findings in Papoulias etal. (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 LC 50 

# ), whichever concentration is lowest.Reference study citation (ifapplicable):Papoulias DM, Tillitt DE, Talykina MG, Whyte JJ,Richter CA. 2014. 

# Atrazine reduces reproduction in Japanese medaka (Oryzias latipes). AquaticToxicology (2014),http://dx.doi.org/10.1016/j.aquatox.2014.05.022.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page21Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsSeparation of testconcentrations:0.1 

# EPA suggests that a concentration separation of between 0.33 (or threefold) and 0.1 (or ten-fold) is scientifically acceptable 1 

# .Number of testconcentrations:3 

# EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?No The reviewer corrected the nominaland mean-measured concentrationsfor the purity of the test material.The standard curve used inanalyzing the analytical sampleswas prepared without correcting forthe purity. Therefore, the calculatedmeasured concentrations were notcorrected for the purity.Indicate the type of valuespresented for measuredconcentrations:Mean-measured concentrationsData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page22Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsLimit 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 ReportedFrequency 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 randomizedcomplete 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page23Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates incontrol:4 

# EPA recommends 4 replicates.Number of replicates insolvent 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 testitem treatment level:4 

# EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:5 

# EPA recommends 2 males per

replicate.Number of female fish perreplicate at test initiation:5 

# EPA recommends 4 females per

replicate.Was a solvent used? NoSolvent type (if applicable): N/AMaximum solventconcentration (if applicable):N/A 

# EPA recommends that the solvent not

exceed 0.02 ml/L3 

# . OECD3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page24Rf46ParameterValue(s)Details or RemarksGuideline 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? NoPositive control (ifapplicable):N/APositive controlconcentration(s) (ifapplicable):N/APhotoperiod: 16 hrs light :8 hrs darkA 30-minute transition period oflow-light intensity was included. 

# EPA recommends photoperiod 16:8

(light:dark).Light intensity at water’ssurface:536-940 lux 

# EPA recommends light intensity 540 – 1080 lux (at water’s surface).Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page25Rf46ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details: NoneTable 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Atrazine.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative 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/ATreatment 2 0.0049 0.0054 4.81 N/ATreatment 3 0.049 0.052 2.42 N/AAbbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints: Daily- survival, general observations (sub-lethal effects), fecundity, fertilityTest termination- sex-specific length and wet weight, liver VTG, necropsy and secondary sex characteristics, and histology andhistopathologyData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page26Rf46Were 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 clinic al signs occur at least daily. At test termination (day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, p lasma 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 beha vior.II. RESULTS AND DISCUSSIONA.ResultsAfter 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 mga.i./L treatment groups, respectively. One female died in the mean-measured 0.0054 mg a.i./L treatment group, and one male diedin the mean-measured 0.052mg a.i./L treatment group (Table 9). No significant differences were reported.Table 9: Adult Fish Survival in 

# O r y z i a s l a t i p e s.Treatment (mg a.i./L)Mean-MeasuredMalesFemalesData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page27Rf46Abbreviations:NANot 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, 

# p0.05). However, a significant increase in female length was detected at the top level relative to the negative control based on the JonckheereTerpstra Trend test ( 

# p0.05).This increase was slight (~4%) and considered to be within the range of biological activity.n# Surviving% Survivaln# Surviving% SurvivalNegative Control20 20 100 20 20 1000.0005920 20 100 20 20 1000.005420 20 100 20 19 950.052 20 19 95 20 20 100Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page28Rf46Table 10: Size at Test Termination in 

# O r y z i a s l a t i p e sTreatment(mg a.i./L)Mean-measuredBody WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDNegative Control 4 0.46 0.02 4 0.62 0.04 4 29 0.7 4 31 0.80.00059 4 0.45 0.03 4 0.600.04 4 29 0.6 4 31 0.80.0054 4 0.49 0.05 4 0.610.03 4 30 0.8 4 31 0.60.052 4 0.46 0.04 4 0.63 0.05 4 29 0.6 4 32a0.6Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.aSignificantly increased relative to the negative control based on the Jonckheere-Terpstra Trend test ( 

# p0.05).

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

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# Version: 22 September 2011 Page 29 R f 46 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 O r y

z

i a

s l

a

t i p

e

s

.

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 Fecundity 1 

# 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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# Version: 22 September 2011 Page 30 R f 46 Table 12: Anal Fin Papillae Count in O r y

z

i a

s l

a

t i p

e

s

.

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation.

Gonado-somatic index was not assessed.

Table 13: Gonado-Somatic Index (GSI) in O r y

z

i a

s l

a

t i p

e

s

.

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 GSI 1



# (%) ±SD n Mean GSI 1



# (%) ±SD

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

0.00059 4 -- -- 4 -- --

0.0054 4 -- -- 4 -- --

0.052 4 -- -- 4 -- --

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# Version: 22 September 2011 Page 31 R f 46 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 O r y

z

i a

s l

a

t i p

e

s

.

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 inTreatment (mg a.i./L) Meanmeasured Males Females

n Median Stage 1 

# n Median Stage 2



# 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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# Version: 22 September 2011 Page 32 R f 46 primary or secondary guideline diagnoses.

Table 15: Gonadal Histopathology in Male O r y

z

i a

s l

a

t i p

e

s

.

Treatment (mg

a.i./L)

Mean-measured Diagnostic Observations 1



# 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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# Version: 22 September 2011 Page 33 R f 46Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page34Rf46Table 16: Additional Gonadal Histopathology Observations in Male 

# O r y z i a s l a t i p e s.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page35Rf46Treatment (mg a.i./L)Mean-measuredAdditional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A1 20 N/A 20 N/A 20N/A 20 N/A 20 N/A2 20 N/A 20 N/A 20N/A 20 N/A 20 N/A3 20 N/A 20 N/A 20N/A 20 N/A 20 N/A4 20 N/A 20 N/A 20N/A 20 N/A 20 N/A0.00059 0 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A1 20 N/A 20 N/A 20N/A 20 N/A 20 N/A2 20 N/A 20 N/A 20N/A 20 N/A 20 N/A3 20 N/A 20 N/A 20N/A 20 N/A 20 N/A4 20 N/A 20 N/A 20N/A 20 N/A 20 N/A0.0054 0 20 N/A 20 N/A 20 N/A 20 N/A 20 N/A1 20 N/A 20 N/A 20N/A 20 N/A 20 N/A2 20 N/A 20 N/A 20N/A 20 N/A 20 N/A3 20 N/A 20 N/A 20N/A 20 N/A 20 N/A4 20 N/A 20 N/A 20N/A 20 N/A 20 N/AData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page36Rf46Treatment (mg a.i./L)Mean-measuredAdditional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.052 0 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A1 19 N/A 19 N/A 19N/A 19 N/A 19 N/A2 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A3 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A4 19 N/A 19 N/A 19 N/A 19 N/A 19 N/A1Gonadal 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 testguideline for reference.

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# Version: 22 September 2011 Page 37 R f 46 Table 17: Gonadal Histop athology in Female O r y

z

i a

s l

a

t i p

e

s

.

Treatment (mg a.i./L)

Mean-measured Diagnostic Observations 1



# 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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# Version: 22 September 2011 Page 38 R f 46 Table 18: Additional Gonadal Histop athology Observations in Female O r y

z

i a

s l

a

t i p

e

s

.

Treatment (mg

a.i./L)

Meanmeasured Additional Diagnostic Observations 1



# 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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# Version: 22 September 2011 Page 39 R f 46 

# seen on reproductive endpoints, secondary sex characteristics, or gonadal histopathology.

Plasma sex steroids were not measured (Table 20).

Table 19: Plasma Vitellogenin in O r y

z

i a

s l

a

t i p

e

s

.

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).Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page40Rf46Table 20: Plasma Sex Steroids in 

# O r y z i a s l a t i p e s(Not Measured).Treatment(mg a.i./L)[measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A0.00059 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A0.0054 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/A0.052 4 N/A N/A 4 N/A N/A 4 N/A N/A 4 N/A N/AAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page41Rf46Aside from the male anal fin papillae, no secondary sex characteristics were reported (Table 21).Table 21: Secondary Sex Characteristics and Clinical Signs in 

# O r y z i a s l a t i p e s.Treatment(mg a.i./L)Mean-measuredSecondary Sex Characteristics and Clinical Signs1MalesFemalesTypenIncidenceTypenIncidenceNegative ControlNone20N/A None20 N/A0.00059None20N/A None20 N/A0.0054None20N/A None19 N/A0.052 None 19 N/A None 20 N/A1Define 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 asnecessary. Note that nuptial tubercle score is reported previously inTable 12.B.Study Author’s Analysis and ConclusionsThe study authors analyzed survival, sex-specific growth (standard length and wet weight), fecundity, fertility, sex-specific hepatic VTG gene expression, occurrenceand number of anal fin papillae, gonad development stage, and gonadal histology (incidence of severity of gonad abnormalities).Unless otherwise noted, the unit ofstatistical analysis was the replicate test vessel. If responses appeared to be monotonic a step-down Jonckheere-Terpstra trendtest was used to evaluate possibleData Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page42Rf46trends in the ranks of replicate means to determine concentration responsive trends. All endpoints, except survival, were analyzed by performing pair-wise comparisonsusing Dunnett’s multiple comparison test to determine which treatment groups differed statistically from the control. For survival, the most suitable unit of statisticalanalysis was the individual animal. Therefore, survival was analyzed using Fishers Exact test. Analyses were conducted atĮ= 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 Japanesemedaka 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 ConclusionsStatistical Methods:The reviewer analyzed survival, sex-specific growth and VTG fold change, fertility, and fecundity using CETIS statistical software version1.8.7.12 with database backend settings implemented by EFED on 3/25/14. Data were tested for normality using the Shaprio-Wilk’stest (Į= 0.01) and forhomogeneity of variance using Levene’s test (Į= 0.01). Survival data failed to meet these assumptions and were therefore analyzed using the Mann-Whitney UTwo-Sample test. All other endpoints met these assumptions and were analyzed using analysis of variance followed by the DunnettMultiple comparison post-hoctest. Female body length data exhibited a suggestive monotonically increasing trend and were therefore also analyzed using theJonckheere-Terpstra Trend test.Unless specified otherwise, all analyses were conducted atĮ= 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 wasslight (3.44% increase). There was a significant increase in male VTG gene expression fold change at middle dose level (Dunnett, 

# p= 0.0090), though thisincrease did not follow a dose-dependent response. Overall, it does not appear that atrazine interferes with the HPG axis of Japanese medaka.Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page43Rf46Table 22: Reproductive and HPG Endpoints1,2for Male 

# O r y z i a s l a t i p e sin the FSTRA with Atrazine.Abbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at 

# p< 0.05.Treatment(mg a.i./L)Mean-measuredTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Median 

# p% Diff. 

# pEffect?(Yes/No)% Diff. 

# p% Diff. 

# p% Diff. 

# pNegative Control-- -- -- -- -- -- -- -- -- -- --0.00059-- -- -- -- No 19.7 0.99 -- -- -- --0.0054-- -- -- -- No 398 0.0090 -- -- -- --0.052-- -- -- -- No 178 0.29 -- -- -- --Statistical Test -- -- -- Dunnett -- --Data Evaluation Record on the Fish Short-Term Reproduction Assay with AtrazineEPA MRID Number 49694001Version: 22 September 2011Page44Rf46Table 23: Reproductive and HPG Endpoints1,2for Female 

# O r y z i a s l a t i p e sin the FSTRA with Atrazine.Abbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at 

# p< 0.05.Treatment(mg a.i./L)Mean-measuredFecundityFert. SuccessTubercle ScoreGSIGonadal Stagingand Histo.Plasma VTGPlasma TPlasma E2%Diff. 

# p%Diff. 

# pMedian 

# p%Diff. 

# pEffect? (Yes/No)%Diff. 

# p%Diff. 

# p% Diff. 

# pNegative 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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# Version: 22 September 2011 Page 45 R f 46 Table 24: Growth Endpoints 1,2 

# 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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# Version: 22 September 2011 Page 46 R f 46 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.