## Data Evaluation Record

ATRAZINE PC Code 080803

EPA Contract No. EP10H001452

Task Assignment No. 4-4-2013

Study Type: Non-Guideline

Citation: Papoulias, D. M., Tillitt, D. E., Talykina, M. G., Whyte, J. J., and Richter, C. A. (2014). Atrazine reduces reproduction in Japanese medaka ( Oryzias latipes ). Aquat Toxicol . 154:230-239.
Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency One Potomac Yard 2777 S. Crystal Drive Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713
Primary Reviewer: Rebecca L. Bryan, B.S. Signature: Date: 11/24/2014 Secondary Reviewer: Kelly Luck, M.S. Signature: Date: 12/8/2014 Program Manager: Jack D. Early, M.S. Signature: Date: 12/15/2014

Disclaimer
This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel.ATRAZINE / 080803 Non-Guideline/Page 1 of 14 EPA Primary Reviewer: Date: _____________ EPA Secondary Reviewer: Date: _____________


### Data Evaluation Record

TXR NO: Not provided

STUDY TYPE: Non-guideline

DP BARCODE: Not provided

PC CODE: 080803

CAS NO.: 1912-24-9

MRID NO.: Not available
TEST MATERIAL (% purity): Atrazine (98%)

CITATION: Papoulias, D. M., Tillitt, D. E., Talykina, M. G., Whyte, J. J., and Richter, C. A. (2014). Atrazine reduces reproduction in Japanese medaka ( Oryzias latipes ). Aquat Toxicol . 154:230-239.

SPONSOR: Not applicable (open literature review)

EXECUTIVE SUMMARY: The purpose of this study was to evaluate the effects of atrazineexposure on egg production and additional reproductive effects along the hypothalamuspituitary-gonad axis of male and female medaka fish (Oryzias latipes) . The biological endpoints included population effects based on egg production, histological effects on reproductive stage and gonad pathology, and biochemical effects on aromatase activity and sex hormone production.
Methods: Mature virgin medaka fish ( Oryzias latipes ) were exposed to atrazine at 0.5, 5.0, and 50 μ g/L (equivalent to 2.3, 23.2, and 231 nM, respectively) in a static renewal system with a 0.02% acetone control for comparison. Atrazine (98% purity; Fluka Chemical, Dorset, UK) stock solutions were prepared in 60% acetone:40% water solution. The fish were obtained from the Columbia Environmental Research Center stock with initial mean weights of 0.57 g for females and 0.64 g for males. The fish were acclimated for one week prior to testing, at which point all females had produced some eggs. One male and four female fish were randomly assigned to each 6.5-L exposure tank, and each treatment group included six replicate tanks for each of the three observation time points, Days 0, 14, and 38. The test water was renewed every 24 hours. Fish were fed live brine shrimp nauplii twice daily, and tanks were siphoned clean once daily. The fish were maintained at 25±1°C with a 14 hour light:10 hour dark photoperiod. Tanks and plumbing were made of glass or Teflon to minimize chemical inferences or adsorption of atrazine. The sides of each tank were covered with opaque contact paper to preventATRAZINE / 080803 Non-Guideline/Page 2 of 14 interactions of fish tank to tank. The atrazine test concentrations were measured weekly using enzyme-linked immunosorbent assay kits, and confirmatory analysis of atrazine stock preparations, and selected tank water samples were performed using GC/NPD analysis. Weekly water quality measurements for oxygen, pH, hardness, alkalinity, and total ammonia were conducted.
Adult mortality and egg production were determined daily. Eggs were collected from each tank (by stripping eggs from females or by examining siphoned waste water), placed in petri dish with exposure water, held in incubator at 25±1°C for 24 hours, and then inspected for viability and counted. On collection days 0, 14, and 38, fish were euthanized with tricaine methanesulfonate and weighed prior to histological and biochemical assays; for all tanks, fish were collected for euthanasia during the same time period (19:00 to 24:00) to minimize the diurnal effects of reproductive hormones. The brain, liver and gonads were collected, and the liver and gonads were weighed. Aromatase protein content was measured from the fresh brain and ovary samples of two female fish from each replicate of each treatment group, using a titrated water assay (Orlando et al. 2002 1 ). The 17 ȕ -estradiol and testosterone levels in eviscerated carcasses were measured by RIA, and the E/T ratio was calculated. The gonad somatic index (GSI) was also calculated.
Histological examinations of medaka gonads were conducted to evaluate reproductive stage and pathological lesions. For each post-exposure collection time point (Days 14 and 38), one whole ovary from one female from each replicate of each treatment and the testis of one male from three replicates of each treatment were preserved in Davidson’s solution and prepared using standard techniques. Ovaries sections were stained with hematoxylin and eosin, and testes sections were stained with Giemsa or hematoxylin/eosin for histological analysis using a compound microscope. Two ovary sections for each fish were evaluated for oogenic stages, which were identified as pre-vitellogenic (stage II), early vitellogenic [stage III; central germinal vesicle (GV)], mid-late vitellogenic (stage IV ; GV moving toward animal pole), and mature (stage V; GV at the animal pole). The oocytes in each section were counted, and the percentages and average were determined for each fish based on the two sections. The testis sections (serial sections of at least one-half of each testis) were evaluated for development stage and pathological lesions. The testis reproductive stages were identified as: tubules containing spermatogonia singly or in clusters predominate (stage II); spermatocytes prevalent (stage III); mostly spermatids with some spermatozoa in duct (stage IV); and duct greatly expanded and filled with spermatozoa (stage V). Clastogenic abnormalities (e.g., bridges, chromosome fragments, micronuclei) were counted for all stages of mitotic spermatogonia but primarily at anaphase and telophase. The percent of cells with chromosomal abnormalities was calculated using 1000 spermatogonial cells in two testicular lobes of each fish and averaged per fish. Abnormalities were visually observed using oil immersion and a compound microscope at 1000x magnification.
Statistical analyses were conducted using SAS statistical software. Cumulative mean egg counts per tank were compared using mixed model procedure with repeated measures, and the

1 Orlando, E.F., Davis, W.P., and Guillette Jr., L.J. (2002) Aromatase activity in the ovary and brain of the eastern mosquito fish (Gambusia holbrooki) exposed to paper mill effluent. Environ. Health Perspect . 110: 429-433.ATRAZINE / 080803 Non-Guideline/Page 3 of 14 differences between control and treatments were determined using least square means. Mean egg production rates (eggs/tank with spawn/day) were compared using GLIMMIX procedures with a Poisson distribution. Weekly spawning rates (number of tanks with eggs/treatment/week) were compared using GLIMMIX procedures with a logit distribution. The total number of spawns per treatment was compared using GLIMMIX procedures and the likelihood of spawning with a binomial distribution. Comparisons of steroid concentrations, aromatase activity in females, abnormal spermatogonia, histological abnormalities, and GSI were conducted using GLM procedures with least square means comparisons for analysis of variance or co-variance, or Kruskal-Wallis non-parametric analysis of ranks when assumptions of normality or heterogeneity of variances were violated. Ratio data were log transformed and proportional data were arcsine transformed for analysis. Data for abnormal spermatogonia were analyzed using a Student's t-test

. When no differences were found, the data from fish collected at different times for a treatment were pooled for analysis. Comparisons between the controls and treatments of the mean percent of pre-vitellogenic oocytes in ovary sections were conducted using an odds ratio and chi-square statistic. Significance was determined at p 0.05.
Results: Mean atrazine concentrations in tank water over the 38-day exposure were <0.05 (below detection limit), 0.56, 5.63, and 51.5 μ g/L for the nominal 0, 0.5, 5.0, and 50 μ g/L atrazine dose groups, respectively. No adult mortality was observed during the study.
Total egg production was lower (36-42%) in all atrazine-exposed groups compared to the controls as represented in Figure 1 (copied without alteration from Papoulias et al. 2014). Significant (p<0.05) decreases in cumulative egg production were observed beginning on Day 24 for the 0.5 and 5.0 μ g/L treatment groups and on Day 25 for the 50 μ g/L treatment groups and remained different until end of study. Weekly egg production rates were significantly reduced in all atrazine treatments for Weeks 3-6 compared to control, as represented in Figure 2 (copied without alteration from Papoulias et al. 2014) . Reduced daily egg production rates were observed in the atrazine-treated groups compared to controls, however, the differences were not statistically significant (Figure 3, copied without alteration from Papoulias et al. 2014). There were no significant differences between the control and treatment groups for mean percent mortality (±SD) of embryos 24 hours after collection (38±18 in control vs. 44±23, 41±19, and 45±20 for the 0.5, 5.0 and 50 μ g/L, respectively).ATRAZINE / 080803 Non-Guideline/Page 4 of 14

Figure 1

. Mean cumulative egg production (number/tank) of medaka exposed to atrazine . Cumulative egg counts were compared with SAS Statistical Software GLM procedure and least square means (p = 0.05). The first day in which cumulative egg numbers were significantly different from controls is designated with an asterisk.

Figure 2

. Mean±SE egg production (eggs/tank with a spawning event/day) of medaka exposed to atrazine . Mean egg production was calculated as the mean of the daily number of eggs produced per tank with a spawning event over a given week. Statistical analysis of mean egg production rates was conducted with SAS statistical software using GLIMMIX procedure with a Poisson distribution and a critical value of p= 0.05. The weeks for which daily egg production in treated tanks was significantly different from controls is designated with an asterisk.ATRAZINE / 080803 Non-Guideline/Page 5 of 14

Figure 3

. Frequency histograms of daily egg production in medaka exposed to four concentrations of atrazine . Ranges of egg production rates (X-axis) reflect the means of daily tank production counted over the 38-d experimental period (Y-axis).

No significant effects were observed for the male end points of steroid hormone concentrations, E/T ratios, or GSI values (Table 1 copied without alteration from Papoulias et al. 2014, Table 2). For testes staging, all fish at all time periods were found to be at stage III or IV. All control fish sampled at Days 14 and 38 were at stage IV. However, for the atrazine treatment groups, a maximum of one out of three sampled fish at Day 14 were at stage IV and two of three sampled fish at Day 38 were at stage IV. The percent of abnormal mitotic events based on aberrant spermatogonia was significantly (p<0.01) greater than controls for all atrazine treatment groups (sampling points combined). Minimal testicular lesions were observed for two out of ten fish in each of the control and 0.5 μ g/L groups, and minimal or moderate lesions were observed for ten out of twelve fish in each of the 5.0 and 50 μ g/L groups. Observed lesions were germ cell syncytia and tissue degeneration.ATRAZINE / 080803 Non-Guideline/Page 6 of 14

Table 1 . Male end points: mean concentrations (SD) of estradiol (E 2 ) and testosterone ( T ), E/T ratio, gonad somatic index (GSI), aberrant spermatogonia and testis histopathology in medaka exposed to atrazine for 14 or 38 days. Values with asterisks ( *) are significantly different from controls (p  ).

No significant effects were observed for the female end points of steroid hormone concentrations, E/T ratios, GSI values, or the gonad and brain aromatase protein content at 14 or 38 days (Table 2 copied without alteration from Papoulias et al. 2014, Table 3). There were significantly (p<0.01) fewer pre-vitellogenic oocytes and more vitellogenic oocytes in the 0.5 and 5.0 μ g/L atrazine groups at 38 days compared to controls. No pathological lesions were observed in the ovaries.

Table 2 . Female end points: mean concentrations (SD) of estradiol (E2), testosterone (T) , E/T ratio, gonad somatic index (GSI), egg staging, and gonad and brain aromatase activity in medaka exposed to atrazine for 14 or 38 days. Values with an asterisk ( *) are significantly different than control values.ATRAZINE / 080803 Non-Guideline/Page 7 of 14

Conclusions: The study authors concluded that significant reductions observed in total egg production for atrazine treatment groups were the result of the reduced number of eggs ovulated by females in atrazine treated tanks. The atrazine treated males had a greater number of abnormal germ cells. There were no effects due to atrazine exposure for the gonadosomatic index, aromatase protein levels, or whole body 17 ȕ -estradiol or testosterone levels. The results suggest that atrazine reduces egg production through alteration of final maturation of oocytes.
The study was conducted at the U.S. Geological Survey, Columbia Environmental Research Center (Columbia, MO) and the Institute of Biology of Inland Waters, Russian Academy of Sciences (Yaroslavl, Russia). The Good Laboratory Practices status of this research is unknown.
The reviewer agrees with the study authors’ conclusions. The study is of sufficient quality to include qualitatively in the risk assessment.

Assessing Quantitative Utility of Reproduction Endpoints for FIFRA Ecological Risk Assessment:
Primary Reviewer: Date: Frank T. Farruggia, PhD. OPP/EFED/ERB3
Secondary Reviewer: Date: Colleen M. Rossmeisl, DVM, MS; OPP/EFED/ERB3

Reviewer Conclustions: The reviewers confirmed that biologically and statistically significant reductions in fecundity were observed for the 50 ug a.i./L treatment group.
NOAEC = 5 ug a.i./L LOAEC = 50 ug a.i./L
Description of Use in Document (QUAN, QUAL, INV): Quantitative
Summary of Results and Statistics Verification by EPA Reviewers: EPA analyzed the data following the methods detailed in the recently finalized 890.2200 Medaka Extended One Generation Reproduction Test (MEOGRT) EPA guideline. This summary reflects a consensus among ORD/NHEERL/MED and OPP/EFED staff, and relies upon the 890.2200 statistical approach, which when applied to this data the results in a LOAEC of 50 ug/L.
EPA was able to replicate the statistical results obtained within the Papoulias paper. However, it should be noted that the study authors’ analyses: 1) censored (excluded) the days in which spawning did not occur from the data for specific aquaria, and 2) also included the data from a 

## 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 11:52:46 -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 12:36:35 -04'00'ATRAZINE / 080803 Non-Guideline/Page 8 of 14 subset of fish that had been sacrificed after only 11 days of reproduction. Also, days without spawning are biologically relevant for evaluating treatment effects on medaka reproduction, thus zeros within the dataset (which were excluded by the authors) are as valid as any other value.
Evaluation of the means for each treatment group shows an apparent reduction from the controls of 39.6%, 44.8%, and 49.4% in the 0.5 μ g/L, 5 μ g/L, and 50 μ g/L treatment concentrations respectively; however this observed reduction is well within the variability observed within the dataset. The large variability in the data is particularly noticeable when viewing cumulative fecundity data for all tanks plotted in one graph. In the graph it is very striking that data for 3 of the 6 control tanks were overlapping with the highly variable and overlapping data from the treatment tanks. Upon analysis as recommended in the 890.2200, the data provides weak statistical evidence of an atrazine effect on fecundity at a 50 μ g/L, with little to no evidence of an effect at 0.5 μ g/l and 5 μ g/l. A topic of concern was the lack of a dose-response even though the treatments spanned a 100-fold concentration range. Finally, it is our opinion that there is little evidence in this study of a significant treatment-related biological response below exposures demonstrated by EPA to be statistically significant. Overall, the resulting LOEC is 50 ug/L. Details of the statistical analyses and EFED/MED’s interpretation of the results using the 890.2200 procedures are presented below.
Limitations of Study contributing to uncertainty : 1. A negative control was not included in the study. Results were compared to a solvent control with acetone (0.02 %). 2. The female to male ratio was 4:1 in the study. The FSTRA EDSP Tier I and draft Tier II recommends a female to male ratio of 1:1. 3. As noted by the author, due to the diluter delivery system, complete randomization of the tank placement was not possible. 4. Plumbing and tank materials were made of both glass and Teflon; no information was provided on which material was used with controls vs. treament groups or if it was random. 5. Although tissue samples were collected at time 0, 14 and 38 days, no results were reported for time zero for these analyses. 6. The overall fecundity of the control fish in the study was low [8847 eggs produced in tanks exposed for 6 weeks (n=6), 4 females per tank (n=24), 38 day exposure = (8847÷ 24 ÷ 38 = 9.7 eggs/female/day]. While it is noted that this study used a group spawningdesign, according to the draft EDSP 890.2200 guideline (medaka extended onegeneration reproduction test (MEOGRT), USEPA 2015) which uses a paired breeding design, the control fecundity should be at least 20 eggs/pair/day. Additionally in the draft (2015) OECD MEOGRT guideline (again using a paired design), the guideline states that medaka may produce 15 to 50 eggs per day and mean daily egg production of control should exceed 15 and may be as high at 40 eggs per day. However, it is noted that this study was conducted prior to the release and during development of the draft EPA and OECD guidelines which encompassed design/culturing elements for optimizing control performance. 7. Additionally, the overall mean embryo percent mortality in the control was relatively high (38%). According to the USEPA draft guideline, fertility in the controls should beATRAZINE / 080803 Non-Guideline/Page 9 of 14 75% or greater and according to the OECD draft guidelne, fertility in controls is typically in the 90% range and should be  80%. 8. The authors did not rank each female’s reproductive strength (number of eggs produced) in the pre-treatement phase of the study in order to divide strong and weak producing females across controls and treatments. Therefore uncertainty lies in the ability to discern whether reductions in observed cumulative egg production across treatments were chemically induced or an effect of random placement of relatively poor producing females in the treatments vs. controls. To address this uncertainty the reviewers used theraw data provide by the authors to explore female egg production both pre- and postculling on day 14. Females that were culled, were compared against those that remained on study (Table 1). The results indicate that there was little difference in mean number of eggs produced per tank across the treatments or within treatments from those that remained on the study versus those that were removed on day 14.

EPA Statistical verifications:
The methods detailed in the 890.2200 Medaka Extended One Generation Reproduction Test (MEOGRT) EPA guideline for the analysis of reproduction endpoint data were applied to the data from this study. The statistical methods recommended within the guidance have been discussed across various Offices of the EPA and with external experts including members of the June 2013 FIFRA SAP on EDSP Tier 2 protocols. As a result of input from the SAP, a statistical workshop was held in December 2013 by EPA/OCSPP/OSCP with additional participants from OPP/EFED, ORD/NHEERL/MED, and the private sector. After reaching consensus at this workshop, the 890.2200 guidance was updated and was further reviewed through processes of both the EPA, including public comment, and the OECD. As a result, the guidance as a whole and more pertinently, the statistics, have been vetted and broadly accepted with the finalized version submitted to the Federal Register, as well as being accepted by OECD member countries. The reproduction data presented in Papoulias et al ., 2014 is qualitatively the same as the reproduction data produced within the MEOGRT 890.2200 protocol. In both, fecundity is recorded daily for several weeks and the unit of replication is the aquarium. While each aquarium in the MEOGRT contains a single breeding pair, and each aquarium in Papoulias et al. , 2014 contains 4 females and a single male, the difference does not change the prescribed statistical method because the data is analyzed at the level of the aquarium ( i.e. , replicate). The preferred method in the MEOGRT 890.2200 guidance recommends the following statistical analysis of fecundity data:

Evaluation of the mean number of eggs produced in the first 17 days for those that were taken off the study vs. those that were kept on for the remaining 6 weeks. Mean Eggs/Tank Tanks Removed Tanks Remaining All tanks on/off Control 11.1 11.4 11.3 0.5 11.3 6.8 8.2

5 9.6 10.6 10.8 50 11.5 15.3 15.9ATRAZINE / 080803 Non-Guideline/Page 10 of 14 “The replicate mean of the raw data should be calculated then a square root transformation applied. A one-way ANOVA on the transformed replicate means should be calculated followed by Dunnett contrasts.”
The outcome of applying the 890.2200 preferred statistical method to the Papoulias et al. 2014 fecundity data is shown in Table 3 . The analysis indicates an effect at a p-value of 0.047 in the 50 ug/L treatment. Although the means of each of the three treatments are estimated to be lower than the controls, the variances are high. The result is weak statistical evidence that the highest concentration of atrazine could cause reduced fecundity.

Table 3

. Analysis of Replicate Means of Cumulative Egg Production for each treatment relative to Controls, including zero spawn days. Columns are: the levels that are being compared (Level), the estimated difference in the levels (Estimate), the standard error ofthe estimate (StdErr), degrees of freedom (df), the value from a t-test (T-Value), the pvalue from the t-test (-Value), the p-value from a Dunnett test (AdjP), and a flag for significance level (Sig) where “*” is p-value < 0.05, “.” is p < 0.1.

Levels Estimate StdErr Df T-Value P-Value AdjP Sig
0.5 μ g/l - 0 μ g/l -1.5535 1.0437 20 -1.4885 0.0761 0.1696

.

5 μ g/l - 0 μ g/l -1.5087 1.0437 20 -1.4455 0.0819 0.1811

. 50 μ g/l - 0 μ g/l -1.8347 1.0437 20 -1.7579 0.047 0.1098 *
As an alternative method, the MEOGRT 890.2200 guidance recommends the following:
“...analyses for fecundity data consist of a step-down Jonckheere-Terpstra or Williams’ test to determine treatment effects, provided the data are consistent with a monotone concentration-response. With a step-down test, all comparisons are done at the 0.05 significance level and no adjustment for the number of comparisons made.”
The results of the alternative analysis on the Papoulias et al. fecundity data is shown in Table 4. The Jonckheere-Terpstra (JT) test confirms the previous result by finding statistically significance evidence of an effect only at the 50 ug/L treatment.
Table 4. The Jonckheere-Terpstra (JT) fecundity analysis showing the estimated test statistic for the JT (JT Statistic), the corresponding p-values for increasing and decreasing tends, and the maximum level of treatment that was included in the test. JT Statistic Decreasing Trend P-Value Increasing Trend P-Value Max Level of Treatment
72.500 0.040 0.959 50 μ g/l

36.000 0.078 0.934 5 μ g/l
Additionally, an analysis of the fecundity between each concentration level within each week was conducted. This analysis was performed using the lme4 package in R, through the use of the glmer (general linear mixed effect regression) function with a Poisson link. ‘Concentration’ andATRAZINE / 080803 Non-Guideline/Page 11 of 14 ‘Week’ were fixed effects while the replicate, and replicate within week, were random effects. There was a slight adjustment of the labels for we eks within the original data set (i.e., the label for week did not align properly with the day in all cases) that was corrected for in this analysis.

Table 5 and Figure 4 show the estimates of the differences in the natural log of fecundity between each concentration with the control, and the estimates of the natural log of mean fecundity for each concentration within each week, respectively. The values were calculated using the lsmean package in R. The P-values below are not adjusted for multiple comparisons and should only be used for exploratory purposes. Table 5 shows that there is a difference between the 50 μ g/l concentration and controls in weeks 2 and 3 only. This analysis further emphasizes the fact that, based on these data, conclusions about atrazine effects on medaka reproduction are difficult to make with confidence due to the high variance.
Table 5. Comparisons of mean fecundity per treatment concentration by week. The table shows the concentrations that are being compared (concentration), the week they are being compared in (week), the estimated difference in the levels (Estimate), the standard error of the estimate (SE), the value from a z-test (Z-V alue), the p-value from the z-test (P-Value), and a flag for significance level (Sig) where “*” is < 0.05, and “.” is p < 0.1. Concentration Week Estimate SE Z-Value P-Value Sig 0 μg/l - 0.5 μ/l 1 0.64 0.74 0.865613 0.193

0 μg/l - 5 μ/l 1 0.29 0.73 0.397589 0.346 0 μg/l - 50 μ/l 1 0.97 0.75 1.286612 0.099

. 0 μg/l - 0.5 μ/l 2 0.55 0.50 1.107657 0.134

0 μg/l - 5 μ/l 2 0.50 0.50 1.002018 0.158 0 μg/l - 50 μ/l 2 0.94 0.50 1.868171 0.031 * 0 μg/l - 0.5 μ/l 3 1.01 0.64 1.578395 0.057

.

0 μg/l - 5 μ/l 3 0.63 0.64 0.990349 0.161 0 μg/l - 50 μ/l 3 1.21 0.64 1.890301 0.029 * 0 μg/l - 0.5 μ/l 4 1.26 0.76 1.643666 0.050

.

0 μg/l - 5 μ/l 4 1.14 0.76 1.509348 0.066

. 0 μg/l - 50 μ/l 4 1.00 0.75 1.331661 0.092

. 0 μg/l - 0.5 μ/l 5 1.90 1.16 1.634579 0.051

.

0 μg/l - 5 μ/l 5 0.88 1.12 0.787951 0.215 0 μg/l - 50 μ/l 5 0.50 1.12 0.443196 0.329 0 μg/l - 0.5 μ/l 6 2.71 1.77 1.528113 0.063

.

0 μg/l - 5 μ/l 6 1.40 1.71 0.816819 0.207 0 μg/l - 50 μ/l 6 0.81 1.70 0.475605 0.317ATRAZINE / 080803 Non-Guideline/Page 12 of 14

Figure 4. Plot of the Least Square Means of Fecundity for Each Concentration by Week with 95% confidence interval. The cumulative egg count for each replicate, color-coded by treatment level, and including zero spawns is shown in Figure 5. Note that while the overall variability is high in all treatments, the variability in the control replicates is exceedingly high. Three of the control replicates have the highest fecundity present in the study but the other three control replicates fall in the middle of the data from all the replicates from all three treatment groups. The high variance within all treatment groups but particularly the control group explains why an apparent large reduction in fecundity in the 50 ug/L treatment (49.4%) is only minimally significant and 39.6% and 44.8% in the 0.5 ug/L and 5 ug/L treatments, respectively, are not statistically significant.ATRAZINE / 080803 Non-Guideline/Page 13 of 14

Figure 5. Cumulative egg count for each of 6 replicates per control and 0.5, 5.0 and 50.0 ug/L atrazine plotted over the days of the study. The Control replicates (black) are highlighted showing the overlap of three of the controls with data from all the treatment levels. Days when no spawning occurred are included for all treatments. Another source of uncertainty in the study is the lack of dose-response across the treatments in the 100-fold concentration range used in the study (0.5 to 50 ug/L). A paired t-test and a JT trend test were used on only the treatment level data to determine if there was a dose-response. Table 6 indicates there is no statistical evidence for a dose- response relationship.
Table 6. Results of a paired t-test and a JT trend test per treatment level showing an Estimated difference in square root of fecundity for the (Effect) of group and the (Estimate) of the JT statistic for the JT-trend test, with (Standard Error), (Degrees of Freedom), the calculated T- value, P-Value, and a flag for significance level (Sig) where “*” is < 0.05, and “.” is p < 0.1. Levels Effect Estimate StdErr Df T-Value P-Value Sig
5 μ g/l - 0.5 μ g/l Group 0.0449 1.022 10 0.0439 0.5171

50 μ g/l - 0.5 μ g/l Group -0.2812 1.119 10 -0.2513 0.4033

50 μ g/l - 5 μ g/l Group -0.326 0.867 10 -0.376 0.3574

ALL JTTrend 46.5 NA NA NA 0.3

A statistical analysis of atrazine effects on the fertility endpoint ( Table 7 and Table 8 ), using the approach specified in the 890.2200 MEOGRT guideline, indicates that atrazine did not affect the proportion of fertile eggs in any treatment compared to the controls. Note that the mean fertility across the entire study is very low.
Table 7. Summary statistics for proportion of fertilized eggs, with (Treatment)ATRAZINE / 080803 Non-Guideline/Page 14 of 14 Concentration, the number of replicates (Count), the Mean, Median, standard deviation (Std_Dev), and standard error (Std_Error)

Concentration Count Mean Median Std_Dev Std_Error 0 μ g/l 6 0.5250 0.545 0.1523 0.06217 0.5 μ g/l 6 0.5967 0.640 0.2310 0.09429

5 μ g/l 6 0.4450 0.515 0.1667 0.06806 50 μ g/l 6 0.5067 0.570 0.1778 0.07260

Table 8. The Jonckheere-Terpstra (JT) test for fertilized eggs, showing the estimated test statistic for the JT (JT Statistic), the corresponding p-values for increasing and decreasing tends, and the max level of treatment that was included in the test. Data Set JT Statistic DecreasingTrend PValue IncreasingTrend PValue Max Level of Treatment
37 Days 96.00 0.27 0.71 50 μ g/l
In summary, using the statistical analyses recommended in the EPA medaka extended onegeneration fish reproduction test protocol (890.2200) for analysis of fecundity data (which has been vetted by the Agency, OECD, and the public) the 50 μ g/L treatment is statistically different from controls at a p = 0.047. The 0.5 μ g/L and 5 μ g/L treatments are not statistically different from the controls. This statistical outcome is the same using both the preferred approach in the 890.2200 and the alternate step-down Jonckheere-Terpstra procedure. While the mean fecundity data suggests that atrazine decreases fecundity in all treatments compared to controls, the very large variability across all the data including controls and the lack of a dose-response argue against this conclusion. Author reported effects reported as statistically significant at the lowest treatment levels cannot be replicated when days with zero spawn are included in the analyses (as per recommendations in 890.2200). Small differences in mean fecundity are observed across the three treatments spanning a 100-fold concentration range making it unlikely that these differences are a treatment-related effect without a compelling toxicological rationale. Using statistical methods appropriate for this type of fecundity data as described in the 890.2200 (with inclusion of biologically-relevant zero spawn days), the fecundity data measured in Papoulias et al., 2014, supports a LOEC of 50 ug/L atrazine.