Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Data Requirement: : EPA DP Barcode D288775 EPA MRID 458675 01 EPA Guideline 70 1( Special Study)

Test material: Purity: not reported Common name Atrazine Chemical name: IUPAC CAS name 6 chloro N ethyl N'( 1 methylethyl) 1,3,5 triazine 2,4 diamine CAS No. 1912 24 9 Synonyms EPA PC Code: 80803 Primary Reviewer: Thomas M. Steeger, Ph. D., Senior Biologist Date: April 1, 2003 Environmental Fate and Effects Division, ERB 4, U. S. Environmental Protection Agency Secondary Reviewer( s): Joseph E. Tietge, M. S., Research Aquatic Biologist Date: Mid Continent Ecology Division, National Health and Environmental Effects Research Laboratory ( Duluth), U. S. Environmental Protection Agency Stephanie Irene, Ph. D., Senior Advisor Date: Environmental Fate and Effects Division, ERB 3, U. S. Environmental Protection Agency Mary J. Frankenberry, Senior Statistician Date: Environmental Fate and Effects Division, ERB 3, U. S. Environmental Protection Agency EPA PC Code 080803 Date Evaluation Completed: 06/ 01/ 2003 CITATION: Giesy, J. P., M. Hecker, and P. D. Jones. 2003. South African Analytical Support   Hormone and Aromatase Analysis ( SA 01C). Aquatic Toxicology Laboratory, Michigan State University, National Food Safety and Toxicology Center, E. Lansing, MI. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number MSU 07. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 2 of 152 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 3 of 152 EXECUTIVE SUMMARY:

A field study was conducted in corn growing ( experimental) and non corn growing ( reference) areas in the Potchefstroom region of South Africa to determine whether exposure to atrazine and related triazines could affect aromatization of testosterone to estradiol in wild populations of African clawed frogs ( Xenopus laevis) In this region, atrazine is typically applied in October to November. Most frogs were collected in April and May after the rainy season; however, in some atrazine use areas, frog collections extended until mid September due to the low sample sizes. Frogs were collected from baited traps after 48 hours, transferred to the lab, and housed individually in 2 L plastic cages for 48 hours before collecting samples. Blood samples were collected for plasma testosterone and estradiol analysis and gonad ( one) samples were collected for aromatase activity analysis. Snout vent length, body weight, and gonad weight were measured. Gonadosomatic index ( GSI = gonad weight ÷ body weight), condition index ( CI = body weight ÷ snout vent length) and the ratio of plasma testosterone to estradiol were calculated.

Mean and median GSI were greater for both males and females in high atrazine exposure ( experimental) sites compared to low atrazine exposure ( reference) sites; however, there was no difference in body weight ( CI) between high and low atrazine exposure sites. When comparisons were made between ponds with the highest atrazine concentrations to those with the lowest atrazine concentrations, males from high atrazine exposure sites had significantly lower median plasma testosterone than males from low atrazine exposure sites. Although male plasma testosterone was not significantly correlated with atrazine concentrations, there was a significant negative correlation between the log of male plasma testosterone levels and log of the atrazine degradate diaminochlorotriazine ( DACT) concentration. Additionally, female plasma testosterone levels were negatively correlated with atrazine concentrations, and females collected from reference ponds had significantly higher testosterone levels than females collected from experimental sites.

Mean plasma estradiol concentrations were significantly lower in experimental site males and females compared to reference site animals. Male plasma estradiol was negatively correlated with log DACT concentrations, while female estradiol concentrations were negatively correlated with atrazine concentrations.

Ideally, a study should be designed based on the variability associated with the measurement endpoints. Sample sizes should reflect the number of test animals required to identify a specified difference within a given level of certainty. Potential sources of variability should also be identified and controlled to the extent possible. The current study did not appear to base its design on the variability associated with the range of measurement endpoints. Animals were collected over some fairly broad periods of time ( up to 6 months) that would almost assure that animals within the same group would vary considerably in terms of development. Based on the number of animals collected, frogs appeared to be more abundant in reference sites than in corn growing regions where half as many frogs were collected. The entire design was questionable, including extended sampling periods in ponds of questionable similarity, baiting with potentially hormone laden liver/ meat, and confinement in cages at unknown loading rates. These factors could confound the study's ability to reliably and reasonably quantify what " typical" steroid hormone levels and/ or aromatase activity is for Xenopus. Additionally, the presence of atrazine and triazine degradates across all sites ( reference and corn growing) and the failure of the study to characterize the suite of pesticides other than triazines in the study sites contributes to the uncertain utility of this study in attempting to document potential effects of atrazine.

In order for such a study to be of value, the authors would need to conduct a pilot study first to establish the necessary sample size required to detect a specified difference in the biological attributes being measured within a certain level of confidence. The sample size would be based on the variability associated with a particular Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 4 of 152 parameter. Unfortunately, the levels of variability associated with the parameters of interest in this study would likely require very high sample sizes. A more practical approach would be to identify major sources of variability and attempt to better control those conditions.

The current data suggest that atrazine and/ or its degradates may be impacting plasma testosterone; however, the data are not conclusive and the mechanism underlying this phenomena can not be identified based on this study.

I. MATERIALS AND METHODS GUIDELINE FOLLOWED: Nonguideline Study COMPLIANCE: Not conducted under full GLP; however, most practices as defined by 40 CFR Part 160, August 19, 1989 were established for this study, including but not limited to:

° Written, authorized protocol ° Written, authorized Standard Operating Procedures for all key procedures.

° Organization and Personnel were sufficient in terms of number, education, training and experience.

° Facilities were of suitable size and construction ° Equipment used was of appropriate design and adequate capacity.

° Independent QA Inspections were conducted.

° Phase Report was written ° Raw data, documentation, records, protocols, and final phase report will be archieved.

A. MATERIALS:

1. Test Material Atrazine Description: Not reported Lot No./ Batch No. : Not reported Purity: Not reported Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 5 of 152 Stability of compound under test conditions: Not reported Storage conditions of test chemicals: Not reported 2. Test organism:

Species: African clawed frog ( Xenopus laevis)

.

Age at test initiation: Adults Weight at study initiation ( mean and range): Not reported Length at study initiation ( mean and range): Not reported Source: Adult X. laevis were field collected in two areas ( non corn growing and corn growing ) in the vicinity of Potchefstrooom, South Africa, using traps baited with liver and meat scraps. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 6 of 152 B. STUDY DESIGN:

Objective: 1. To determine whether exposure to atrazine and related triazines under field conditions in a major corn growing area in South Africa could affect aromatization of testosterone and estradiol in wild populations of clawed frogs ( X. laevis) and more specifically:

a. to determine if exposure to triazines used in corn production affects plasma concentrations of the sex steroid hormones T and E2 in male and female X. laevis adults;

b. to determine whether there are alterations in aromatase activity in adult X. laevis in both sexes in response to exposure to triazines;

c. to evaluate the range of triazine concentrations, if any, that might lead to changes in plasma hormone concentrations and aromatase activity synthesis; and d. to evaluate the possible relevance of triazine mediated alterations in the aromatization of T to E2 to reproduction.

1. Experimental Conditions a) Range finding Study:

b) Definitive Study Table 1 . Experimental Parameters Sampling was conducted in April and May approximately 6 months after atrazine was applied ( October /

November); water samples to characterize exposure was collected one month prior to frog sampling.

Environmental parameters measured at each site included habitat description ( vegetative cover and water depth). Water samples were analyzed for atrazine, related triazines, other pesticides and metals. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 7 of 152 A total of eight sites in two adjacent regions ( Viljoenskroon corn growing region = E for experimental were sampled; Potchefstroom non corn growing region = C or R or control/ reference) in South Africa.

Compounds of interest included atrazine, its metabolites desethylated atrazine ( DEA), desisopropyl atrazine ( DIA), diaminochlorotriazine ( DACT), plus terbutylazine, simazine and acetochlor.

Non corn growing sites had secchi disc readings ranging from 6.5 to 32 cm; pH ranged from 5.1 to 8.8; some of the control ponds were subject to drying ( semi permanent).

Corn growing sites had surface areas ranging from 2,400 m2 to 68,000 m2; pH ranged from 7.2  10.8 and upper secchi disc value of 207 cm.

Frogs were sampled after the rainy season in April and May; however, at atrazine site E8, frogs were sampled over a six  month period ( April  September) due to low sample sizes. Sampling at all other sites took place once or twice within a " few days." Frogs were collected in 10 baited traps and harvested after two days.

Afterwards, animals were housed individually in 2 L plastic containers for 48 hours to recover from capture stress. Blood was collected by cardiac puncture; gonads were removed, weighed and measured. One gonad was fixed in 10% neutral buffered formalin or Bouin's, while the second was snap frozen. Gonadosomatic index ( weight of the gonad ÷ snout vent length) and the condition index ( body weight ÷ snout vent length) were calculated.

Concentrations of testosterone ( T) and estradiol ( E2) in blood plasma were measured by competitive ELISA as described by Cuisset et al. 1994 with modifications by Hecker et al. 2002 using COSTAR high binding plates. Working ranges of the assays were from 0.78 to 800 pg/ well for both testosterone and estradiol.

Aromatase activity was measured following the protocol from Lephart and Simpson 1991 with minor modifications ( Sanderson 2000). Less than 0.5 g of gonad was homogenized. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 8 of 152 II. RESULTS and DISCUSSION: [ All results discussed in this section and the next are those reported by the study authors. Although supplemental data are typically used in a qualitative manner only, EFED verified spreadsheet data and ran basic statistical analyses on the major study parameters. See attached appendix. If results appeared to differ in any substantive way, the difference was reported in the text below.]

Atrazine Concentrations With the exception of site E8 ( peak concentration in late January at 9

g/ l), the greatest concentrations of atrazine in pond water were observed at the end of the rainy season ( March through May); highest atrazine concentrations were measured at sites E1 ( 4.1

g/ L), E6 ( 3.9

g/ L) and E8 ( 3.5

g/ L). Concentrations at C1,

C3, C6 and E4 were low but could be quantified. The log concentrations of triazines DIA, DEA and atrazine were significantly correlated with pH of the water. ( p < 0.034, r range: 0.770  0.802). Log DIA also showed a positive linear regression with log temperature ( 7 = 0.801; p= 0.017) and log visual depth ® = 0.814; p =

0.014). Monitoring data ( Table 1) indicated that measurable levels of atrazine were detected at all sampling sites ( range 0.2  3.53

g/ L). The atrazine degradate desisopropyl atrazine ( DIA) was as high in C3 ( 0.61

g/ L) as in many of the corn growing sites, while diaminochlorotriazine ( DACT) in C1 ( 3.91

g/ L) was the second highest concentration of the degradate detected at any of the sampling sites. Mean measured atrazine concentrations, which were determined four weeks before frogs were collected and averaged over November 2001 to June 2002 ( Table 2), indicated that atrazine in reference sites was generally an order of magnitude lower than sites located in corn growing areas. However, all concentrations were apparently readily quantified.

Table 1. Concentrations of pesticides (

g/ L) at the different sampling sites. Values were calculated from two individual samplings that were conducted with a time period in 2002 of 4 weeks before collecting X.

laevis.

Site C1 C3 C6 E1 E3 E4 E6 E8 4/ 2 4/ 15 3/ 18 4/ 2 5/ 1 5/ 13 4/ 15 5/ 1 4/ 1 4/ 15 4/ 2 4/ 15 4/ 15 5/ 1 4/ 15 5/ 1 Simazin e < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 1.7 1.2 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 9 of 152 DACT 3.91 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 0.38 < 0.1 < 0.1 < 0.1 5.45 < 0.1 1.24 < 0.1 DIA 0.23 0.27 0.3 0.61 0.13 0.15 0.7 0.93 0.47 0.28 0.18 0.64 0.49 0.32 0.7 0.88 DEA 0.18 < 0.1 0.15 < 0.1 0.12 0.13 1.29 1.16 0.43 0.38 0.11 < 0.1 1.09 0.9 1.04 0.91 Atrazine 0.28 0.26 0.36 0.57 < 0.1 0.2 4.14 3.5 1.05 1.01 0.32 0.29 3.78 3.9 3.53 3.12 Terbuty l < 0.1 < 0.1 < 0.1 < 0.1 0.51 0.59 3.66 2.95 0.97 1.07 0.19 0.24 2.46 2.4 0.86 0.72 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 10 of 152 Table 2. Mean concentrations of atrazine at the sampling locations measured during four weeks collecting frogs, and time weighted means of atrazine measured between November 2001 and June 2002.

Values listed in ascending order of concentration Location Mean Atrazine Concentration 4 weeks Before Sampling Location Time weighted Mean Atrazine Concentration C6 0.13 C1 0.15 C1 0.27 C6 0.19 E4 0.31 C3 0.25 C3 0.47 E4 0.48 E3 1.0 E3 0.82 E8 3.3 E6 2.6 E1 3.8 E1 3.2 E6 3.8 E8 3.4 Gonadosomatic Index ( GSI)

The means of the median GSI values for both female ( t test; p = 0.0640) and male ( t test; p = 0.0725) from corn growing areas were greater than the female and male means from the non corn growing areas. The GSI of females form E1 and E8 were more variable than those at other locations.

Condition Index No statistically significant differences were observed in the means of the site specific CI values between the corn growing and non corn growing sites regardless of whether the analysis was based on means or medians.

Testosterone ( T) Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 11 of 152 Plasma T concentrations were greater in males than in females except in reference pond C1 where mean male plasma testosterone (  105 pg/ mL) was roughly an order of magnitude lower than females' mean plasma testosterone (  1005 pg/ mL). At sites C6 and E6, males and females contained roughly similar amounts of plasma testosterone. For males, there was no significant difference between plasma T in frogs from corngrowing and non corn growing sites, and there was no correlation between plasma T and atrazine. However,

there was a significant negative correlation between the log of male plasma T concentration and the log of the DACT concentration ( r=  0.839, p = 0.009) Median and mean plasma T concentrations of males were not significantly different between the experimental and reference sites. However, when the sites were grouped into either those with the four greatest and four least atrazine concentrations or into groups where atrazine concentrations were greater than 3

g/ L vs those with concentrations that were less than 1

g/ L, there were significant differences, with the frogs from the greater atrazine sites having less median concentrations of plasma T.

Although the report states that mean and median female plasma testosterone levels were significantly higher in experimental ponds than at reference sites ( t test, mean p = 0.018; median 0.0061), the figure ( Figure 4)

depicting this relationship suggests the opposite, i. e., that median female plasma testosterone concentrations were higher in reference sites. This would better support the fact that female plasma T concentrations were negatively correlated with atrazine concentrations ( r =  0.725; p = 0.042).

Estradiol ( E2)

Median female plasma concentrations of E2 from experimental sites were significantly less than those of females from reference sites ( t test; p = 0.0018), but there was no significant difference for males. Mean plasma estradiol concentrations were less at experimental sites for both males ( t test, p = 0.0186) and females ( t test; p = 0.0052). Concentrations of E2 in the plasma of both males and females were more variable in reference sites than those from experimental sites. Male plasma E2 were not related to atrazine but were negatively correlated with the log of DACT concentrations ( r =  0.779; p = 0.023). In females, plasma E2 was negatively correlated with atrazine concentration ( r =  0.833; p = 0.010). Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 12 of 152 E2/ T Ratio Because of the magnitude of variability associated with this index, E2/ T ratios for both males and females were not significantly different either between the experimental and reference sites or between other location groupings regardless of whether they were based on means or medians. E2/ T ratio in males was negatively correlated with DIA concentrations ( r =  0.830; p = 0.011). There were no significant relationships for E2/ T ratio for females and any triazine residues.

Aromatase Activity Aromatase levels in testes were not measurable at most sites. There were no significant differences in median aromatase activity in ovaries between experimental and reference or when locations were grouped based on the highest and lowest atrazine concentrations. The greatest variability in ovarian aromatase activity was at site E8 and was attributed to the extended sampling period ( April to September). Log ovarian aromatase activity was correlated with the log of the 4WM water temperature ( 7 = 0.786; p = 0.021); aromatase activity was not correlated with atrazine concentration but was correlated with with the log of the DIA concentration ( r =

0.857; p = 0.007).

There were statistically significant linear regressions between the logarithms of T and E2 in both males ( r =

0.757; p < 0.001) and females ( r = 0.868; p < 0.001). Both sexes also exhibited positive relationships between log E2 and log E2/ T ( males: r = 0.804, p < 0.001; females: r = 0.582, p < 0.001). However,

aromatase in females was not related to sex steroid concentrations.

C. REPORTED STATISTICS: Data used in statistical tests were stratified by sex, i. e., male and female data analyzed separately. Studies were designed to be analyzed by both fixed effects models and by regression type statistics because specific locations could not be classified as exposed or unexposed since atrazine residues were also detected in non corn growing " reference sites". Statistical comparisons were made between the median concentrations of plasma testosterone and estradiol as well as the E2/ T ratio, the GSI, the EI and Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 13 of 152 gonadal aromatase; regression analysis between atrazine concentration and these parameters was also conducted.. For statistical tests, locations were grouped several ways: grouped based on whether they were in corn growing ( E) or non corn growing ( C) areas; grouped with a mean atrazine concentration  1.0

g/ L ( C1, C3, C6, E3, E4) versus > 3

g/ L ( E1, E6, E8). Finally, study sites were grouped with the four sites with the 4 smallest means in one group, and the remaining four sites with the four largest means in the other group.

Two sided two sample t test was used to compare the mean of the site values in the first group to the mean of the site values in the second group. Two sample Kolmogorov Smirnov test was used to compare individual locations. Linear model comparisons of four  week average atrazine and/ or metabolites were used for each parameter.

D. VERIFICATION OF STATISTICAL RESULTS: See attached SAS © output ( Statistical Analysis System, Release 8.01, Cary, North Carolina.

;

E. STUDY DEFICIENCIES:

Atrazine and/ or triazine degradates were present in control sites.

While the low sample size at sites E1 and E8 may have been explained by the sudden introduction of predatory catfish, it does not explain why the remaining corn growing sites ( E3, E4 and E6) also yielded fewer frogs than non corn growing sites.

Because frogs were sampled over disproportionate periods of time, e. g., 6 months for site E8, samples would likely contain animals at different stages of development Frog traps were baited with liver/ meat scraps that may have contained hormones.

Following capture, frogs were housed individually in " clean water" for 48 hours. The volume and characteristics of this water were not described in the study. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 14 of 152 F. REVIEWER'S COMMENTS:

The large amount of variability associated with aromatase and plasma hormone data in laboratory studies make it difficult to understand the utility of conducting a field study with these parameters. Collecting animals over a six month period would also contribute to extremely high variability.

Atrazine is typically applied in October / November when corn is planted in South Africa. Frog sampling was conducted after the rainy season in April to May and water samples to occurred within a time span of four weeks before frogs were sampled. At exposure site E8, frogs were sampled over a 6 month time frame. The fewer number of frogs was attributed to a high runoff event that washed catfish into the pond; the catfish preyed on the frogs.

Reference and atrazine exposure sites appeared to be widely divergent, ranging from 5.1  8.8 at reference sites and 7.2  10.8 at atrazine sites.

Frogs were attracted into baited traps containing liver and/ or scrap meat which may have contained hormones.

Apparently trapped animals were held in cages for two days which should have provided sufficient time to consume all of the bait. Also, depending on catch rates, the loading within the cages could have stressed the frogs. Although it doesn't state the desired sample size in the report's methodology, apparently ( based on the raw data), 20 male and 20 female frogs were to be sampled at each site. Table 3 shows that while the desired sample size was generally available for frogs collected in non corn growing areas, sample sizes were minimally 50% lower in corn growing areas. In site E1, sampling for female frogs extended into late June, and at site E8 sampling extended into mid August for females and into mid September for males. Extended sampling periods at these two sites may have resulted in collecting animals that were at considerably different states of development. The gonadosomatic index and ovarian aromatase activity in females collected from sites E1 and E8 expressed the highest variability relative to any of the other treatment sites.

Table 3. Summary of total number of Xenopus laevis collected from April through May 2002 at non corn growing ( C) and corn growing ( E) sites in South Africa. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 15 of 152 Site Males Females C1 20 ( Apr 21  Apr 23) 20 ( Apr 21  May 1)

C3 17 ( Apr 4  Apr 11) 20 ( Apr 4  Apr 11)

C6 20 ( May 13  May 15) 20 ( May 13  May 14)

E1 8 ( April 29  May 7) 8 ( April 29  June 29)

E3 10 ( Apr 9  Apr 17) 10 ( Apr 9  Apr 18)

E4 10 ( Apr 13  Apr 15) 10 ( Apr 14  Apr 16)

E6 10 ( May 5  May 9) 10 ( May 5  May 6)

E8 6 ( Apr 29  Sep 17) 15 ( Apr 29  Aug 17)

Statistical analyses were more regression based because specific sampling locations could not be classified as exposed or unexposed. There was a range of concentrations of primary residues of concern, with greater concentrations in the corn growing areas; however, residues were also detected in non corn growing areas.

In Phase II of the study ( Smith et al. 2003), the authors suggested that atrazine residues in reference ponds may be a result of atmospheric deposition ( wind).

The GSI and aromatase ovarian activity of females from E8 were more variable than those at other locations.

This variability may have resulted from the long sampling period ( six months) and the considerable changes in the frogs' reproductive state.

Gonadosomatic index suggests that both males and females collected in corn growing areas were more sexually developed than their counterparts in " reference" sites.

Comparisons between male and female plasma testosterone levels were also are hindered by the considerable variability within pond and within treatment groups. Although median plasma testosterone levels were qualitatively different. Males in reference ponds had higher median plasma testosterone levels than males in experimental ponds by approximately a factor of 2. Similarly, females in reference ponds had higher median Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 16 of 152 plasma testosterone levels than females in experimental ponds by approximately a factor of 8. However,

given the magnitude of variability, it wasn't possible to document statistical differences in means or medians.

Only when the samples were grouped based on the three highest and three lowest atrazine exposures could statistical differences be documented in median T levels. Also, the text may be in error when it concludes that female plasma testosterone levels were higher in corn growing areas ( experimental) than in non corn growing areas because the statement appears to contradict the figure ( Figure 4) depicting the data and from the significant correlation showing that female plasma testosterone is negatively related to atrazine.

Based on Figure 5 , median plasma E2 concentrations in males from reference sites were approximately similar to female plasma estradiol concentrations in reference sites, while median male plasma estradiol at reference sites was approximately five fold higher than plasma estradiol concentrations of males from experimental sites.

A similar pattern existed for females where reference site animals had roughly six fold higher estradiol concentrations than females at experimental sites.

Although the study authors conclude that there were no statistical differences in the ratio of estradiol to testosterone ( E2/ T), in a qualitative sense, the median values for females were relatively consistent across experimental and reference sites. However, the median ratio for males tended to be roughly double in animals collected from reference sites compared to those collected at experimental sites.

Although not considered very predictive ( correlation coefficients ranged from 0.22 to 0.38), there were significant correlations ( p< 0.026) between condition index and both gonadosomatic index and log aromatase activity in females. This correlation is intuitively obvious because more gravid females should weigh more and have higher aromatase activity than less developed animals.

The report discussed a number of statistical differences that were detected and how misleading this could be based on the number of statistical comparisons that were run. The authors suggested that if Bonferroni's correction had been made, none of the p values would have been significant. The Bonferroni correction is used to control experiment error rates and is obtained by dividing the overall   level for the study by the number of groups being compared. If the study has a large number of means, however, this process can be very Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 17 of 152 conservative. It may also fail to detect significant differences between groups and increase the likelihood of making a Type II error.

In this study, the authors discuss the relevancy of the Tavera Mendoza et al. ( 2002a and 2002b) studies which showed decreased testicular volume in animals exposed to 21

g/ L and how the current study demonstrated increases in GSI. At the same time, though, they note that the current study's maximum exposure values were roughly five fold lower than those used in the Tavera Mendoza study and that improved food sources may have resulted in increased gonadal weight . Others differences in the Tavera Mendoza study include: the laboratory study was presumably conducted on animals that were probably in the same state of development, the environmental conditions in the laboratory were better defined, and the laboratory study was an acute exposure ( 48 hour) as opposed to the current chronic study.

The authors state that there is no plausible mechanism that could be postulated to explain the negative correlation between plasma E2 concentrations in females and concentrations of atrazine in pond water;

however, they note that correlations do not imply causality.

Since there were no differences in aromatase activities between locations or significant correlations between exposure to atrazine and aromatase activity, the authors believe that these findings do not support the Hayes et al. 2002a, b hypothesis that atrazine causes an increase in the production of estrogen by inducing aromatase activity. The fact that plasma T concentrations were less in groups with the higher atrazine exposure values was viewed as being consistent with the hypothesis that atrazine decreased plasma T, but the fact that atrazine concentrations were not correlated with plasma T was viewed as suggesting that the statistical significance was artifactual. The significant negative correlation between the E2/ T ratio of males and DIA was viewed as being inconsistent with Sanderson et al. 2001, showing that DIA significantly increased CYP 19 mRNA expression as well as aromatase activity. They concluded, though, that the differences in plasma steroid hormone concentrations were not caused by up regulation of aromatase activity. The report states " although significant negative correlations were observed between exposure to atrazine and its metabolite DACT and plasma concentrations of sex steroids in wild X. laevis, it is impossible from this study to conclude the exact cause or accuracy of these differences." The experimental corn growing regions were subject to a range of chemicals there were not fully characterized and that may have contributed to the observed statistical differences. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 18 of 152 G. CONCLUSIONS:

This study indicates that plasma hormone levels and gonadal aromatase activity are highly variable and depend on a number of factors that were not anticipated when the study was designed. Reference ponds in areas of South Africa where corn is not grown were subject to variable levels of atrazine contamination that approached exposure values for ponds in corn growing regions. Collecting frogs over protracted periods of time may have constituted one of the greatest sources of variability because the frogs were probably at different stages of their sexual cycles. It is unclear what effect the bait used to attract frogs into traps may have had on hormone levels. In spite of the fact that in some comparisons the study was unable to differentiate male and female hormone levels, some evidence suggested that males subjected to higher atrazine concentrations tended to have reduced plasma testosterone and that higher concentrations of the atrazine degradate DACT were associated with reduced plasma testosterone levels. Also, contrary to what the authors state, median female plasma testosterone appeared to be significantly higher in reference sites where atrazine exposure was reduced. This conclusion is consistent with the authors' observation that female plasma testosterone was negatively correlated with atrazine concentration. Mean plasma estradiol concentrations in both males and females were significantly lower in high atrazine exposure sites, and like testosterone, male estradiol concentrations were negatively correlated with concentrations of the atrazine degradate DACT. Female estradiol concentrations were negatively correlated with atrazine concentrations. Gonadal aromatase activity in males could not be accurately characterized because of its low level across the majority of collection sites,

while high variability in female aromatase activity made all the comparisons insignificant. Aromatase activity,

though, was negatively correlated with concentrations of the atrazine degradate DIA.

Ideally, a study should be designed based on the variability associated with the measurement endpoints.

Sample sizes should reflect the number of test animals required to identify a specified difference within a given level of certainty. Potential sources of variability should also be identified and controlled to the extent possible.

The current study did not appear to base its design on the variability associated with the range of measurement endpoints. Animals were collected over broad periods of time ( up to six months) which would assure variation within the same group in terms of development. Based on the number of animals collected, frogs Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 19 of 152 appeared to be more abundant in reference sites than in corn growing regions where half as many frogs were collected. The entire design was questionable, including: the extended sampling periods in ponds of questionable similarity, baiting with potentially hormone laden liver/ meat, and confinement in cages at unknown loading rates. These factors could confound the study's ability to reliably and reasonably quantify " typical" steroid hormone levels and/ or aromatase activity in Xenopus. Additionally, the presence of atrazine and triazine degradates across all sites ( reference and corn growing) and the failure to characterize other pesticides at the study sites limits the usefulness of this study in determining potential effects of atrazine and in supporting the study's hypothesis.

Before a field study of this scope is undertaken, researchers need to conduct a pilot study to establish the necessary sample size for detecting a specified difference in the biological attribute being measured within a certain level of confidence. The sample size would be based on the variability associated with a particular parameter. Unfortunately, the levels of variability associated with the parameters of interest in this study would likely require very high sample sizes. A more practical approach would be to identify major sources of variability and then try to control those conditions.

The current data suggest that atrazine and/ or its degradates may be impacting plasma testosterone; however,

the data are not conclusive for the reasons cited above.

H. REFERENCES:

Cuisset, G., P. Pradelles, D. E. Kime, E. R. Kühn, P. Barbin, S. Davail, F. Le Menn. 1994. Enzyme immunoassay for 11 ketotestosterone using acetylcholineesterase as label: application to the measurement of 11 ketotestosterone in plasma of Siberian sturgeon. Comp. Biochem. Physiol. 108: 229  241.

Hayes, T. B., A. Collins, M. Lee, M. Mendoza, N. Noriega, A. S. Stuart, and A. Vonk. 2002a.

Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences of the United States of America 99( 8): 5476  5480. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 20 of 152 Hayes, T. B., K. Haston, M. Tsui, A. Hoang, C. Haeffele, and A. Vonk. 2002b. Atrazine induced hermaphroditism at 0.1 ppb in American leopard frogs ( Rana pipiens): laboratory and field evidence.

Environmental Health Perspectives.

Hecker, M., C. R. Tyler, S. Maddix, and L. Karbe. 2002. Plasma biomarkers in fish provide evidence for endocrine modulation in th Elbe River, Germany. Environ. Sci. Technol. 36 2311  2321.

Kennedy, S. W. and S. P. Jones. 1994. Simultaneous measurement of cytochrome P4501A catalyitc activity and total protein concentration with fluorescence plate reader. Analytical Biochemistry 222( 1): 217  223.

Laphart, E. D. and E. R. Simpson. 1991. Assay of aromatase activity. Methods in Enzymology 206: 477

483.

Sanderson, J. T., W. Seinen, J. P. Giesy and M. van den Berg. 2000. 2 chloro S triazine herbicides induce aromatase activity in H295R human adreno cortical carcinoma cells. A novel mechanism for estrogenicity.

Toxicol. Sci., 53: 121  127.

Sanderson, J. t., R. J. Letcher, M. Heneweer, J. P. Giesy, M. van den Berg. 2001. Effects of chloro S triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes. Environ. Health Perspect. 109: 1  6.

Smith, E., L DuPreez and K. Solomon. 2003. Field exposure of Xenopus laevis to atrazine and other triazines in South Africa: exposure characterization and assessment of laryngeal and gonadal responses. The Institute of Environmental & Human Health, Texas Tech University, Lubbock, Texas 79490 ( USA) and School of Environmental Sciences and Development, Potchefstroom University for CHE, Private Bag X6001,

Potchefstroom 2520 ( South Africa). Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number SA 01B Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 21 of 152 Tavera Mendoza, L., S. Ruby, P. Brousseau, M. Fournier, D. Cyr and D. Marcogliese. 2001. Response of the amphibian tadpole Xenopus laevis to atrazine during sexual differentiation of the ovary. Environmental Toxicology and Chemistry 21 ( 6): 1264  1267.

Tavera Mendoza, L., S. Ruby, P. Brousseau, M. Fournier, D. Cyr and D. Marcogliese. 2001. Response of the amphibian tadpole Xenopus laevis to atrazine during sexual differentiation of the testes. Environmental Toxicology and Chemistry 21 ( 3): 527  531. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 22 of 152 MEAN LENGTHS OF FROGS COLLECTED BY POND AND SEX 391 Obs POND Sex GROUP _ TYPE_ _ FREQ_ LENGTH L_ SD L_ CV 1 C1 F REF 0 20 69.5450 8.0156 11.5258 2 C1 M REF 0 20 58.0000 6.6373 11.4436 3 C3 F REF 0 20 68.9100 14.4936 21.0326 4 C3 M REF 0 20 56.9882 10.9170 19.1565 5 C6 F REF 0 20 76.9500 7.0433 9.1531 6 C6 M REF 0 20 72.1100 7.1908 9.9721 7 E1 F EXP 0 10 64.4833 14.3272 22.2185 8 E1 M EXP 0 10 59.2000 9.4870 16.0253 9 E3 F EXP 0 10 74.3900 6.9437 9.3341 10 E3 M EXP 0 10 66.6300 6.9863 10.4853 11 E4 F EXP 0 10 77.5400 10.9218 14.0853 12 E4 M EXP 0 10 63.8300 3.3549 5.2561 13 E6 F EXP 0 10 88.5000 9.5274 10.7654 14 E6 M EXP 0 10 62.6600 9.5755 15.2816 15 E8 F EXP 0 15 64.4733 13.8195 21.4344 16 E8 M EXP 0 10 60.7167 7.2841 11.9968 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 23 of 152 ANOVA FOR LENGTH OF FROGS BETWEEN PONDS BY SEX 392  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 115 NOTE: Due to missing values, only 111 observations can be used in this analysis.

Dependent Variable: LENGTH Sum of Source DF Squares Mean Square F Value Pr > F Model 7 5026.98373 718.14053 6.07 <. 0001 Error 103 12178.98817 118.24260 Corrected Total 110 17205.97189 R Square Coeff Var Root MSE LENGTH Mean 0.292165 14.96340 10.87394 72.67027 Source DF Anova SS Mean Square F Value Pr > F POND 7 5026.983725 718.140532 6.07 <. 0001 Levene's Test for Homogeneity of LENGTH Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 432098 61728.3 3.51 0.0020 Error 103 1810469 17577.4 Bartlett's Test for Homogeneity of LENGTH Variance Source DF Chi Square Pr > ChiSq POND 7 17.4523 0.0147 Dunnett's t Tests for LENGTH NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 24 of 152 Alpha 0.05 Error Degrees of Freedom 103 Error Mean Square 118.2426 Critical Value of Dunnett's t 2.69261 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits E6  C1 18.955 7.615 30.295 ***

E4  C1 7.995  3.345 19.335 C6  C1 7.405  1.854 16.664 E3  C1 4.845  6.495 16.185 C3  C1  0.635  9.894 8.624 E1  C1  5.062  18.690 8.567 E8  C1  5.072  15.072 4.929 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 25 of 152 ANOVA FOR LENGTH OF FROGS BETWEEN PONDS BY SEX 396  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 110 NOTE: Due to missing values, only 101 observations can be used in this analysis.

Dependent Variable: LENGTH Sum of Source DF Squares Mean Square F Value Pr > F Model 7 3055.667841 436.523977 6.78 <. 0001 Error 93 5987.449980 64.381183 Corrected Total 100 9043.117822 R Square Coeff Var Root MSE LENGTH Mean 0.337900 12.78217 8.023789 62.77327 Source DF Anova SS Mean Square F Value Pr > F POND 7 3055.667841 436.523977 6.78 <. 0001 Levene's Test for Homogeneity of LENGTH Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 92002.7 13143.2 2.03 0.0591 Error 93 601693 6469.8 Bartlett's Test for Homogeneity of LENGTH Variance Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 26 of 152 Source DF Chi Square Pr > ChiSq POND 7 14.5701 0.0419 Dunnett's t Tests for LENGTH NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 93 Error Mean Square 64.38118 Critical Value of Dunnett's t 2.70384 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits C6  C1 14.110 7.249 20.971 ***

E3  C1 8.630 0.228 17.032 ***

E4  C1 5.830  2.572 14.232 E6  C1 4.660  3.742 13.062 E8  C1 2.717  7.382 12.815 E1  C1 1.200  7.876 10.276 C3  C1  1.012  8.169 6.145 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 27 of 152 NONPARAMETRIC COMPARISON OF FROG LENGTH BETWEEN PONDS 400  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable LENGTH Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 896.00 1120.0 130.323757 44.800000 C3 20 975.50 1120.0 130.323757 48.775000 C6 20 1361.00 1120.0 130.323757 68.050000 E1 6 209.00 336.0 76.675775 34.833333 E3 10 593.00 560.0 97.084207 59.300000 E4 10 656.00 560.0 97.084207 65.600000 E6 10 945.50 560.0 97.084207 94.550000 E8 15 580.00 840.0 115.922875 38.666667 Average scores were used for ties.

Kruskal Wallis Test Chi Square 28.5206 DF 7 Pr > Chi Square 0.0002 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable LENGTH Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 7.0 9.909910 2.033723 0.350000 C3 20 6.0 9.909910 2.033723 0.300000 C6 20 14.0 9.909910 2.033723 0.700000 E1 6 1.0 2.972973 1.196537 0.166667 E3 10 6.0 4.954955 1.515014 0.600000 E4 10 5.0 4.954955 1.515014 0.500000 E6 10 10.0 4.954955 1.515014 1.000000 E8 15 6.0 7.432432 1.808995 0.400000 Average scores were used for ties. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 28 of 152 Median One Way Analysis Chi Square 21.6625 DF 7 Pr > Chi Square 0.0029 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 29 of 152 NONPARAMETRIC COMPARISON OF FROG LENGTH BETWEEN PONDS 403  Sex= M

Wilcoxon Scores ( Rank Sums) for Variable LENGTH Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 713.00 1020.0 117.339834 35.650000 C3 17 559.00 867.0 110.167138 32.882353 C6 20 1600.50 1020.0 117.339834 80.025000 E1 8 312.50 408.0 79.519644 39.062500 E3 10 644.50 510.0 87.944495 64.450000 E4 10 552.50 510.0 87.944495 55.250000 E6 10 494.00 510.0 87.944495 49.400000 E8 6 275.00 306.0 69.602588 45.833333 Average scores were used for ties.

Kruskal Wallis Test Chi Square 35.4807 DF 7 Pr > Chi Square <. 0001 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable LENGTH Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 6.0 9.900990 2.012363 0.300000 C3 17 3.0 8.415842 1.889352 0.176471 C6 20 20.0 9.900990 2.012363 1.000000 E1 8 2.0 3.960396 1.363751 0.250000 E3 10 6.0 4.950495 1.508236 0.600000 E4 10 6.0 4.950495 1.508236 0.600000 E6 10 4.0 4.950495 1.508236 0.400000 E8 6 3.0 2.970297 1.193675 0.500000 Average scores were used for ties. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 30 of 152 Median One Way Analysis Chi Square 33.1792 DF 7 Pr > Chi Square <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 31 of 152 ANOVA FOR LENGTH OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 404  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: LENGTH Sum of Source DF Squares Mean Square F Value Pr > F Model 1 8.0782352 8.0782352 0.11 0.7524 Error 6 444.1424033 74.0237339 Corrected Total 7 452.2206385 R Square Coeff Var Root MSE LENGTH Mean 0.017863 11.76994 8.603705 73.09896 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 8.07823521 8.07823521 0.11 0.7524 Levene's Test for Homogeneity of LENGTH Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 8547.2 8547.2 1.76 0.2326 Error 6 29101.8 4850.3 Bartlett's Test for Homogeneity of LENGTH Variance Source DF Chi Square Pr > ChiSq GROUP 1 1.1508 0.2834 Dunnett's t Tests for LENGTH Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 32 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 74.02373 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference Simultaneous GROUP Between 95% Confidence Comparison Means Limits REF  EXP  2.076  17.451 13.299 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 33 of 152 ANOVA FOR LENGTH OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 408  Sex= M

Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: LENGTH Sum of Source DF Squares Mean Square F Value Pr > F Model 1 0.1091324 0.1091324 0.00 0.9533 Error 6 175.7919208 29.2986535 Corrected Total 7 175.9010531 R Square Coeff Var Root MSE LENGTH Mean 0.000620 8.658181 5.412823 62.51686 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 0.10913236 0.10913236 0.00 0.9533 Levene's Test for Homogeneity of LENGTH Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 3162.6 3162.6 5.27 0.0615 Error 6 3600.4 600.1 Bartlett's Test for Homogeneity of LENGTH Variance Source DF Chi Square Pr > ChiSq GROUP 1 2.7649 0.0964 Dunnett's t Tests for LENGTH NOTE: This test controls the Type I experimentwise error for comparisons of all treatments Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 34 of 152 against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 29.29865 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference GROUP Between Simultaneous 95%

Comparison Means Confidence Limits REF  EXP  0.2413  9.9140 9.4315 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 35 of 152 NONPARAMETRIC COMPARISON OF FROG LENGTH BETWEEN REFERENCE AND EXPERIMENTAL SITES 412  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable LENGTH Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 23.0 22.50 3.354102 4.600000 REF 3 13.0 13.50 3.354102 4.333333 Wilcoxon Two Sample Test Statistic 13.0000 Normal Approximation Z 0.0000 One Sided Pr < Z 0.5000 Two Sided Pr > | Z| 1.0000 t Approximation One Sided Pr < Z 0.5000 Two Sided Pr > | Z| 1.0000 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 0.0222 DF 1 Pr > Chi Square 0.8815 Median Scores ( Number of Points Above Median) for Variable LENGTH Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 REF 3 1.0 1.50 0.731925 0.333333 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 36 of 152 Median Two Sample Test Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 37 of 152 NONPARAMETRIC COMPARISON OF FROG LENGTH BETWEEN REFERENCE AND EXPERIMENTAL SITES 414  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable LENGTH Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 25.0 22.50 3.354102 5.000000 REF 3 11.0 13.50 3.354102 3.666667 Wilcoxon Two Sample Test Statistic 11.0000 Normal Approximation Z  0.5963 One Sided Pr < Z 0.2755 Two Sided Pr > | Z| 0.5510 t Approximation One Sided Pr < Z 0.2849 Two Sided Pr > | Z| 0.5698 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 0.5556 DF 1 Pr > Chi Square 0.4561 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable LENGTH Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 38 of 152 REF 3 1.0 1.50 0.731925 0.333333 Median Two Sample Test Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 39 of 152 MEAN WEIGHTS OF FROGS COLLECTED BY POND AND SEX 416 Obs POND Sex GROUP _ TYPE_ _ FREQ_ WEIGHT W_ SD W_ CV 1 C1 F REF 0 20 32.8315 11.1449 33.9459 2 C1 M REF 0 20 19.4445 6.1022 31.3826 3 C3 F REF 0 20 35.3455 20.2334 57.2446 4 C3 M REF 0 20 20.7541 13.2996 64.0818 5 C6 F REF 0 20 41.2495 10.4261 25.2757 6 C6 M REF 0 20 37.2590 12.0340 32.2982 7 E1 F EXP 0 10 29.1450 19.2315 65.9856 8 E1 M EXP 0 10 23.3888 14.4006 61.5704 9 E3 F EXP 0 10 39.3810 8.4697 21.5071 10 E3 M EXP 0 10 32.5740 8.9938 27.6105 11 E4 F EXP 0 10 48.8210 18.5235 37.9417 12 E4 M EXP 0 10 29.9000 4.1673 13.9376 13 E6 F EXP 0 10 66.9560 22.6873 33.8838 14 E6 M EXP 0 10 26.1050 12.3935 47.4758 15 E8 F EXP 0 15 29.8047 18.8819 63.3521 16 E8 M EXP 0 10 23.0917 8.7925 38.0765 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 40 of 152 ANOVA FOR WEIGHT OF FROGS BETWEEN PONDS BY SEX 417  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 115 NOTE: Due to missing values, only 111 observations can be used in this analysis.

Dependent Variable: WEIGHT Sum of Source DF Squares Mean Square F Value Pr > F Model 7 11751.86955 1678.83851 6.31 <. 0001 Error 103 27410.49679 266.12133 Corrected Total 110 39162.36634 R Square Coeff Var Root MSE WEIGHT Mean 0.300081 41.51186 16.31323 39.29775 Source DF Anova SS Mean Square F Value Pr > F POND 7 11751.86955 1678.83851 6.31 <. 0001 Levene's Test for Homogeneity of WEIGHT Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 2120258 302894 2.53 0.0190 Error 103 12311095 119525 Bartlett's Test for Homogeneity of WEIGHT Variance Source DF Chi Square Pr > ChiSq POND 7 20.4080 0.0048 Dunnett's t Tests for WEIGHT Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 41 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 103 Error Mean Square 266.1213 Critical Value of Dunnett's t 2.69261 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits E6  C1 34.124 17.112 51.137 ***

E4  C1 15.990  1.023 33.002 C6  C1 8.418  5.472 22.308 E3  C1 6.549  10.463 23.562 C3  C1 2.514  11.376 16.404 E8  C1  3.027  18.030 11.976 E1  C1  3.687  24.133 16.760 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 42 of 152 ANOVA FOR WEIGHT OF FROGS BETWEEN PONDS BY SEX 421  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 110 NOTE: Due to missing values, only 101 observations can be used in this analysis.

Dependent Variable: WEIGHT Sum of Source DF Squares Mean Square F Value Pr > F Model 7 4500.49282 642.92755 5.75 <. 0001 Error 93 10393.96375 111.76305 Corrected Total 100 14894.45657 R Square Coeff Var Root MSE WEIGHT Mean 0.302159 39.57071 10.57180 26.71624 Source DF Anova SS Mean Square F Value Pr > F POND 7 4500.492823 642.927546 5.75 <. 0001 Levene's Test for Homogeneity of WEIGHT Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 339909 48558.4 1.40 0.2144 Error 93 3223997 34666.6 Bartlett's Test for Homogeneity of WEIGHT Variance Source DF Chi Square Pr > ChiSq Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 43 of 152 POND 7 22.0595 0.0025 Dunnett's t Tests for WEIGHT NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 93 Error Mean Square 111.7631 Critical Value of Dunnett's t 2.70384 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits C6  C1 17.815 8.775 26.854 ***

E3  C1 13.130 2.059 24.200 ***

E4  C1 10.455  0.615 21.526 E6  C1 6.661  4.410 17.731 E1  C1 3.944  8.013 15.902 E8  C1 3.647  9.658 16.953 C3  C1 1.310  8.120 10.739 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 44 of 152 NONPARAMETRIC COMPARISON OF FROG WEIGHT BETWEEN PONDS 425  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable WEIGHT Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 895.50 1120.0 130.332621 44.775000 C3 20 971.00 1120.0 130.332621 48.550000 C6 20 1266.00 1120.0 130.332621 63.300000 E1 6 215.50 336.0 76.680990 35.916667 E3 10 601.00 560.0 97.090810 60.100000 E4 10 724.00 560.0 97.090810 72.400000 E6 10 950.00 560.0 97.090810 95.000000 E8 15 593.00 840.0 115.930760 39.533333 Average scores were used for ties.

Kruskal Wallis Test Chi Square 28.2346 DF 7 Pr > Chi Square 0.0002 Median Scores ( Number of Points Above Median) for Variable WEIGHT Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 7.0 9.909910 2.033723 0.350000 C3 20 8.0 9.909910 2.033723 0.400000 C6 20 12.0 9.909910 2.033723 0.600000 E1 6 1.0 2.972973 1.196537 0.166667 E3 10 6.0 4.954955 1.515014 0.600000 E4 10 6.0 4.954955 1.515014 0.600000 E6 10 10.0 4.954955 1.515014 1.000000 E8 15 5.0 7.432432 1.808995 0.333333 Average scores were used for ties. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 45 of 152 Median One Way Analysis Chi Square 18.3589 DF 7 Pr > Chi Square 0.0105 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 46 of 152 NONPARAMETRIC COMPARISON OF FROG WEIGHT BETWEEN PONDS 427  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable WEIGHT Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 649.0 1020.0 117.344960 32.450000 C3 17 568.0 867.0 110.171950 33.411765 C6 20 1525.0 1020.0 117.344960 76.250000 E1 8 311.0 408.0 79.523118 38.875000 E3 10 681.0 510.0 87.948337 68.100000 E4 10 658.0 510.0 87.948337 65.800000 E6 10 484.0 510.0 87.948337 48.400000 E8 6 275.0 306.0 69.605629 45.833333 Average scores were used for ties.

Kruskal Wallis Test Chi Square 36.5882 DF 7 Pr > Chi Square <. 0001 Median Scores ( Number of Points Above Median) for Variable WEIGHT Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 4.0 9.900990 2.012363 0.200000 C3 17 4.0 8.415842 1.889352 0.235294 C6 20 18.0 9.900990 2.012363 0.900000 E1 8 2.0 3.960396 1.363751 0.250000 E3 10 6.0 4.950495 1.508236 0.600000 E4 10 9.0 4.950495 1.508236 0.900000 E6 10 4.0 4.950495 1.508236 0.400000 E8 6 3.0 2.970297 1.193675 0.500000 Average scores were used for ties. Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 47 of 152 Median One Way Analysis Chi Square 33.6219 DF 7 Pr > Chi Square <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 48 of 152 ANOVA FOR WEIGHT OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 429  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: WEIGHT Sum of Source DF Squares Mean Square F Value Pr > F Model 1 75.510261 75.510261 0.44 0.5307 Error 6 1024.136445 170.689408 Corrected Total 7 1099.646706 R Square Coeff Var Root MSE WEIGHT Mean 0.068668 32.30525 13.06482 40.44177 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 75.51026075 75.51026075 0.44 0.5307 Levene's Test for Homogeneity of WEIGHT Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 64108.8 64108.8 1.83 0.2246 Error 6 209888 34981.3 Bartlett's Test for Homogeneity of WEIGHT Variance Source DF Chi Square Pr > ChiSq GROUP 1 2.4716 0.1159 Dunnett's t Tests for WEIGHT Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 49 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 170.6894 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference Simultaneous GROUP Between 95% Confidence Comparison Means Limits REF  EXP  6.346  29.693 17.001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 50 of 152 ANOVA FOR WEIGHT OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 433  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: WEIGHT Sum of Source DF Squares Mean Square F Value Pr > F Model 1 2.6671491 2.6671491 0.06 0.8143 Error 6 265.7568743 44.2928124 Corrected Total 7 268.4240234 R Square Coeff Var Root MSE WEIGHT Mean 0.009936 25.05318 6.655285 26.56463 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 2.66714907 2.66714907 0.06 0.8143 Levene's Test for Homogeneity of WEIGHT Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 5070.2 5070.2 4.36 0.0818 Error 6 6973.5 1162.3 Bartlett's Test for Homogeneity of WEIGHT Variance Source DF Chi Square Pr > ChiSq GROUP 1 1.8380 0.1752 Dunnett's t Tests for WEIGHT Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 51 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 44.29281 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference Simultaneous GROUP Between 95% Confidence Comparison Means Limits REF  EXP  1.193  13.086 10.700 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 52 of 152 NONPARAMETRIC COMPARISON OF FROG WEIGHT BETWEEN GROUPS 437  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable WEIGHT Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 23.0 22.50 3.354102 4.600000 REF 3 13.0 13.50 3.354102 4.333333 Wilcoxon Two Sample Test Statistic 13.0000 Normal Approximation Z 0.0000 One Sided Pr < Z 0.5000 Two Sided Pr > | Z| 1.0000 t Approximation One Sided Pr < Z 0.5000 Two Sided Pr > | Z| 1.0000 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 0.0222 DF 1 Pr > Chi Square 0.8815 Median Scores ( Number of Points Above Median) for Variable WEIGHT Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 REF 3 1.0 1.50 0.731925 0.333333 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 53 of 152 Median Two Sample Test Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 54 of 152 NONPARAMETRIC COMPARISON OF FROG WEIGHT BETWEEN GROUPS 439  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable WEIGHT Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 25.0 22.50 3.354102 5.000000 REF 3 11.0 13.50 3.354102 3.666667 Wilcoxon Two Sample Test Statistic 11.0000 Normal Approximation Z  0.5963 One Sided Pr < Z 0.2755 Two Sided Pr > | Z| 0.5510 t Approximation One Sided Pr < Z 0.2849 Two Sided Pr > | Z| 0.5698 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 0.5556 DF 1 Pr > Chi Square 0.4561 Median Scores ( Number of Points Above Median) for Variable WEIGHT Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 REF 3 1.0 1.50 0.731925 0.333333 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 55 of 152 Median Two Sample Test Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 56 of 152 MEAN TESTICULAR WEIGHTS FOR FROGS COLLECTED BY POND 441 Obs POND GROUP _ TYPE_ _ FREQ_ TESTES T_ SD T_ CV 1 C1 REF 0 20 0.024585 0.010550 42.9144 2 C3 REF 0 20 0.030953 0.023958 77.4001 3 C6 REF 0 20 0.047570 0.016667 35.0366 4 E1 EXP 0 10 0.036925 0.014242 38.5703 5 E3 EXP 0 10 0.053570 0.017665 32.9753 6 E4 EXP 0 10 0.057670 0.009618 16.6769 7 E6 EXP 0 10 0.033560 0.018074 53.8565 8 E8 EXP 0 10 0.044200 0.019885 44.9884 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 57 of 152 ANOVA FOR TESTICULAR WEIGHT FOR FROGS BETWEEN PONDS 442 The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 8 Dependent Variable: TESTES Sum of Source DF Squares Mean Square F Value Pr > F Model 7 0.00093152 0.00013307 . .

Error 0 0.00000000 .

Corrected Total 7 0.00093152 R Square Coeff Var Root MSE TESTES Mean 1.000000 . . 0.041129 Source DF Anova SS Mean Square F Value Pr > F POND 7 0.00093152 0.00013307 . .

Levene's Test for Homogeneity of TESTES Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 0 0 . . .

Error 0 0 .

Bartlett's Test for Homogeneity of TESTES Variance Source DF Chi Square Pr > ChiSq POND 0 0 .

ANOVA FOR TESTICULAR WEIGHT FOR FROGS BETWEEN PONDS 445 The ANOVA Procedure Level of  TESTES Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 58 of 152 POND N Mean Std Dev C1 1 0.02458500 .

C3 1 0.03095294 .

C6 1 0.04757000 .

E1 1 0.03692500 .

E3 1 0.05357000 .

E4 1 0.05767000 .

E6 1 0.03356000 .

E8 1 0.04420000 . Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 59 of 152 ANOVA FOR TESTICULAR WEIGHT FOR FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITE 446 The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: TESTES Sum of Source DF Squares Mean Square F Value Pr > F Model 1 0.00021934 0.00021934 1.85 0.2229 Error 6 0.00071218 0.00011870 Corrected Total 7 0.00093152 R Square Coeff Var Root MSE TESTES Mean 0.235460 26.48934 0.010895 0.041129 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 0.00021934 0.00021934 1.85 0.2229 Levene's Test for Homogeneity of TESTES Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 1.14E 10 1.14E 10 0.02 0.8818 Error 6 2.831E 8 4.719E 9 Bartlett's Test for Homogeneity of TESTES Variance Source DF Chi Square Pr > ChiSq GROUP 1 0.0415 0.8386 Dunnett's t Tests for TESTES Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 60 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 0.000119 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference GROUP Between Simultaneous 95%

Comparison Means Confidence Limits REF  EXP  0.010816  0.030285 0.008653 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 61 of 152 NONPARAMETRIC COMPARISON OF FROG TESTICULAR WEIGHT BETWEEN TREATMENT GROUPS 450 The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable TESTES Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 27.0 22.50 3.354102 5.40 REF 3 9.0 13.50 3.354102 3.00 Wilcoxon Two Sample Test Statistic 9.0000 Normal Approximation Z  1.1926 One Sided Pr < Z 0.1165 Two Sided Pr > | Z| 0.2330 t Approximation One Sided Pr < Z 0.1359 Two Sided Pr > | Z| 0.2719 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 1.8000 DF 1 Pr > Chi Square 0.1797 Median Scores ( Number of Points Above Median) for Variable TESTES Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 REF 3 1.0 1.50 0.731925 0.333333 Median Two Sample Test Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 62 of 152 Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 63 of 152 MEAN GONADOSOMATIC INDEX ( GSI) OF FROGS COLLECTED BY POND AND SEX 452 Obs POND Sex GROUP _ TYPE_ _ FREQ_ GSI GSI_ SD GSI_ CV 1 C1 F REF 0 20 1.20519 0.59529 49.3938 2 C1 M REF 0 20 0.12568 0.02906 23.1225 3 C3 F REF 0 20 2.32511 1.78135 76.6135 4 C3 M REF 0 20 0.13554 0.04066 29.9998 5 C6 F REF 0 20 1.56896 1.23447 78.6810 6 C6 M REF 0 20 0.12992 0.03488 26.8432 7 E1 F EXP 0 10 3.84820 3.58562 93.1766 8 E1 M EXP 0 10 0.17529 0.04832 27.5681 9 E3 F EXP 0 10 5.74161 2.38193 41.4853 10 E3 M EXP 0 10 0.16518 0.03373 20.4210 11 E4 F EXP 0 10 5.39806 1.45422 26.9397 12 E4 M EXP 0 10 0.19433 0.03069 15.7908 13 E6 F EXP 0 10 2.52463 1.94072 76.8714 14 E6 M EXP 0 10 0.13001 0.04169 32.0636 15 E8 F EXP 0 15 4.60581 3.38835 73.5669 16 E8 M EXP 0 10 0.18583 0.02224 11.9674 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 64 of 152 ANOVA FOR GSI OF FROGS BETWEEN PONDS BY SEX 453  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 115 NOTE: Due to missing values, only 111 observations can be used in this analysis.

Dependent Variable: GSI Sum of Source DF Squares Mean Square F Value Pr > F Model 7 292.3774342 41.7682049 10.12 <. 0001 Error 103 424.9868075 4.1260855 Corrected Total 110 717.3642417 R Square Coeff Var Root MSE GSI Mean 0.407572 68.15863 2.031277 2.980220 Source DF Anova SS Mean Square F Value Pr > F POND 7 292.3774342 41.7682049 10.12 <. 0001 Levene's Test for Homogeneity of GSI Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 1421.7 203.1 7.65 <. 0001 Error 103 2732.9 26.5335 Bartlett's Test for Homogeneity of GSI Variance Source DF Chi Square Pr > ChiSq Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 65 of 152 POND 7 52.6276 <. 0001 Dunnett's t Tests for GSI NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 103 Error Mean Square 4.126086 Critical Value of Dunnett's t 2.69261 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits E3  C1 4.5364 2.4181 6.6547 ***

E4  C1 4.1929 2.0746 6.3112 ***

E8  C1 3.4006 1.5325 5.2688 ***

E1  C1 2.6430 0.0971 5.1889 ***

E6  C1 1.3194  0.7989 3.4377 C3  C1 1.1199  0.6097 2.8495 C6  C1 0.3638  1.3658 2.0934 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 66 of 152 ANOVA FOR GSI OF FROGS BETWEEN PONDS BY SEX 457  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 110 NOTE: Due to missing values, only 101 observations can be used in this analysis.

Dependent Variable: GSI Sum of Source DF Squares Mean Square F Value Pr > F Model 7 0.06119627 0.00874232 6.84 <. 0001 Error 93 0.11878563 0.00127726 Corrected Total 100 0.17998190 R Square Coeff Var Root MSE GSI Mean 0.340013 24.34181 0.035739 0.146821 Source DF Anova SS Mean Square F Value Pr > F POND 7 0.06119627 0.00874232 6.84 <. 0001 Levene's Test for Homogeneity of GSI Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 0.000018 2.513E 6 0.86 0.5406 Error 93 0.000272 2.92E 6 Bartlett's Test for Homogeneity of GSI Variance Source DF Chi Square Pr > ChiSq Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 67 of 152 POND 7 5.9559 0.5449 Dunnett's t Tests for GSI NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 93 Error Mean Square 0.001277 Critical Value of Dunnett's t 2.70384 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits E4  C1 0.06865 0.03122 0.10607 ***

E8  C1 0.06015 0.01517 0.10513 ***

E1  C1 0.04961 0.00918 0.09003 ***

E3  C1 0.03950 0.00208 0.07693 ***

C3  C1 0.00986  0.02202 0.04173 E6  C1 0.00433  0.03309 0.04176 C6  C1 0.00424  0.02632 0.03480 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 68 of 152 NONPARAMETRIC COMPARISON OF GSI BETWEEN PONDS BY SEX 461  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable GSI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 640.0 1120.0 130.332907 32.000000 C3 20 1005.0 1120.0 130.332907 50.250000 C6 20 780.0 1120.0 130.332907 39.000000 E1 6 347.0 336.0 76.681158 57.833333 E3 10 885.0 560.0 97.091023 88.500000 E4 10 887.0 560.0 97.091023 88.700000 E6 10 577.0 560.0 97.091023 57.700000 E8 15 1095.0 840.0 115.931014 73.000000 Kruskal Wallis Test Chi Square 42.0856 DF 7 Pr > Chi Square <. 0001 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable GSI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 3.0 9.909910 2.033723 0.150000 C3 20 11.0 9.909910 2.033723 0.550000 C6 20 4.0 9.909910 2.033723 0.200000 E1 6 3.0 2.972973 1.196537 0.500000 E3 10 9.0 4.954955 1.515014 0.900000 E4 10 10.0 4.954955 1.515014 1.000000 E6 10 4.0 4.954955 1.515014 0.400000 E8 15 11.0 7.432432 1.808995 0.733333 Median One Way Analysis Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 69 of 152 Chi Square 36.9250 DF 7 Pr > Chi Square <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 70 of 152 NONPARAMETRIC COMPARISON OF GSI BETWEEN PONDS BY SEX 463  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable GSI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 714.0 1020.0 117.345643 35.700000 C3 17 783.0 867.0 110.172592 46.058824 C6 20 757.0 1020.0 117.345643 37.850000 E1 8 548.0 408.0 79.523581 68.500000 E3 10 645.0 510.0 87.948849 64.500000 E4 10 829.0 510.0 87.948849 82.900000 E6 10 394.0 510.0 87.948849 39.400000 E8 6 481.0 306.0 69.606034 80.166667 Kruskal Wallis Test Chi Square 34.3083 DF 7 Pr > Chi Square <. 0001 Median Scores ( Number of Points Above Median) for Variable GSI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 5.0 9.900990 2.012363 0.250000 C3 17 9.0 8.415842 1.889352 0.529412 C6 20 4.0 9.900990 2.012363 0.200000 E1 8 6.0 3.960396 1.363751 0.750000 E3 10 6.0 4.950495 1.508236 0.600000 E4 10 10.0 4.950495 1.508236 1.000000 E6 10 4.0 4.950495 1.508236 0.400000 E8 6 6.0 2.970297 1.193675 1.000000 Median One Way Analysis Chi Square 30.7445 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 71 of 152 DF 7 Pr > Chi Square <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 72 of 152 ANOVA FOR GSI OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 465  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: GSI Sum of Source DF Squares Mean Square F Value Pr > F Model 1 13.91190688 13.91190688 11.42 0.0149 Error 6 7.30985217 1.21830869 Corrected Total 7 21.22175905 R Square Coeff Var Root MSE GSI Mean 0.655549 32.44289 1.103770 3.402195 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 13.91190688 13.91190688 11.42 0.0149 Levene's Test for Homogeneity of GSI Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 2.3261 2.3261 1.69 0.2409 Error 6 8.2425 1.3737 Bartlett's Test for Homogeneity of GSI Variance Source DF Chi Square Pr > ChiSq GROUP 1 1.1609 0.2813 Dunnett's t Tests for GSI Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 73 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 1.218309 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference GROUP Between Simultaneous 95%

Comparison Means Confidence Limits REF  EXP  2.7239  4.6963  0.7515 *** Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 74 of 152 ANOVA FOR GSI OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 469  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: GSI Sum of Source DF Squares Mean Square F Value Pr > F Model 1 0.00296251 0.00296251 6.99 0.0383 Error 6 0.00254148 0.00042358 Corrected Total 7 0.00550399 R Square Coeff Var Root MSE GSI Mean 0.538248 13.25886 0.020581 0.155225 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 0.00296251 0.00296251 6.99 0.0383 Levene's Test for Homogeneity of GSI Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 4.36E 7 4.36E 7 1.49 0.2676 Error 6 1.753E 6 2.921E 7 Bartlett's Test for Homogeneity of GSI Variance Source DF Chi Square Pr > ChiSq GROUP 1 3.4829 0.0620 Dunnett's t Tests for GSI Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 75 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 0.000424 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference GROUP Between Simultaneous 95%

Comparison Means Confidence Limits REF  EXP  0.03975  0.07653  0.00297 *** Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 76 of 152 NONPARAMETRIC COMPARISON OF GSI BETWEEN TREATMENT GROUPS BY SEX 473  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable GSI Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 30.0 22.50 3.354102 6.0 REF 3 6.0 13.50 3.354102 2.0 Wilcoxon Two Sample Test Statistic 6.0000 Normal Approximation Z  2.0870 One Sided Pr < Z 0.0184 Two Sided Pr > | Z| 0.0369 t Approximation One Sided Pr < Z 0.0377 Two Sided Pr > | Z| 0.0753 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 5.0000 DF 1 Pr > Chi Square 0.0253 Median Scores ( Number of Points Above Median) for Variable GSI Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 4.0 2.50 0.731925 0.80 REF 3 0.0 1.50 0.731925 0.00 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 77 of 152 Median Two Sample Test Statistic 0.0000 Z  2.0494 One Sided Pr < Z 0.0202 Two Sided Pr > | Z| 0.0404 Median One Way Analysis Chi Square 4.2000 DF 1 Pr > Chi Square 0.0404 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 78 of 152 NONPARAMETRIC COMPARISON OF GSI BETWEEN TREATMENT GROUPS BY SEX 475  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable GSI Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 29.0 22.50 3.354102 5.800000 REF 3 7.0 13.50 3.354102 2.333333 Wilcoxon Two Sample Test Statistic 7.0000 Normal Approximation Z  1.7889 One Sided Pr < Z 0.0368 Two Sided Pr > | Z| 0.0736 t Approximation One Sided Pr < Z 0.0584 Two Sided Pr > | Z| 0.1168 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 3.7556 DF 1 Pr > Chi Square 0.0526 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable GSI Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 4.0 2.50 0.731925 0.80 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 79 of 152 REF 3 0.0 1.50 0.731925 0.00 Median Two Sample Test Statistic 0.0000 Z  2.0494 One Sided Pr < Z 0.0202 Two Sided Pr > | Z| 0.0404 Median One Way Analysis Chi Square 4.2000 DF 1 Pr > Chi Square 0.0404 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 80 of 152 MEAN CONDITION INDEX OF FROGS COLLECTED BY POND AND SEX 477 Obs POND Sex GROUP _ TYPE_ _ FREQ_ CONDITION CI_ SD CI_ CV 1 C1 F REF 0 20 0.46055 0.10789 23.4271 2 C1 M REF 0 20 0.32900 0.06735 20.4721 3 C3 F REF 0 20 0.47447 0.19746 41.6174 4 C3 M REF 0 20 0.33864 0.14453 42.6786 5 C6 F REF 0 20 0.53024 0.09217 17.3826 6 C6 M REF 0 20 0.50706 0.10983 21.6600 7 E1 F EXP 0 10 0.41857 0.18672 44.6087 8 E1 M EXP 0 10 0.37327 0.15799 42.3275 9 E3 F EXP 0 10 0.52443 0.06631 12.6435 10 E3 M EXP 0 10 0.48108 0.09040 18.7901 11 E4 F EXP 0 10 0.61236 0.14012 22.8822 12 E4 M EXP 0 10 0.46696 0.04620 9.8932 13 E6 F EXP 0 10 0.74118 0.17227 23.2423 14 E6 M EXP 0 10 0.40045 0.12809 31.9870 15 E8 F EXP 0 15 0.42604 0.19041 44.6937 16 E8 M EXP 0 10 0.37014 0.11131 30.0735 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 81 of 152 ANOVA FOR CONDITION INDEX OF FROGS BETWEEN PONDS BY SEX 478  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 115 NOTE: Due to missing values, only 111 observations can be used in this analysis.

Dependent Variable: CI Sum of Source DF Squares Mean Square F Value Pr > F Model 7 0.87825562 0.12546509 5.65 <. 0001 Error 103 2.28870294 0.02222042 Corrected Total 110 3.16695856 R Square Coeff Var Root MSE CI Mean 0.277318 29.03514 0.149065 0.513396 Source DF Anova SS Mean Square F Value Pr > F POND 7 0.87825562 0.12546509 5.65 <. 0001 Levene's Test for Homogeneity of CI Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 0.0167 0.00238 3.16 0.0046 Error 103 0.0775 0.000753 Bartlett's Test for Homogeneity of CI Variance Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 82 of 152 Source DF Chi Square Pr > ChiSq POND 7 23.0265 0.0017 Dunnett's t Tests for CI NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 103 Error Mean Square 0.02222 Critical Value of Dunnett's t 2.69261 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits E6  C1 0.28063 0.12518 0.43608 ***

E4  C1 0.15181  0.00365 0.30726 C6  C1 0.06969  0.05723 0.19662 E3  C1 0.06388  0.09157 0.21933 C3  C1 0.01392  0.11300 0.14085 E8  C1  0.03451  0.17161 0.10258 E1  C1  0.04198  0.22881 0.14485 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 83 of 152 ANOVA FOR CONDITION INDEX OF FROGS BETWEEN PONDS BY SEX 482  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values POND 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 110 NOTE: Due to missing values, only 101 observations can be used in this analysis.

Dependent Variable: CI Sum of Source DF Squares Mean Square F Value Pr > F Model 7 0.50980220 0.07282889 6.01 <. 0001 Error 93 1.12670273 0.01211508 Corrected Total 100 1.63650493 R Square Coeff Var Root MSE CI Mean 0.311519 27.00248 0.110069 0.407624 Source DF Anova SS Mean Square F Value Pr > F POND 7 0.50980220 0.07282889 6.01 <. 0001 Levene's Test for Homogeneity of CI Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F POND 7 0.00421 0.000602 1.65 0.1310 Error 93 0.0339 0.000365 Bartlett's Test for Homogeneity of CI Variance Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 84 of 152 Source DF Chi Square Pr > ChiSq POND 7 20.4868 0.0046 Dunnett's t Tests for CI NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 93 Error Mean Square 0.012115 Critical Value of Dunnett's t 2.70384 Comparisons significant at the 0.05 level are indicated by ***.

Difference POND Between Simultaneous 95%

Comparison Means Confidence Limits C6  C1 0.17805 0.08394 0.27217 ***

E3  C1 0.15208 0.03682 0.26734 ***

E4  C1 0.13795 0.02269 0.25322 ***

E6  C1 0.07145  0.04381 0.18672 E1  C1 0.04426  0.08024 0.16876 E8  C1 0.04114  0.09739 0.17967 C3  C1 0.00964  0.08853 0.10782 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 85 of 152 NONPARAMETRIC COMPARISON OF CONDITION INDEX BETWEEN PONDS BY SEX 486  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable CI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 901.0 1120.0 130.332907 45.050000 C3 20 982.0 1120.0 130.332907 49.100000 C6 20 1200.0 1120.0 130.332907 60.000000 E1 6 216.0 336.0 76.681158 36.000000 E3 10 607.0 560.0 97.091023 60.700000 E4 10 762.0 560.0 97.091023 76.200000 E6 10 946.0 560.0 97.091023 94.600000 E8 15 602.0 840.0 115.931014 40.133333 Kruskal Wallis Test Chi Square 28.0380 DF 7 Pr > Chi Square 0.0002 Median Scores ( Number of Points Above Median) for Variable CI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 7.0 9.909910 2.033723 0.350000 C3 20 8.0 9.909910 2.033723 0.400000 C6 20 11.0 9.909910 2.033723 0.550000 E1 6 1.0 2.972973 1.196537 0.166667 E3 10 6.0 4.954955 1.515014 0.600000 E4 10 8.0 4.954955 1.515014 0.800000 E6 10 10.0 4.954955 1.515014 1.000000 E8 15 4.0 7.432432 1.808995 0.266667 Median One Way Analysis Chi Square 22.5214 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 86 of 152 DF 7 Pr > Chi Square 0.0021 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 87 of 152 NONPARAMETRIC COMPARISON OF CONDITION INDEX BETWEEN PONDS BY SEX 488  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable CI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 632.0 1020.0 117.345643 31.600000 C3 17 572.0 867.0 110.172592 33.647059 C6 20 1476.0 1020.0 117.345643 73.800000 E1 8 319.0 408.0 79.523581 39.875000 E3 10 701.0 510.0 87.948849 70.100000 E4 10 700.0 510.0 87.948849 70.000000 E6 10 480.0 510.0 87.948849 48.000000 E8 6 271.0 306.0 69.606034 45.166667 Kruskal Wallis Test Chi Square 36.7915 DF 7 Pr > Chi Square <. 0001 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable CI Classified by Variable POND Sum of Expected Std Dev Mean POND N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 20 2.0 9.900990 2.012363 0.100000 C3 17 4.0 8.415842 1.889352 0.235294 C6 20 17.0 9.900990 2.012363 0.850000 E1 8 3.0 3.960396 1.363751 0.375000 E3 10 7.0 4.950495 1.508236 0.700000 E4 10 10.0 4.950495 1.508236 1.000000 E6 10 3.0 4.950495 1.508236 0.300000 E8 6 4.0 2.970297 1.193675 0.666667 Median One Way Analysis Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 88 of 152 Chi Square 41.3124 DF 7 Pr > Chi Square <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 89 of 152 ANOVA FOR CONDITION INDEX OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 490  Sex= F

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: CONDITION Sum of Source DF Squares Mean Square F Value Pr > F Model 1 0.00589954 0.00589954 0.46 0.5212 Error 6 0.07630211 0.01271702 Corrected Total 7 0.08220165 R Square Coeff Var Root MSE CONDITION Mean 0.071769 21.54234 0.112770 0.523480 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 0.00589954 0.00589954 0.46 0.5212 Levene's Test for Homogeneity of CONDITION Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 0.000358 0.000358 2.43 0.1703 Error 6 0.000884 0.000147 Bartlett's Test for Homogeneity of CONDITION Variance Source DF Chi Square Pr > ChiSq GROUP 1 2.5067 0.1134 Dunnett's t Tests for CONDITION Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 90 of 152 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 0.012717 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference GROUP Between Simultaneous 95%

Comparison Means Confidence Limits REF  EXP  0.05609  0.25761 0.14543 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 91 of 152 ANOVA FOR CONDITION INDEX OF FROGS BETWEEN REFERENCE ( REF) AND EXPERIMENTAL ( EXP) SITES 494  Sex= M

The ANOVA Procedure Class Level Information Class Levels Values GROUP 2 EXP REF Number of observations 8 Dependent Variable: CONDITION Sum of Source DF Squares Mean Square F Value Pr > F Model 1 0.00134800 0.00134800 0.26 0.6279 Error 6 0.03102745 0.00517124 Corrected Total 7 0.03237545 R Square Coeff Var Root MSE CONDITION Mean 0.041636 17.61129 0.071911 0.408325 Source DF Anova SS Mean Square F Value Pr > F GROUP 1 0.00134800 0.00134800 0.26 0.6279 Levene's Test for Homogeneity of CONDITION Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F GROUP 1 0.000038 0.000038 3.08 0.1298 Error 6 0.000074 0.000012 Bartlett's Test for Homogeneity of CONDITION Variance Source DF Chi Square Pr > ChiSq GROUP 1 1.0139 0.3140 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 92 of 152 Dunnett's t Tests for CONDITION NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 0.005171 Critical Value of Dunnett's t 2.44695 Comparisons significant at the 0.05 level are indicated by ***.

Difference GROUP Between Simultaneous 95%

Comparison Means Confidence Limits REF  EXP  0.02681  0.15532 0.10169 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 93 of 152 NONPARAMETRIC COMPARISON OF CONDITION INDEX BETWEEN GROUPS BY SEX 498  Sex= F

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable CONDITION Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 23.0 22.50 3.354102 4.600000 REF 3 13.0 13.50 3.354102 4.333333 Wilcoxon Two Sample Test Statistic 13.0000 Normal Approximation Z 0.0000 One Sided Pr < Z 0.5000 Two Sided Pr > | Z| 1.0000 t Approximation One Sided Pr < Z 0.5000 Two Sided Pr > | Z| 1.0000 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 0.0222 DF 1 Pr > Chi Square 0.8815 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable CONDITION Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 94 of 152 REF 3 1.0 1.50 0.731925 0.333333 Median Two Sample Test Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 95 of 152 NONPARAMETRIC COMPARISON OF CONDITION INDEX BETWEEN GROUPS BY SEX 500  Sex= M

The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable CONDITION Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 25.0 22.50 3.354102 5.000000 REF 3 11.0 13.50 3.354102 3.666667 Wilcoxon Two Sample Test Statistic 11.0000 Normal Approximation Z  0.5963 One Sided Pr < Z 0.2755 Two Sided Pr > | Z| 0.5510 t Approximation One Sided Pr < Z 0.2849 Two Sided Pr > | Z| 0.5698 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 0.5556 DF 1 Pr > Chi Square 0.4561 The NPAR1WAY Procedure Median Scores ( Number of Points Above Median) for Variable CONDITION Classified by Variable GROUP Sum of Expected Std Dev Mean GROUP N Scores Under H0 Under H0 Score âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ EXP 5 3.0 2.50 0.731925 0.600000 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 96 of 152 REF 3 1.0 1.50 0.731925 0.333333 Median Two Sample Test Statistic 1.0000 Z  0.6831 One Sided Pr < Z 0.2473 Two Sided Pr > | Z| 0.4945 Median One Way Analysis Chi Square 0.4667 DF 1 Pr > Chi Square 0.4945 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 97 of 152 MEAN FEMALE PLASMA ESTRADIOL CONCENTRATIONS BY SITE AND ANIMAL NUMBER 82 Obs Site Ind_ No _ TYPE_ _ FREQ_ E2 STD CV 1 C1 21 0 3 4748.49 330.25 6.9549 2 C1 22 0 3 97.48 21.83 22.3947 3 C1 23 0 3 22.77 12.41 54.5215 4 C1 24 0 3 33025.69 7581.68 22.9569 5 C1 25 0 3 51.59 27.63 53.5657 6 C1 26 0 3 3137.18 267.56 8.5286 7 C1 27 0 3 3877.23 780.16 20.1215 8 C1 28 0 3 13.78 2.62 19.0307 9 C1 29 0 3 2781.17 285.40 10.2618 10 C1 30 0 3 110.61 25.45 23.0079 11 C1 31 0 3 25.21 10.25 40.6502 12 C1 32 0 3 5463.82 1002.76 18.3527 13 C1 33 0 3 49.35 24.61 49.8719 14 C1 34 0 3 292.85 184.51 63.0053 15 C1 35 0 3 55.43 25.69 46.3413 16 C1 36 0 3 6696.34 1406.15 20.9987 17 C1 37 0 3 . . .

18 C1 38 0 3 29043.30 0.00 0.0000 19 C1 39 0 3 16585.36 2876.22 17.3419 20 C1 40 0 3 14608.67 1333.87 9.1307 21 C3 21 0 3 18963.76 6968.94 36.7487 22 C3 22 0 3 69.64 8.83 12.6770 23 C3 23 0 3 242.59 62.08 25.5892 24 C3 24 0 3 7865.85 343.34 4.3649 25 C3 25 0 3 . . .

26 C3 26 0 3 367.45 87.33 23.7661 27 C3 27 0 3 6028.80 244.98 4.0635 28 C3 28 0 3 . . .

29 C3 29 0 3 145.50 58.21 40.0054 30 C3 30 0 3 6402.32 782.25 12.2182 31 C3 31 0 3 8629.88 1789.24 20.7330 32 C3 32 0 3 5017.56 551.41 10.9895 33 C3 33 0 3 33218.85 0.00 0.0000 34 C3 34 0 3 33.97 3.34 9.8194 35 C3 35 0 3 23.23 10.79 46.4362 36 C3 36 0 3 249.98 8.50 3.4014 37 C3 37 0 3 169.56 36.07 21.2708 38 C3 38 0 3 15342.62 4160.93 27.1200 39 C3 39 0 3 5834.65 1516.16 25.9854 40 C3 40 0 3 108.12 63.16 58.4186 41 C6 21 0 3 8718.85 770.64 8.8388 42 C6 22 0 3 112.90 30.66 27.1571 43 C6 23 0 3 . . . Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 98 of 152 44 C6 24 0 3 4578.96 512.44 11.1912 45 C6 25 0 3 546.55 199.05 36.4200 46 C6 26 0 3 26.31 19.35 73.5299 47 C6 27 0 3 4376.85 163.55 3.7366 48 C6 28 0 3 31.89 5.44 17.0485 49 C6 29 0 3 5682.53 431.56 7.5946 50 C6 30 0 3 4741.45 502.20 10.5918 51 C6 31 0 3 73.75 21.62 29.3201 52 C6 32 0 3 3646.29 166.95 4.5785 53 C6 33 0 3 4181.47 . .

54 C6 34 0 3 19.63 16.47 83.9291 55 C6 35 0 3 26.74 14.71 55.0063 56 C6 36 0 3 56754.90 0.00 0.0000 57 C6 37 0 3 4586.95 357.81 7.8006 58 C6 38 0 3 34923.37 1076.57 3.0827 59 C6 39 0 3 7233.54 943.02 13.0367 60 C6 40 0 3 105573.43 9858.48 9.3380 61 E1 1 0 3 260.32 44.45 17.0755 62 E1 2 0 3 1859.49 92.60 4.9797 63 E1 3 0 3 3229.19 374.05 11.5835 64 E1 4 0 3 680.29 79.79 11.7284 65 E1 5 0 3 46.21 19.26 41.6735 66 E1 6 0 3 . . .

67 E3 1 0 3 128.90 23.59 18.3008 68 E3 2 0 3 198.40 45.41 22.8901 69 E3 3 0 3 162.61 19.74 12.1382 70 E3 4 0 3 266.59 4.57 1.7142 71 E3 5 0 3 . . . Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 99 of 152 MEAN FEMALE PLASMA ESTRADIOL CONCENTRATIONS BY SITE AND ANIMAL NUMBER 83 Obs Site Ind_ No _ TYPE_ _ FREQ_ E2 STD CV 72 E3 6 0 3 206.28 25.87 12.5389 73 E3 7 0 3 89.89 11.12 12.3714 74 E3 8 0 3 296.97 13.75 4.6306 75 E3 9 0 3 367.43 44.06 11.9904 76 E3 10 0 3 122.72 13.99 11.3976 77 E4 1 0 3 1041.18 134.13 12.8821 78 E4 2 0 3 2967.96 495.96 16.7104 79 E4 3 0 3 1810.90 319.77 17.6580 80 E4 4 0 3 659.32 146.41 22.2065 81 E4 5 0 3 561.15 83.25 14.8360 82 E4 6 0 3 3533.16 209.97 5.9427 83 E4 7 0 3 781.20 25.54 3.2692 84 E4 8 0 3 76.74 4.14 5.3991 85 E4 9 0 3 7830.98 1283.47 16.3897 86 E4 10 0 3 2956.11 376.50 12.7363 87 E6 1 0 3 95.92 40.27 41.9873 88 E6 2 0 3 124.57 27.11 21.7657 89 E6 3 0 3 7.63 0.00 0.0000 90 E6 4 0 3 138.21 47.09 34.0689 91 E6 5 0 3 87.17 2.84 3.2539 92 E6 6 0 3 166.49 32.02 19.2329 93 E6 7 0 3 182.97 58.74 32.1016 94 E6 8 0 3 10.56 0.00 0.0000 95 E6 9 0 3 140.54 29.13 20.7254 96 E6 10 0 3 128.53 31.29 24.3410 97 E8 1 0 3 392.23 31.03 7.9118 98 E8 2 0 3 1894.28 290.91 15.3573 99 E8 3 0 3 248.29 16.21 6.5302 100 E8 4 0 3 739.52 140.60 19.0130 101 E8 5 0 3 3581.29 223.32 6.2359 102 E8 6 0 3 594.92 53.15 8.9345 103 E8 7 0 3 567.99 61.997 10.9152 104 E8 8 0 3 8292.57 562.256 6.7802 105 E8 9 0 3 253.48 13.662 5.3897 106 E8 10 0 3 245.23 35.042 14.2896 107 E8 11 0 3 490.67 159.633 32.5339 108 E8 12 0 3 114.38 23.707 20.7265 109 E8 13 0 3 . . .

110 E8 14 0 3 . . .

111 E8 15 0 3 . . . Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 100 of 152 ANOVA FOR FEMALE PLASMA ESTRADIOL BETWEEN SITES 85 The ANOVA Procedure Class Level Information Class Levels Values Site 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 111 NOTE: Due to missing values, only 102 observations can be used in this analysis.

Dependent Variable: E2 Sum of Source DF Squares Mean Square F Value Pr > F Model 7 1995951818 285135974 1.69 0.1218 Error 94 15898863008 169136841 Corrected Total 101 17894814826 R Square Coeff Var Root MSE E2 Mean 0.111538 253.2194 13005.26 5135.965 Source DF Anova SS Mean Square F Value Pr > F Site 7 1995951818 285135974 1.69 0.1218 Levene's Test for Homogeneity of E2 Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F Site 7 6.25E18 8.929E17 1.19 0.3154 Error 94 7.045E19 7.495E17 Bartlett's Test for Homogeneity of E2 Variance Source DF Chi Square Pr > ChiSq Site 7 250.4 <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 101 of 152 Dunnett's t Tests for E2 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 94 Error Mean Square 1.6914E8 Critical Value of Dunnett's t 2.69946 Comparisons significant at the 0.05 level are indicated by ***.

Difference Simultaneous Site Between 95% Confidence Comparison Means Limits C6  C1 6587  4803 17977 C3  C1  312  11860 11235 E4  C1  4130  17846 9586 E8  C1  4901  17846 8045 E1  C1  5137  22783 12509 E3  C1  6147  20354 8059 E6  C1  6244  19959 7472 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 102 of 152 NONPARAMETRIC COMPARISON OF FEMALE PLASMA ESTRADIOL BETWEEN SITES 89 The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable E2 Classified by Variable Site Sum of Expected Std Dev Mean Site N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 19 985.0 978.50 116.343958 51.842105 C3 18 1059.0 927.00 113.921025 58.833333 C6 19 1142.0 978.50 116.343958 60.105263 E1 5 247.0 257.50 64.520669 49.400000 E3 9 331.0 463.50 84.759955 36.777778 E4 10 603.0 515.00 88.863191 60.300000 E6 10 235.0 515.00 88.863191 23.500000 E8 12 651.0 618.00 96.280839 54.250000 Kruskal Wallis Test Chi Square 14.9116 DF 7 Pr > Chi Square 0.0371 Median Scores ( Number of Points Above Median) for Variable E2 Classified by Variable Site Sum of Expected Std Dev Mean Site N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 19 10.0 9.50 1.975719 0.526316 C3 18 9.0 9.00 1.934573 0.500000 C6 19 13.0 9.50 1.975719 0.684211 E1 5 3.0 2.50 1.095671 0.600000 E3 9 0.0 4.50 1.439369 0.000000 E4 10 9.0 5.00 1.509049 0.900000 E6 10 0.0 5.00 1.509049 0.000000 E8 12 7.0 6.00 1.635013 0.583333 Median One Way Analysis Chi Square 28.2849 DF 7 Pr > Chi Square 0.0002 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 103 of 152 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 104 of 152 FEMALE PLASMA ESTRADIOL CONCENTRATIONS BY TREATMENT 91 Obs TREAT _ TYPE_ _ FREQ_ E2 STD CV 1 ATRAZINE 0 153 1057.20 1810.05 171.211 2 CONTROL 0 180 8612.35 17414.02 202.198 ANOVA FOR FEMALE PLASMA ESTRADIOL ACROSS TREATMENTS 92 The ANOVA Procedure Class Level Information Class Levels Values TREAT 2 ATRAZINE CONTROL Number of observations 333 NOTE: Due to missing values, only 302 observations can be used in this analysis.

Dependent Variable: E2 Sum of Source DF Squares Mean Square F Value Pr > F Model 1 4277610072 4277610072 25.73 <. 0001 Error 300 49878301383 166261005 Corrected Total 301 54155911454 R Square Coeff Var Root MSE E2 Mean 0.078987 249.8882 12894.22 5159.997 Source DF Anova SS Mean Square F Value Pr > F TREAT 1 4277610072 4277610072 25.73 <. 0001 Levene's Test for Homogeneity of E2 Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F TREAT 1 6.662E18 6.662E18 7.08 0.0082 Error 300 2.821E20 9.404E17 Bartlett's Test for Homogeneity of E2 Variance Source DF Chi Square Pr > ChiSq TREAT 1 438.5 <. 0001 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 105 of 152 Dunnett's t Tests for E2 NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 300 Error Mean Square 1.6626E8 Critical Value of Dunnett's t 1.96790 Comparisons significant at the 0.05 level are indicated by ***.

Simultaneous Difference 95%

TREAT Between Confidence Comparison Means Limits CONTROL  ATRAZINE 7555 4624 10486 *** Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 106 of 152 NONPARAMETRIC COMPARISON OF FEMALE PLASMA ESTRADIOL BETWEEN TREATMENTS 96 The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable E2 Classified by Variable TREAT Sum of Expected Std Dev Mean TREAT N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ ATRAZINE 138 18405.0 20907.0 755.946352 133.369565 CONTROL 164 27348.0 24846.0 755.946352 166.756098 Average scores were used for ties.

Wilcoxon Two Sample Test Statistic 18405.0000 Normal Approximation Z  3.3091 One Sided Pr < Z 0.0005 Two Sided Pr > | Z| 0.0009 t Approximation One Sided Pr < Z 0.0005 Two Sided Pr > | Z| 0.0010 Z includes a continuity correction of 0.5.

Kruskal Wallis Test Chi Square 10.9545 DF 1 Pr > Chi Square 0.0009 Median Scores ( Number of Points Above Median) for Variable E2 Classified by Variable TREAT Sum of Expected Std Dev Mean TREAT N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ ATRAZINE 138 57.0 69.0 4.335590 0.413043 CONTROL 164 94.0 82.0 4.335590 0.573171 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 107 of 152 Average scores were used for ties.

Median Two Sample Test Statistic 57.0000 Z  2.7678 One Sided Pr < Z 0.0028 Two Sided Pr > | Z| 0.0056 Median One Way Analysis Chi Square 7.6607 DF 1 Pr > Chi Square 0.0056 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 108 of 152 MEAN FEMALE PLASMA TESTOSTERONE CONCENTRATIONS BY SITE AND ANIMAL NUMBER 98 Obs Site Ind_ No TREAT _ TYPE_ _ FREQ_ T STD CV 1 C1 21 CONTROL 0 6 1934.52 262.42 13.565 2 C1 22 CONTROL 0 6 321.86 293.90 91.313 3 C1 23 CONTROL 0 6 86.37 34.90 40.408 4 C1 24 CONTROL 0 6 14971.64 3255.59 21.745 5 C1 25 CONTROL 0 6 139.76 121.89 87.217 6 C1 26 CONTROL 0 6 103.94 85.34 82.100 7 C1 27 CONTROL 0 6 7023.04 900.09 12.816 8 C1 28 CONTROL 0 6 120.39 143.49 119.182 9 C1 29 CONTROL 0 6 3169.52 186.57 5.887 10 C1 30 CONTROL 0 6 173.79 21.49 12.363 11 C1 31 CONTROL 0 6 21.74 4.23 19.473 12 C1 32 CONTROL 0 6 2863.16 374.89 13.094 13 C1 33 CONTROL 0 6 7710.56 11725.47 152.070 14 C1 34 CONTROL 0 6 882.00 742.98 84.238 15 C1 35 CONTROL 0 6 19.60 6.26 31.937 16 C1 36 CONTROL 0 6 2153.11 262.17 12.176 17 C1 37 CONTROL 0 6 . . .

18 C1 38 CONTROL 0 6 14521.65 0.00 0.000 19 C1 39 CONTROL 0 6 6802.20 479.18 7.045 20 C1 40 CONTROL 0 6 4473.92 192.11 4.294 21 C3 21 CONTROL 0 6 7195.61 2147.40 29.843 22 C3 22 CONTROL 0 6 182.50 51.98 28.479 23 C3 23 CONTROL 0 6 208.80 18.94 9.072 24 C3 24 CONTROL 0 6 2036.82 371.23 18.226 25 C3 25 CONTROL 0 6 . . .

26 C3 26 CONTROL 0 6 217.95 46.48 21.327 27 C3 27 CONTROL 0 6 2243.51 289.97 12.925 28 C3 28 CONTROL 0 6 177.61 172.35 97.040 29 C3 29 CONTROL 0 6 491.07 415.26 84.562 30 C3 30 CONTROL 0 6 2786.38 1293.81 46.433 31 C3 31 CONTROL 0 6 9437.32 1086.34 11.511 32 C3 32 CONTROL 0 6 2538.57 225.46 8.882 33 C3 33 CONTROL 0 6 13840.63 3033.06 21.914 34 C3 34 CONTROL 0 6 67.85 32.83 48.383 35 C3 35 CONTROL 0 6 71.99 48.21 66.959 36 C3 36 CONTROL 0 6 899.73 253.65 28.191 37 C3 37 CONTROL 0 6 198.41 0.00 0.000 38 C3 38 CONTROL 0 6 238.65 15.39 6.449 39 C3 39 CONTROL 0 6 6217.78 4372.15 70.317 40 C3 40 CONTROL 0 6 102.76 79.92 77.772 41 C6 21 CONTROL 0 6 1706.25 400.48 23.471 42 C6 22 CONTROL 0 6 278.77 15.39 5.521 43 C6 23 CONTROL 0 6 1442.72 311.10 21.564 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 109 of 152 44 C6 24 CONTROL 0 6 627.55 65.07 10.369 45 C6 25 CONTROL 0 6 175.93 67.73 38.501 46 C6 26 CONTROL 0 6 36.73 16.11 43.849 47 C6 27 CONTROL 0 6 2129.90 518.64 24.351 48 C6 28 CONTROL 0 6 60.51 20.16 33.320 49 C6 29 CONTROL 0 6 1756.10 227.47 12.953 50 C6 30 CONTROL 0 6 1697.45 526.60 31.023 51 C6 31 CONTROL 0 6 14.63 3.13 21.411 52 C6 32 CONTROL 0 6 1888.82 361.66 19.147 53 C6 33 CONTROL 0 6 2307.73 238.14 10.319 54 C6 34 CONTROL 0 6 74.49 123.46 165.746 55 C6 35 CONTROL 0 6 45.12 8.36 18.519 56 C6 36 CONTROL 0 6 14815.70 5414.25 36.544 57 C6 37 CONTROL 0 6 1428.39 413.12 28.922 58 C6 38 CONTROL 0 6 3102.61 411.83 13.274 59 C6 39 CONTROL 0 6 3876.90 735.67 18.976 60 C6 40 CONTROL 0 6 3085.45 900.82 29.196 61 E1 1 ATRAZINE 0 6 34.64 4.44 12.830 62 E1 2 ATRAZINE 0 6 1532.07 142.84 9.323 63 E1 3 ATRAZINE 0 6 2127.42 184.66 8.680 64 E1 4 ATRAZINE 0 6 63.66 31.31 49.174 65 E1 5 ATRAZINE 0 6 44.52 33.72 75.747 66 E1 6 ATRAZINE 0 6 . . .

67 E3 1 ATRAZINE 0 6 119.73 13.69 11.430 68 E3 2 ATRAZINE 0 6 244.45 99.19 40.576 69 E3 3 ATRAZINE 0 6 154.14 42.81 27.772 70 E3 4 ATRAZINE 0 6 129.56 43.94 33.913 71 E3 5 ATRAZINE 0 6 6.00 0.00 0.000 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 110 of 152 MEAN FEMALE PLASMA TESTOSTERONE CONCENTRATIONS BY SITE AND ANIMAL NUMBER 99 Obs Site Ind_ No TREAT _ TYPE_ _ FREQ_ T STD CV 72 E3 6 ATRAZINE 0 3 . . .

73 E3 7 ATRAZINE 0 6 63.25 42.27 66.832 74 E3 8 ATRAZINE 0 6 110.76 55.10 49.745 75 E3 9 ATRAZINE 0 6 136.19 118.46 86.983 76 E3 10 ATRAZINE 0 6 519.34 783.60 150.883 77 E4 1 ATRAZINE 0 6 501.40 214.98 42.876 78 E4 2 ATRAZINE 0 6 2196.90 244.47 11.128 79 E4 3 ATRAZINE 0 6 663.50 97.44 14.685 80 E4 4 ATRAZINE 0 6 396.55 37.25 9.392 81 E4 5 ATRAZINE 0 6 239.31 50.18 20.969 82 E4 6 ATRAZINE 0 6 4495.01 2233.98 49.699 83 E4 7 ATRAZINE 0 6 371.69 32.59 8.767 84 E4 8 ATRAZINE 0 6 103.61 44.09 42.550 85 E4 9 ATRAZINE 0 6 4473.15 870.89 19.469 86 E4 10 ATRAZINE 0 6 1838.00 170.56 9.280 87 E6 1 ATRAZINE 0 6 72.81 15.26 20.956 88 E6 2 ATRAZINE 0 6 37.52 10.78 28.742 89 E6 3 ATRAZINE 0 6 . . .

90 E6 4 ATRAZINE 0 6 99.46 19.78 19.888 91 E6 5 ATRAZINE 0 6 103.05 42.38 41.123 92 E6 6 ATRAZINE 0 6 154.92 30.48 19.675 93 E6 7 ATRAZINE 0 6 29.62 17.58 59.351 94 E6 8 ATRAZINE 0 6 . . .

95 E6 9 ATRAZINE 0 6 199.67 112.48 56.334 96 E6 10 ATRAZINE 0 6 70.86 10.91 15.391 97 E8 1 ATRAZINE 0 6 338.68 254.52 75.151 98 E8 2 ATRAZINE 0 6 2491.05 1026.81 41.220 99 E8 3 ATRAZINE 0 6 74.43 22.66 30.452 100 E8 4 ATRAZINE 0 6 186.29 26.58 14.270 101 E8 5 ATRAZINE 0 6 2214.21 1006.46 45.455 102 E8 6 ATRAZINE 0 6 110.45 18.60 16.842 103 E8 7 ATRAZINE 0 6 183.41 15.589 8.4997 104 E8 8 ATRAZINE 0 6 4381.40 601.186 13.7213 105 E8 9 ATRAZINE 0 6 74.89 28.512 38.0707 106 E8 10 ATRAZINE 0 6 34.50 15.136 43.8774 107 E8 11 ATRAZINE 0 6 109.70 24.201 22.0601 108 E8 12 ATRAZINE 0 6 348.47 37.360 10.7213 109 E8 13 ATRAZINE 0 5 . . .

110 E8 14 ATRAZINE 0 5 . . .

111 E8 15 ATRAZINE 0 5 . . . Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 111 of 152 ANOVA FOR FEMALE PLASMA TESTOSTERONE BETWEEN SITES 101 The ANOVA Procedure Class Level Information Class Levels Values Site 8 C1 C3 C6 E1 E3 E4 E6 E8 Number of observations 111 NOTE: Due to missing values, only 102 observations can be used in this analysis.

Dependent Variable: T Sum of Source DF Squares Mean Square F Value Pr > F Model 7 134465197 19209314 1.97 0.0673 Error 94 916631986 9751404 Corrected Total 101 1051097183 R Square Coeff Var Root MSE T Mean 0.127928 168.4578 3122.724 1853.713 Source DF Anova SS Mean Square F Value Pr > F Site 7 134465196.7 19209313.8 1.97 0.0673 Levene's Test for Homogeneity of T Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F Site 7 5.972E15 8.531E14 1.23 0.2963 Error 94 6.54E16 6.958E14 Bartlett's Test for Homogeneity of T Variance Source DF Chi Square Pr > ChiSq Site 7 114.0 <. 0001 Dunnett's t Tests for T NOTE: This test controls the Type I experimentwise error for comparisons of all treatments Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 112 of 152 against a control.

Alpha 0.05 Error Degrees of Freedom 94 Error Mean Square 9751404 Critical Value of Dunnett's t 2.70019 Comparisons significant at the 0.05 level are indicated by ***.

Difference Site Between Simultaneous 95%

Comparison Means Confidence Limits C3  C1  965.2  3700.9 1770.5 C6  C1  1524.7  4225.9 1176.6 E4  C1  2024.3  5318.5 1269.9 E8  C1  2673.3  5782.4 435.8 E1  C1  2791.8  7029.9 1446.3 E3  C1  3387.4  6799.4 24.6 E6  C1  3456.3  7010.0 97.5 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 113 of 152 NONPARAMETRIC COMPARISON OF FEMALE PLASMA TESTOSTERONE ACROSS SITES 105 The NPAR1WAY Procedure Wilcoxon Scores ( Rank Sums) for Variable T Classified by Variable Site Sum of Expected Std Dev Mean Site N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 19 1173.0 978.50 116.343958 61.736842 C3 19 1145.0 978.50 116.343958 60.263158 C6 20 1122.0 1030.00 118.645129 56.100000 E1 5 171.0 257.50 64.520669 34.200000 E3 9 287.0 463.50 84.759955 31.888889 E4 10 634.0 515.00 88.863191 63.400000 E6 8 178.0 412.00 80.340940 22.250000 E8 12 543.0 618.00 96.280839 45.250000 Kruskal Wallis Test Chi Square 20.0577 DF 7 Pr > Chi Square 0.0054 Median Scores ( Number of Points Above Median) for Variable T Classified by Variable Site Sum of Expected Std Dev Mean Site N Scores Under H0 Under H0 Score ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ C1 19 12.0 9.50 1.975719 0.631579 C3 19 10.0 9.50 1.975719 0.526316 C6 20 13.0 10.00 2.014797 0.650000 E1 5 2.0 2.50 1.095671 0.400000 E3 9 1.0 4.50 1.439369 0.111111 E4 10 8.0 5.00 1.509049 0.800000 E6 8 0.0 4.00 1.364326 0.000000 E8 12 5.0 6.00 1.635013 0.416667 Median One Way Analysis Chi Square 20.5428 DF 7 Pr > Chi Square 0.0045 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 114 of 152 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 115 of 152 MEAN FEMALE PLASMA TESTOSTERONE CONCENTRATIONS BY TREATMENT 107 Obs TREAT _ TYPE_ _ FREQ_ T STD CV 1 ATRAZINE 0 300 724.55 1270.60 175.364 2 CONTROL 0 360 2942.16 4636.87 157.601 ANOVA FOR FEMALE PLASMA TESTOSTERONE ACROSS TREATMENTS 108 The ANOVA Procedure Class Level Information Class Levels Values TREAT 2 ATRAZINE CONTROL Number of observations 111 NOTE: Due to missing values, only 102 observations can be used in this analysis.

Dependent Variable: T Sum of Source DF Squares Mean Square F Value Pr > F Model 1 98659132 98659132 10.36 0.0017 Error 100 952438051 9524381 Corrected Total 101 1051097183 R Square Coeff Var Root MSE T Mean 0.093863 166.4853 3086.160 1853.713 Source DF Anova SS Mean Square F Value Pr > F TREAT 1 98659131.80 98659131.80 10.36 0.0017 Levene's Test for Homogeneity of T Variance ANOVA of Squared Deviations from Group Means Sum of Mean Source DF Squares Square F Value Pr > F TREAT 1 4.797E15 4.797E15 6.77 0.0107 Error 100 7.081E16 7.081E14 Bartlett's Test for Homogeneity of T Variance Source DF Chi Square Pr > ChiSq Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 116 of 152 TREAT 1 50.9636 <. 0001 Dunnett's t Tests for T NOTE: This test controls the Type I experimentwise error for comparisons of all treatments against a control.

Alpha 0.05 Error Degrees of Freedom 100 Error Mean Square 9524381 Critical Value of Dunnett's t 1.98398 Comparisons significant at the 0.05 level are indicated by ***.

Difference TREAT Between Simultaneous 95%

Comparison Means Confidence Limits CONTROL  ATRAZINE 1985.8 761.7 3209.9 *** Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 117 of 152 MEAN MALE PLASMA ESTRADIOL CONCENTRATIONS BY SITE AND ANIMAL NUMBER 112 Obs Site Ind_ No TREAT _ TYPE_ _ FREQ_ E2 STD CV 1 C1 1 CONTROL 0 3 15.87 9.19 57.913 2 C1 2 CONTROL 0 3 4492.83 294.58 6.557 3 C1 3 CONTROL 0 3 2404.51 250.12 10.402 4 C1 4 CONTROL 0 3 8.01 2.40 29.951 5 C1 5 CONTROL 0 3 4130.31 228.71 5.537 6 C1 6 CONTROL 0 3 4077.53 326.52 8.008 7 C1 7 CONTROL 0 3 5541.93 1066.63 19.247 8 C1 8 CONTROL 0 3 12.86 4.63 35.962 9 C1 9 CONTROL 0 3 41.91 8.78 20.956 10 C1 10 CONTROL 0 3 31.25 11.07 35.417 11 C1 11 CONTROL 0 3 64380.80 0.00 0.000 12 C1 12 CONTROL 0 3 35.75 17.69 49.488 13 C1 13 CONTROL 0 3 . . .

14 C1 14 CONTROL 0 3 98.39 46.18 46.939 15 C1 15 CONTROL 0 3 24.46 3.23 13.187 16 C1 16 CONTROL 0 3 63399.39 0.00 0.000 17 C1 17 CONTROL 0 3 21.41 0.54 2.518 18 C1 18 CONTROL 0 3 13.60 7.82 57.470 19 C1 19 CONTROL 0 3 . . .

20 C1 20 CONTROL 0 3 55.10 19.47 35.334 21 C3 1 CONTROL 0 3 . . .

22 C3 2 CONTROL 0 3 4683.85 269.53 5.754 23 C3 3 CONTROL 0 3 6290.86 1152.91 18.327 24 C3 4 CONTROL 0 3 48.31 55.54 114.965 25 C3 5 CONTROL 0 3 40.76 9.89 24.262 26 C3 6 CONTROL 0 3 25.63 0.70 2.715 27 C3 7 CONTROL 0 3 23262.42 2510.43 10.792 28 C3 8 CONTROL 0 3 4743.00 257.49 5.429 29 C3 9 CONTROL 0 3 28.66 7.32 25.555 30 C3 10 CONTROL 0 3 6568.09 1080.88 16.456 31 C3 11 CONTROL 0 3 5361.80 893.98 16.673 32 C3 12 CONTROL 0 3 4603.38 113.50 2.466 33 C3 13 CONTROL 0 3 10.13 5.90 58.246 34 C3 14 CONTROL 0 3 62.73 23.13 36.871 35 C3 15 CONTROL 0 3 98.45 . .

36 C3 16 CONTROL 0 3 315.76 84.04 26.617 37 C3 17 CONTROL 0 3 . . .

38 C6 1 CONTROL 0 3 . . .

39 C6 2 CONTROL 0 3 433.93 54.03 12.452 40 C6 3 CONTROL 0 3 4619.07 1398.84 30.284 41 C6 4 CONTROL 0 3 29462.37 0.00 0.000 42 C6 5 CONTROL 0 3 7268.35 . .

43 C6 6 CONTROL 0 3 13.29 6.66 50.074 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 118 of 152 44 C6 7 CONTROL 0 3 4376.36 307.80 7.033 45 C6 8 CONTROL 0 3 7893.73 619.58 7.849 46 C6 9 CONTROL 0 3 18.34 7.19 39.233 47 C6 10 CONTROL 0 3 5383.19 1515.08 28.145 48 C6 11 CONTROL 0 3 16.80 9.09 54.124 49 C6 12 CONTROL 0 3 26817.92 834.57 3.112 50 C6 13 CONTROL 0 3 3.37 0.00 0.000 51 C6 14 CONTROL 0 3 12815.92 2261.06 17.643 52 C6 15 CONTROL 0 3 10.54 0.00 0.000 53 C6 16 CONTROL 0 3 6065.52 199.53 3.290 54 C6 17 CONTROL 0 3 9.45 0.00 0.000 55 C6 18 CONTROL 0 3 4763.25 1793.75 37.658 56 C6 19 CONTROL 0 3 82865.65 . .

57 C6 20 CONTROL 0 3 5527.13 292.49 5.292 58 E1 1 ATRAZINE 0 3 193.25 37.43 19.368 59 E1 2 ATRAZINE 0 3 238.75 12.20 5.109 60 E1 3 ATRAZINE 0 3 6.11 0.00 0.000 61 E1 4 ATRAZINE 0 3 106.33 44.92 42.243 62 E1 5 ATRAZINE 0 3 187.66 47.96 25.559 63 E1 6 ATRAZINE 0 3 6.85 0.00 0.000 64 E1 7 ATRAZINE 0 3 8.03 0.00 0.000 65 E1 8 ATRAZINE 0 3 9991.09 2055.96 20.578 66 E3 1 ATRAZINE 0 3 201.97 70.70 35.004 67 E3 2 ATRAZINE 0 3 182.84 7.29 3.987 68 E3 3 ATRAZINE 0 3 507.43 53.75 10.592 69 E3 4 ATRAZINE 0 3 9399.57 1177.61 12.528 70 E3 5 ATRAZINE 0 3 244.15 39.68 16.254 71 E3 6 ATRAZINE 0 3 263.68 31.09 11.790 Data Evaluation Report on South African Analytical Support   Hormone and Aromatase Analysis EPA MRID Number 458675 01 Page 119 of 152 MEAN MALE PLASMA ESTRADIOL CONCENTRATIONS BY SITE AND ANIMAL NUMBER 113 Obs Site Ind_ No TREAT _ TYPE_ _ FREQ_ E2 STD CV 72 E3 7 ATRAZINE 0 3 7777.23 759.22 9.762 73 E3 8 ATRAZINE 0 3 182.68 27.28 14.934 74 E3 9 ATRAZINE 0 3 5.14 0.00 0.000 75 E3 10 ATRAZINE 0 3 149.09 13.68 9.179 76 E4 1 ATRAZINE 0 3 7943.10 3964.57 49.912 77 E4 2 ATRAZINE 0 3 150.47 18.66 12.403 78 E4 3 ATRAZINE 0 3 79.94 52.95 66.242 79 E4 4 ATRAZINE 0 3 36.94 21.52 58.246 80 E4 5 ATRAZINE 0 3 3410.68 436.81 12.807 81 E4 6 ATRAZINE 0 3 3640.90 352.58 9.684 82 E4 7 ATRAZINE 0 3 . . .