UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY 

Research Triangle Park, NC 27711

July 8, 2010 OFFICE OF 

RESEARCH AND 

DEVELOPMENT 

MEMORANDUM: 

SUBJECT: Internal Report Identifying Effects of Atrazine on Luteinizing Hormone 

FROM: Ralph L. Cooper, Chief, MD-72 

 Endocrine Toxiciology Branch 

Toxicity Assessment Division

 

TO: Elizabeth Mendez, 

Senior Scientist, OPP 7509 P 

THRU: John Rogers, Acting Directory, MD--72 

 Toxicity Assessment Division 

THRU: Julian Preston, 

 Associate Director of Health, 

 NHEERL 

THRU: Hal Zenick, 

 Director, NHEERL 

 

Attached please find a report authored by Ralph Cooper et al., describing the effects of 

the chlorotriazines herbicide Atrazine on the pre-ovulatory surge of luteinizing hormone 

in the rat, an endocrine event necessary for normal ovulation. As you are aware, 

several studies have shown that atrazine and atrazine metabolites will inhibit the 

estrogen-, or estrogen plus progesterone-induced surge of luteinizing hormone in the 

ovariectomized female rat. Furthermore, atrazine has been shown to alter the 

spontaneous surge of LH released during the afternoon of vaginal proestrus in the intact 

female rat. These studies have typically employed a 3- or 4-day oral dosing period with 

the hormone measured on the last day of dosing. To date, the LOEL or NOEL for the 

atrazine-induced suppression of the LH surge has not been identified. To our 

knowledge, the lowest published oral dose of atrazine found to suppress LH secretion is 

6.25 mg/kg (Cooper et al., 2007), administered to the intact, Long-Evans female once a 

1 
day for four consecutive days. Again, in that study a NOEL was not identified. This 

report summarizes studies conducted to confirm the Cooper et al., 2007 study and 

identify a NOEL. In addition, this report provides a more detailed description of the 

experimental procedures employed in this work. 

Enc: Internal Report 

Cc: Tina Levine, Director HED, OPP 7509P 

John R. Fowle, Deputy Director, HED, OPP 7509P 

Anna Lowitt, OPP, 7509P 

Steven Bradbury, Office Directory, OPP 7501P 

2 
Summary Report: 

Evaluating the Effect of the Chlorotriazine Herbicide Atrazine on the Amplitude of 

the Pre-ovulatory LH surge in the Long-Evans Rat. 

By 

Ralph Cooper, Angela Buckalew, Melanie Fraites, Jerome Goldman, Susan Laws, 

Michael Narotsky and Tammy Stoker 

Endocrine Toxicology Branch, Toxicity Assessment Division, 

National Health and Environmental Effects Research Laboratory, ORD, US EPA, 

RTP, NC, 27711 

 

 

Please address all correspondence to Ralph Cooper, Chief, Endocrine Toxicology 

Branch, Toxicology Assessment Division, National Health and Ecological Effect 

Laboratory, ORD, USEPA, RTP, NC, 27711. Phone: 919 541-0173, Fax: 919 541-

5138, E-mail: Cooper.ralph@epa.gov 

This Internal Report was prepared for the use by the Office of Pesticide Programs, 

Office of Chemical Safety and Pollution Prevention. July 1, 2010 

Disclaimer: This manuscript has been reviewed in accordance with the policy of the 

National Health and Environmental Effects Research Laboratory, Office of Research 

and Development, U.S. EPA, and approved for publication. Approval does not signify 

that the contents necessarily reflect the views or policy of the Agency, nor does mention 

of trade names or commercial products constitute endorsement or recommendation for 

use. 

3 
Abstract: 

Several studies have shown that atrazine and atrazine metabolites will inhibit the 

estrogen-, or estrogen plus progesterone-induced surge of luteinizing hormone in the 

ovariectomized female rat. Furthermore, atrazine has been shown to alter the 

spontaneous surge of LH released during the afternoon of vaginal proestrus in the intact 

female rat. These studies have typically employed a 3- or 4-day oral dosing period with 

the hormone measured on the last day of dosing. To date, the LOEL or NOEL for the 

atrazine-induced suppression of the LH surge has not been identified. To our 

knowledge, the lowest published oral dose of atrazine found to suppress LH secretion is 

6.25 mg/kg (Cooper et al., 2007), administered to the intact, Long-Evans female once a 

day for four consecutive days. Again, in that study a NOEL was not identified. This 

report summarizes studies conducted to confirm the Cooper et al., 2007 study and 

identify a NOEL. In addition, this report provides a more detailed description of the 

experimental procedures employed and identifies a NOEL of 1.56 mg/kg when the LH 

surge was analyzed at several different time points on afternoon of vaginal proestrus. 

In addition, when the 1800 h peak values were analyzed, a NOEL of 3.12 mg/kg was 

identified. 

4 
Background: 

The effects of atrazine (ATR) and its metabolites on mammalian health have been 

studied extensively, with the most consistent effects being changes in the hormonal 

control of the female reproductive axis (Cooper et al. 2000; Eldridge et al. 1999). Oral 

gavage exposure to ATR disrupted estrous cyclicity (Cooper et al. 1996) and diminished 

the surge of luteinizing hormone (LH). For example, in ovariectomized rats, atrazine 

and its metabolites have been shown to decrease the estrogen- or estrogen- plus 

progesterone- induced LH surge (Cooper et al., 2000, McMullin et al. 2004, Foradori et 

al., 2009). In general, these studies have employed relatively high doses of atrazine 

(i.e., 50–300 mg/kg/day administered for a period of 3–4 days). Using a similar

ovariectomized, estrogen-primed model, Morseth et al., (1996) reported significantly 

lower doses of atrazine were required to decrease the LH surge in females treated for a 

period of 6 months prior to measuring the hormone. This led to the belief that if the 

animal was exposed to atrazine for a longer duration, the lowest observed effect level 

(LOEL) or no observed effect level (NOEL) would be lower than that observed in an 

acute or short-term exposure study. This was supported in part by the observation that 

a single treatment with 300 mg/kg atrazine was required to lower the LH surge in 

ovariectomized, estrogen-primed Long-Evans (LE) rats (Cooper et al. 2000). We also 

found that this dose was without effect on similarly treated Sprague-Dawley (SD) rats. 

In contrast, three daily doses of atrazine (50-300 mg/kg) did reduce the estrogeninduced LH surge in LE but not SD females, indicating that multiple days of exposure 

appeared to be necessary to affect LH secretion and that the SD strain was less 

sensitive to the effects of atrazine on LH. Atrazine was found to suppress the 

estrogen-induced LH surge in ovariectomized SD females following a 21-day exposure, 

but with a NOEL and LOEL of 75 mg/kg and 150 mg/kg, respectively. Again, a LOEL 

was not identified in the LE females after 21 days, further indicating a strain difference 

in sensitivity to this compound. 

In a study of the intact, regularly cycling female, Cooper et al (2007), reported that oral 

atrazine exposure for four consecutive days (one estrous cycle) reduced the 

spontaneous, pre-ovulatory surge of LH with a LOEL of 6.25 mg/kg. In that study, we 

tested atrazine doses of 6.25, 12.5, 25 and 75 mg/kg. The 75 mg/kg dose was selected 

because it was identified as the dose that would marginally interfere with regular ovarian 

cycles in the four-day cyclic female treated for two weeks. We hypothesized that a 

dose below 75mg/kg would alter the LH surge, but it was somewhat surprising that a 

NOEL was not identified at the doses tested. Subsequent to the publication of that 

manuscript, we repeated the evaluation of atrazine on the LH surge in regularly cycling 

females and compared vehicle-treated animals with those exposed to 6.25, 3.12 and 

1.56 mg/kg. The results of this work are reported below. 

5 
Methods 

All experiments were approved by the U.S. Environmental Protection Agency, 

National Health and Environmental Effects Research Laboratory’s (NHEERL) 

Institutional Animal Care and Use Committee and were conducted in accordance with 

National Institute of Health standards for laboratory animal research. The research was 

conducted within the IRP entitled: “Effects of Selected Pesticides and their Metabolites 

on Female Reproduction and the Neuroendocrine System in Rats”. NHEERL-HRTP/RTD-EB-RLC-2007-01-r0. This research was also reviewed and included as a 

part of the Office of Research and Development, EPA Multi-Year Plan for Human 

Health, Long Term Goal 1 (mode and mechanism of action), Annual Performance Goal 

(APG 2012-01) and Annual Performance Measure APM 148. 

Animals 

Female Long-Evans Hooded rats (60 days old) were purchased from Charles River 

Laboratories (Raleigh, NC) and housed singly in a room maintained at 22 ± 2°C with a 

14:10h light:dark schedule (lights on at 0500 hours: lights out at 1900 hours). Purina 

Laboratory Rat Chow 5001 and water were provided ad libitum. Beginning at 80 daysof-age, the ovarian cycle of each female was monitored for a period of 2 weeks by 

evaluating daily vaginal lavages as described in detail by Cooper et al. (1993). Only 

females displaying regular, 4-day estrous cycles during the two-week pretreatment 

period were included in the experiments. 

Dosing solutions and procedures

Atrazine (purity 97.1%) was a gift from Syngenta Crop Protection, Inc. (Greensboro, 

NC). All dosing was administered by oral gavage in a suspension of 1% methyl 

cellulose/distilled water (M-7140, Sigma Chemical Co., St. Louis, MO) in a volume of 

5.0 ml dosing solution / kg body weight. Control animals received 1% methyl cellulose 

vehicle (5.0 ml / kg body weight) only. 

Experimental Procedures 

To evaluate the pituitary-ovarian hormone response to ATR exposure over the course of 

one estrous cycle (e.g., 4 consecutive days), females were gavaged once per day at 

0900 hours beginning on the day of vaginal estrus and ending on the following day of 

proestrus. 

Animals assigned to treatments were ranked by body weight and placed into control or 

atrazine groups such that the mean body weights for all groups were similar. The 

experiment was conducted in three blocks. Animals in the first block received either 

vehicle, 25 or 75 mg/kg. As a NOEL was not established, three additional groups of 

animals, receiving either 25, 12.5 or 6.25 mg/kg, were subsequently tested in a second 

block, along with a concurrent control group. These data were analyzed and published 

by Cooper et al. (2007). However, as the 6.25 mg/kg dose group was still not a NOEL, a 

6 
subsequent experiment was conducted in which two additional dose groups were 

examined (3.12 mg/kg and 1.56 mg/kg) along with a concurrent control and another 

6.25 mg/kg group. Thus, for each block, concurrent controls were included, along with 

the lowest dose from the previous block. The number of animal in each group is 

presented in the results, as each animal included in the data analysis had to meet three 

criteria at the time of kill: (1) a proestrous vaginal smear, (2) increased uterine weight 

and (3) elevated concentration of progesterone. 

To fully evaluate the spontaneous ovulatory surge of LH, animals from each dose group 

were killed by decapitation at 1200, 1400, 1600, 1800 or 2000 hours (h). The animal 

was removed from its cage to an adjacent necropsy room, where it was decapitated. 

This process was routinely accomplished within 10-15 seconds from the time the home 

cage was opened. After decapitation, the trunk blood was collected on ice in 16-x 100-

mm serum-separation tubes (Becton-Dickinson, Rutherford, NJ) and centrifuged at 

3000 rpm for 30 min at 4°C. Serum was subsequently divided into two aliquots and 

stored at -80°C until assayed for hormone concentrations. 

Radioimmunoassay 

Radioimmunoassay for serum LH was performed using kit materials obtained from the 

National Hormone and Pituitary Program through Dr. A. Parlow (UCLA, Los Angeles, 

California). Tracer iodination with 125I was performed in-house, and the assays 

conducted according to recommendations provided, with the sensitivity optimized by a 

24 h co-incubation of sample and first antibody prior to the addition of 125I-labeled tracer. 

The sensitivity of the LH assay is 10 pg/ml and the intra- and inter-assay coefficients of 

variation were 7.7% and 5.9% respectively. Serum progesterone for each animal was 

analyzed using a Coat-A-Count Progesterone Kit (Coat-A-Count, Siemens Healthcare 

Diagnostic, Deerfield, IL) that was performed according to the manufacturer’s 

instructions with an assay sensitivity of 0.1 ng/ml and an intra- and inter- assay 

coefficient of variation of less than 10%. 

Statistics

Comparisons of the number of animals achieving the criteria set for a proestrous female 

were done using a Chi square test. Comparisons of the hormone values in this 

experiment were conducted using a one-or two-way analysis of variance followed by the 

appropriate t-tests comparing individual groups (Graph Pad Prism 4, (La Jolla, CA). with 

the significance level set at a probability of less than 0.05. 

 

 

 

7 
Results 

Identification of proestrus females: The original goal was to achieve a minimum sample 

size of 8 rats per dose/time treatment group, this aim was not achieved for all cells 

because the number of animals that actually met the criteria for achieving proestrus 

varied as the experiment progressed. In order for the female to be classified as 

proestrus the animal had to have a proestrous vaginal smear, have a uterine weight of 

greater than 500 mg, and for the later sample times (1600-2000 h), elevated serum 

progesterone. These criteria are based on our laboratory’s extensive experience 

evaluating the female rat. 

A total of 470 females displaying regular 4-day cycles were selected from a larger pool 

of animals. Of those selected, 351 animals (for all dose/time groups) actually met all 

three criteria defining proestrus. Figure 1 depicts the percentage of females in each 

dose group that achieved proestrus. For each dose group the number of females not 

meeting the criteria ranged between 10.4-38.6% with the greater percentage of 

excluded animals being in the two higher dose groups (figure 1). A chi square analysis 

revealed that a significantly greater proportion of females did not meet the outlined 

criteria in the 75 mg/kg dose. Although there was a loss of animals across all groups a 

sufficient number of animals within all but two groups (1.56 mg/kg at 1400 h and 1600 

h) met these criteria. 

Uterine weight: Of the animals that met the established criteria for being proestrus, 

there was no difference among the time by dose groups in uterine weight. This was 

true for both absolute uterine weights and for the weight of this tissue when corrected 

for body weight. Figure 2 depicts the absolute weight at 1800 h. A similar lack of 

difference was noted at all time points tested. 

While Figure 2 demonstrates that the absolute uterine weight of the females was not 

different across the group. Table 1 depicts the relative uterine weight expressed as 

mg/100g body weight. Table 1 also shows the actual numbers of animals used in each 

of the dose x time groups for which the LH surge was analyzed. Note that an N=2 was 

present in the 1.56 mg/kg dose group at 14 and 1600 h. This low number was the result 

of the small number of animals meeting the criteria for proestrus. Because the LH 

values for these animals did not influence the outcome of the overall data analysis and 

the LH values of the animals receiving a greater dose of atrazine revealed no difference 

in LH at this time point, it was felt that increasing the N would not be a wise use of 

resources. 

Serum Progesterone: In control animals, as expected serum progesterone revealed a 

dramatic increase over the course of the day of proestrus (Figure 3) with the highest 

concentration observed at 2000h. 

The concentration of serum progesterone observed for each treatment group at each 

time point was not different than that observed in the control groups for any of the dose 

8 
groups (Figure 4) when the concentration of this hormone for all the time points 

measured was analyzed. However, when the progesterone concentration for 1800 hour 

groups was analyzed separately, the 25 mg/kg/day group was statistically lower than 

the controls (Figure 5). 

Serum LH: As described in the methods section, this experiment was conducted in 

three consecutive blocks. Therefore, the control groups from each of the three blocks 

were compared to identify any potential block effects. Table 2 depicts the number of 

observations, mean and variance (SEM & SD) of each of these groups and shows that 

all three groups of vehicle treated animals were not statistically different. Thus the 

control groups were combined for the subsequent statistical analysis. It should also be 

noted that a comparison of the treated groups with their concurrent control revealed the 

same statistical difference at each dose x time level. 

Effect of atrazine on the LH surge: Figure 6 shows the results of treatment with 

atrazine for four consecutive days on the LH surge observed on the day of vaginal 

proestrus. The peak LH values in control and each of the atrazine-treated groups were 

observed at 1800 h, indicating that the onset of the surge was not modified. Analysis of 

LH secreted in the control vs. the treated females revealed a significant suppression of 

LH at 3.12 mg/kg/day and above. This analysis identified 1.56 mg/kg/day as the 

NOEL. 

By way of comparison, these data extend and support the earlier published data 

comparing the effect of atrazine on the LH surge in intact females by Cooper et al., 

(2007). However, as mentioned previously, the 2007 study did not identify a NOEL 

(Figure 7). 

Comparison of the1800 h LH values only: Because the 1800 h time point represented 

the peak for all dose groups tested, this time point was analyzed separately by one-way 

ANOVA. This type of analysis indicates that the LOEL for 1800 h was 6.25 mg/kg/day 

and the NOEL was 3.12 mg/kg/day (Figure 8). 

9 
Discussion: 

The results of this study confirm and extend our understanding of the dose required to 

alter the amplitude of the pre-ovulatory surge of LH in the rat. In this study, we 

selected a high dose of 75 mg/kg. This dose was based on the results of our previous 

observation (Cooper et al., 1996) that it would disrupt, but not completely abolish 

ovarian cycles in the LE female. In the present study, the 75 mg/kg/day dose resulted 

in a significant decrease in the number of females that met the criteria for classification 

as proestrus on the day of kill. This observation indicates that four days of treatment at 

this dose was sufficient to modify the pituitary gonadal axis in the female controlling 

ovarian function. Importantly, doses of atrazine that were substantially lower than 75 

mg/kg were also found to significantly reduce the peak amount of LH secreted during 

the pre-ovulatory surge. Thus, we found that the peak concentration of LH was 

statistically lower in females dosed at concentrations as low as 3.12 mg/kg or 6.25 

mg/kg depending on the way the surge was analyzed (i.e., 2-way ANOVA for entire day 

vs. one-way ANOVA at 1800 h). 

The observation that doses as low as 1.56 or 3.12 mg/kg/day for 4 days will lower peak 

LH concentrations demonstrates that the amount of atrazine required to modify the LH 

surge is lower that what had been previously believed. However, it should be noted that 

a NOEL/LOEL dose was generally not determined in the previous, short-term 

evaluations of the effect of atrazine on LH in ovariectomized females (i.e., Cooper et al., 

2000; McMullin et al. 2004 and Foradori et al., 2009). Using this model, the only study 

in which a NOEL for a 3-day exposure was identified was that of Cooper et al (2000). In 

that study, a LOEL of 150 mg/kg and a NOEL and 75 mg/kg was identified in the 

Sprague-Dawley female. This was not the case for the identically treated LE female. In 

this strain, all doses tested suppressed the surge (lowest dose = 75 mg/kg) and thus a 

LE female NOEL was not identified. These results also indicate that the LE female is 

more sensitive than the SD female to the short-term effects of atrazine on LH in the 

ovariectomized model and perhaps in the intact female as well. In the intact, regularly 

cycling female, Cooper et al. (2007) reported that a dose of 6.25 mg/kg/day for 4-day 

(the same treatment period in the current study), significantly reduced LH at 1800 h on 

vaginal proestrus. The data in the present study confirm this finding and identify a 

NOEL. 

The mechanisms underlying the change in LH observed in the present study are still not 

understood. It is interesting that doses less than 300 mg/kg administered on the day of 

an expected LH surge in the estrogen-primed, ovariectomized female was without effect 

on the amount of LH released. Similarly, when proestrous females were exposed to a 

single dose of atrazine on the morning of vaginal proestrus, it did not affect ovulation in 

the LE female. Why a single large dose is without effect while four-days of dosing in the 

same strain will decrease serum LH at doses as low as 3.12 mg/kg remains to be 

determined. 

10 
Literature cited: 

Cooper, R. L., Goldman, J. M. and Vandenbergh, J. G.: Monitoring of estrus cyclicity in 

the laboratory rodent by vaginal lavage. In: Methods in Toxicology. Vol III, Part 

B. Female Reproductive Toxicology ed. by R.E. Chapin and J. Heindel, 

Academic Press, Orlando, pp 45-56, 1993. 

Cooper, R.L., Stoker, T.E., Goldman, J.M, Parrish, M.B. and Tyrey, L. Effect of atrazine 

on ovarian function in the rat. Reproductive Toxicology 10: 257-264, 1996. 

Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., and McElroy W.K. Atrazine 

disrupts the hypothalamic control of pituitary-ovarian function. Toxicological 

Sciences 53: 297-307, 2000. 

Cooper, R.L., Laws, S.C., Das, P.C., Narotsky, M.G., Goldman, J.M, Tyrey, E.L. and 

Stoker, T.E. Atrazine and reproductive function: Mode and Mechanism of action 

studies. Birth Defects Research (Part B) 80:98–112, 2007. 

Eldridge, J. C., Wetzel, L. T., and Tyrey, L. Estrous cycle patterns of Sprague-Dawley 

rats during acute and chronic atrazine administration. Reprod Toxicol 13, 491-9, 

1999. 

Foradori CD, Hinds LR, Hanneman WH, Legare ME, Clay CM, Handa RJ. Atrazine 

inhibits pulsatile luteinizing hormone release without altering pituitary sensitivity 

to a gonadotropin-releasing hormone receptor agonist in female Wistar rats. Biol 

Reprod. 2009 Jul;81(1):40-5, 2009. 

McMullin, T. S., Andersen, M. E., Nagahara, A., Lund, T. D., Pak, T., Handa, R. J., and 

Hanneman, W. H. Evidence that atrazine and diaminochlorotriazine inhibit the 

estrogen/progesterone induced surge of luteinizing hormone in female SpragueDawley rats without changing estrogen receptor action. Toxicol Sci 79, 278-86, 

2004. 

Morseth, SL; LT Wetzel; JC Eldridge; and L Tyrey. Evaluation of the Luteinizing Hormone 

(LH) Surge in Atrazine-Exposed Female Sprague-Dawley Rats - 6 Month Report to

Environmental Protection Agency. MRID 44152102. p.727, 1996.

11 
Table 1: Relative Uterine Weight (mg/100 g bw) of Females Included in Proestrous LH 

Surge Experiment 

Control 1.5 mg/kg 3.12 mg/kg 6.25 mg/kg 

Time Weight N Weight N Weight N Weight N

1200 235.4 ±

11.4 18

211.4 ± 

15.8 5 238.2 ± 16.8 4 

243.4 ± 

14.7 8

1400 

278.3 ± 

12.8 13

231.4 ± 

10.3 2^ 238.0 ± 16.9 5 

278.4 ± 

20.9 12

1600 

257.3 ± 

15.7 14

243.5 ± 

43.3 2^ 256.7 ± 8.7 4 

276.0 ± 

20.4 6

1800 

297.2 ± 

13.5 16

295.9 ± 

20.2 11 313.3 ± 11.2 12 

322.9 ± 

17.7 17

2000 

299.3 ± 

14.6 18

313.2 ± 

42.0 5

275.0 ± 

31.0 5

318.4 ± 

15.4 8

12.5 mg/kg 25 mg/kg 75 mg/kg 

N Weight N Weight N

1200 227.9 ±

12.0 11 233.8 ± 17.1 8 

261.6 ± 

21.3 8

1400 

274.1 ± 

15.7 6 260.6 ± 14.5 14 

245.7 ± 

13.6 6

1600 

258.5 ± 

11.7 6 298.1 ± 12.4 11 

269.2 ± 

14.6 6

1800 

297.3 ± 

19.5 7 289.5 ± 11.4 14 

296.9 ± 

20.2 8

2000 

332.9 ± 

17.7 9 316.5 ± 15.9 13 

292.5 ± 

13.3 7 

* N= the number of animals included in the LH analysis at each dose and time point. 

^ Groups for which a sufficient number of animals were not observed.

12 
TABLE 2: Serum LH means (ng/ml) in Control Animals from Three Different Blocks 

Observation Block 1 Block 2 Block 3 

1 33.36 14.29 20.29

2 8.40 20.25 17.91

3 23.85 26.21 11.93

4 28.41 13.03 15.52

5 19.63 17.75 14.80

6 28.13 24.03 28.52

7 18.96 28.44 18.85

8 29.54 15.30 20.66

9 20.39

10 24.6

11 24.54

Mean 23.785 19.913 19.679

Standard Deviation 7.949 5.786 5.299

Number 8 8 10

SEM 3 2.187 1.707

13 
Figure Legends 

Figure 1. Percentage of Females Not Meeting Criteria for Proestrus. This is the 

percentage of rats for each dose examined collapsed across different time 

points. 

 

Figure 2. Absolute uterine weight for females killed at 1800 h. 

Figure 3 Serum Progesterone in Control females. 

 

Figure 4. Serum progesterone in females dosed with atrazine (mg/kg) and killed at 

time points noted. There were no statistical differences between control 

and treated animals at any of the time points. Note that the 1.5 mg/kg 

group killed at 1400 h and 1600h contained only two animals each. 

Figure 5. Serum progesterone in all females killed at 1800 h. Asterisk indicates that 

the concentration of this hormone in the 25 mg/kg dose group was 

significantly below that of the controls. However, the progesterone for the 

75 mg/kg was not statistically different. 

Figure 6. Serum LH in atrazine-treated proestrous females. Asterisk depict groups 

that are statistically different from control at the same time point using a 2-

way ANOVA and Bonferroni posttests. Note that the 1.56 dosed animals 

only had an N=2 at the 1400 and 1600 h time points and thus these data 

were not included in the analysis.

Figure 7. Effects of atrazine on serum LH published by Cooper et al., 2007. 

Figure 8. Serum LH levels at 1800 h. 

14 
Figure 1: 

Proportion of Rats Excluded Because 

Proestrous Criteria was not Achieved 

Veh 1.5 3.12 6.25 12.5 25 75

0

10

20

30

40

Perc

e

nt

*

Dose 

Figure 2 

Veh 1.56 3.12 6.25 12.5 25 75

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Time (h)

gram

s

15 
Figure 3. 

Serum Progesterone

1200 1400 1600 1800 2000

0

5

10

15

20

25

30

35

40

45

50

55

Time (h)

ng/

ml

Figure 4 

Serum Progesterone

1200 1400 1600 1800 2000

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Control

25

75

1.56

3.12

6.25

12.5

Time (h)

ng/

ml

16 
Figure 5. Serum progesterone at 1800 h. 

*

1800 h Progesterone

Control

1.56

3.12

6.25

12.5

25

75

0

5

10

15

20

25

30

35

40

45

50

55

mg/kg ATR

ng/ml

Figure 6 

Serum LH

1200 1400 1600 1800 2000

0

5

10

15

20

25

Control

25

75

1.56

3.12

6.25

12.5

Time (h)

ng/

ml

*

* *

*

*

*

*

17 
Figure 7. 

 

Figure 8. 

18