UNITED ST ATES E N VI RON M E N T A L PR OTE C TION AGE NCY

NAT I ONAL HEALT H AND ENVIRONMENT A L EFFEC T S R ESEARCH LA BORATORY

Rese arch T r iangl e Park, NC 277 11

July 8, 2010 OFFI CE OF

RESEARCH AND

DEVELOPMENT

MEMORANDUM:

SUBJ ECT: Internal Report Identifying Effect s 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

, NHE ERL

THRU: Hal Zen i ck, Director, NHEE R L

Attached please find a report authored by Ral ph Cooper et al., describ ing 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 s hown that atrazine an d atrazine metabolites will inhib i t the estrogen-, or estrogen plus pr ogesterone-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 a fternoon of vaginal proestrus in the intact female rat. These studies have typically em ployed 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 su rge has not been identified. To our knowledge, the lowest published or al dose of atrazine found to suppress LH secretion is 6.25 mg/kg (Cooper et al. , 2007), administer ed to the intact, Long-Evans female once a

1day for four consecutive days. Again, in t hat study a NOEL was not identified. This report summarizes studies c onducted to confirm the Cooper et al. , 2007 study and identify a NOEL. In addition, this report pr ovides a more detailed description of the experimental procedur es employed in this work.

Enc: Internal Report

Cc: Tina Levine, Director HED, OPP 7509P John R. Fowle, Deput y Director, HED, OPP 7509P

Anna Lowit t, OPP, 7509P Steven Bradbury, Office Directory, OPP 7501P

2

### Summar y Report :

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

the Pre-ovulator y LH surge in the Long-Evans Rat.

By Ralph Cooper, Angela Buckalew, Melanie Fr aites, Jerome Goldman, Susan Laws, Michael Narotsky and Tammy Sto k er

Endocrine Toxicology Branch, Toxic i ty Assessment Division,

National Health and Environmental Effects Research Laboratory, ORD, US EPA, RTP, NC, 27711

Please address all correspondence to Ralp h Cooper, Chief, Endoc r ine Toxicology Branch, Toxic o logy Assessm ent Divis ion, National Hea l th and Ecologic a l 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 us e by the Office of Pesticide Progr ams, Office of Chemical Safety and Pollution Prevention . July 1, 2010

Disclaimer : This manuscript has been reviewed in acc o rdance wit h the policy of the

National Health and Environmental Effects Re search Laboratory, O ffice of Research

and Deve lo pment, U.S

. EPA, and approved for public ation. Approval does not signify that the contents necessarily reflect the view s or polic y of the Agen cy, nor does mention of trade names or commercial products cons titute endorsement or recommendation for

use.

3Abstract :

Several studies have s hown that atrazine an d atrazine metabolites will inhib i t the estrogen-, or estrogen plus pr ogesterone-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 a fternoon of vaginal proestrus in the intact female rat. These studies have typically em ployed 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 su rge has not been identified. To our knowledge, the lowest published or al dose of atrazine found to suppress LH secretion is 6.25 mg/kg (Cooper et al. , 2007), administer ed to the intact, Long-Evans female once a day for four consecutive days. Again, in t hat study a NOEL was not identified. This report summarizes studies condu cted to confirm the Cooper et al. , 2007 study and identify a NOEL. In addition, this report pr ovides a more detailed description of the experimental procedur es employed and ident if ies a NOEL of 1.56 mg/kg when the LH surge was analyzed at several different time points on afternoon of v aginal proestrus. In addition, when the 1800 h peak values we re analyz ed, a NOEL of 3.12 mg/kg was

identified.

4Backgrou nd:

The effects of atrazine (ATR) and its metabolites on mammalian health have been studied extensively, with the most consis tent effects being changes in the hormonal control of the female reproductiv e axis (Cooper et al. 2000; Eldridge et al. 1999). Oral gavage exposure to ATR disrupt ed estrous cyclicity (Cooper et al. 1996) and diminis h ed the surge of luteinizing hormone (LH). Fo r example, in ovariectomized rats, atrazine

and its metabolites have been shown to dec rease 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 employ ed relatively high doses of atrazine (i.e., 50–300 mg/kg/day administered for a period of 3–4 days). Using a similar ovariectomized, estrog en-primed model, Morseth et al. , (1996) reported significantly lower doses of atrazine were required to decr ease the LH surge in females treated for a period of 6 months prior to m easuring the hormone. This l ed to the belief that if the

animal was exposed to atrazine for a longer dur ation, 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 wa s supported in part by the observation that a single treatment with 300 mg/kg atrazine wa s required to lower the LH surge in ovariectomized, estrogen-prim ed Long-Evans (LE) rats (Cooper et al. 2000). We also

found that this dose was without effect on si milarly treated Sprague-Dawley (SD) rats. In contrast, three daily doses of atrazi ne (50-300 mg/kg) did reduce the estrogeninduced LH surge in LE but not SD females, indic a ting that multiple days of exposure appeared to be necessary to affect LH secr etion and that the SD strain was less

sensitive to the effects of atrazine on LH. Atrazine was found to suppress the estrogen-induced LH s u rge in ovariectomized SD females following a 21-day expos ure, 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 sensitiv ity to this compound.

In a study of the intact, regularly cycling fe male, Cooper et al (2007) , reported that oral atrazine exposure for four consecutive days (one estrous cycle) red u ced 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 t he 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 t he doses tested. Subsequent to the publication of that manuscript, we repeated the evaluation of atra zine on the LH surge in regular l y cycling females and compared vehicle-treated anim a ls with those expos ed to 6.25, 3.12 and 1.56 mg/kg. The results of this work are reported below.

5Methods

All exper iments were approved by the U.S. Environmental Protection Age n cy

, National Health and Environmental Effe cts Research La boratory’s (NHEERL)

Institutional Animal Care and Use Committe e and were conducted in accordance with

National Institute of Health standards for labo ratory animal research. The research was

conducted within the IRP entitled: “Effects of Selected Pestic ides and their Metabolites on Female Reproduction and the Neur oendocrine System in Rats”. NHEE R L-HRTP/RT D-EB-RLC-2 0 07-01-r0 . This research was als o reviewed and included as a part of the Office of Res earch and Development, EPA Mu lti-Year Plan for Human Health, Long Term Goal 1 (mode and mechanis m of acti on), Annual Performance Goal (APG 2012-01) and Annual Perfo rmance Measure APM 148.

Animals

Female Long-Evans Hooded rats (60 days ol d) were purchased from Charles River Laboratories (Raleigh, NC) and ho used singly in a room main tained at 22 ± 2°C with a 14:10h light:dark schedule (light s on at 0500 hours: lights out at 1900 hours). Purina Laboratory Rat Chow 5001 and water were provided a d libitu m . 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 wer e included in the exper iments.

Dosing solutions and procedures

Atrazine (purity 97.1%) was a gift from Syngenta Crop Prot ection, Inc. (Greensboro, NC). All dosing was administered by or al gavage in a suspension of 1% methyl cellulose/distilled water (M-7140, Sigma Chem ical Co., St. Louis, MO) in a volume of 5.0 ml dosing solution / kg body weight. Cont rol animals received 1% methyl cellulose vehicle (5.0 ml / kg body weight) only.

Experimental Procedur es

To evaluat e the pituitary-ovarian hormone res ponse 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 es trus and ending on the following day of proestrus.

Animals as signed to treatments were ranked by body weight and placed into control or atrazine groups such that the mean body wei ghts for all groups were similar. The experiment was conducted in thre e blocks. Animals in the first block received either vehicle, 25 or 75 mg/kg. As a N O EL was not established, three additional groups of animals, receiving either 25, 12.5 or 6.25 mg/kg, were subs equently tested in a second block, along with a concurrent control gr oup. These data were analyzed and publis hed by Cooper et al. (2007). However, as the 6.25 mg/k g dose group was still not a NOEL, a

6subsequent experiment was conducted in wh ich two additional dos e 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, concu rrent 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 in c l uded in the data analysis had to meet three criteria at the time of kill: (1) a proestr ous vagina l smear, (2) increased uterin e weight and (3) elevated concentration of progesterone.

To fully evaluate the spontaneous ovulatory su rge 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 nec ropsy room, where it was decapitated. This process was routinely accomplished with in 10-15 seconds from the time the home cage was opened. After decapitation, the tr unk blood was collected on ice in 16-x 100- mm serum-separation tubes (Becton-Dickins on, Rutherford, NJ) and centrifuged at 3000 rpm for 30 min at 4°C. Serum was subsequently divided into two aliquot s 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 thr ough Dr. A. Parlow (UCLA, Los Angeles, California). Tracer iodination with 125 I was performed in-house, and the assays conducted according to recommendations pr ov ided, wit h the sensitivity optimized by a 24 h co-incubation of sample and firs t antibody prior to the addition of 125 I-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 per formed 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. Compar isons 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 in dividual groups (Graph Pad Prism 4, (La Jolla, CA). with the signific ance level set at a probability of less than 0.05.

7Results

Identification of proestrus females : The original goal was to achieve a minimum sample size of 8 rats per dose/time treatment group, this aim w a s 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 sa mple times (1600-2000 h), elevated serum progesterone. These criteria are based on our laboratory’s extensive experience evaluating the female rat.

A total of 4 70 females display i ng regular 4- day cycles were selected from a larger pool of animals. Of those selected, 351 animals (f or all dose/time groups) actually met all

three criteri a defining proestr us. Figure 1 depicts the per centage of females in each dose group that achieved proestrus. For each dose group the number of females not meeting the criteria ra nged between 10.4-38.6% with t he greater percentage of

exc l uded animals being in the two higher dos e groups (fi gure 1). A chi square analys is revealed that a significantly greater propor tion of females did not meet the outlined criteria in the 75 mg/kg dose. Although ther e 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 estab lished criteria for being proestrus, there was no difference among the time by dos e 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 depic t s the absol ute weight at 1800 h. A similar lack of difference was noted at all time points tested.

While Figure 2 demonstrates that the absolut e uterine weight of the females was not different across the group. Table 1 depicts t he relative uterine weight expres sed as mg/100g body weight. Table 1 also shows t he actual numbers of animals us ed in each of the dose x time groups for which the LH su rge 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. B e cause the LH

values for these anim a ls did not influence t he outcome of the over all data analysis and the LH values of the animals receiving a gr eater dose of atrazine revealed no difference in LH at this time point, it was felt that increasing t he N would not be a wise use of

resources.

Serum Progesterone: In control animals, as expec ted s e rum progesterone revealed a

dramatic increase over the c ourse 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 observ ed in the control groups for any of the dose

8groups (Figure 4) when the concentration of this hormone for all the time points measured was analyz ed. However, when the progesterone concentration for 1800 hour

groups was analyzed s eparately, the 25 mg/k g/day group was statistically lower than the controls (Figure 5).

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

three consecutive blocks. T here f ore, the control groups fr om each of the three blocks were compared to identify any potential bloc k effects. Table 2 de picts the number of observations, mean and varianc e (SEM & SD) of each of these groups and shows that all three groups of vehicle treated animals we re not statistically different. Thus the control groups were combined for the subsequent statistical analys is. It should also be noted that a comparison of t he treated groups with their conc urrent control revealed the

same statistical difference at each dose x time level.

Effect of atrazine on the LH surge : Figure 6 shows the re sults 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. Analys is 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 ext end and support the earlier publis hed data comparing the effect of atrazine on the LH surge in intact females by Cooper et al.

, (2007). However, as menti oned 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 se parately by one-way ANOVA. This type of analys is indicates t hat the LOEL for 1800 h was 6.25 mg/kg/day and the NOEL was 3.12 mg/kg/day (Figure 8).

9Discussio n :

The results of this study confirm and ex tend our understanding of the dose required to alter the amplitude of the pre-ov ulatory surge of LH in the rat. In this study, we selected a high dose of 75 mg/kg. This dos e 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 pr esent study, th e 75 mg/kg / day dose resulted in a signific ant decrease in the number of fema les that met the criteria for classification as proestrus on the day of kill. This observa tion indic a tes that four days of treatment at

this dose was sufficient to m odify the pituitary gonadal axis in the female controllin g ovarian function. Importantly, doses of atra zine that were substantially lower than 75 mg/kg were also found to significantly r educe the peak amount of LH secreted during the pre-ovulatory surge. Thus, w e f ound 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 anal yzed (i.e., 2-way A N OVA 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 concent rations 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 gener ally not dete rmined in the previous, short-term

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

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

in whic h a NOEL for a 3-day exposure was ident ified 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 re sults also indic a te that the LE female is

more sensitive than the SD female to the sh ort-term effects of atra zine on LH in the ovariectomized model and perhaps in the intact fe male 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 unde rlying the change in L H obs erved in the present study are still not

understood. It is interesti ng that doses less than 300 mg/k g 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 expos ed 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 wit hout effect while four-days of dosing in the

same strai n will decrease serum LH at dos es as low as 3.12 mg/kg remains to be

determined.

10Literature cited:

Cooper, R. L., Goldman, J. M. and Vandenbergh, J. G.: Moni toring of estrus cyclicity in the laboratory rodent by vagina l lavage. In: Methods in Toxicology. Vol III, P a rt B. Female Reproductive Toxicology ed. by R.E. Chapin and J. Heindel,
Academic Press, Or lando, pp 45-56, 1993.

Cooper, R.L., Stoker, T.E., Goldm an, J.M, Parrish, M.B. and Tyre y, L. Effect of atrazine

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

Cooper, R.L., Stoker, T.E., Tyrey, L., Go ldman, J.M., and McElroy W.K. Atrazine disrupts the hypothalamic control of pitu itary-ovarian function. Toxicological Sciences 53: 297-307, 2000.

Cooper, R.L., Laws, S

. C., Das, P.C., Narotsky, M.G., Gold man, J.M, Tyrey, E.L. and Stoker, T.E

. Atrazine and reproductive f unction: Mode and Mechanism of acti on studies. B i rth Defects Res earch (Part B) 80:98–112, 2007.

Eldridge, J. C., Wetzel, L. T., and Tyrey, L. E s trous cycle patterns of Sprague-Dawley rats during acute and chronic atrazine admin istration. Reprod To xic o l 13, 49 1-9,

1999.

Foradori CD, Hinds LR, Hanneman WH, Legare ME, C l ay CM, Handa RJ. Atrazine inhibits pulsatile luteinizing horm one rel eas e 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., Nagah ara, A., Lund, T. D., Pak, T., Handa, R. J., and Hanneman, W. H. Evidence that atrazine and diaminoc hlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female SpragueDawley rats without changing es trogen rec eptor action. Toxic o l Sci 79, 278-86,

2004.

Mors eth, SL; LT Wetzel; JC Eldridge; a nd L Ty rey. Ev aluatio n of th e Lute i nizin g Hor m on e (LH) S u rg e in Atraz i ne

- E x posed Fe male Spra g ue- Da wle y Rats - 6 M o nth Re port t o Env i ron m en tal Protect i o n Age n cy. MRID 4 4 1 5 210 2. p.72 7

, 1996.

11Table 1: Relative Uterine Weight (mg/100 g bw ) of F e mal es Included in Proestro us LH Surge Expe riment

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

T i m e W e i g h t N W e i g h t N W e i g h t N W e i g h t N

1 2 0 0 2 3 5

. 4 ± 1 1

. 4 1 8 211.4 ±

15.8 5 238.2 ± 16.8 4 243.4 ±

1 4

. 7 8

1400 278.3 ±

1 2

. 8 1 3 231.4 ±

10.3 2^ 238.0 ± 16.9 5 278.4 ±

2 0

. 9 1 2

1600 257.3 ±

1 5

. 7 1 4 243.5 ±

43.3 2^ 256.7 ± 8.7 4 276.0 ±

2 0

. 4 6

1800 297.2 ±

1 3

. 5 1 6 295.9 ±

20.2 11 313.3 ± 11.2 12 322.9 ±

1 7

. 7 1 7

2000 299.3 ±

1 4

. 6 1 8 313.2 ±

4 2

. 0 5 275.0 ±

3 1

. 0 5 318.4 ±

1 5

. 4 8

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

N W e i g h t N W e i g h t N

1 2 0 0 2 2 7

. 9 ± 12.0 11 233.8 ± 17.1 8 261.6 ±

2 1

. 3 8

1400 274.1 ±

15.7 6 260.6 ± 14.5 14 245.7 ±

1 3

. 6 6

1600 258.5 ±

11.7 6 298.1 ± 12.4 11 269.2 ±

1 4

. 6 6

1800 297.3 ±

19.5 7 289.5 ± 11.4 14 296.9 ±

2 0

. 2 8

2000 332.9 ±

17.7 9 316.5 ± 15.9 13 292.5 ±

13.3 7

* N= the number of animals include d in the LH analysis at e a ch dose an d time point. ^ Groups for which a su fficient num ber of animals were not observed.

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

Observation Block 1 Block 2 Block 3

1 3 3

. 3 6 1 4

. 2 9 2 0

. 2 9

2 8

. 4 0 2 0

. 2 5 1 7

. 9 1

3 2 3

. 8 5 2 6

. 2 1 1 1

. 9 3

4 2 8

. 4 1 1 3

. 0 3 1 5

. 5 2

5 1 9

. 6 3 1 7

. 7 5 1 4

. 8 0

6 2 8

. 1 3 2 4

. 0 3 2 8

. 5 2

7 1 8

. 9 6 2 8

. 4 4 1 8

. 8 5

8 2 9

. 5 4 1 5

. 3 0 2 0

. 6 6

9 2 0

. 3 9

1 0 2 4

. 6

1 1 2 4

. 5 4

Mean 2 3

. 7 8 5 1 9

. 9 1 3 1 9

. 6 7 9

Standard Deviation 7

. 9 4 9 5

. 7 8 6 5

. 2 9 9

Number 8 8 1 0

SEM 3 2

. 1 8 7 1

. 7 0 7

13Figure Legends

Figure 1

. Percentage of Females Not Meeti ng Cr iteria for Proestr us. This is the percentage of rats for each dose exam ined c o llapsed ac ross different time points.

Figure 2. Absolute ut erine 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 st atistical 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 cont ained only two animals each.

Figure 5. Serum progesterone in all female s killed at 1800 h. Asterisk indica tes that

the concentration of this horm one in the 25 mg/kg dose group was signific antly below that of the controls . However, the progesterone for the 75 mg/kg was not stati s tically different.

Figure 6

. Serum LH in atrazine-treated proestr ous females. Asterisk depict groups that are statistically diffe rent from control at the same time point using a 2- way ANOVA and Bonf erroni 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.

14Figure 1:

Proportion of Rats E xcluded Because

Proestrou s Criteria w as not A c hieved

Ve h 1. 5 3. 12 6. 25 12. 5 25 75 0 10 20 30 40Percent 

# *

Dose

Figure 2

Ve h 1. 56 3. 1 2 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



### Ti m e ( h )grams

15Figure 3.



### S e ru m P r o g e s t e ro n e

1200 1400 1600 1800 2000 0 5 10 15 20 25 30 35 40 45 50 55



### Ti m e ( h )ng/ml

Figure 4



### Ser u m Pr ogest er one

1200 1400 1600 1800 2000 0 5 10 15 20 25 30 35 40 45 50 55 60 65



### C ont r o l

25

75 1. 56

3. 12

6. 25

12. 5

Ti m e ( h )ng/ml

16Figure 5. Serum progesterone at 1800 h.



# * 180 0 h P r og es t e r o n e

Co

nt

r ol 1. 5 6 3. 1 2 6. 2 5 12 . 5 25 75 0 5 10 15 20 25 30 35 40 45 50 55

mg / k g A T Rng/ml

Figure 6



## Se r u m L H

1200 

### 1400 

### 1600 

### 1800 

### 2000 

### 0 

### 5 

### 10 

### 15 

### 20 

### 25



### C ont r o l



### 25



### 75 

### 1. 56



### 3. 12



### 6. 25



### 12. 5



### Ti m e ( h )ng/ml 

### *

* *

*

*

*

*

17Figure 7.

Figure 8.

18