Data Evaluation Record

ATRAZINE

PC Code 080803

EPA Contract No. EP10H001452

Task Assignment No. 4-4-2013

Study Type: Non-Guideline

Citation: Brodeur, J.C., Sassone, A., Hermida, G.N., and Codugnello, N. (2013). 

Environmentally-relevant concentrations of atrazine induce non-monotonic 

acceleration of developmental rate and increased size at metamorphosis in 

Rhinella arenarum tadpoles. Ecotoxicology and Environmental Safety. 92:10-

17.

Prepared for

Environmental Fate and Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency

One Potomac Yard 

2777 S. Crystal Drive

Arlington, VA 22202

Prepared by

Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B

Durham, NC 27713

Primary Reviewer:

Sandra Hastings Signature:

Date: 11/25/2014

Secondary Reviewer:

Kelly Luck, M.S. Signature:

Date: 12/3/2014

Program Manager:

Jack D. Early, M.S. Signature:

Date: 12/15/2014

Disclaimer

This Data Evaluation Record may have been altered by the Environmental Fate and Effects 

Division subsequent to signing by Dynamac Corporation personnel.
ATRAZINE / 080803 Non-Guideline/Page 1 of 8

EPA Primary Reviewer: Amy Blankinship Date: __7/15___________

EPA Secondary Reviewer: Date: _____________

Data Evaluation Record

ECOTOX NO: 164768

TXR NO: Not provided

STUDY TYPE: Non-guideline

DP BARCODE: Not provided

PC CODE: 080803

CAS NO.: 1912-24-9

MRID NO.: Not available

TEST MATERIAL (% purity): Atrazine (technical grade, 98%)

CITATION: Brodeur, J.C., Sassone, A., Hermida, G.N., and Codugnello, N. (2013). 

Environmentally-relevant concentrations of atrazine induce non-monotonic 

acceleration of developmental rate and increased size at metamorphosis in 

Rhinella arenarum tadpoles. Ecotoxicology and Environmental Safety. 92:10-17.

SPONSOR: Not applicable (open literature review)

EXECUTIVE SUMMARY: The purpose of this study was to examine the effects of

environmentally-relevant concentrations of atrazine on the timing of metamorphosis and body

size at metamorphosis in the common South American toad Rhinella arenarum. The 

experimental design executed in the present study is a follow-up of an earlier study (Brodeur et 

al. 20091) which demonstrated a non-monotonic acceleration of the time to metamorphic climax 

(Gosner stage 42) and delayed tail resorption. Exposures examined in the current study were 

longer (from stage 25 instead of stage 38) and the tested concentrations were lower and more 

similar to levels detected in the environment.

Methods: Adult South American toads (R. arenarum), weighing approximately 200-250 g, were

collected from pasture fields in Buenos Aires Province, Argentina. The ovulation of female 

toads was induced by an intraperitoneal injection of homologous hypophysis suspended in 

 1 Brodeur, J.C., Svartz, G., Perez-Coll, C.S., Marino, D.J.G., and Herkovits, J. (2009). Comparative susceptibility 

to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis; Nonmonotonous acceleration of the time to climax and delayed tail resorption. Aquat Toxicol. 91:161-170.
ATRAZINE / 080803 Non-Guideline/Page 2 of 8

AMPHITOX solution (AS; Herkovits and Perez-Coll, 20032). Oocytes were fertilized in vitro 

using fresh sperm suspended in AS, and the resulting embryos were maintained in AS at 20±2 °C

until reaching stage 25. When tadpoles began feeding at stage 24-25, they were offered boiled 

Swiss chard ad libitum.

Tadpoles were pooled from four different clutches, and twenty replicates were performed for 

each concentration tested. In each replicate, ten tadpoles (at stage 25) were placed in AS with or 

without test substance. Tadpoles were exposed to atrazine (98% purity, Chem Service, West 

Chester, PA) at 0.1, 1, 10, 100 or 1000 μg/L. A stock solution of atrazine (10 mg/L) was 

prepared in AS using acetone as a solvent, and the test solutions were prepared by sequentially 

diluting the stock solution with AS; the atrazine concentration in the stock solution was verified 

by LC/MS/MS analysis. Additionally, the study included a control group exposed to AS only; a 

solvent control group exposed to AS containing 0.02% (v/v) acetone; and a positive control 

group exposed to 100 μg/L of 17β-estradiol (E2; prepared in AS from a stock solution in 

acetone). To avoid evaporation of test solution, experimental tanks consisted of two superposed 

glass Petri dishes. Test solutions were replaced entirely every 48 h, and the temperature was 

maintained at 20±2 °C throughout the experiment. Dead tadpoles were removed and the Gosner

stage and survival were evaluated every other day when renewing the solutions. Following 40 

days of exposure, tadpoles were transferred to two bigger superposed glass Petri dishes 

containing test solution to provide space to the growing tadpoles. Frogs completing 

metamorphosis were examined for malformations, and their body mass and snout-vent length

(SVL) were measured.

The study authors used a linear regression of body mass on SVL to evaluate body condition. 

Data from the control groups were used to perform this regression analysis. The theoretical body 

weight value of frogs from both control and treated groups (obtained by introducing the length of 

the animal in the equation of the regression line) was then subtracted from the measured body 

weights to obtain a “residual" value, representing an expression of the difference between the 

measured weight of the metamorph and that of an average healthy subject with the same SVL.

An individual with a positive residual was considered to be in a good condition whereas an 

individual with a negative residual was regarded as having a reduced body condition. 

Comparisons between the control and solvent control groups were made using a t-test or a rank 

sum test if normality and equal variance could not be obtained. Because there were no

differences between the two control groups, data from both groups were combined for further 

analyses. Four-parameter logistic regression analysis was conducted for each treatment on the

cumulative numbers of animals reaching stage 39, stage 42 or completing metamorphosis as a

function of exposure duration (GraphPad Prism software). For each curve, the time for 50% of

the individuals to reach the stage being examined, stage 39 (T39), stage 42 (T42) or completion 

of metamorphosis (TCM), was calculated. The duration of the intervals between stage 39 and 

stage 42, and between stage 42 and completion of metamorphosis were calculated by subtracting 

the values (the value of T39 from the value of T42, or the value of T42 from the value of TCM)

 2 Herkovits, J. and Perez-Coll, C.S. (2003) AMPHITOX: a customized set of toxicity tests employing amphibian 

embryos. In: Linder G., Sparling, D.W., Krest, S., Little, E. (Eds.), Multiple Stressor Effects in Relation to 

Declining Amphibian Populations, American Society for Testing and Materials International, Bridgeport, NJ,

USA. pp. 46-60.
ATRAZINE / 080803 Non-Guideline/Page 3 of 8

for each replicate of each treatment. The values of T39, T42 and TCM, the SVLs of the 

metamorphs, and the intervals between stage 42 and completion of metamorphosis were

compared between treatments using one-way analysis of variance (ANOVA) followed by a 

Holm-Sidak test for multiple comparisons. A non-parametric Kruskal-Wallis one-way ANOVA 

on Ranks was used to compare the following parameters for which normality and equal variance 

could not be obtained: survival of tadpoles; intervals between stage 39 and stage 42; proportions 

of individuals reaching stage 39, stage 42 or completing metamorphosis; body mass of 

metamorphs; and residuals describing body condition. All t-tests, ANOVAs, and multiple 

comparison tests were conducted using SigmaStat 3.11 statistical software. Significance was 

determined at p<0.05.

Results: The only concentration of atrazine that had significant mortality was 1000 μg/L, at 

which survival was reduced to 52.0 ± 10.0%, compared to 74.2 ± 6.5% for the control group and 

78.0 ± 8.5% for the E2 group. Survival rates in the 0.1, 1, 10, and 100 μg/L treatment groups 

(75.7-82.5%) were not significantly different from the control or E2 groups. In all atrazine 

treatment groups, most of the mortalities occurred during the first 20 days of exposure (data not 

reported).

The progression of the metamorphosing tadpoles to stage 39, stage 42, and completion of 

metamorphosis is presented in Figures 1, 2, and 3, respectively (copied without alteration from 

Brodeur et al. 2013), along with the time required for 50% of the tadpoles to reach that stage for 

each treatment group. Tadpoles exposed to atrazine at 1000 μg/L took significantly (p<0.05) 

more time than controls to reach stage 39 (increased T39), whereas exposure to 1, 10 and 100 

μg/L significantly reduced T39 (Figure 1b) compared to the controls. This acceleration of 

development with atrazine exposure had a U-shaped concentration-response relationship as 10

μg/L caused a greater reduction of T39 than 1 or 100 μg/L (which generated comparable effects). 

A similar U-shaped pattern of acceleration of development as a function of atrazine

concentration was observed when considering T42 (Figure 2b) and TCM (Figure 3b).

Tadpoles exposed to 17-estradiol (100 μg/L) showed acceleration of development of the same 

amplitude and characteristics as those observed in the atrazine 1 and 100 μg/L treatment groups 

(Figures 1-3).
ATRAZINE / 080803 Non-Guideline/Page 4 of 8

 Figure 1. (a) Cumulative number of individuals reaching stage 39 over time for control larvae of Rhinella 

arenarum and larvae exposed to atrazine and 17β-estradiol. The lines represent the sigmoidal curve models 

calculated for every treatment (b) Time required (mean ± SE) for 50 percent of the larvae of Rhinella arenarum to 

reach stage 39 for controls and larvae exposed to atrazine and estradiol. Controls are denoted by C. The number 

indicated at the side of the abbreviation ''At" represents the concentration of atrazine in μg/L. E2=tadpoles exposed 

to 100 μg/L of 17β-estradiol. Bars with the same letter are not significantly different (p<0.05).

 Figure 2. (a) Cumulative number of individuals reaching stage 42 over time for control larvae of Rhinella 

arenarum and larvae exposed to atrazine and 17β-estradiol. The lines represent the sigmoidal curve models 

calculated for every treatment (b) Time required (mean ± SE) for 50 percent of the larvae of Rhinella arenarum to 

reach stage 42 for controls and larvae exposed to atrazine and estradiol. Controls are denoted by C. The number 

indicated at the side of the abbreviation ''At" represents the concentration of atrazine in μg/L. E2=tadpoles exposed 

to 100 μg/L of 17β-estradiol. Bars with the same letter are not significantly different (p<0.05).
ATRAZINE / 080803 Non-Guideline/Page 5 of 8

 Figure 3. (a) Cumulative number of individuals completing metamorphosis over time for control larvae of Rhinella 

arenarum and larvae exposed to atrazine and 17β-estradiol. The lines represent the sigmoidal curve models 

calculated for every treatment (b) Time required (mean ± SE) for 50 percent of the larvae of Rhinella arenarum to 

complete metamorphosis for controls and larvae exposed to atrazine and estradiol. Controls are denoted by C. The 

number indicated at the side of the abbreviation ''At" represents the concentration of atrazine in μg/L. E2=tadpoles

exposed to 100 μg/L of 17β-estradiol. Bars with the same letter are not significantly different (p<0.05).

The duration of the interval between stage 39 and stage 42, and between stage 42 and completion 

of metamorphosis are presented in Figure 4 (copied without alteration from Brodeur et al. 2013). 

The interval between stages 39 and 42 was significantly decreased at 1000 μg/L compared to 

controls. There were no significant differences between treatment groups and controls for the 

interval between stage 42 and completion of metamorphosis. The study authors concluded that 

the impact of atrazine on metamorphosis occurred entirely between stage 25 and stage 39 for 

atrazine concentrations between 1 and 100 μg/L. 

It was noted that the effects on metamorphosis in the 1000 μg/L group differed from effects

caused by atrazine exposure at lower concentrations. The initial significant slowing of 

development observed between stages 25 and 39 (Figure 1b) was followed by a significant 

decrease in the duration of the interval between stage 39 and stage 42 (Figure 4), which caused 

the overall duration of metamorphosis to be similar to controls (Figure 3b). 

The only tested concentration of atrazine that did not affect the timing of metamorphosis was 0.1 

μg/L; therefore, it was concluded that this concentration represented the no observed effect 

concentration (NOEC).
ATRAZINE / 080803 Non-Guideline/Page 6 of 8

 Figure 4. (a) Duration of the interval (mean ± SE) between when 50% of Rhinella arenarum larvae reach stage 39

and when 50% reach stage 42. (b) Duration of the interval (mean ± SE) between when 50% of Rhinella arenarum

larvae reach stage 42 and when 50% complete metamorphosis. Controls are denoted by C. The number indicated at 

the side of the abbreviation ''At" represents the concentration of atrazine in μg/L. E2=tadpoles exposed to 100 μg/L

of 17β-estradiol. * = significantly different from controls (p<0.05).

Body mass and SVL at completion of metamorphosis for control and exposed tadpoles are 

presented in Figure 5 (copied without alteration from Brodeur et al. 2013). Tadpoles exposed to 

17β-estradiol or atrazine at 1 or 10 μg/L grew into significantly (p<0.05) longer and heavier 

metamorphs than controls. A similar result was obtained when body size data were examined in 

terms of body condition (Figure 6, copied without alteration from Brodeur et al. 2013); for these 

data, a significant increase was also found for tadpoles exposed to atrazine at 100 μg/L. The

concentration response curve took the form of an inverted U in this case. It was concluded that 

the NOEC for size and condition at metamorphosis was 0.1 μg/L.

 Figure 5. (a) Body mass at completion of metamorphosis (mean ± SE) for control larvae of Rhinella arenarum and 

larvae exposed to atrazine and 17β-estradiol. (b) Snout-vent length at completion of metamorphosis (mean ± SE) for 

control larvae of Rhinella arenarum and larvae exposed to atrazine and 17β-estradiol. Controls are denoted by C. 

The number indicated at the side of the abbreviation "At" represents the concentration of atrazine in μg/L. E2 = 

tadpoles exposed to 100 μg/ L of 17β-estradiol. * = significantly different from controls (p<0.05).
ATRAZINE / 080803 Non-Guideline/Page 7 of 8

 Figure 6. (a) Distribution of body mass in function of snout-vent length for recently metamorphosed control larvae 

of Rhinella arenarum and larvae exposed to atrazine and 17β-estradiol. The regression line describes the 

relationship observed between body mass and snout-vent length in control animals. (b) Residual (mean ± SE)

between measured body mass and the theoretical body mass of a control animal calculated by applying the equation 

of the regression line presented above (a) to snout-vent length data of animals exposed to atrazine and 17β-estradiol.

Controls are denoted by C. The number indicated at the side of the abbreviation "At" represents the concentration of 

atrazine in μg/L. E2 = tadpoles exposed to 100 μg/ L of 17β-estradiol. * = significantly different from controls 

(p<0.05).

Conclusions: The study authors concluded that low atrazine concentrations in the range of 1-100 

μg/L were found to accelerate the developmental rate in a non-monotonic U-shaped 

concentration-response relationship, and that this observed acceleration of the metamorphic 

process occurred entirely between stages 25 and 39 (treated tadpoles proceeded through 

metamorphosis similar to control animals beyond this point). Furthermore, tadpoles exposed to 

atrazine concentrations in the range of 1-100 μg/L transformed into significantly larger 

metamorphs than controls, the concentration-response curve taking the form of an inverted U in 

this case. The NOEC was 0.1 μg/L atrazine for both size at metamorphosis and timing of 

metamorphosis. Tadpoles exposed to 100 μg/L 17β-estradiol presented the exact same 

alterations of developmental rate and body size as those exposed to 1, 10 and 100 μg/L of 

atrazine. 

The study was conducted in Buenos Aires. Argentina at the Instituto de Recursos Biológicos, 

Centro Nacional de lnvestigaciones Agropecuarias, lnstituto Nacional de Tecnología 

Agropecuaria; the Amphibian Biology Laboratory - Animal Histology Biodiversity and 

Experimental Biology Department, Faculty of Exact Sciences, University of Buenos Aires; and 

the Ecology Laboratory, University National of Lujan. The Good Laboratory Practices status of 

this research is unknown.

The study is of sufficient quality to include qualitatively in the weight of evidence for EDSP. 

However, the following study limitations are noted:

• The adult frogs used to generate tadpoles for this study were caught in the wild (pastures 

in Buenos Aires); any potential previous pesticide exposure is not known.

• The source of the fresh sperm used to fertilize the oocytes was not specifically stated, 

although it is assumed that the wild-caught adult male frogs were the source of the sperm.

• Individual data for mortalities in each group were not reported.
ATRAZINE / 080803 Non-Guideline/Page 8 of 8

• No quantitative data were provided for the study results; all results were presented in bar

graphs.