## 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, B uilding 100, S uit e 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

E PA Primary Reviewer: Amy Blankinship Date: __ 7 /15

___________ E PA 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. Ecotoxicolo gy and E nvironmental S afety . 92:10

- 17.

SPONSOR : Not applicable (open literature review)

E XECUTIVE 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

e xperimental design executed in the present study is a follow

- up of an earlier study ( Brodeur et al. 2009 1 ) which demonstrated a non

- monotonic acceleration of the time to metamorphic climax ( Gosner stage 42) and delayed tail resor ption

. Exposures examined in th e current study we re longer (from stage 25 instead of stage 38) and the tested concentrations we re lower and more similar to levels detected in the environment

.

Met hods : Adult South American toad s ( R

. arenarum )

, weighing approximately 200

- 250 g

, were collected from pasture fields in Buenos Aires Province, Argentina . The o vulation of female toad s was induced by an intraperitoneal injection of homologous hypoph y sis suspended in

1 Brodeur

, J.C

. , Svartz, G., Perez

- C o ll, 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; Non

monotonous acceleration of the time to climax and delayed tail resorption

. Aquat Toxicol

. 91 : 161

- 170

.ATRAZINE / 080803 Non

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AMPHITOX solution ( AS ; Herkovits and Perez

- Col l , 2003 2 )

. Oocytes were fertilized in vitro using fresh sperm suspended in AS

, and t he resulting embr yos were maintained in AS at 20± 2 ° C until reaching stage 25. W hen tadpoles began feeding at stage 24

- 25 , they were offered boiled Swiss chard ad libitum

. Tadpoles were pooled from four different clutches , and t wenty replicates were performed for

e ach concentration tested

. In e ach 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 μ g/L . A stock solution of atrazine (10 mg/L) was prepared in AS using acetone as a solvent , and the test solutions were p repared by sequentially diluting the stock solution with AS ; the atrazine concentration in the stock solution was verified by LC/MS/M S analysis

. Additionally

, the study included a control group exposed to AS only ; a solvent control group expose d to AS con taining 0.02% (v/v) acetone ; and a positive control
group exposed to 100 μ g/L of 17 β

- estradiol (E2 ; prepar ed 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 G osner 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 an d snout

- vent length (SVL) were measured

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

. D ata from the control groups were used to perform th is regression analysis

. The theoretical body weight value of frogs fr om 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

, represent ing an expression of the difference between the measured weight of the metamorph and that of an average healthy subject w ith the same SVL

. A n indiv idual with a positive residual wa s considered to be in a good condition whereas an individual with a negative residual wa s 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

. F our

- parameter logistic regression analysis wa s conducted for each treatment on the cumulative numbers of animals reaching stage 39, stage 42 or completing metamorphosis as a function of exposure duration ( G raphPad Prism software )

. For each curve, the time for 50 % of the individuals to reach the stage being examined , s tage 39 (T39), stage 42 (T42) or completion of metamorphosis (TCM)

, was calculate d

. The duration of the inter vals 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

- Col l

, 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

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for each replicate of e ach treatment

. Th e values of T 39, T 42 and TCM, the SVL s of the metamorphs, and the intervals between s tage 42 and completion of metamorphosis were compared between treatments using o n e

- way analysis of v ariance (ANOVA) followed by a Holm

- Sidak test for multiple comparisons

. A non

- parametric Kruska l

- Wall is one

- way ANOVA on Ranks was used to compare the following parameters for which normality and equal variance could not be obtained : s urvival of tadpole s ; intervals between stage 39 and stage 42 ; proportions of individuals r eaching stage 39, stage 42 or completing metamorphos is ; 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 determ ined at p<0.05.

Results : T he only concentration of atrazine that had significant mortality was 1000 μ g/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 μ g /L treatment groups (75.7

- 82.5%) were not significant ly 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)

.

T he pr ogression of the metamorphosing tadpoles to stage 39, stage 42

, and completion of metamorphosis is presented in Fig ures 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 μ g /L took significantly (p<0.05) more time than controls to reach stage 39 (increased T39)

, whereas expos ure to 1, 10 and 100 μg/L significantly reduced T39 (Fig ure 1b) compar ed to the controls

. Th is acceleration of development with atrazine exposure ha d a U

- shaped concentration

- response relationship as 10
μ g /L caus ed a greater reduction of T 39 than 1 or 100 μ g/L ( which generat ed comparable effect s )

. A simila r U

- shaped pattern of acceleration of development as a function of at r azine concentration was observed when considering T42 (Fig ure 2b) and TCM (Fig ure 3b).

Tadpoles exposed to 17

- estradiol (100 μg/L) showed acceleration of development of the same amplit ude and characteristics as those observed in the atrazine 1 and 100 μg/L treatment groups (Figures 1

- 3).ATRAZINE / 080803 Non

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Figure 1

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

arenarum and larvae exposed to atrazine and 17 β

- 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 μ g / L

. E2=tadpoles exposed to 100 μ g /L of 17 β

- 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 17 β

- 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" rep resents the concentration of atrazine in μ g / L

. E2=tadpoles exposed to 100 μ g /L of 17 β

- estradiol

. Bars with the same letter are not significantly different (p<0.05)

.ATRAZINE / 080803 Non

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Figure 3

. (a) Cumulative number of individuals c ompleting metamorphosis over time for control larvae of Rhinella

arenarum and larvae exposed to atrazine and 17 β

- 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 compl ete 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 μ g / L

. E2=tadpoles exposed to 100 μ g /L of 17 β

- estr adiol

. Bars with the same letter are not significantly different (p<0.05)

.

T he duration of the interval between stage 39 and stage 42, and between stage 42 and completion of metamorphosis are presented in Fig ure 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 we re no significant differences between treatment groups and controls for the interval between stage 42 and completion of metamorphos is. 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 μ g/L

.

It was noted that t he effects on metamorphosis in the 1000 μg/L group diffe red from effects caused by atrazine exposure at lower concentrations. The initial significant slowing of development observed between stage s 25 and 39 ( F igure 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

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Figure 4

. (a) Duration of the interval (mean ± SE) between when 50% of Rhine lla arenaru m 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 μ g / L

. E2 =tadpoles exposed to 100 μ g /L of 17 β

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

. Body mass and SVL at completion of metamorphosis for control and exposed tadpoles are presented in Fig ure 5 (copied without alteration from Brodeur et al. 2013)

. Tadpoles exposed to 17 β

- estradiol or atrazine at 1 or 10 μ g/ L grew into significantly (p<0.05) longer and heavier metamorphs than controls

. A similar result was obtained when body size data were exam ined in terms of body condition ( Fig ure 6
, copied without alteration from Brodeur et al. 2013 ); for these data, a significant increase was also found f o r tadpoles exposed to atrazine at 100 μ g/L . T he concentration response curve t ook the form of an invert ed U in this case. It was concluded that
t he NOEC f o r size and condition at metamorphosis was 0.1 μ g/L

.

Figure 5

. (a) Body mass at completion of metamorphosis (mean ± SE) for control larvae of Rhine ll a arenarum and larvae exposed to atrazine and 17 β

- estradiol. (b) Snout

- vent length at completion of metamorphosis (mean ± SE) for control larvae of Rhinella arenarum and larvae exposed to atrazine and 17 β

- estradiol

. Controls are denoted by C. The number indicated at the side of the abbreviation "At" r epresents the concentration of atrazine in μ g/L. E2 = tadpoles exposed to 100 μ g/ L of 17 β

- estradiol

. * = significantly different from controls ( p <0.05)

.ATRAZINE / 080803 Non

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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 17 β

- estradiol . T he regression line describes the relationship observed between body mass and snout

- vent length in control animals. (b) Residual (mean ± SE) between measure d 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 17 β

- estradiol

. Controls are denoted by C. The number indicated at the side of the abbreviation "At" represents the concentration of atrazine in μ g/L. E2 = tadpoles exposed to 100 μ g/ L of 17 β

- estradiol

. * = significantly different from controls ( p <0.05)

.

Conclusions : The study authors concluded that l ow atrazine concentrations in the range of 1

- 100
μ g/ L were found to accelerate the developmental rate in a non

- monotonic U

- shaped concentration

- response relationship , and that t his observed acceleration of the metamorphic
process occurred entirely between stages 25 and 39 ( treated tadpoles proceed ed through metamorphosis similar to control animals beyond this point )

. Furthermore

, tadpoles exposed to

a tr azine concentrations in the range of 1

- 100 μ g/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 μ g /L atrazine for both size at metamorphosis and timing of metamorphosis. Tadpoles exposed t o 100 μ g/ L 17 β

- estradiol presented the exact same alterations of developmental rate and body size as those exposed to 1

, 10 and 100 μ g/ L of atrazine.

The study was conducted in Buenos Aires

. Argentina at the I nstituto de Re cursos Bio l ó gicos, Centro Naci ona l de lnvestiga ci ones Agrope cuarias , lnstituto Nacional de Tecnolog í a Agrope cuaria; the Amphibian Biology La boratory

- Animal Histology Biodiversity and Experimental Biology Department

, Fa cu lty of Exact Sciences, University of Buenos Aires ; and the Ecology Laboratory

, University National of L ujan

. 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, t he following study limitations are noted :

• The a dult 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 da ta for mortalities in each group were not reported.ATRAZINE / 080803 Non

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