APPENDIX B.4 Additional Amphibian Studies reviewed since 2012 SAP

The following studies are those that have been reviewed since the 2012 SAP and were included in the 

weight of evidence analysis for effects to aquatic amphibians. Previous amphibian study reviews can be 

found in Appendix B.2. Studies are listed below starting with the most recent date and the open 

literature reviews immediately follow. 

Ref # Author Title Source Publication 

Year

Page No.

166446 Dornelles 

MF;Oliveira GT

Effect of Atrazine, 

Glyphosate and 

Quinclorac on 

Biochemical 

Parameters, Lipid 

Peroxidation and 

Survival in 

Bullfrog Tadpoles 

(Lithobates 

catesbeianus)

Arch. Environ. Contam. 

Toxicol. 66(3): 415-429

2014 3

164768 Brodeur JC;

Sassone A;

Hermida GN;

Codugnello N

EnvironmentallyRelevant 

Concentrations of 

Atrazine Induce 

Non-Monotonic 

Acceleration of 

Developmental 

Rate and 

Increased Size at 

Metamorphosis 

in Rhinella 

arenarum 

Tadpoles

Ecotoxicol. Environ. Saf. 

92: 10-17

2013 5 (review 

conducted 

under 

EDSP 

program)

164775 McMahon 

TA;Romansic 

JM;Rohr JR

Nonmonotonic 

and Monotonic 

Effects of 

Pesticides on the 

Pathogenic 

Fungus 

Batrachochytrium 

dendrobatidis in 

Culture and on 

Tadpoles

Environ. Sci. Technol. 

47(14): 7958-7964

2013 15

161314 Paetow LJ;

McLaughlin JD;

Pauli BD;

Marcogliese DJ

Mortality of 

American Bullfrog 

Tadpoles 

Lithobates 

catesbeianus 

Infected by 

J. Aquat. Anim. Health 

25(1): 15-26

2013 17
2

Gyrodactylus 

jennyae and 

Experimentally 

Exposed to 

Batrachochytrium 

dendrobatidis

None Rohr JR, Raffel 

TR, Halstead 

NT, McMahon 

TA, Johnson 

SA, Boughton 

RK,Martin LB.

Early-life 

exposure to a 

herbicide has 

enduring effects 

on pathogen has 

enduring effects 

on pathogen 

induced 

mortality.

Proc R Soc B 280 

:20131502.

2013 19

165358 Rohr JR;Palmer 

BD

Climate Change, 

Multiple 

Stressors, and the 

Decline of 

Ectotherms

Conserv. Biol. 27(4): 741-

751

2013 23

160519 Paetow 

LJ;McLaughlin 

JD;Cue RI;Pauli 

BD;Marcogliese 

DJ

Effects of 

Herbicides and 

the Chytrid 

Fungus 

Batrachochytrium 

dendrobatidis on 

the Health of 

Postmetamorphic 

Northern 

Leopard Frogs 

(Lithobates 

pipiens)

Ecotoxicol. Environ. Saf. 

80: 372-380

2012 26

160887 Svartz 

GV;Herkovits 

J;Perez-Coll CS

Sublethal Effects 

of Atrazine on 

Embryo-Larval 

Development of 

Rhinella 

arenarum (Anura: 

Bufonidae)

Ecotoxicology 21(4): 

1251-1259

2012 29
3

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 166446

Dornelles M.F. and G.T. Oliveira. 2014. Effect of Atrazine, Glyphosate and Quinclorac on Biochemical 

Parameters, Lipid Peroxidation and Survival in Bullfrog Tadpoles (Lithobates catesbeianus). Arch. 

Environ. Contam. Toxicol. 66(3): 415-429.

Purpose of Review: Registration Review

Date of Assessment: 10/23/2015

Brief Summary of Study Findings: This study examined the effects of atrazine at 5, 10 and 20 μg/L on 

bullfrog tadpoles (Lithobates catesbeianus) on biochemical parameters, lipid peroxidation and survival. 

Biochemical parameters analyzed included glycogen, total lipids, triglycerides, cholesterol and total 

proteins in liver, gill and muscle. Atrazine exposure induced significant changes in all biochemical 

parameters analyzed and increased lipid peroxidation levels, with significant impacts to glycogen stores 

in all compartments. No significant changes in growth or survival in treatment groups as compared to 

controls were noted at the end of 14 days.

Methods: Live tadpoles were acquired from a commercial frog at 4 months of age and were in the larval

Gosner stage 26 according to Gosner (1960). Animals were placed in 22 aquariums each containing 12 L 

of water (n = 12), with constant aeration, a water temperature of 22 ± 2 oC, pH 6.2 ± 0.3, and a 12-h 

light-to-dark cycle. The tadpoles were fed (5 % of their biomass) once daily with the same fish feed used 

in the commercial frog farm. Animals were acclimated for 7 days with exposure occurring on day 8. 

Experimental design consisted of two replicates for each exposure concentration of 5, 10 and 20 ug/L 

atrazine. Commercial formulation of atrazine was used (Primo ́leo, 400 g/L; Syngenta). The exposure 

period was 7 days with atrazine being applied only once in liquid formulation and dissolved in distilled 

water. Animals were sacrificed b the freezing method in treatment groups at day 14 and in control 

groups at days 7 and 14. Left and right gills, liver and muscle were removed and tissue specimens were 

then used for biochemical analysis of the levels of glycogen, total protein, lipids, cholesterol, and 

triglycerides as well as evaluation of lipid peroxidation levels for each treatment level and controls.

Results: Significant differences (p<0.05) from controls were noted in the atrazine test concentrations 

below:

Tissue 

tested

Glycogen Total 

protein

Lipids Cholesterol Triglycerides Lipid 

Peroxidation

(ug/L)

Gill 5, 10, 20 None 5, 10, 20 None 5, 10 5, 10, 20

Liver 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20
4

Muscle 5, 10, 20 5, 10, 201 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20 1 Significant difference only noted when compared to control group sacrificed at 7 days.

No significance differences were noted in weight or survival of atrazine treated animals as compared to 

controls. 

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment.

Rationale for Use: The study provides information on sublethal effects of atrazine on biochemical 

parameters as well as growth and survival in bullfrog tadpoles.

Limitations of the Study: Nominal concentrations are only reported; there is no inclusion of measured 

concentrations in control or treatment groups.

Reported water quality parameters were dissolved oxygen, pH and temperature. No water renewal was 

noted during the 7 day treatment period. Constant aeration was reported. 

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division
5

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). Environmentallyrelevant 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
6

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

subsequent to signing by Dynamac Corporation personnel.
7

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). Environmentallyrelevant 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 environmentallyrelevant 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 AMPHITOX solution

 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.
8

(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) 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

 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.
9

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 Ushaped 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).
10

 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).
11

 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).
12

 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).
13

 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.
14

• Individual data for mortalities in each group were not reported.

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

graphs.
15

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 164775

McMahon TA; Romansic JM; Rohr JR. 2013. Nonmonotonic and Monotonic Effects of Pesticides on the 

Pathogenic Fungus Batrachochytrium dendrobatidis in Culture and on Tadpoles. Environ. Sci. Technol. 

47(14): 7958-7964.

Purpose of Review: Registration Review

Date of Assessment: 4/5/2015

Brief Summary of Study Findings: This study examined the effects of atrazine and chlorothalonil on the 

survival of Bd both in culture and on Osteopilus septentrionalis (Cuban treefrog) tadpoles. In the 

concentration range tested (0.011 to 212 μg/L) atrazine reduced levels of Bd growth in culture and 

reduced Bd growth on tadpoles as compared to controls. Overall tadpole survival was impacted by Bd

but not atrazine exposure. The atrazine treated tadpoles had significant decreases in the time to death 

as compared to controls and increased mass of tadpoles as compared to the chlorothalonil group.

Methods: Bd culture was prepared using Bd stock isolated from Rana Catesbenia from Savanah River 

site, SC and culturing in tryptone broth. Zoospores were grown on agar plates and used for experimental 

inoculation. 

Effects of the two pesticides on Bd growth in culture were measured after one and two weeks. 

Chlorothalonil was tested at 0.0176, 0.176, 1.76, and 17.6 μg/L and atrazine at 1.06, 10.6, 58, and 106 

μg/L; both pesticides were technical grade, purity >98% (Chemservice, West Chester, PA) and two 

controls were prepared for each pesticide (water and solvent; acetone). Actual concentrations of the 

stock solutions were measured at 176 ug/L chlorothalonil and 106 ug/L atrazine. Culture tubes were

incubated at 23 °C for either one or two weeks and Bd concentrations were quantified at the one and 

two week time increments based on 20-uL aliquots on a hemocytometer.

Effects of the two pesticides on Bd growth in culture were also measured based on daily Bd growth for 8 

days and 10 treatments based on 10-fold serial dilutions of the pesticide (chlorothalonil: 0.000176, 

0.00176, 0.0176, 0.176, 1.76, 8.8, 17.6, 35.2, 90.2, and 176 μg/L; and atrazine: 0.011, 0.106, 1.06, 10.6, 

23, 58, 77, 106, 159, and 212 μg/L). Effects were determined by measuring changes in Bd density using 

spectrophotometric analysis. 

Effects of the two pesticides on Bd growth in Osteopilus septentrionalis (Cuban treefrog) tadpoles were 

also determined based on four concentrations of technical grade chemical (chlorothalonil: 0.0176,0.176, 

1.76, and 17.6 μg/L; and atrazine: 1.06, 10.6, 58, and 106
16

μg/L). Tadpoles were collected from three clutches naturally oviposited in outdoor wading pools and 

were completely mixed before being distributed among treatment groups. All tadpoles were Gosner 

stage 25 with an average tadpole weight at the start of the experiment of 0.086 ± 0.01 g (mean± SEM 

based on 10 randomly selected tadpoles). Tadpoles were housed in 500 mL glass mason jars and kept on 

a 14:10 light/dark cycle. Water was exchanged weekly and Bd and pesticide treatments re-dosed after 

each water change. Bd exposure was attained through inoculation with 3 mL of Bd+ inoculum 

(concentration of Bd+ was 6.0 x 104 zoospores/mL). Bd was quantified based on PCR analysis of tadpole 

mouthparts. 

Results: Atrazine exposure resulted in a monotonic negative impact on Bd growth in culture, but only 

the highest concentration tested 17.6 ug/L significantly reduced concentrations compared to controls. 

For daily Bd growth as measured using spectrophotometric analysis after correcting for pesticide effect 

on absorbance, all atrazine concentrations significantly reduced Bd growth as compared to controls 

except for the 0.1 ug/l concentration. For Bd growth on tadpoles, atrazine exposure resulted in 

significantly less Bd growth at all concentrations as compared to controls. The author also reported that 

tadpoles exposed to atrazine died sooner than control animals or those exposed to chlorothalonil (512.4 

±11.18, 533.8 ± 10.06, 534.5 ± 10.38 h, respectively; F1, 136 =4.98, p = 0.03) and higher mass than 

tadpoles exposed to chlorothalonil treatments (mean ± SEM: 0.26 ±0.02 g and 0.21 ± 0.01 g, 

respectively; F1, 129 = 8.21, p = 0.005).

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment.

Rationale for Use: The study provides information on effects of atrazine on Bd growth and some limited 

information on tadpole growth and time to death after atrazine exposure.

Limitations of the Study: Regarding the effect of atrazine on survival time (tadpoles died sooner) and 

increased mass, no supporting raw data was reported to support this analysis and the effect is not 

associated with a definitive concentration. 

Water quality parameters not reported in study. 

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division
17

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 161314

Paetow LJ; McLaughlin JD; Pauli BD; Marcogliese DJ. 2013. Mortality of American Bullfrog Tadpoles 

Lithobates catesbeianus Infected by Gyrodactylus jennyae and Experimentally Exposed to 

Batrachochytrium dendrobatidis. J. Aquat. Anim. Health 25(1): 15-26.

Purpose of Review: Registration Review

Date of Assessment: 4/6/2015

Brief Summary of Study Findings: This study investigated the effects of atrazine on larval American 

bullfrogs (Lithobates catesbeianus) experimentally exposed to Bd. After the start of the experiment, the 

sample population was found to be infected with gyrodactylus jennyae leading to mild to severe skin 

erosions in the experimental population. Increased mortality was observed between control groups and 

Bd and Bd+ atrazine groups, but no difference was found between Bd and Bd + atrazine groups. The 

relationship between the co-infections and the severity of skin lesions was correlated with their 

influence on survival.

Methods: 102 bullfrog tadpoles ((Gosner stages 26–37) were obtained from an Environment Canada 

laboratory located in Moncton, New Brunswick (originally purchased from a commercial source in the 

United States). A virulent strain of Bd was used (JEL423) provided by J.E. Longcore

(University of Maine, Orono). Tadpoles were randomly selected for each treatment group. 

Four experimental treatment and control groups were established: (1) a group of tadpoles acutely 

exposed to two doses of Bd zoospores (2) a group exposed for 14 d to atrazine, followed by two acute 

doses of zoospores (3) a group exposed for 14 d to glyphosate, followed by two acute doses of 

zoospores and (4) a group that received no experimental exposures. There were four replicates for each 

group, except for the control group, which had five; each replicate consisted of six tadpoles. Nominal 

chemical exposure concentrations were 21 μg of atrazine, and 1.5 mg acid equivalents (a.e.) of 

glyphosate, per liter of water. In the herbicide exposed animals, the two acute exposures to Bd occurred 

at 14 and 16 d after the start of the pesticide exposures, and each Bd exposure lasted 24 h.

Each replicate group was maintained in a 33-L aquarium containing 8 L of aerated, dechlorinated water. 

Three-quarters of the water in each tank was changed every 3 d. During the pesticide

exposure phase, the herbicides were replenished with each water change. Water volume was 

maintained at 1.3 L per tadpole and reduced by 1 L with each tadpole’s death, to a minimum of

4 L. At day 45, all surviving tadpoles were killed by immersion in a 0.8% solution of MS-222 (tricaine 

methanesulfonate). Tadpoles that died or were killed were immediately fixed in Bouin’s
18

solution, in a 70% solution of ethanol, or in a 10% solution of buffered formalin (two tadpoles in each 

solution per aquarium).

At day 0, a stock solution of each herbicide was prepared. During the pesticide exposure phase of the 

study, each liter of water that was siphoned from the exposure tanks every third day was replaced with 

1 mL of pesticide stock solution. At two time points (at 12 h after herbicide addition and at day 14), 

water samples were drawn directly from two randomly selected pesticide-containing aquariums (one 

containing atrazine and one containing glyphosate)

to compare the actual and nominal concentrations.

The concentrations of glyphosate measured at 12 h and at 14 d post addition of the pesticides to the 

aquariums were both 2.1 mg/L a.e., which was 1.4 times higher than the nominal

concentration of 1.5 mg/L a.e. The concentrations of atrazine measured on the same days were 140 

μg/L and 77 μg/L, respectively, which were 6.7 times and 3.7 times higher than the

nominal concentration of 21 μg/L.

Results: By day 30 of the experiment, a consistent pattern of dermal depigmentation was observed 

around the eyes and at the base of the tail of tadpoles in every group, including the control group. At 

days 30 and 31, two tadpoles that displayed severe depigmentation (each from the group exposed to 

both glyphosate and Bd) were removed from the experiment for examination. Depigmentation was 

found to be the result of skin erosion caused by the tadpoles’ infection by the monogenean ectoparasite 

G. jennyae. Further examination revealed that G. jennyae was present in each aquarium.

Increased mortality was observed between control groups and Bd and Bd+ atrazine groups, but no 

difference was found between Bd and Bd + atrazine groups. Increased susceptibility to Bd due to 

atrazine exposure was not demonstrated in the study. 

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment, but with limited utility.

Rationale for Use: The study provides information on effects of Bd on survival and the effects of coinfection of tadpoles with monogenean ectoparasite G. jennyae. However, effects due to atrazine alone 

could not be discerned from those due to Bd alone.

Limitations of the Study: The presence of the co-infection in all experimental animals with the

monogenean ectoparasite G. jennyae confounded interpretation of results. 

There is uncertainty in the concentrations of atrazine based on the measured concentrations (14 ug/L 

and 77 ug/L) as compared to the nominal concentration of 21 ug/L. 

Water quality parameters not reported in study. 

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division
19

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 165358

Rohr J.R. and B.D. Palmer. 2013. Climate Change, Multiple Stressors, and the Decline of Ectotherms. 

Conservation Biology. 27(4): 741-751.

Purpose of Review: Registration Review

Date of Assessment: 10/23/2015

Brief Summary of Study Findings:

This study examined the effects of temperature (22oC or 27 oC), moisture (wet or dry) and atrazine (0, 4, 

40 and 400 μg/L) on the survival, growth, behavior and foraging of streamside salamanders (Ambystoma 

barbouri). Changes in temperature and moisture alone caused significant loss of mass and mortality with 

atrazine exposure having an additive effect by decreasing water conserving behavior, foraging efficiency, 

mass and time to death. Compared to controls, at the end of the experiment there was a significant 

negative effect on the percent of mass change at all atrazine concentrations tested as well as a negative 

association between atrazine concentration and time of survival. Temperature had the greatest impact 

on individual fitness, with higher temperatures having a higher impact on survival than moisture. 

Methods: Streamside salamanders were exposed to atrazine during their larval stage from the embryo 

stage (April 2002) to metamorphosis (August 2002). No further atrazine exposure occurred after 

metamorphosis. Methods were briefly summarized in the study and the reader is referred to Rohr 2004 

for further details. 31 eggs were placed into 48 aquaria containing 9.5 L at pH of 8 and temperature of 

15◦C. Egg clutches were collected from Fossil Creek (Jessamine County, KY). Four atrazine 

concentrations, each containing solvent DMSO, were tested at 0, 4, 40, and 400 ppb (80% pure 

atrazine). Water was changed weekly and the chemical reapplied with weekly measurements of atrazine 

taken and reported as 4.95 μg/L (SE 0.30), 38.70 μg/L (SE 2.14), and 386.04 μg/L (SE 23.24). 

For the 11 week post metamorphosis observation period 160 terraria were constructed. 3 metamorphs 

from each treatment group were placed in a terrarium. Two incubators were established at 22◦C and 

27◦C. The experimental layout consisted of:
20

Atrazine aquatic exposure 

concentration prior to 

metamorphosis (ug/L)

Terrestrial temperature/moisture exposure matrix

Incubator 1: 

22◦C

Incubator 2: 

22◦C

Incubator 3: 

27◦C

Incubator 4: 

27◦C

Wet Dry Wet Dry Wet Dry Wet Dry

0

4

40

400

1 dry = 42% moisture by weight, wet = 78% moisture by weight 

Temperature selection was based on the natural conditions of salamanders in Kentucky (22◦C) with the 

27◦C temperature intended to be both representative of a possible range of temperatures associated 

with global warming but also within the bounds of current natural conditions (Kentucky temperatures 

reach 30.5◦C in summer). Cool and moist conditions were anticipated to provide the most favorable 

conditions and warm and dry conditions expected to be associated with the most significant adverse 

effects.

Each terrarium was provided one cricket per salamander per week. Individual salamanders were 

denoted by toe clipping. Thirteen times (approximately once weekly) during the procedure observations 

were made if salamanders were in refuge (under a refuge plate or buried), moving, huddled, or climbing 

up the walls of the terrarium. Mass and length were measured during the last week of the experiment. 

Survival was assessed once weekly when the terraria were weighed as it sometimes required disturbing 

buried salamanders. Foraging trials were conducted during the last week of the experiment where 4 

crickets were placed in each terraria and number of crickets eaten and number of salamanders buried 

was recorded hourly for 4 hours. This was only conducted in the 22◦C incubators due to high mortality at 

this point in the experiment in the 27◦C terraria.

Results: From the summary of results, the following interactions were noted:

Endpoint measured NOAEC

(ug/L)

LOAEC

(ug/L)

Behavior – Huddling 4 40

Behavior – Inactivity <4 4

Behavior – In refuge 4 40

Mass change <4 4*

Survival >400 NA

*p=0.054 at 40 ug/L atrazine

Each square represents separate 

incubator conditions that contain 5 

replicates (terrariums) with 3 

metamorphs in each replicate 
21

Significant effect was noted on certain behaviors at all atrazine concentrations; however, as presented, 

results were pooled from all temperature and moisture test conditions together making it difficult to 

separate out effects due to atrazine alone. Significant differences on behavior were also reported due to 

temperature and moisture changes when all atrazine exposure concentrations pooled. A significant 

effect on mass change was noted with atrazine exposure overall. A 4% change with each log 10 

difference was noted at 27oC. 

In foraging trials (performed only on 22oC incubators due to high mortality in 27◦C incubator groups), 1 

hr after exposure most salamanders were not buried in wet or dry conditions. Under dry conditions, 1 hr 

after release of crickets, atrazine exposed salamanders had reduced foraging efficiency as evidenced by 

the number of crickets eaten per salamander. However, due to the effect of atrazine to reduce the 

number of salamanders in refuge under dry conditions, at 2-4 hrs there was an increase in foraging 

efficiency as less salamanders were buried by 4 hrs. Under wet conditions, atrazine exposure was 

associated with a negative effect in foraging efficiency at all time points. The author noted the 

cumulative number of crickets eaten depended significantly on time, moisture and atrazine exposure. 

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment.

Rationale for Use: The study provides information on amphibian growth, mortality and behaviors under 

different and changing environmental conditions; implications for multiple stressors in the environment; 

uncertainties identified are described below.

Limitations of the Study: DMSO was used as the solvent without a negative control. Uncertainty exists 

in the use of DMSO as a solvent as it may accelerate the movement of chemicals across cell membranes. 

However, the author reported that no negative control was included as the solvent has not been shown 

to have effects on behavior, growth or survival in this species in a previous study (Rohr et al., 2003a). 

All results were only presented graphically, making it difficult to validate or further analyze the reported 

results. In addition, for certain endpoints, it was difficult to discern if the reported effects associated 

with atrazine exposure were from pooled results of all temperature and exposure concentrations or 

individual exposure regimens. As significant effects were seen due to changes in temperature and 

moisture alone, this made it challenging to differentiate a clear causation from atrazine exposure alone. 

The aqueous phase exposure portion of this study was previously reported under Rohr 2004. The 2004 

study was previously reviewed (Open literature summary review, A. Pease and A. Blankenship, 2012). 

Concern was noted in the study review due to a possible fugal pathogen forming a white film on 

embryos, use of DMSO, no negative control, no raw data reported, unknown study duration, variation in 

results from 2002 to 2003, possible prior exposure of test organisms to atrazine, high loading rates and 

the lack of ammonia and nitrate reporting. 

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division
22

References: 

Rohr, J. R., A. A. Elskus, B. S. Shepherd, P. H. Crowley, T. M. Mc-Carthy, J. H. Niedzwiecki, T. Sager, A. Sih, 

and B. D. Palmer.2003a. Lethal and sublethal effects of atrazine, carbaryl, endosulfan, and octylphenol 

on the streamside salamander, Ambystoma barbouri. Environmental Toxicology and Chemistry 22:2385–

2392.

Rohr, J. R., A. A. Elskus, B. S. Shepherd, P. H. Crowley, T. M. McCarthy, J. H. Niedzwiecki, T. Sager, A. Sih, 

and B. D. Palmer. 2004. Multiple stressors and salamanders: effects of an herbicide, food limitation, and 

hydroperiod. Ecological Applications 14:1028–1040.
23

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: None

Rohr JR, Raffel TR, Halstead NT, McMahon TA, Johnson SA, Boughton RK,Martin LB. 2013. Early-life 

exposure to a herbicide has enduring effects on pathogen induced mortality. Proc R Soc B 280 

:20131502.

Purpose of Review: Registration Review

Date of Assessment: 4/6/2015

Brief Summary of Study Findings: This study examined the effects of 6 day atrazine exposure at 65.9 

μg/L to Cuban tree frog tadpoles (Osteopilus septentrionalis) on the response to Bd exposure both 

immediately after atrazine exposure as tadpoles and 46 days later (post-metamorphosis). Tadpoles were 

exposed to atrazine during the first week (window 1; Gosner stage 26-28) and the second week (window 

2; Gosner stage 35-37) of development. A significant difference was found in tadpoles exposed during 

window 2 but not window 1 in SVL and time to metamorphosis; those tadpoles exposed during window 

2 were smaller and morphed earlier than window 1. Mortality was significantly higher in atrazine 

exposed groups when exposed to Bd both immediately and 46 days later (post metamorphosis) as 

compared to solvent controls exposed to Bd.

Methods: 60 outdoor mesocosms (1.8 m diameter, 60 cm deep, approx. 1100 L) were established, each

containing 800 l of water and inoculations of local zooplankton, phytoplankton and periphyton, and 

covered with 60% shade cloth. Forty tanks received 50 O. septentrionalis tadpoles (all at Gosner stages 

[25–28]) from five clutches collected from pools at the University of South Florida (USF) campus (the

clutches were mixed to homogenize genetic variation before being distributed among the tanks). Pools 

were filled with municipal water and could not receive any run-off that might contain contaminants. This 

experiment was conducted in outdoor tanks, so that the food resources of the tadpoles might be more 

natural than in a laboratory.

The study uses a completely randomized 2x2x2x2 factorial design with five replicates of each test group. 

Tanks received either 25 ml of ethanol solvent or atrazine (Chemservice, West Chester, PA;technical 

grade, purity more than 98%) dissolved in 25 ml of ethanol. A water control was not included due to 

previous studies by the author that never detected any immediate or later-life effects of the solvent on 

any measured traits. Water samples were taken from every tank 1 h after dosing and atrazine was 

quantified using the Abraxis (Abraxis LLC,Warminster, PA) ELISA microtiter plate kit, with each sample 

processed in duplicate (measured concentrations were 65.9+3.48 ug/L3, mean+s.e.).

 

3 This concentration was erroneously reported as 65.9 mg/L in the originally manuscript but is corrected to 65.9 

ug/L in a correction issued to the article. 
24

Tadpoles were exposed to the solvent control or atrazine for 6 days at one of two periods during their

development, either in the first week (Gosner stage 26–28) or second week (Gosner stage 35–37) of the 

experiment. After exposure to atrazine or solvent, Gosner stage and snout–vent lengths (SVLs) of all 

tadpoles was characterized and tadpoles were exposed to Bd or not. To test for short- and long-term 

effects of atrazine exposure on disease risk, the pathogen treatments occurred less than 18 h or a mean

of 46 days after the end of the chemical exposure period. The latter Bd exposure time was postmetamorphosis. If a tank of tadpoles was designated to be exposed to the Bd treatment post

metamorphosis, then all the tadpoles from the tank were counted and transferred to an adjacent tank 

after the 6-day atrazine- or solvent-exposure period and then reared to metamorphosis. These adjacent 

tanks were established the same way as the ‘exposure’ tanks but were free of atrazine or solvent. 

Metamorphs were pulled from the tanks every other day and maintained in the laboratory (12 h light 

cycle, 238C) on vitamin- and mineral-dusted crickets (fed ad libitum) until their use in the Bd-exposure 

portion of the experiment. In summary, there were 60 mesocosms total; 40 served as the primary tanks 

for the 2x2x2 portion of the experiment (atrazine or solvent x early or late in development x immediate 

or delayed Bd exposure), and 20 mesocosms had freshwater to house the delayed Bd exposure animals

in the absence of atrazine or solvent.

Tadpoles were exposed to 2 ml of the Bd+ or Bd- (prepared identically to Bd+ but lacked Bd zoospores) 

inocula in 80 ml of artificial spring water for 2 days. Metamorphic frogs were exposed to the Bd by 

pipetting 2 ml of 3 x 10-4 zoospores ml-1 onto the frog’s ventral side. In general, five tadpoles or 

metamorphs were exposed to Bd from each tank, but these numbers

varied depending on survival. Post-metamorphic frogs were fed five vitamin- and mineral-dusted 

crickets (5–15 mm) twice per week, and tadpoles were fed organic spinach ad libitum. During the Bd+ or 

Bd- exposure periods, tadpoles and metamorphs were maintained at 23◦C on a 12 h light cycle and their 

survival was monitored daily for 14 days. PCR analysis was used to quantify Bd from tadpole mouthparts 

(dissected) and hindlimbs.

Results: No factors had significant effects on tadpole survival during the 6-day atrazine or solvent 

exposure. Atrazine had no significant effects on survival to metamorphosis in the absence of Bd nor any 

interactions of other factors with atrazine were significant predictors of mortality. Atrazine exposure 

early in life significantly increased mortality in the presence of Bd indicating a significant interaction 

between Bd and earlier exposure to atrazine. Risk of death was two times more likely for individuals 

exposed to atrazine and Bd and atrazine then those exposed to solvent and Bd. There was a similar 

magnitude of mortality when frogs were exposed to Bd immediately after the 6-day exposure to

atrazine or later in life (mean = 46 days), suggesting there was no evidence of recovery from early-life 

exposure to atrazine. The authors provide discussion on factors possibly contributing to the observed 

results, including the impact of atrazine exposed individuals being of reduced size and accelerated 

development at the time of Bd exposure and also the possible direct impact of atrazine on the endocrine 

and immune systems. The latter theory was further investigated by controlling for development and SVL 

and still demonstrating increased Bd-mortality in atrazine exposed individuals. 

Despite atrazine-exposed frogs having significantly greater Bd-induced mortality than solvent-exposed 

frogs, there was no evidence that the atrazine-exposed frogs were less resistant to Bd.
25

Tolerance differed significantly among the atrazine- and solvent-exposed frogs; at the same Bd load,

atrazine-exposed frogs were more likely to die than solvent exposed frogs. 

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment.

Rationale for Use: The study provides information on the impacts of atrazine exposure in early life 

stages to mortality from Bd infection.

Limitations of the Study: Although tanks started with 50 tadpoles each, it is unclear the number of frogs 

that were ultimately exposed to Bd for each group, although it was noted generally 5 frogs from each 

tank were exposed. Summary statistics but no raw data were provided.

Municipal water was used to fill tanks; water quality data was not reported. 

Nominal concentrations not reported; atrazine concentrations only monitored on Day 1 (1 hour post 

treatment).

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division
26

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 160519

Paetow LJ, McLaughlin JD, Cue RI, Pauli BD, and DJ Marcogliese. 2012. Effects of Herbicides and the 

Chytrid Fungus Batrachochytrium dendrobatidis on the Health of Post-metamorphic Northern Leopard 

Frogs (Lithobates pipiens). Ecotoxicol. Environ. Saf. 80: 372-380.

Purpose of Review: Registration Review

Date of Assessment: 11/4/2015

Brief Summary of Study Findings: The effects of atrazine and the chytrid fungus Batrachochytrium 

dendrobatidi (Bd) were investigated. Post-metamorphic northern leopard frogs (Lithobates pipiens) 

were exposed to atrazine at 2.1 μg/L (actual levels 1.70 to 4.28 μg/L) alone for 21 days then followed by 

Bd and observed for 94 days. Significant effects on mass gain were seen in the atrazine treated group 

over the course of the experiment (Days 1-94) with 11% and 8% decrease in mass gain as compared to 

control means in atrazine alone and atrazine + Bd groups collectively. No significant effects were seen 

on survival, biomarkers of animal health or immune function. There was no evidence of active Bd

infection in the frogs at the end of the exposure period, which the author attributed to a potentially 

resistant strain of frogs and significantly increased skin shedding in exposed frogs which may have 

helped resist or clear infection. 

Methods: Recently metamorphed northern leopard frogs were captured from a wildlife reserve near 

Boucherville, Quebec. Water samples were taken from the pond the frogs were captured from and 

analyzed for 39 pesticides, including atrazine. Twenty random individuals were immediately swabbed to 

test for Bd infection. During the 8 week acclimation period, frogs were housed in individual 1.84 L plastic 

containers containing 50 mL of dechlorinated, UV-treated water and two platforms permitted frogs to 

exit the water. Acclimation included a 16 h:8 h light:dark photoperiod and room temperature of 21 ±1.3 

◦C. Frogs were measured and weighed prior to the start of the experiment. Sixty-four frogs were 

exposed to atrazine at 2.1 ug/L (Aatrex© Liquid 480, Syngenta Crop Protection Canada, Inc.) for 21 days 

using 1 L mason jars containing 50 mL of treatment solution. Jars were held at a slant (except days of Bd 

exposure) to allow frogs to enter and leave the liquid and mimic a semi-terrestrial environment. Water 

was changed daily during the acclimation and exposure period. Pesticide was diluted from stock solution 

to achieve the desires test concentrations. Diluted stock solution on Day 20 and samples from 6 

randomly selected exposure vessels from the treatment group were analyzed for atrazine concentration. 

At the end of exposure period on Day 21, frogs were again weighted and measured. Frogs were 

subdivide into three groups (n=20-22 frogs per group). One group was euthanized and necropsied, one 

was selected as the Bd exposure group and one served as the post atrazine exposure Bd negative 
27

control group. Bd exposure occurred on Day 22 and every eighth day of the next 25 –day period (4 Bd

exposures total). Exposure to Bd was through cultured Bd distributed in 50 uL aliquots into each control 

vessel. Concentration of zoospores in the aliquots was measured for each exposure day. Vessels were 

held upright for the 24 hour bd exposure day, then test solution was replaced and the jars were 

returned to a slanted position so frogs could exit the water. Frogs were then observed and held until 94 

days post initial atrazine exposure (or 74 days post initial Bd-exposure). At Day 94, all remaining frogs 

were weighed, measured, euthanized and necropsied for final effects measures. In addition to weight 

and length, The measured biomarkers of health included hepatosomatic index HSI, densities and mean 

sizes of hepatic and splenic melanomacrophage aggregates (HMAs and SMAs, respectively), density and 

mean size of hepatic granulomas (HGAs), the neutrophil to lymphocyte ratio (N/L ratio), and immune 

function measures including splenosomatic index (SSI), differential leucocyte counts, and the ratio of 

leucocytes to erythrocytes (WBC/RBC).

To monitor for Bd infection, toe samples from right hindfoot were collected from each frog killed at Day 

94 and examined histologically for Bd and hyperkeratosis. Toe sample from the left hind foot were used 

for diagnosis and quantification of Bd infection by real time PCR. Full body swabs were also taken at 

several time points to monitor for infection. As frogs were housed individually, the frequency with which 

they shed their skin was also quantified as this can be an indicator of Bd infection.

Results: Atrazine was not detected in samples collected from the frogs’ natal pond and none of the frogs 

initially tested were positive for Bd infection. The concentration of atrazine in the stock solution was 

4.28±0.04 ug/L and the water samples collected from the exposure vessel was 1.70±0.26 ug/L. 

At the end of the 21 day exposure period, no difference in SVL or mass was detected in the atrazine 

exposure group. At the end of the 94 day period, there was a significant difference in mass of the 

atrazine (11% decrease) and atrazine-Bd (8% decrease) exposure group from their respective controls. 

There was no difference in the SVL between treatment and control groups. 

Survival was not impacted by atrazine or Bd exposure. No significance difference was found in any 

biomarkers analyzed, but blood cell ratios wre almost affected by atrazine exposure and occasionally by 

Bd exposure. 

None of the samples showed microscopic evidence of Bd infection, either through the presence of 

zoospores or hyperkeratosis and only one sample tested positive for Bd via PCR analysis with a low DNA 

copy number (1.6 DNA copies). Ecdysis (skin molting) was significantly affected by Bd exposure, with Bd

exposed frogs shedding skin 1.03±1.03 during the 4 week period and unexposed frogs shedding an 

average of 0.19±0.54 times. 

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment.

Rationale for Use: The study provides information on sublethal effects of atrazine on growth and 

biochemical parameters in leopard frogs. Information on the interaction of atrazine and Bd infection 

rate is of limited use. 
28

Limitations of the Study: Although intended to test the interaction and effect of atrazine on Bd

infection, none of the frogs in the study ultimately became infected with Bd. Although some conclusions 

are drawn in the article on the lack of impact of prior exposure to atrazine on the susceptibility to Bd 

infection, this conclusion is hard to confirm as Bd infection was not induced in any of the frogs exposed 

including controls. This could be due to a resistant population of frogs used in the study, the impact of 

increased skin shedding on disease resistance in the Bd exposed frogs or other factors. 

Atrazine was not measured in the control samples. Atrazine in stock solution and exposure vessels was 

measured only on Days 20 and 21, respectively. 

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division
29

Open literature Review Summary

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: E160887

Svartz GV; Herkovits J; Perez-Coll CS. 2012 Sublethal Effects of Atrazine on Embryo-Larval Development 

of Rhinella arenarum (Anura: Bufonidae). Ecotoxicology 21(4): 1251-1259.

Purpose of Review: Registration Review

Date of Assessment: 4/6/2015

Brief Summary of Study Findings: This study examined the effects of atrazine exposure on embryolarval development of the common South American toad, Rhinella arenarum,

Methods: Three mating pairs of adult common South American toad, Rhinella arenarum

weighing approximately 200-250 g were acquired in Lobos (Buenos Aires province, Argentina). Ovulation 

of females was induced by means of an intraperitoneal injection of a suspension of one homologous 

hypophysis in 1 mL of AMPHITOX solution (AS) per female. Oocytes were fertilized in vitro using fresh 

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

coat removal, embryos were kept in AS and maintained at 20 ± 2 °C. The AS was replaced entirely every 

three days and monitored weekly to ensure that the pH was at acceptable levels (7 ± 0.5).

For treatments during embryonic stages (S4-S25), ten embryos were randomly placed in triplicate in 10 

cm diameter glass Petri dishes containing 40 mL of test solution. For treatments during 

prometamorphosis (S28), ten larvae were placed in triplicate in 20 cm-diameter glass. Petri dishes 

containing 100 mL of test solution. Test solutions were entirely replaced every 48 h. Test groups 

included:

l) Continuous exposure from early blastula (S4) up to complete operculum stage (S25); 336 h (14 days) at

concentrations between 5 and 30 mg/L.

2) Continuous exposure from early complete operculum stage (S25) up to late complete operculum 

stage (S25); 336 h (14 days) at concentrations between 1 and 30 mg/L.

3) Continuous exposure from completed back legs (S28) until completion of metamorphosis; 1080 h (45 

days) at concentrations between 1 and 30 mg/L.

4) 24 h pulse-exposure with concentrations ranging between 1 and 40 mg/L in blastula (S4), neural plate 

(S13), muscular activity (S18), gill circulation (S20), opercular folds (S23), and complete operculum (S25). 

After the treatments, the embryos were thoroughly washed and kept in AS until 168 h post-exposure.

Toxicity tests were performed using technical-grade atrazine with a purity of 98 %, purchased from 

Chem Service (West Chester, PA, USA). Due to precipitation of atrazine at higher test concentrations, 

0.2% acetone was added to the test concentrations. Solvent and blank controls were maintained. 
30

Results: At the blastula stage, sublethal effects were noted in atrazine concentrations over 5 mg/L. 

Malformations were observed from 48 hours onward and percent affected increased with dose, up to 

67% at 30 mg/L. The main malformations were: delayed development, reduced body size, tail and axial 

flexures, irregular border, different types of edema, wavy tail, microcephaly, malformed mouth and 

adhesive structures, gut miscoiling, underdeveloped gills, cloacal protrusion, and cellular dissociation. 

During short-term chronic (168 h) exposure, the NOEC value for sublethal effects was 10 mg/L. The 

percentage of affected embryos was proportional to atrazine concentration, with a maximal of 50 %

affected at 30 mg/L. From 96 hour onwards, malformations, mainly tail, axial flexures, different types of 

edema, malformed mouth and adhesive structures, and cellular dissociation in specific regions such as 

the stunted tail, were observed. In acute exposure, treated larvae showed neurotoxic effects such as

spasmodic contractions and general weakness at concentrations equal or higher than 15 mg/L. A 

reduction in food intake was also observed. Lethality was recorded at concentrations higher than 20 

mg/L starting from 72 h of exposure onwards.

For the 24 hour pulse exposure, lethality in embryos occurred at 40 mg/L 168 hours post exposure. A 

range of sublethal teratogenic and neurotoxic effects occurred with LOAECs ranging from 15 to >40 

mg/L, with the blastula stage being the most sensitive and showing the widest range of effects.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk 

assessment.

Rationale for Use: The study provides information on the potential for teratogenic and neurotoxic 

effects due to atrazine exposure during embryo-larval development.

Limitations of the Study: Wild caught test animals were used for sources of eggs with unknown 

exposure history.

Author reported that measured concentrations were within 5% of nominal concentration but no 

measured concentrations were reported or the frequency of measurement. Some concentrations 

exceed the water solubility of atrazine (30 mg/L).

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division