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 මC. 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 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. 

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 COLLEEN 

ROSSMEISL

Digitally signed by COLLEEN ROSSMEISL 

DN: c=US, o=U.S. Government, ou=USEPA, 

ou=Staff, cn=COLLEEN ROSSMEISL, 

dnQualifier=0000069649 

Date: 2016.04.07 17:55:39 -04'00'