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 μ g/L (actual levels 1.70 to 4.28 μ g/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 s een 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 expose d 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 ම 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 mi mic 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 concent rations. Diluted stock solution on Day 20 and samples from 6 randomly selected exposure vesse ls 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 Bddistributed 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 ra tio of leucocytes to erythrocytes (WBC/RBC).

To monitor for Bd infection, toe samples from right hind foot 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 th eir skin was also quantifi ed as this can be an

indicator of Bd infection.

Results: Atrazine was not detected in samples co llected 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 samp les 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 wr e 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 st udy ultimately became infected with Bd . Although someconclusions 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 Da ys 20 and 21, respectively.

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

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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'