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. Earlylife 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 (postmetamorphosis). 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/L1

, mean+s.e.). 

1 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. 
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 post-metamorphosis. 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 mineraldusted 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 23මC 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 solventexposed frogs, there was no evidence that the atrazine-exposed frogs were less resistant to Bd. 

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 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 17:53:58 

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