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 Chan ge, 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 μ g/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 atra zine 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 test ed 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 briefl y 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 ම 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 κ͘ερʅŐͬ>;^Ϭ͘ϯϬͿ͕ϯΘ͘ϳϬʅŐͬ> (SE Ϯ͘ϭκͿ͕ĂŶĚϯΘς͘ϬκʅŐͬ>; 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 ම and 27 ම . The experimental layout consisted of:Atrazine aquatic exposure concentration prior to metamorphosis (ug/L) Terrestrial temperature/moisture exposure matrix

Incubator 1:

22 ම Incubator 2:

22 ම Incubator 3:

27 ම Incubator 4:

27 ම

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 මͿ
ǁŝƚŚƚŚĞϮϳම 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 ම 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 sala mander 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 ම C incubators due to high mortality at this point in the experiment in
the 27 ම 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 replicateSignificant effect was noted on certain behavior s 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 wa s noted with atrazine exposure overall. A 4% change with each log 10 difference was noted at 27oC.

In foraging trials (performed only on 22 oC incubators due to high mortality in 27 ම 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 effici ency 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 note d 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; uncertai nties 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 validat e or further analyze the reported results. In addition, for certain endpoint s, 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, th is 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

References: 

### 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 17:58:15 -04'00'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 stre amside salamander, Ambystoma barbouri. Environmental Toxicology and Chemistry 22:2385–2392.

Rohr, J. R., A. A. Elskus, B. S. Shepherd, P. H. Crow ley, T. M. McCarthy, J. H. Niedzwiecki, T. Sager, A. Sih, and B. D. Palmer. 2004. Multiple stressor s and salamanders: effects of an herbicide, food limitation, and hydroperiod. Ecological Applications 14:1028–1040.