Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation:

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......**·_~_1_** L**L~_~_1** 1 DUUllt::,lVl, lJ. dllU**~.** lVl, Jdlllt:::s. L.UV.J.**Illlt::ld~UUll:S** UI**illl1l1:St::~U~lUt::, llt::lUI~IUt::,**  dllU natural stressors in amphibian community mesocosms. Ecological Applications. 13(3): 829-841. (MRID 48261104)

Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associated determination ofaLevel ofConcern (LOC).

Date ofReview: September4, 2009

Purpose ofStudy: This study examines the effects ofatrazine and carbaryl (study design included individual and combined exposure treatments) onphytoplankton and four amphibian species in outdoormesocosms. Tadpole density and photoperiod were also manipulated.
Summary ofStudy Findings: The focus ofthe summary is on atrazine treatments (treatments including carbaryl are not reported).

Methods: Egg masses were collected for southern leopard frogs (Rana sphenocephala; 3 egg masses) and spotted salamanders (Ambystoma maculatum; 21 egg masses) on March 14, 2000 and March 27,2000, respectively, from the BasketWildlife Areain Boone County, Ashland, Missouri. Egg masses were also collected for Americantoads (Bufo americanas; 7 egg masses) and small-mouth salamanders (Ambystoma texanum; ca. 30 egg masses) on March 30,200 from the ForumNature Areain Boone County, Columbia, Missouri and April 9, 2001 from the Basket Wildlife Area, respectively. Eggs were hatched in the laboratory at 23-25°C and held until all larvae were free-swimming. Clutches were mixed within each species to homogenize genetic variation.

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# 111••••111 2087802The aquatic communities were prepared by filling polyethylene cattle tankponds (1.85 m in diameter, 1480 L volume) with 1000 L oftap water, 1 kg ofleaflitterfrom a mixed, deciduous forest and plankton from natural ponds (500 mL ofplanktonperpond at six differenttimes) in mid-March 200 and 2001. The planktonpopulations were wellestablishedpriorto addition oflarvae. The aquatic communities were maintained outdoors during the experiments to simulate natural conditions. In orderto protectthe test systems, the cattle pond tanks were located inside a fenced field and covered with mesh lids during the experiments.

For Experiment I, the effects ofcompetition, atrazine and carbaryl were studiedby a fully crossed design with three replicates (36 ponds) where initial anuran density was either low (20 tadpoles/lOOO L) orhigh (60 tadpoles/lOOO L), carbaryl concentration was 0,3.5 or 7.0 mg/L, and atrazine concentration was 0 or 200 f.lg/L. Twelve spotted salamanders were also in eachpond. Carbaryl (liquid Sevin, 21.3% carbaryl) and atrazine (liquid Aatrex, 40.8% atrazine) were added via mixing with 5 L ofpond water and pouring evenly overthe pond surface without stirring. Uncontaminated pond water (5 L) was added in the same mannerto each control pond. The herbicide/insecticide treatment was added between 1000 and 1100 hours Central Standard Time (pond water: pH 7.9 ± 0.01, 14.6 ± 0.06°C). Ponds with only carbaryl were sampled (triplicate 2-L aliquots from each ofthe three ponds) at 1, 24, 48 and 96 hours post-treatment. Samples from each pond were combined, and an aliquot (20 mL) was refrigerated for analysis. Ponds with only atrazine were sampled (triplicate 2-L aliquots from each ofthe three ponds) at 1, 15 and 57 days post-treatment. Samples from eachpond were combined, and an aliquot (l L) was refrigerated for analysis. The half-lives were determined after HPLC analysis via linear regression ofthe determined amounts: 

### ca. 4.5 days for carbaryl and 34 days for atrazine. After 56-58 days (39-41 days ofexposure), the experimentwas terminated. Most ofthe tadpoles and salamander larvae had notreached metamorphosis. Mean body mass, developmental stage, snout-vent length (SVL, salamander larvae only) and pond survival was determined. Data were analyzed for effects ofcarbaryl, atrazine, density (fortadpoles) and all interactions on mass, developmental stage and SVL using multivariate analysis ofcovariance (MANCOVA) for ponds with both species. Univariate analysis ofcovariance (ANCOVA) was used for determining which responses were the strongest contributors to the multivariate effects. Survival was analyzed using analysis ofvariance (ANOVA). ANOVA was also used to determine differences in the control ponds with one or both species. To normalize data and stabilize variances, the study authors angularly transformed all proportion data, log transformed mass and SVL, and used rankprocedure (PROC RANK) on developmental stage databefore analyses. Conservative pair-wise comparisons for significant main effects were performedusing Scheffe's multiple comparison tests.

For Experiment II, the effects ofhydroperiod, atrazine, and carbaryl on amphibians reared through metamorphosis were studied by a fully crossed design with four replicates (32 ponds) where hydroperiod was either constant or dried with a movable standpipe, carbaryl concentration was 0 or 5.0 mg/L, and atrazine concentration was 0 or 200 f.lg/L. Twelve small-mouthed salamanders and 45 American toads were also in each pond;

2initial anuran density was not variable since the results ofthe first experimentindicated that competitionbetween anurans and salamanders had no effect. Carbaryl (liquid Sevin, 22.5% carbaryl) and atrazine (liquid Aatrex, 40.8% atrazine) were added. The herbicide/insecticide treatment was on day 8 (pond water: pH 7.7 ± 0.03, 13.3 ± 0.04°C). Ponds with only carbaryl were sampled (triplicate 2-L aliquots from each ofthe three ponds) at 1 and 48 hours post-treatment. Samples from eachpond were combined, and an aliquot (20 mL) was refrigerated for analysis. Ponds with only atrazine were sampled (triplicate 2-L aliquots from each ofthe three ponds) at 1 day post-treatment. Samples**.£'..~_.** __ __1 ..1

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__ 1.: 4.- /1 T'\ .£'..•.: ._.L_..J**.£'_~** 1 .:_ HUlU**I;;a~u** pUUU Wl;;ll;;**~UlUUUlI;;U,** auu au alHiuUl "-I L} wa::> 1l;;Hlgl;;lalcu lUI aualy::>l::>. Atrazine concentration was confirmed as 197 ug/L. After 88 days (80 days ofexposure), the experiment was terminated. Meanbody mass, time to metamorphosis, and pond survival was determined. Toad datawere analyzed for effects ofcarbaryl, atrazine, hydroperiod and all interactions on mass and time to metamorphosis using MANCOVA; survival was the covariant. ANCOVA was used for determining which responses were the strongest contributors to the multivariate effects. Survival was analyzed using analysis ofvariance (ANOVA). Salamander data were analyzed for effects ofcarbaryl, atrazine, hydroperiod, carbaryl by hydroperiod, and atrazine by hydroperiod interactions on mass andtime to metamorphosis using MANCOVA; survival was the covariant. ANCOVA was used for determining which responses were the strongest contributors to the multivariate effects. Priorto analysis, the following transformations were performed: angulartransformation (all proportion data) and log transformation (mass and time to metamorphosis)

. Chlorophyll contentwas measured by taking a l-L composite sample (from three 2-L samples) from each pond. An aliquot (l00 mL) was filtered and incubated with 15 mL of neutralized 90% acetone in the dark at 5°C for 24 hours. Chlorophyll was measured using fluorometry. Water samples were taken before chemical application on day 15 and then on days 22,29, and 42 (all post-application) for Experiment 

### 1. For experiment II, water samples were taken before chemical application on day 8 and then on day 50 (42 days ofexposure). Datawere analyzed for effects oftime, carbaryl, and atrazine for both experiments in additionto density (Experiment I) or hydroperiod (Experiment II) using ANCOVA.



### Results: ExperimentI: effects of competition, atrazine, andcarbarylon larvalamphibians Atrazine exposure at 200 ug/L did not influence the mass, SVL, developmental stage, or survival ofspotted salamanders. There was a significantinteraction ofatrazine and carbaryl on SVL thatthe authors attributed to atrazine acting as an ameliorating agent for the carbaryl treatments. Similar observations occurred on body mass and developmental stage, although the interaction was not significant (P>0.05). For leopard frogs, atrazine exposure did not significantly influence survival. However, survival was significantly influenced by an atrazine by population density interaction where atrazine exposure decreased survival in the high-density ponds but notthe low-density ponds relative to the

3control ponds (treatments without atrazine). The multivariate responses ofleopard frogs were significantly affected by atrazine exposure; mass significantly decreased with atrazine exposure compared to the controls (P=0.0052). Results ofthe competitive controls indicated that no difference occurred between spotted salamanders reared alone or with southern leopard frogs.



### ExperimentII: effects of hydroperiod, atrazine, andcarbarylon amphibians reared th,.nJloh**""otfl""n,.nhn~d~** 

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_ ....,.. ..·_·....·_·r..**-~·~** Multivariate analyses on salamanders indicated that atrazine exposure and hydroperiod (individually and their interaction) significantly affected mass and time to metamorphosis. The effect ofatrazine exposure was attributed to increasing time to metamorphosis. Time and mass to metamorphosis were affected by the atrazinehydroperiod interaction, resulting in longer larval periods in constant hydroperiods and smallermass at metamorphosis in drying hydroperiods. ForAmerican toads, multivariate responses were significantly affected by atrazine exposure. Atrazine exposure significantly reduced toad mass at metamorphosis.


### Chlorophyllcontent In Experiment I, atrazine exposure had a significanteffect on chlorophyll content. After 12 days ofexposure, significantly lower chlorophyll content was observed in ponds with atrazine exposure of200 f.lg/L (1.31 ± 0.48 f.lg/L) versus the control ponds (3.88 ± 0.48 f.lg/L; F1,24 = 15.65, P= 0.0006). Examination of

### Figure 1 suggests that the chlorophyll content (3.88 f.lg/L) reported by the authors for control ponds included all ponds without atrazine (i.e., some ponds contained carbaryl). The authors state that there was no difference between treatments and controls on day 42 (after 25 days ofexposure); however, it was not stated if'no difference' meant lack ofa statistically significant difference. However, chlorophyll content was higher than controls after 25 days of exposure (day 42; See 

### Figure 1 below).

In Experiment II, the effects ofatrazine exposure on chlorophyll content were not discussed, but Table 5 (summary in Table 1 below) ofthe study report indicates that atrazine did nothave significant effects on chlorophyll at the end ofthe study (P = 0.62). However, chlorophyll content was only measured after42 days ofexposure in experiment II. In experiment I, chlorophyll contenthad already recovered after 25 days ofexposure so it is reasonable tp assume that any effect in experiment II may have been missed due to insufficient sampling. No figures were provided which illustrated the chlorophyll contentthrough Experiment II.

4Table 1. Summary ofrepeated-measures ANOVA for effects oftime and atrazine exposure on chlorophyll content in pond water in Experiments I and II (from Table 5 in Boone and James, 2003). Wilks' Source ofvariation lambda df F P Experiment I Time 0.0351 3,22 201.69 0.0001 Time x atrazine 0.6858 3,22 3.36 0.0371**Y;_r_~_~_** 

## ....._+- TT LAp'CJ**1111~lH** .lJ. Time 0.1974 1,24 97.59 0.0001 Time x atrazine 0.9895 1,24 0.25 0.6190



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Figure 1. Pond chlorophyll measures for an atrazine-by-carbaryl interaction over time from Experiment I. Numbers representthe concentration ofcarbaryl (in milligrams per liter) and atrazine (in micrograms per liter), respectively, to which the ponds were originally exposed (Figure 10 in Boone and James, 2003).



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### Data Summary Table:

Durmg expenment 1, atrazme was measured at day 1 (207 uglL) followmg exposure. Time Nominal Weighted Magnitude Atrazine Average Did Effect of Effect Concen- Day of Study Atrazine Recovery Comments relative to tration Effect Occurred Concentration Occur-if controls (pg/L) from Day Oto so, when? When Effect

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_____ \....Ull11Vl**It;Vt;;J~** Wt,;lC higherthan all treatment levels at Day 12 in Experiment I (Figure 1). However, there were no apparent decreases in chlorophyll in othertreatment levels with atrazine and chlorophyll in the atrazine-only treatment was higherthatthe control on Day 25. Reduction in Not chlorophyll None 200 1 Not applicable Not applicable applicable There were no content apparent effects in Experiment 2 (Table 1) although this analysis appears to have included treatments containing both atrazine and carbaryl, thus is not considered for the summary table (see reviewer comments).
No experimental variability was available for this effect. 1



### Reviewer Comments: The study was mainly an amphibian study in which chlorophyll content was measured in orderto examine effects ofatrazine on amphibians via diminishing their food source, the aquatic plant life. The study authors were looking for statistical trends and relationships, and a limited amount ofthe dataused in the analyses was reported.

6The study authors reportedthatprevious studies by other groups had shown that phytoplanktonwas diminished by atrazine by 50-200 !lg/L although recovery of phytoplankton was common.

It appears that most ofthe statistics presented did not look at the individual effects of atrazine exposure, thatis, in the absence ofcarbaryl exposure. Therefore, the atrazine exposure results as presented in the article may be confounded by inclusion in the analysis oftreatments containing atrazine and carbaryl and 'control ponds' without atrazine but containing carbaryl. There was little raw datapresentedto tease outthe individual effects ofatrazine, therefore the results should be interpreted with caution.

Background concentrations ofthe pesticides in neitherthe source water nor the controls were reported.

The treatment was limited to one test concentration ofatrazine (200 ug atrazine/L). Atrazine was applied as part ofa formulated product (Aatrex). It is unknown ifthere were any effects due to other ingredients in the formulation.

There was an acclimation period but its duration is unclear. Natural phytoplankton communities appearto have been added to each mesocosm twice. An initial 500 ml of plankton was used to establishthe ponds and acclimate. Then, the pesticides were mixed with 5 L ofpond water ofunknown source and added to the mesocosms. It is unclear if the pond waterused for adding the chemical treatment had phytoplankton in it and ifso how much. Based onthe mean chlorophyll concentrations presented in Figure 1, chlorophyll concentrations appear similarpriorto and a few days after application. It would be helpful to know more about the methodology to assess ifthe results may have been confounded by addition ofmore phytoplankton withpesticide application.
Description ofUse in Document: Qualitative

Rationale for Use: There was one endpointrelevantto aquatic plants measured inthis study (chlorophyll content). There was an observation ofreduced chlorophyll relative to controls at Day 12; however, the reduction was somewhat slight and transient (effect observed at Day 12, but not at the next measurement on Day 25). In addition, there was no indication ofstatistical significance at Day 12 (i.e., ifatrazine-only treatments were statistically analyzed separately) and there was no indication ofvariability. Chlorophyll content was higherthan controls at Day 25. Therefore, this observation was not considered to be treatment related.

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### Limitations ofStudy: Results have good applicability to mesocosms ornatural aquatic systems; however, the focus ofthe study was the effects ofatrazine and carbaryl on the amphibian community and notthe effects onphytoplankton. Also, the study design, the analysis, and the presentation and discussion ofresults ofthe study focused on interaction offactors, as opposed to the single effect ofatrazine. No statistics or information about variation among replicates was provided regarding atrazine-only treatments. In addition, lake water was used, and there was no indication ofwhether ornot atrazine may have beenpresentin controls.


### Primary Reviewer: LisaMuto, M.S., StaffScientist, Cambridge Environmental Inc 

### Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 

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Tertiary Reviewer: BrianAnderson, Biologist, EPA

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