### Open Literature Review Summary Chemical Name: Atrazine 

### CAS No: 1912-24-9


### ECOTOX Record Number and Citation: Rohr, J. R., A. M. Schotthoefer, T. R. Raffel, H. J. Carrick, N. Halstead, J. T. Hovennan, C. M. Johnson, L. B. 

### Johnson, C. Lieske, M. D. Piwoni, P. K. 

### Schoff, and V. R. Beasley. 2008. Agrochemicals increase trematode infections in a declining amphibian species. Nature. 455(7217): 1235-1239. (MRID48261131) 

### Purpose ofReview: Detennine 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 ofa Level ofConcern (LOC).


### Date ofReview: October 10, 2009 

### Purpose ofStudy: This study examines the relationship between environmental concentrations ofthe herbicide atrazine andthe number oftrematode infections in the decliningnorthern leopard frog Ranapipiens by studying the effects ofatrazine onR. palustris and R. pipiens in prepared community-level mesocosms and by conducting a wetland survey to identify the bestpredictors oftotal larval trematode abundance inR. pipiens. 

### Summary ofStudy Findings: The focus ofthe summary is on the mesocosm experiment. Methods and results from the wetland survey are briefly mentioned.


### Methods: Mesocosm experiment The experiment was conducted at Pennsylvania State University using a randomized blockdesign. The Pennsylvania State University area was in the southern range ofR. 

### pipiens where, according to the study authors, the species is declining. The test container was a 1,100-Lplastic cattle tank filled with 800 L ofwater and covered with shade cloth. Twelve tanks were prepared for the test (atrazine 

### =4, controls 

### = 8). Three weeks prior to dosing, each tank was prepared with 300 g ofmixed hardwood leaves, plexiglass to

1 

# 11111111111 2087815sample snail egg masses and hatchlings, periphyton samplers, and inoculations of zooplankton, periphyton, and phytoplankton from four ponds (location ofponds not reported). Justbefore dosing, each testtank was prepared with four larval amphibians (5 Ambystoma maculatum, 20 Hyla versicolour, 20 Ranapalustris and 20 Rana clamitans), two snail (11 Planorbella trivolvis and 10 Physa gyrina), one beetle (5 Hydrochara sp.), two water bug (2 Belostomaflumineum and 7Notonecta undulate) and one dragonfly species (2Anaxjunius). The R. palustris (pickerel frog) andR. clamitans (green frog) are congeners ofthe declining northern leopard frog R. pipiens. All snails which were used inthe experiment were screenedtwice for patent (shedding) trematode infections before being placed in the tanks. Tanks were exposed to atrazine via a single dose oftechnical grade atrazine (from ChemService; % purity was notreported) at 102 !J.g/L. According to the study authors, 102 !J.g/L atrazine was chosen as the expected environmental concentration, based on calculations using the U.S. EPA's GENEEC (version 2) software. One hour after dosing, water samples were taken and shipped on ice for analysis ofactual applied atrazine (mean 117 !J.g/L). In orderto simulate exposure to trematodes, each tank also received a cagedplanorbid snail shedding cercariae from the family Plagiorchidae (the timing ofthis addition was notreported). The caged snails were rotated among the tanks every other day for homogenization. Snails were thoroughly rinsed between tanks; tanks were not rotated. The experiment ran for 4 weeks (one generation) from the end ofJune 2007 to July 2007. Both water and solvent control tanks were prepared (unreported number oftanks per control). The solventused and its final concentration were not reported.
Atthe end ofthe experiment, the study authors took water samples for phytoplankton analyses, scrapedperiphyton from the periphyton samplers, and scored water clarity on a scale of 1 to 5. Additionally, the study authors countedthe snail egg masses on the walls ofeach tank, the snails hatchlings from the plexiglass placed in each tank and the surviving and dead animals. Chlorophyll 

### a measurements were made using standard fluorometry methods (not specified). Five preserved R. clamitans and five R. palustris tadpoles from each tank were selected randomly and melanomacrophage and eosinophils cells were counted per field ofview from their sectioned and stained livers (zero-to-five scale, where zero equatedto none and five equated to more than 90% ofthe liver contained melanomacrophage or eosinophils cells). Each tadpole was also cleared and stained and their larval trematodes were counted under a compound scope (zero-to-five scale). All variables were quantified blind to the mesocosm treatment. The mean ofthe mesocosm unit data was used for statistical analysis.
The effect ofatrazine on chlorophyll 

### a concentration in phytoplankton and periphyton and on percent survival (after angulartransformation) was analyzed using the general linearmodel. Log eosinophils and log melanomacrophages per field ofview and trematode loads were analyzed using the general linearmodel controlling for the biomass ofplagiorchid-infected snails pertank. In leukocyte and trematode load analyses for 

### R. clamitans only, the study authors controlled for mean Gosner development stage due to the fact thattheirprevious work demonstrated that susceptibility to trematodes changes can occurin different developmental stages for this species. The effect ofatrazine on
2

### counts ofsnail egg masses and snail hatchlings was analyzed using the generalized linear model with a Poisson errordistribution and a log link. The effect ofatrazine on water clarity was analyzed using the generalized linear model with an ordinal multinomial error distribution and a logit link. The physid snail datawas not analyzed due to lack of experimental data. For each ofthe mesocosm analyses, the tank was the replicate and the blockwas included in the model ifits probability value was less than 0.25. To determine ifeosinophils and melanomacrophages were significantpredictors ofplagiorchid loads, the study authors conducted a regression analysis for eachpredictor using individual tadpoles as replicates and blocking by tank. The water and solvent control tanks were pooled for analyses because no significant difference was found betweenthese treatments; however, the study authors did not specify the statistics used and ifthe lack of significance was true for all response variables.
The study authors rescreened the snails atthe end ofthe experiments and noted that some were pre-patent (infected but not shedding trematodes) atthe beginning ofthe experiment and began shedding plagiorchid cercariae during the experiment. Upon dissection, the study authors discovered that one ofthe snails was infected with strigeidtrematodes; therefore, the tank from which the snail was taken was eliminated from the trematode load analyses.
Wetland survey Eighteen (18) wetlands in the BroadleafForestEcoregion in Minnesota, USA, were surveyed regarding more than 240 plausible predictors ofthe abundance oflarval trematodes in northern leopard frogs (R. 

### pipiens) living in those wetlands. During 1999, the study authors characterizedbiological diversity (three visits: March-April, May-June and, July-August) and obtained water, sediment, and amphibiantissue samples to quantify various analytes (two visits: April-May and June-July). Analyte quantification was conducted using standard US EPA protocols. During each visitto the wetland, the study authors attempted to collect 15 recently metamorphosed R. 

### pipiens for parasite quantification and 25 for pathology. Faunal diversity was conducted using daytime, visual time and area-constrained dip-net sampling methods with effort adjusted according to wetland size. Relative plant cover ofeachplant species was estimatedusing three 10- m transects and the line-interceptmethod. Landscape structure was summarized at l-km and 10-km extents using Fragstats. Frogs were inspected for parasites (buccal and abdominal cavities, visceral organs, skin, and eyes). After necropsy, a dissecting microscope was used to identify encysted metacercariae. Frog livers (frogs in Gosner stage 46) were examined for melanomacrophage and eosinophils cells under a light microscope.
The statistical analysis for the wetland survey was done in four steps: 1) calculatedthe coefficient ofdetermination (R 2) 

### ofeachpredictor inthe absence ofcovariates; 2) used the general linearmodel to generate the 20 best subset models for mean larval trematode loads in R. 

### pipiens (maximum of3 predictors, ranked by R2); 

### 3) used a structural equation model, followed by path analysis to evaluate the amount ofsupport for the
3specific proposed casual mechanisms underlying the significantpositive relationship between atrazine concentration and larval trematode abundance; and 4) assessed whether atrazine was correlated with any other variables that might explain the results more concisely. Atrazine and desethylatrazine (one ofthe transformationproducts ofatrazine) were measured separately; however, they were combined together as "atrazine" for the analysis since their combination was the bestpredictor oftotal larval trematode abundance in

### R. pipiens.
Results: 

### Mesocosm Experiment Inthe mesocosm experiment, atrazine significantly reduced phytoplankton chlorophyll 

### a concentrations (mean ± s.e.m.; atrazine treatment, 2.52 ± 0.45; water and solvent controls, 7.14 ± 1.67; general linear model, 

### F= 4.88, 

### P = 0.031). The study author's state that this reduction resulted in greatly increased nutrient availability, water clarity, and sunlightpenetration to attached algae, however, only data on water clarity are reported (Table 1). The concentration ofperiphyton chlorophyll 

### a was significantly elevated in the atrazine-treated mesocosms (1.04 ± 0.18), compared to the water and solvent controls (0.70 ± 0.08; general linear model, 

### F

### = 7.17, 

### P 

### = 0.032).
The snail population ofthe atrazine-treated tanks was more than four times that ofthe control tanks injust one generation (P 

### < 0.001).
For

### R. palustris, specimens from the atrazine-treated mesocosms showed much lower livermelanomacrophages and eosinophil counts than specimens from the control tanks. Survival of

### R. palustris was also much lower in the atrazine-treated tanks (47.5% survival in the atrazine treatment group versus 70% survival in the controls), preventing the study authors from performing an unconfounded test ofthe effect ofatrazine on trematode loads (since the most infected individuals were most likely those which did not survive). For

### R. clamitans, the study authors did not observe significant differences between the atrazine-treated tanks and the control tanks regarding livermelanomacrophages or elevated mortality; however, atrazine-treated mesocosms showed significantreductions in liver eosinophil counts. 

### R. clamitans in atrazine treated tanks had significantly more plagiorchid trematodes than in control tanks when controlling for the relationship between plagiorchid loads and eosinophils. Plagiorchid trematode infections were not reported for

### R. palustris.
The findings ofthe mesocosm experiment and statistic analysis results are reported in Table 1.

4Table 1. Findings from mesocosm experiment examining the effectofatrazine (mean ± s.e.m.) versus the control (water and solvent combined, mean ± s.e.m.) on factors proposed to influence larval trematode loads in frogs. Table 1 from Rohr et

### at. 2008.

n-4. 2 n=8; the solvent and water controls were pooledbecause there was no significant difference between the two treatments (details ofstatistical testing notreported). 3 Block was includedin the model ifits P value was less than 0.25. 4 Fstatistic from analyses using the general linear model. 5 One-tailedtest. 6

## i statistic from analyses using the generalized linear model with an ordinal multinomial error distribution and logit link. 7 X 2 statistic from analyses using the generalized linear model with a Poisson error distribution and a log link. 8 The power ofthe test was 0.646; the minimum sample size necessary per group to detect a significant difference given the powerwas six. 9 The power ofthe test was 0.326. 10 The powerofthe test was 0.056. 11 Controlling for biomass ofplagiorchid-infected snails (FI,7= 5.45, P = 0.026) and mean log eosinophil counts in tadpoles (F1,7 = 3.58, P = 0.050). Response variable Atrazine1 Control:': Test statistic P' Chlorophyll a in 2.52 ± 0.45 7.14 ± 1.67 4.884 0.031 5 phytoplankton (JlgIL) Chlor0r-hyll a in periphyton 1.04±0.18 0.70 ± 0.08 7.174 0.032 (mg/m) Water clarity (scale 1-5) 4.75 ± 0.25 3.88 ± 0.23 5.25" 0.011' Snail hatchings (per mol) 468.09 ± 205.85 110.64 ± 29.31 20.19 <0.001 Snail egg masses (per m:l) 30.45 ± 3.03 12.37 ± 4.34 4.98 0.026 Melanomacrophages per field 0.16 ± 0.06 0.36 ± 0.06 4.504 0.031 5 ofview for R palustris (log) Eosinophils perfield ofview 0.03 ± 0.14 0.21 ± 0.06 1.884 0.1025,8 for R. palustris (log) Survival ofR palustris (%) 47.50 ± 7.50 70.00 ± 3.90 9.774 0.017 Melanomacrophages per field 0.25 ± 0.13 0.38 ± 0.09 0.6"t 0.2175,9 ofview for R clamitans (log) Eosinophils per field ofview 0.11 ± 0.10 0.39 ± 0.07 4.754 0.0295 for R clamitans (log) Survival ofR. clamitans (%) 76.25 ± 3.15 76.25 ± 2.80 <0.001 4 0.971 1v No. ofplagiorchidtrematode 20.61 ± 4.52 7.51 ± 3.30 4.904 0.031 5•11 cysts in R. clamitans
I -



### WetlandSurvey The study authors' analysis of 18 wetlands in Minnesota, USA, indicatedthat atrazine combined with desethylatrazine (one ofits transformation products) was the best predictor oftotal larval trematode abundance in R. 

### pipiens out ofmore than 240 measured plausible predictors ofamphibian trematode infections. Atrazine alone (R2 =

50.46, F 1,15 

### = 12.77, P

### = 0.003, see 

### Figure 1) and desethylatrazine alone 

### (If

### =0.46, F1,15 

### = 12.93, P = 0.003) were significantpredictors ofincreasedtrematode infections inR. 

### pipiens. The sum ofatrazine and desethylatrazine accounted for more variation than any other single predictor (51%, Monte Carlo randomization test, 

### P = 0.001; see 

### Figure 2a). Atrazine combined with desethylatrazine was a significant negative predictor of melanomacrophages aggregate scores (R2 

### =0.17, F 1,16 

### = 3.17, P

### =0.047; See 

### Figure 3). Melanomacrophage scores were from 0 (not present) to 5 (> 90% ofliver filled with melanomacrophage aggregates). 

# i 2.0 1.5 1.0 

# A 

# ••

0.04 0.08 0.12 Atrazine {k)g (JJgtL+1» 

# •

0.16
Figure 1. The relationship between larval trematode abundance in northern leopard frogs, Rana pipiens, and detectable concentrations ofatrazine. Each plotted point represents a wetland and 95% confidence bands are shown. Figure S3A from Rohr etal., 2008.

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Figure 2. Proposed mechanisms for the relationship between atrazine and larval trematodes in amphibians. a, The relationship between detectable concentrations ofatrazine plus desethylatrazine in water samples and larval trematode abundance ('probit' transformed, that is, the normal standard deviate ofmean abundance/l,OOO) in northern leopard frogs, R.p;Piens. Each data point represents a wetland, and a 95% confidence band is shown. b, Results ofa path analysis examining the suggested mechanisms by which atrazine plus desethylatrazine increase larval trematode abundance. Unstandardized and standardized coefficients, respectively, are provided next to each path. Solid arrows represent significant paths. The model chi-square was 0.339 (d.f. 

### = 1, P

### =0.561), indicating that the model was a good fit to the data. Figure 1 from Rohr etal., 2008.

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## M t 1) Figure 3. The relationship between melanomacrophage aggregate score in R. pipiens and concentrations ofatrazine plus desethylatrazine in water samples. In the graph and analyses, the study authors used the residuals from the relationship among mean melanomacrophage aggregate scores and the richness and abundance ofnematodes and adult trematodes in leopard frogs. Each data point represents a wetland, and 95% confidence bands are shown. Individual data for atrazine was not illustrated. Figure 2a from Rohr etal., 2008. The study authors also noted that larval trematode loads in amphibians must depend on the abundance ofdefmitive hosts, such as amphibian-eating birds (a source oftrematode eggs in wetlands). This dependence was supported by the significant interaction between atrazine and the number ofsuitable hosthabitatpatches (forest, wetlands and open water) around each wetland (Monte Carlo randomization test: 1 km radius, P = 0.004; 10 km radius, 

### P = 0.019).
The analyses for the wetlands survey were mainly based on atrazine combined with desethylatrazine in the study report. However, in the supplemental information (found onlineathttp://www.nature.cgrnlnature/journallv455/n7217/extre£lnature07281-s1.pdt), some results were presented for atrazine and desethylatrazine individually. In addition, supplementary details about the models and results from the models are presented.


### OverallResults Overall, based on the results ofthe study, the study authors concluded that atrazine (plus desethylatrazine) can be a primary driver oflarval trematode infections for a declining

8amphibian species at concentrations commonly occurring in freshwater ecosystems (they also suggestedthatphosphate played a complementary role). The study authors proposed that atrazine increased exposure and susceptibility to larval trematodes by increasing the amounts ofsnail intermediate hosts (via increasing the periphyton) and by suppressing amphibian immune responses. The scheme ofthe proposedpath ofatrazine to increased larval trematode infections and mortality in amphibians is illustrated in Figure 4.



### 9Reduces suspended algae

Increases waterclarity and thus sunlight reaching attached algae

Increases attached algae, the food source for snails

Inaeases snails, the 

### rlM intermediate host oftrematodes

Increases amphibian exposure to trematode

cercariae that infect amphibians Compromises amphibian immune responses

Increases amphibian

SlJS('eptibility to trematode cercariae that infect amphibians

Increases amphibian larval trematode loads

Increases the risk of aq»hibian mol'bility Figure 4. A simple scheme which summarizes the main findings ofthe study authors. Figure 84 from Rohr etal., 2008.

10Data Summary Table: Time Nominal Weighted Average Did Magnitude of Atrazine Day of Atrazine Recovery Effect Effectrelative Concen- Study Effect Concentration Occur-if Comments to controls tration Occurred from Day Oto so, when? (p,glL/ When Effect Occurred Chlorophyll a in Fourweeks, Could notbe Could notbe P=0.031 2 phytoplankton 65% lower 102 Jlg/L study detennined. detennined. tennination. Chlorophyll a in Fourweeks, Could notbe Could notbe P= 0.0322 periphyton 49% higher 102 Jlg/L study detennined. detennined. tennination. Four weeks, Couldnotbe Could not be P < 0.001 2 Snail hatchings 323% higher 102 !lg/L study detennined. detennined. tennination. Snail egg Fourweeks, Could not be Could notbe P= 0.0262 masses 146% higher 102 Jlg/L study detennined. detennined. termination.MelanomacroFourweeks, phages perfield Could not be Could notbe P =0.031 2 ofview for R 56% lower 102 Jlg/L study detennined. detennined. palustris tennination. Eosinophils per Four weeks, Could not be Could not be P =0.1022 field ofview for 86% lower 102 Jlg/L study R. palustris tennination. detennined. detennined. Survival ofR. Four weeks, Could notbe Could notbe P= 0.0172 palustris 32% lower 102 Jlg/L study detennined. detennined. termination.MelanomacroFour weeks, phages per field Could not be Could notbe P =0.2172 ofview for R. 34% lower 102 Jlg/L study detennined. detennined. clamitans tennination. Eosinophils per Fourweeks, Could notbe Could notbe P =0.0292 field ofview for 72% lower 102 Jlg/L study R. clamitans tennination. detennined. detennined. Survival ofR. Fourweeks, Could notbe Could notbe P=0.971 2 clamitans No effect 102 !lg/L study detennined. detennined. tennination. No. of Four weeks, plagiorchid Could notbe Could notbe P=0.031 2 trematode cysts 174% higher 102 Jlg/L study detennined. detennined inR. clamitans tennination.

Mean actual concentration was 117 Jlg/L. Samples taken one hour after applIcatIOn, however, samples were sentto another lab to be analyzed at an unknown time after application.
2 See Table 1 for infonnation about statistics.

11Reviewer Comments: (on mesocosm experiments)
Several details ofthe mesocosm methodology were missing. Given that this study was a "Letters" communication in thejournal 

### Nature, it was expected that the study report and methodology would be abbreviated. However, no details were provided regarding the water used for the mesocosm experiment (the pond characteristics and locations ofthe four ponds were not specified; freshwater source or otherwise). The solvent used and its final concentration were notreported. In addition, it is unclear whetherthe larval amphibian, snail, beetle, water bug, and dragonfly species may have been previously exposed to other contaminants (including pesticides) priorto the experiment, given that no previous contaminant history orthe location ofthe collection sites was provided.
There was a three week acclimation period for zooplankton, phytoplankton, and periphyton; however, all othertaxa were added to the tanksjustbefore dosing with atrazine.
Atrazine concentrations were not monitored throughout the experiment. Mesocosms were sampled one hour after dosing and shipped to another lab for measurement of atrazine. It was not stated how long after sampling that atrazine was actually measured.
It was notreported ifatrazine was present in the source ponds orthe control mesocosms.
It was not reported ifthe algal communities (i.e. chlorophyll 

### a concentrations) were similar among the mesocosms priorto dosing.
The generalized linear model was used to test for significant differences between the atrazine treatment and the controls. The study authors did not explain why this analysis was chosen instead ofa simpler statistical method for comparing two treatments.
The study authors suggested that the potentially important relationship between atrazine (pollution) and amphibian parasitism would not have been detected in the standard tests conducted in the United States and Europe for chemical registration since the tests are typically conducted on solitary species (isolated from other species with which they coexist, such as theirparasites). Description ofUse in Document: Qualitative

12

### Rationale for Use: The study contributes to the weight ofevidence regarding effects of atrazine on aquatic plant communities.


### Limitations ofStudy: Although the experimental mesocosms containedboth phyt<?plankton and periphytic algae, as well as several species from higher levels ofthe food web (i.e., larval amphibian, snail, beetle, water bug, and dragonfly species); there were a number oflimitations associated withthe study. The study authors did notreport the solvent orthe concentration inwhich it was used. Actual pesticide (atrazine) concentrations were not monitored throughout the 4-week experiment. Water samples collected one hour after dosing measurement ofatrazine concentration; however, samples were shippedto another lab and it is unclear when the analysis occurred. The source ponds andthe control mesocosms were not analyzed for atrazine, and the contaminant history oflocations where the animals were collected, priorto the experiment, was not provided. In addition, it is unknown ifthe algal communities were similar among the mesocosms priorto dosing. 

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

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

# eM

# M Tertiary Reviewer: AnitaPease, M.S., Senior Biologist, EPA 

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13 

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