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 of atrazine on an aquatic community. Endpoints from this

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant

communities and associated determination of a Level ofConcern (LOC).

Date ofReview:

October 10, 2009

Purpose of Study:

This study examines the relationship between environmental concentrations ofthe

herbicide atrazine and the number oftrematode infections in the declining northern

leopard frog Rana pipiens by studying the effects of atrazine on R. palustris and R.

pipiens in prepared community-level mesocosms and by conducting a wetland survey to

identify the best predictors oftotal larval trematode abundance in R. pipiens.

Summary of Study 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

block design. The Pennsylvania State University area was in the southern range of R.

pipiens where, according to the study authors, the species is declining. The test container

was a 1,100-L plastic 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

2087815
sample snail egg masses and hatchlings, periphyton samplers, and inoculations of

zooplankton, periphyton, and phytoplankton from four ponds (location of ponds not

reported). Just before dosing, each test tank was prepared with four larval amphibians (5

Ambystoma maculatum, 20 Hyla versicolour, 20 Rana palustris and 20 Rana clamitans),

two snail (11 Planorbella trivolvis and 10 Physa gyrina), one beetle (5 Hydrochara sp.),

two water bug (2 Belostomaflumineum and 7 Notonecta undulate) and one dragonfly

species (2 Anaxjunius). The R. palustris (pickerel frog) and R. clamitans (green frog) are

congeners ofthe declining northern leopard frog R. pipiens. All snails which were used

in the experiment were screened twice 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 not reported) 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 of actual applied atrazine (mean 117 !J.g/L). In order to simulate exposure to

trematodes, each tank also received a caged planorbid snail shedding cercariae from the

family Plagiorchidae (the timing ofthis addition was not reported). 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 solvent used

and its final concentration were not reported.

At the end ofthe experiment, the study authors took water samples for phytoplankton

analyses, scraped periphyton from the periphyton samplers, and scored water clarity on a

scale of 1 to 5. Additionally, the study authors counted the snail egg masses on the walls

of each 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 of view from their sectioned and stained livers (zero-to-five

scale, where zero equated to 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 of atrazine on chlorophyll a concentration in phytoplankton and periphyton

and on percent survival (after angular transformation) was analyzed using the general

linear model. Log eosinophils and log melanomacrophages per field ofview and

trematode loads were analyzed using the general linear model controlling for the biomass

of plagiorchid-infected snails per tank. In leukocyte and trematode load analyses for R.

clamitans only, the study authors controlled for mean Gosner development stage due to

the fact that their previous work demonstrated that susceptibility to trematodes changes

can occur in different developmental stages for this species. The effect of atrazine on

2
counts ofsnail egg masses and snail hatchlings was analyzed using the generalized linear

model with a Poisson error distribution and a log link. The effect of atrazine on water

clarity was analyzed using the generalized linear model with an ordinal multinomial error

distribution and a logit link. The physid snail data was not analyzed due to lack of

experimental data. For each ofthe mesocosm analyses, the tank was the replicate and the

block was included in the model ifits probability value was less than 0.25. To determine

if eosinophils and melanomacrophages were significant predictors of plagiorchid loads,

the study authors conducted a regression analysis for each predictor 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 between these

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 at the end ofthe experiments and noted that some

were pre-patent (infected but not shedding trematodes) at the beginning ofthe experiment

and began shedding plagiorchid cercariae during the experiment. Upon dissection, the

study authors discovered that one ofthe snails was infected with strigeid trematodes;

therefore, the tank from which the snail was taken was eliminated from the trematode

load analyses.

Wetland survey

Eighteen (18) wetlands in the BroadleafForest Ecoregion 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 characterized biological diversity (three visits: March-April, May-June

and, July-August) and obtained water, sediment, and amphibian tissue samples to

quantify various analytes (two visits: April-May and June-July). Analyte quantification

was conducted using standard US EPA protocols. During each visit to 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 of each plant species was estimated using three 10-

m transects and the line-intercept method. 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) calculated the

coefficient of determination (R2

) of each predictor in the absence of covariates; 2) used

the general linear model to generate the 20 best subset models for mean larval trematode

loads in R. pipiens (maximum of 3 predictors, ranked by R2

); 3) used a structural

equation model, followed by path analysis to evaluate the amount ofsupport for the

3
specific proposed casual mechanisms underlying the significant positive 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 transformation products of atrazine)

were measured separately; however, they were combined together as "atrazine" for the

analysis since their combination was the best predictor oftotal larval trematode

abundance in R. pipiens.

Results:

Mesocosm Experiment

In the 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 sunlight penetration to attached algae, however, only data on water clarity are

reported (Table 1). The concentration of periphyton 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 in just one generation (P < 0.001).

For R. palustris, specimens from the atrazine-treated mesocosms showed much lower

liver melanomacrophages and eosinophil counts than specimens from the control tanks.

Survival ofR. 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 of atrazine 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 liver melanomacrophages or

elevated mortality; however, atrazine-treated mesocosms showed significant reductions

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.

4
Table 1. Findings from mesocosm experiment examining the effect ofatrazine (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 pooled because there was no significant difference between the

two treatments (details ofstatistical testing not reported).

3 Block was included in the model ifits P value was less than 0.25.

4 F statistic from analyses using the general linear model.

5 One-tailed test.

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 power was six.

9 The power ofthe test was 0.326.

10 The power ofthe 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

of view for R palustris (log)

Eosinophils per field 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

of view for R clamitans (log)

Eosinophils per field of view 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 <0.0014 0.971 1v

No. ofplagiorchid trematode 20.61 ± 4.52 7.51 ± 3.30 4.904 0.031 5

•

11

cysts in R. clamitans

I -

Wetland Survey

The study authors' analysis of 18 wetlands in Minnesota, USA, indicated that 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 of amphibian trematode infections. Atrazine alone (R2 =

5
0.46, 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 significant predictors ofincreased trematode infections in R.

pipiens. The sum of atrazine 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 of atrazine. Each plotted point represents a wetland and 95%

confidence bands are shown. Figure S3A from Rohr et al., 2008.

6
a

- 2.0 Z

0 1.5 ...

Do -

b

P=o.026

13.9&1

0.993

P=O.048

4.244

0.411

GIIItrapad

riDtnII8

,,"= 0.170

p= 0.01.

44.671

0.4&2

~1nIiI

ibundllnce

~=0.217

' ....................... ... P=O..DS1 ' ...

0.233 "" ... 0.012 ..... l.M¥aJ tnInudDde

atuld8nI»

f1!= 0.611

Figure 2. Proposed mechanisms for the relationship between atrazine and larval trematodes in

amphibians. a, The relationship between detectable concentrations of atrazine 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 of a 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 et al., 2008.

7
a 0.4 ... ....

"- .... ...

0.0

•

•

... • ...

..... ....

..... '"

"'.... ......

,.

.. - --....... ' ..J_-

-'"""--'~'''.-----'''''~-~'''''-' ",.........

.... "-.... .. '.... .... ..... .... .... ....

0.0

0.2

-0.2

= 0.17, Ft ,11= 9.17,.

P =0.047

-0.4 '-----------------

-2.0 -1.6 -1.2 -0.9 -0.4

Atrazine + ~rametog M t 1)

Figure 3. The relationship between melanomacrophage aggregate score in R. pipiens and

concentrations of atrazine 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 of nematodes 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 et al., 2008.

The study authors also noted that larval trematode loads in amphibians must depend on

the abundance of defmitive 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 host habitat patches (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.

Overall Results

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

8
amphibian species at concentrations commonly occurring in freshwater ecosystems (they

also suggested that phosphate 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 proposed path of atrazine to increased

larval trematode infections and mortality in amphibians is illustrated in Figure 4.

9
Reduces suspended algae

Increases water clarity

and thus sunlight

reaching attached algae

Increases attached

algae, the food source

for snails

Inaeases snails, the

rlM intermediate host

of trematodes

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 of the study authors. Figure 84 from

Rohr et al., 2008.

10
Data Summary Table:

Time

Nominal Weighted

Average Did Magnitude of Atrazine Day of Atrazine Recovery Effect Effect relative Concen- Study Effect Concentration Occur-if Comments

to controls tration Occurred from Day Oto so, when? (p,glL/ When Effect

Occurred

Chlorophyll a in Four weeks, Could not be Could not be P=0.031 2

phytoplankton 65% lower 102 Jlg/L study detennined. detennined. tennination.

Chlorophyll a in Four weeks, Could not be Could not be P= 0.0322

periphyton 49% higher 102 Jlg/L study detennined. detennined. tennination.

Four weeks, Could not be Could not be P < 0.001 2 Snail hatchings 323% higher 102 !lg/L study detennined. detennined. tennination.

Snail egg Four weeks, Could not be Could not be P= 0.0262

masses 146% higher 102 Jlg/L study detennined. detennined. termination.

MelanomacroFour weeks, phages per field Could not be Could not be P = 0.0312

of view 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 of view for 86% lower 102 Jlg/L study

R. palustris tennination. detennined. detennined.

Survival ofR. Four weeks, Could not be Could not be P= 0.0172

palustris 32% lower 102 Jlg/L study detennined. detennined. termination.

MelanomacroFour weeks, phages per field Could not be Could not be P = 0.2172

of view for R. 34% lower 102 Jlg/L study detennined. detennined.

clamitans tennination.

Eosinophils per Four weeks, Could not be Could not be P = 0.0292 field of view for 72% lower 102 Jlg/L study

R. clamitans tennination. detennined. detennined.

Survival ofR. Four weeks, Could not be Could not be P=0.971 2

clamitans No effect 102 !lg/L study detennined. detennined. tennination.

No. of

Four weeks, plagiorchid Could not be Could not be P=0.031 2

trematode cysts 174% higher 102 Jlg/L study detennined. detennined

in R. clamitans tennination.

Mean actual concentration was 117 Jlg/L. Samples taken one hour after applIcatIOn, however, samples

were sent to another lab to be analyzed at an unknown time after application.

2 See Table 1 for infonnation about statistics.

11
Reviewer Comments: (on mesocosm experiments)

Several details ofthe mesocosm methodology were missing. Given that this study was a

"Letters" communication in the journal 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 not reported. In addition, it is unclear whether the larval

amphibian, snail, beetle, water bug, and dragonfly species may have been previously

exposed to other contaminants (including pesticides) prior to the experiment, given that

no previous contaminant history or the location ofthe collection sites was provided.

There was a three week acclimation period for zooplankton, phytoplankton, and

periphyton; however, all other taxa were added to the tanks just before 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 not reported if atrazine was present in the source ponds or the control mesocosms.

It was not reported ifthe algal communities (i.e. chlorophyll a concentrations) were

similar among the mesocosms prior to 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 of a 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 their parasites).

Description of Use in Document: Qualitative

12
Rationale for Use: The study contributes to the weight of evidence regarding effects of

atrazine on aquatic plant communities.

Limitations of Study: Although the experimental mesocosms contained both

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 with the study. The study authors did not report

the solvent or the concentration in which it was used. Actual pesticide (atrazine)

concentrations were not monitored throughout the 4-week experiment. Water samples

collected one hour after dosing measurement of atrazine concentration; however, samples

were shipped to another lab and it is unclear when the analysis occurred. The source

ponds and the control mesocosms were not analyzed for atrazine, and the contaminant

history oflocations where the animals were collected, prior to the experiment, was not

provided. In addition, it is unknown ifthe algal communities were similar among the

mesocosms prior to dosing.

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc

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

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA Afu5<-

13

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