### Open Literature Review Summary
ChemicalName: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Rohr, 

### J. R. and P. W. Crumrine. 2005. Effects ofan herbicide and an insecticide onpond community structure and processes. Ecological Applications. 15(4): 1135-1147. (MRID 48261130) 

### 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 (LaC).



### Date ofReview: October 5, 2009 

### Purpose ofStudy: This study examines the effect ofthe herbicide atrazine and the insecticide endosulfan (tested independently) on constructed outdoorfreshwater pond communities whenthe presence ofcommon community members (wood frog tadpoles, adult snails, and caged dragonfly larvae) was varied. 

### Summary ofStudy Findings: The focus ofthe summary is on treatments with exposure to atrazine.



### Methods: Twenty wood frog (Rana sylvatica) clutches were collected in late February 2003 from Gray's Arch Pond in Kentucky. Tadpoles were hatched andreared in outdoorwading pools (containing aged tap water and covered with 60% shade cloth). They were fed rabbitchow ad libitum. Clutches were thoroughly mixedpriorto experimentation. Adult snails (PlanorbeUa trivolvis) were collected in early March 2003 from a pond atthe University ofKentucky's Ecological Research Facility. They were raised inpond water in the laboratory until their use 

### in the experiment. Late-instar dragonfly larvae (Anax junius) were collected 

### in early March 2003 from various ponds atthe University of Kentucky's Ecological Research Facility. Priorto the experiment, the dragonflies were held individually in the laboratory and fed aquatic invertebrates and R. sylvatica tadpoles.

1 

# 11111••111 2087816The study authors employed a 3 x 2 x 2 x 2 completely randomized factorial design with three "pesticide" treatments (acetone solvent control, 25 Ilg/L ofatrazine, and 10 1lg!L of endosulfan), two tadpole treatments (presence or absence oflOR. sylvatica tadpoles, Gosner stage 23-26), two snail treatments (presence or absence oflOP. trivolvis), and two dragonfly treatments (presence or absence ofone caged, late-instarA.junius). The experimental densities ofthe community members were atthe high-end ofregional densities (based on the personal observation ofthe study authors). The experiment lasted for four weeks; the entire design was replicated four times in four spatial blocks (96 experimental units in total). No water (negative) control was included. According to the study authors, acetone was not expected to be toxic at the level used in the experiment (0.0246 ml acetone/L final concentration in the experimental tub was estimated by the secondary reviewer).

Each experimental unit (mesocosm) was a polyethylene tub (11.3 L capacity, 40.6 x 28.5
x 17.5 em) containing a perforated, transparentplastic cup (0.296 L capacity) securedto one end ofthe tub with monofilament line and five microscope slides attached to the side ofthe tub with silicone. The tubs were located on gravel atthe University ofKentucky's Ecological Research Facility. One March 31, 2003, each tub received 9.0 L ofaged tap water, 1.0 L ofwater from the pond from which the snails were collected, 15 g ofleaves from the mixed deciduous forest surrounding the pond from which the wood frogs were collected, and 3 g ofrabbit chow (to stimulate growth ofperiphyton). The pond water was the source ofphytoplankton, zooplankton, and insect larvae/eggs. The tubs were covered with 60% shade clothto preventpredation and allowed to mature for two weeks in order to establish aperiphyton community prior to addition ofthe pesticide, tadpoles, snails, and dragonflies.

On April 14, 2003, the three "pesticide" treatments (acetone control, atrazine, and endosulfan) were added to the tubs. Forthe solvent control, 246 ilL ofacetone was added to the appropriate tubs. Forthe atrazine treatment (25 Ilg/L), 27.8 ilL ofatrazine stock solution (8.984 mg atrazine permL ofacetone) was added. The atrazine was 99% pure. The concentration ofthe atrazine stock solution was verified by flame ionization detection gas chromatography. The tubs were redosed with atrazine two weeks later, midway in the experiment, in orderto simulate a second run-offevent. The maximum nominal concentration ofatrazine was 50 Ilg/L (i.e., for the secondtwo weekperiod of the experiment). The authors do not state ifa second dose ofacetone was added to the acetone control tubs. Atrazine concentration was not monitored during the experiment. However, the study authors estimatedthatthe nominal concentration was higherthan the actual concentration due to heavy rainfall that occurred during the experiment.
Tadpole, snail, and dragonfly masses were obtained before they were added into each tub (0.219 ± 0.002,0.692 ± 0.007, and 0.960 ± 0.032 g/individual, respectively). Dragonflies were placed in the transparent, perforated cup ofeach tub so thatthey were nonlethal to tadpoles outside the cup but could be visually and chemically detected. Dragonflies were

2fed two randomly chosenR. sylvatica tadpoles from the wading pool stocks three times perweekthroughoutthe experiment. Empty cups were picked up and gently dropped to control for feeding disturbance across tanks. Dragonfly mortality was assessed during these feedings.
Periphytic algae in each mesocosm were assessed by measuring the chlorophyll 

### a concentration ofthe periphyton scraped offa microscope slide. Chlorophyll 

### a was measured using American Public HealthAssociation standardprotocols. Samples for chlorophyll measurements were takenjustpriorto treatment (all tubs) and then from half ofthe spatial blocks every other weekthroughoutthe experiment. Dissolved oxygen (DO) and pH measurements were taken weekly from each tub using meters (lCM Model 31200 and Corning Model 320).

Twice a week and once during the last week, the study authors conducted four observations on each mesocosm, recording the number oftadpoles observed (those not hiding under leaves), the proportion oftadpoles avoiding the cup (on the opposite halfof the mesocosm), and the number oftadpoles moving during a 15-s interval. Metamorphs (Gosner stage 42-46) were observed and removed only during the lastweek ofthe experiment. The experimentwas ended on May 12th for blocks 1 and 2 and on May 13th for blocks 3 and 4. At experimenttermination, surviving snails and tadpoles were counted and weighed and snail egg masses were counted for eachmesocosm. Surviving tadpoles were euthanized with a 0.01% solution ofbenzocaine and preserved in 10% neutral buffered formalin for later determination ofGosner developmental stage. Water clarity was quantified by the study authors using a scale of1 to 5. Chironomid larvae (dipterans) ofthe genus Polypedilum (periphytivores and detritivores) and cladoceran zooplankton ofthe genus Daphnia (primarily Daphniapulex; phytoplanktivores) were the most common invertebrates in the mesocosms. Their abundance was quantified on a scale of 1 to 5. The study authors proposedthattheir introduction into the mesocosms was viathe pond water aliquots. The presence ofinsects and crustaceans in genera other than Polypedilum and Daphnia were noted as the presence or absence of"otherinsects and crustaceans" (one lumped category for statistical analysis); these observations were rare.

All statistical analysis was conducted using Statistica 5.5a, using the mesocosm as the experimental unit. Proportion data were angularly transformed. Covariates andblock effects were included when significant. Three separate multivariate analyses ofvariance (MANOVA) were conductedto analyze tadpole behavior (% hiding: % observed avoiding cup, % observedmoving), tadpole growth and development (mass/tadpole and Gosner stage), and snail.growth andreproduction (mass per snail and egg masses per snail). Univariate analyses ofvariance (ANOVA) were subsequently performed on those parameters. DO and pH were regressed versus tadpole growth and development (mass pertadpole and Gosner stage) and snail growth and reproduction (mass per snail and egg masses per snail). One-way ANOVA was also used to assess the influence ofthe pesticide treatments on the duration ofdragonfly survival. Repeated-measures ANOVA

3was used to evaluate the effect ofpesticide treatments on chlorophyll 

### a in the first and second halfofthe experiment. Treatment effects on the number ofsnails that died and the number oftadpoles that reached metamorphosis were evaluated using Poisson regression (PR; with a log link). Water clarity and the abundance ofPolypedilum and Daphnia were analyzed using logistic regression for an ordinal multinomial distribution (OMR; with a logit link). The presence ofother insects and crustaceans and dragonfly survival were evaluated using logistic regression for a binomial distribution (BR; with a logit link). Whenthe main effect ofthe pesticide was significant (P

### < 0.05) in ANOVAs, the study authors performed a Turkey's honestly significant difference multiple comparisontest (Tukey's hsd) for all pairwise comparisons. When the main effect ofthe pesticide was significant (P 

### < 0.05) using logistic regression (pR, OMR, BR), the study authors performed Bonferroni-adjusted linear contrasts for all pairwise comparisons.

Results: Effects on PeriphyticAlgae Periphytic algae amounts were significantly reduced in mesocosms with atrazine treatment (5.575 ± 0.292 mg/m2

, mean ± SE) relative to the solvent control (6.575 ± 0.411 mg/m2

, mean ± SE, Tukey's hsd, P 

### < 0.001).
Effects on Water Clarity According to the study authors, water clarity was greater in mesocosms with atrazine (OMR, P 

## =0.045), although it is unclear whetherthe statistical comparison was made relative to the solvent control or endosulfan treated mesocosms. Effects on Insects and Crustaceans Relative to mesocosms containing solvent control, mesocosms with atrazine treatment had lower densities ofPolypedilum larvae (OMR, P = 0.004). Atrazine treatment did not have a significant effect relative to the control on Daphnia abundance or % of mesocosms containing 'other' insects and crustaceans (Figure 1).

Pesticide treatments (atrazine and endosulfan) did not significantly reduce the percentage ofdragonfly larvae which survived until the end ofthe experiment (atrazine, 12.5%; endosulfan, 25%; solvent, 25%; BR, df= 2, 45,

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= 1.08, P = 0.582) orthe mean number ofdays that dragonfly larvae were alive (atrazine, 17.50 ± 2.43 days; endosulfan, 21.56 ± 1.96 days; solvent 22'.13 ± 1.72 days; ANOVA, F2,45 = 1.60, P = 0.213). The study authors did notprovide a statistical pairwise comparison betweenthe atrazine treatment and the control for either dragonfly survival ormean number ofdays alive.

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### EndOIuIan Figure 1. Effects ofpesticidetreatments (atrazine, endosulfan, solvent control) on (A) Polypedilum (chironomid) larvae abundance, (B) Daphnia abundance, and (C) the percentage ofmesocosms containing insects or crustaceans otherthan Polypedilum and Daphnia. Bars representmeans (± SE) of32 mesocosms. Differentlowercase letters above bars reflect significant differences at P< 0.05 among treatments according to Tukey's honestly significant difference multiple comparison tests. Figure 2 from Rohr and Crumrine, 2005.

5

### Effects on Snails Snail final mass was reduced by atrazine relative to the solvent control (Tukey's hsd, P 

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### Figure 2). When controlling for final mass, fewer egg masses per surviving snail were deposited in mesocosms with atrazine compared to the control; however, the difference was not significant (Tukey's hsd, P> 0.05, 

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Figure 2. (A) Effects ofpesticide (atrazine, endosulfan, solvent control) and dragonfly treatments (presence and absence) on snail mass and (B) effects ofpesticide and snail treatments (presence and absence) on tadpole mass. Values represent means and SE ofeight mesocosms, and probability values are for the interaction between displayed treatments when controlling for significant predictors [see Tables 1 and 3 ofRohr and Crumrine (2005) for predictors]. Figure 4 from Rohr and Crumrine, 2005.

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## EndoalMln SdYent Figure 3. Effects ofpesticide treatments (atrazine, endosulfan, solvent control) on (A) the number of egg masses deposited per surviving snail and (B) tadpole Gosner development stage. Bars represent means (± SE) of16 mesocosms. Different lowercase letters above bars reflect significant differences atP < 0.05 among treatments according to Tukey's honestly significantdifference mUltiple comparison tests when controlling for significant predictors [see Tables 1 and 3 ofRohr and Crumrine (2005) for predictors]. Figure 6 from Rohr and Crumrine, 2005.


### Effects on WoodFrogs The study authors detennined thatthe majortemporal changes observed in wood frog behavioral responses throughtime were primarily due to dragonfly mortality. Since there were no significanttreatment differences in dragonfly mortality ortiming ofmortality, the study authors used the mean ofthe seven days ofwood frog behavior observations for subsequent analyses. A greater percentage oftadpoles were hiding in the solvent controls than the atrazine treatment (Tukey's hsd, 

### P = 0.028). Atrazine treatment did nothave a



### 7significant effect on the number oftadpoles avoiding the cup comparedto the control (Tukey's hsd, 

### P > 0.05, 

### Figure 4). Atrazine treatment did not appearto affecttadpole activity relative to the control 

### (Figure 5). Wood frog development (Gosner stage) was slowed by atrazine compared to the control but the difference was not statistically significant (Tukey's hsd, 

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### Figure 3). Atrazine treatment did not have a significant effectrelative to the control on tadpole final mass (Tukey's hsd, 

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Figure 4. Effects ofpesticide treatments (atrazine, endosulfan, solvent control) on (A) the percentage oftadpoles hiding (measured as the percentage oftotal individuals that were not observed) and (B) the percentage ofindividuals observed that were avoiding the cup. Bars represent means (± SE) of16 mesocosms. Different lowercase letters above bars reflect significant differences at P < 0.05 among treatments according to Tukey's honestly significant difference multiple comparison tests when controlling for significant predictors [see Table 2 ofRohr and Crumrine (2005) for predictors]. Figure 7 from Rohr and Crumrine, 2005.

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### .... PfII88't SaMnt Figure 5. Interaction among pesticide (atrazine, endosulfan, solvent control), snail, and dragonfly treatments (presence and absence) on the percentage oftadpoles observed that were moving during a 15-s observation period. Values represent means (± SE) offour mesocosms. All main effects and twoway interactions were significant (P< 0.05) when controlling forDaphnia abundance [see Table 2 of Rohr and Crumrine (2005)]. Figure 9 from Rohr and Crumrine, 2005.



### Data Summary Table: Time Weighted Magnitude Nominal Average Did

of Effect Atrazine Day of Study Atrazine Recovery Effect relative to Concentration Effect Concentration Occur-if Comments controls (pglL) Occurred fromDayOto so, when? When Effect

Occu"ed Mean atrazine, (5.575 Mean ofdata mg/m2); 15% 50 IlgIL collected over Could notbe Could not mean Chlorophyll a decrease (maximum)! the four week determined. be solvent, determined. (6.575 study period mg/m2). Tukey's hsd, p < 0.001 Polypedilum Could not Figure 1 abundance -24% 50 IlgIL Unknown2 Could notbe be decrease (maximum)! determined. determined. OMR, p= score 0.004 Could not Figure 1 Daphnia No effect 50 IlgIL Unknown2 Could notbe be abundance observed (maximum)! determined. determined. Tukey's hsd, P> 0.05

9Time Weighted Magnitude Nominal Average Did Day of Study Atrazine Effect of Effect Atrazine Effect Concentration Recovery Comments relative to Concentration Occurred fromDayOto Occur-if controls (p.glL) When Effect so, when? Occurred Other insects/ Could not Figure 1 crustaceans No effect 50**~g/L** Unknown2 Could not be be abundance observed (maximum)! determined. determined. Tukey's hsd, P> 0.05 Atrazine (12.5%); solvent, (25%) Unknown if difference Survival was through the Could not significant Dragonfly 50% 50**~g/L** Could notbe (no larvae survival decrease (maximum)! end ofthe determined. be statistical experiment determined. (days 29/30i comparison provided), variation among replicates unknown (only mean values provided). Atrazine (mean± SE; 17.50 ± 2.43); solvent (mean± SE, 22.13 ± 1.72).
Mean ofdata Unknown if Could not difference Dragonfly 21% 50**~g/L** collected over Could notbe be larvae lifespan decrease (maximum)! the four week determined. was determined. significant study period. (no statistical comparison provided), SE values suggestthat means may not be significantly different.

10Time Weighted Magnitude Nominal Average Did Day of Study Atrazine Effect of Effect Atrazine Effect Concentration Recovery Comments relative to Concentration Occur-if controls (pgIL) Occurred from Day Oto so, when? When Effect

Occurred Figure 2 Snail final 5OIlg!L Days 29/30 Could notbe Could not Reduction (maximum)! (study detennined. be Tukey's hsd, mass tenninationi detennined. P<O.OOI (text). Days 29/30 Could not Figure 3 No. egg mass No effect 5OIlg!L Could notbe per snail observed (maximum)! (study detennined. be Tukey's hsd, tenninationi detennined. p> 0.05 Figure 2 No effect judgedby overlapping Days 29/30 Could not errorbars Tadpole final No effect 5OIlg!L Could not be for atrazine observed (maximum)' (study detennined. be treatment mass tennination)3 detennined. and control.
No statistical comparison provided Figure 3 Tadpole 5OIlg!L Days 29/30 Could notbe Could not Gosner stage No effect (maximum)' (study detennined. be Tukey's hsd, tennination)3 detennined. P = 0.067 (text) Mean ofdata Figure 4 Tadpoles

- 6% less 5OIlg!L collected over Could notbe Could not be Tukey's hsd, hiding hiding (maximum)' the four week detennined. detennined. P= 0.028 study period. (text) Mean ofdata Could not Figure 4 Tadpoles 5OIlg!L collected over Could notbe avoiding cup No effect (maximum)! the four week detennined. be study period. detennined. P> 0.05

11InItIal dose ofatrazme was 25 l!g!L, (nommal) then aftertwo weeks the ponds were redosed WIth 25 l!g!L (nominal). Thus the exposure for the fIrst two weeks was 25 l!g!L (nominal) and the exposure for the

. secondtwo weeks was assumed by the study authors to have been a maximum of50 l!g!L (nominal). For the purposes ofthe associated endpoint summary spreadsheet, it was assumed that 25 l!g!L was the initial test concentration.

2 Not specifIed by study authors. Primary reviewer assumed study termination (day 29/30).
3 Experiment was terminated in halfofthe ponds on day 29 and the other halfon day 30. Time Weighted Magnitude Nominal Average Did

of Effect Atrazine Day of Study Atrazine Recovery Effect relative to Concentration Effect Concentration Occur-if Comments controls (pg/L) Occurred from Day Oto so, when? When Effect

Occurred Figure 5
No effect judged by overlapping Tadpoles Mean ofdata Could not errorbars No effect 50 l!g!L collected over Could notbe for atrazine moving! observed (maximum)l the four week determined. be treatment mobility study period. determined. and control. No statistical comparison provided I 

### ..

Reviewer Comments: Much ofthe study results as presented by the study authors were not specific to atrazine treatment effects. Results ofMANCOVA, ANCOVA, ANOVA, and Poissonregression did not distinguish the individual effects ofatrazine. In general, these analyses examined the effects of"pesticide" treatment (atrazine, endosulfan, or solvent control) orthe effects ofnon-pesticide treatments (e.g. effects ofsnail absence or presence on chlorophyll 

### a with the "pesticide" treatments combined). The study authors presented the simple effect ofatrazine treatments for some but not all results, that is, apairwise comparison between atrazine treated and solvent control mesocosms. This study review focuses on and presents results specific to atrazine treatment effects, unless otherwise noted (i.e., analyses and results that include endosulfan treatments are generally excluded). It should also be noted thatmesocosms treated with atrazine (endosulfan and control as well) varied in regards to the presence or absence ofsnails, dragonflies, and tadpoles, and that these non-pesticide treatment effects were integrated into the atrazine treatment effects presented in this study.

The study authors stated that direct effects ofatrazine (i. e., reduction ofperiphyton chlorophyll a) resulted in indirect effects on snails, tadpoles, and Polypedilum chironomid larvae.

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### Description ofUse in Document: Qualitative



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



### Limitations ofStudy: The results have good applicability to mesocosms ornatural aquatic systems since the experimental mesocosms contained not only periphytic algae, but also several species from higher levels ofthe food web, however, there were limitations. Actual pesticide (atrazine) concentrations were notmonitored throughoutthe experiment. Although a nominal dose was reported, exposure was complicatedbecause a second dose of25**~g** atrazine/L was made two weeks afterthe initial application of25**~g!L** and the study authors reported heavy rainfall (timing not reported) during the experimentwhich wouldhave diluted the pesticide in the mesocosms. Nominal concentrations were verified in the stock solution and atrazine shouldhave been stable over a period oftwo weeks; therefore, the study authors made a conservative assumption that 50**~g** atrazinelL was in each mesocosm Itwas not clearifthe solvent control pond received a second application ofacetone aftertwo weeks; effects ofacetone wouldhave beenunderestimated ifa second application was not made. There was no effortto determine ifatrazine or other contaminants were presentin the source water for the experiments orthe control mesocosms. Although there was an acclimation period for the periphyton, it does not appearthatthere was an acclimationperiod for the snails, tadpoles, and dragonflies. In addition, it is unclear whether wood frog tadpoles, adult snails, and dragonfly larvae may have been previously exposedto other contaminants (including pesticides) priorto the experiment, giventhat no previous contaminanthistory ofthe collection sites was provided. 

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

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

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Tertiary Reviewer: AnitaPease, M.S., SeniorBiologist, EPA

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