### Open Literature Review Summary

ChemicalName: 

### Atrazine CAS No: 

### 1912-24-9

ECOTOX Record Number and Citation: 

### Diana, S. G., W. J. Resetarits, Jr., D. 1. 

### Schaeffer, K. B. 

### Beckmen and V. R. Beasley. 2000. Effects of atrazine on amphibian growth and survival in artificial aquatic communities. Environmental Toxicology and Chemistry. 19 (12): 2961-2967. (MRID 48261107)
Purpose'ofReview: 

### Determinepotential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plantcommunities and associated determination ofaLevel ofConcern (LOC).

Date 

### ofReview: October 5, 2009
Summary ofStudy Findings
Purpose ofStudy: 

### This study examines the effects ofatrazine on a simple aquatic community including aquatic plants and the larval gray tree frog (Hyla versicolor) 

### inexperimental outdoormicrocosms. The focus ofthis reviewis ontreatment-related effects to the aquatic plant community, althollgh possible indirecteffects onHyla versicolor 

### are noted.

Methods: 

### Plastic wading pools were used to construct sixteen outdoorexperimentalponds (microcosms) in mid-May 1996 atthe experimental pond facility ofthe Illinois Natural History Survey (Urbana, Illinois). Eachmicrocosmmeasured 1.22-mdiameter; depthwas notreported. The microcosms were arranged in a 4 x 4 Latin square design on abedofgravel. Each was filled with 90 Lwater from anearby pond, notfurther described, and amended with 150 gdry wheat straw (unreported source) as a slow-release nutrient source. The microcosms were covered with fiberglass screen (3.2-mmmesh) to retain added amphibians and excludepredators. Clear, greenhouse, polyethylene film covers (6-llmthickness) were suspended43 cm above the microcosms to exclude rainwater, with one cover shielding two microcosms. The microcosms were allowedto acclimate 5 weeks priorto treatment. Waterlostthrough evaporationwas replaced twice weekly with deionized (DI) water.

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### Study organisms were obtainedfrom nearby natural locations, notfurther described. Eighty stems ofthe aquatic macrophyte marsh-pepperknotweed 

### (Polygonum hydropiper) were collected, potted, and distributed five plants permicrocosm. Captive-born

### Hyla versicolorlarvae were initiallymaintained in pond water indoors. 40, 9-day-old larvae (5 days post-hatch) were then randomly assigned to each ofthe outdoormicrocosms. Larvae were 15 days old (11 days post-hatch) atthe time ofatrazine application.

Atrazine stock solution was preparedthrough drop-wise addition oftechnical grade atrazine (purity unspecified) in acetone (5.002 mg/ml) to DI water, with continuous stirring. The final atrazine concentration ofthepremix was 30.01 mg/L. A vehicle control solution was similarly preparedby adding an equal volume ofacetone to OI water. Boththe test and vehicle control solutions were purged with air overnightto remove acetone through volatilization. Each microcosm was treated with 6 L ofan appropriate combination ofaqueous atrazinetest solution and/orvehicle control solutionto yield nominal atrazine treatmentrates of0, 20, 200 and 2,000 Jlg/L. Fourreplicates were includedpertreatment. Solutions were evenly sprinkled overthe water surface using awatering can, after whichthe waterwas briefly mixed.

Three series ofwaterquality measurements were taken to analyze general water quality parameters, atrazine concentrations, and chlorophyll 

### a. Dissolved oxygen (DO), temperature, andpH measurements were recorded 

### in situ atpre-dawn and pre-dusk, daily, from Dayll -1 through Day 10. Measurements were continued every 3 days thereafterthrough Day 51. Water samples (unreported volume) for atrazine analysis were collected atDay 0 and 3 weeks posttreatment. Concentrations were quantified using the Ohmicron Atrazine RaPID Assay (Ohmicron Environmental Diagnostics), with intra- and inter-assay coefficients ofvariance of 3.91% and 1.24%, respectively. Additional water samples (1 L) were collectedfor chlorophyll 

### a analysis priorto treatment and at 7, 14, 21, 28 and 42 days post-treatment (Figure 1). Chlorophyll 

### a was extracted from the waterwith acetone and quantified by measuring absorbance at 630,647,664 and 740 nm with a split-beam spectrophotometer (Lambda 3, Perkin-Elmer) with quartz cuvettes (1-cmpath length). Intra- and inter-assay coefficients of variance were 1.2% and 1.58%, respectively. Extractionrecovery efficiency was 89.29 ± 

### 2.77% (USEPA Method 446.0, EPA/600/R-97/072).
Periphytonthathad adhered to duplicate glass microscope slides suspended inthe microcosm waterwere similarly analyzed for chlorophyll 

### a on aweeklybasis. To determined macrophyte biomass, plants were cut atthe soil line, blotted dry, and immediately weighed (wet weight) as microcosms were drained at study termination. During the study, metamorphosing frog larvae were collected nightly from microcosm sidewalls and maintained in alaboratory at26.7 ± 

### laC until completion ofmetamorphosis (tail resorption). Following tail resorption, the frogs were sacrificedby immersion in a 0.1 % aqueous solution of3-amino benzoic acid ethyl ester. Snoutventlength (to nearest 0.05 mm), bodymass (to nearest 0.001 g) and date ofmetamorphosis

I Day 0 = Date ofatrazine application (treatment) 

### - 2 -completion were recorded. Survival through metamorphosis was calculated as the percentage of frogs that completed metamorphosis, once no additional larvae couldbe found in the microcosms.

Statistical tests were conducted using SYSTAT v 7.0 software. Repeated measures analysis of variance (ANOVA) was used to evaluate potential effects ofpool position on temperature. Phytoplankton chlorophyll 

### a concentrations, DO, pH, and temperature were also analyzed by repeated-measures ANOVA. One-way ANOVA was usedto analyze response values for macrophyte biomass and arcsin-transformed survival values. Frog larval duration was analyzed by life-table analysis and a second method (unreported, presumably ANOVA).

For all analyses, aprobability value (P) of0.05 was selectedto detect significant differences in responses. When ANOVA detected an overall difference betweentreatment groups, responses were compared using the Tukey HSD multiple comparisontest and linear contrasts. Leastsignificant differences were determined for repeated-measures ANOVA, using a 95% confidence level, to compare responses atthe same interval as well as responses between consecutive intervals.

One control microcosm was excluded from data analysis because it experienced an early and sustained zooplanktonbloom, resulting in a clearing ofthe phytoplankton, which did notrecover. Adherentperiphyton chlorophyll 

### a was excludedbecause itwas "almostuniformly" less thanthe limit ofdetection.



### Results: At Day 0, measured atrazine concentrations in the water were 1.64 ± 0.09 Ilg/L, 20.13 ± 0.58 Ilg/L, 195.50 ± 8.74 Ilg/L and 2,035.83 ± 51.54 Ilg/L for the 0, 20, 200 and 2,000 Ilg/L treatment rates, respectively. At 3 weeks post-treatment, atrazine concentrations in the 20, 200 and 2,000 Ilg/L treated microcosms had decreased 21 %, 9% and 16%, respectively, from initial concentrations

..
Based on ANOVA, phytoplankton chlorophyll 

### a changed significantly over the study duration within the treatment groups (p<O.OOI) and differed significantly between treatment groups (p=O.OI, Figure 1). There were no clear treatment-related changes in chlorophyll a at Day 7. Chlorophyll a levels at 200 Ilg/L were higher than controls, and chlorophyll levels at 20 Ilg/L and 2000 Ilg/L were lower than controls. Chlorophyll a levels ranged from approximately 5 to 10 Ilg/L to 40 Ilg/L at Day 7. By Day 14, a more consistentpattern was observed. Chlorophyll a levels at 200 Ilg/L and 2000 Ilg/L atrazine were approximately 2- to 4-fold higher than controls until the end of study. The highest chlorophyll a levels were observed in the 2000 Ilg/L treatment group from Day 7 to Day 21, and inthe 200 Ilg/L onDay 28 and thereafter.

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### The overall effect of atrazine on the macrophyte 

### Polygonum hydropiper was highly significant (p<O.OOl) with biomass decreased 30%, 98% and 99% in the 20, 200 and 2,000 Ilg/L treated microcosms, respectively, as comparedto controls at study termination.

No data or analysis results were provided with respect to temperature. Figure 4 shows effects on dissolved oxygen (DO). Decreasing concentrations ofDO levels occurred inpre-dusk water with increasing atrazine concentrations, as compared to controls, with significant differences between treatment groups (p<O.OOI), as well as in the change of DO over time (p<O.OOl). The same trends were seen in pre-dawn DO levels (p=0.007 and p=0.002, respectively). In 20 Ilg/L treated microcosms, pre-dusk DO was decreased 10-20% at 5-7 days, as compared to controls, then recovered at 10 days and remained near control levels thereafter. At the 200 and 2,000 Ilg/L treatment rates, pre-dusk DO was decreased**~50%** 

### and 75%, respectively, at 1-7 days, as compared to controls. DO recovered to control levels by 14 days, then again decreased to**~20-40%** 

### and**~40-50%,** 

### respectively, from 30 days to studytermination.

There were significant differences in pre-dawn and pre-dusk pH levels between treatment groups, as well as change in pH over time (both p<O.OOI), and although there were significant decreases in pH in the 200 and 2,000 Ilg/L treated microcosms, as compared to controls, pH levels generally remainedbetween approximately 7 to 9.

At the 200 and 2,000 Ilg/L treatment rates, frogs at metamorphosis were significantly shorter (5%) with lower body mass (7%), as compared to frogs in the control and 20 Ilg/L treated microcosms (p<O.OOI,Table 1, attached Worksheet). Results ofanalyses differed with respect to larval period duration. Life table analysis indicated no significant differences across treatments (calculations not reported). However, in the 2,000 Ilg/L treatment, larval period duration was significantly longer than in the 200 Ilg/L treatment (+5%, p=0.025, method unreported). No statistically significant differences in survival were seen across treatments, although survival in the 2000 Ilg/L treatment was notably lower at 70.7% ±4.9 (attached Worksheet). For all treatment groups, mean snout-vent length and mass initially decreased at 2-3 weeks posttreatment and increased at 3-5 weeks. The increase continued at 5-6 weeks in control and 20 Ilg/L treated microcosms, but decreases occurred in the 200 and 2,000 Ilg/L treated microcosms (Figure 3).

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## so Days from atrazine addition Figure 1. Mean phytoplankton chlorophyll a concentrations (± standard error) in water; n = 4 for each atrazine treatment level, n = 3 for controls. Figure 1 from Diana etal. 2000.

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Figure 2. Mean macrophyte biomass (± standard error), "A" and "B" indicate that groups differed significantly (p<0.05); n

### =4 for each atrazine treatment level, n

### =3 for controls. Figure 2 from Diana etal. 2000.



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7 Weeks from atrazine addition Figure 3. Mean (± standard error) snout-vent length and mass at metamorphosis for gray tree frog (Hyla versicolor); statistically indistinguishable groups are combined, n = 7 for pooled 0- and 20-ltglL treated groups and n = 8 for pooled 200- and 2,000-ltg/L treated groups. For 0- and 20-ltg/Ltreated groups at 2 weeks, frogs had emerged from only one microcosm; consequently, no standard error is presented. Figure 5 from Diana etal. 2000.



### - 6 -Table 1. Frog survival through metamorphosis, larval duration, mass and length at metamorphosis (mean ± st. dev., n = 97,134,135 and 112 individual for 0-, 20-, 200- and 2,000-l-lg/L treatment rates, respectively; from Table 1 ofstudy).

Atrazine Microcosm Larval period duration

(Ilg/L) replicates Percent survivall (days) 0 3 80.8 ± 5.6 13.38 ± 0.59 20 4 83.1 ± 7.9 13.55 ± 0.59 200 4 84.4 ± 4.1 12.39 ± 0.76 2,000 4 70.7 ± 4.9 13.99 ± 0.693 1 Differences were not statistically significant atp<0.05. 2 Significantly (p<O.OO1) less thanthe 0- and 20-llg/Ltreatment groups. 3 Significantly (p=0.025) greaterthan the 200-llg/L treatment group.**~'~"I** I o·f

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Fig. 4. Mean (± standard error ofmean [SED (a) Predusk and (b) predawn dissolved oxygen (DO) concentrations as a function ofatrazine concentrations and time N

## =3 for the control group and4 for each atrazine-treated group.



### - 7 - Mass at metamorphosis (g) 0.482 ± 0.026 0.484 ± 0.021 0.449 ± 0.0192 0.448 ± 0.021 2 Length at metamorphosis (mm) 19.00 ± 0.28 18.9 ± 0.20 18.0 ± 0.202 18.1 ± 0.202d 

### hObO d 

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aa ummary a e: illOpJan on, macropJryies an amp, I tans In out oormicrocosms Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration StudyEffect Concentration Occur

- 

### if so, Comments to controls (pgIL/ Occurred from Day Oto when? When Effect Occurred The study authors reported that "no significant differences in phytoplankton 20 /lg/L; initial chlorophyll a No clear effect NA NA NA concentration were 20.13 ± 0.58 /lg/L detected between control and 20 /lg/L treatment groups at any time." The reviewers agree with this conclusion. Magnitude ofeffect estimated from Not determined Figure 1. Approximately 200 /lg/L; initial (measured No; effect Chlorophyll a levels a 3-fold concentrations was present increased at all Phytoplankton Stimulation 195.50 ± 8.74 14 were only at the end of intervals as compared chlorophyll a above controls /lg/L reported at Day the study. to controls. (/lg/L) 

### oand Day 21) Maximum effect was statistically significant (p"S0.05). Magnitude ofeffect estimated from Figure 1. Initial decrease was Not determined Yes, observed that was not Approx

.. 2- (measured chlorophyll a statistically fold increase 2,000 /lg/L; initial concentrations levels were significant, p value relative to 2,035 ± 51.54 14 were only similarto notreported), controls. /lg/L reported at Day controls by followed by 

### oand Day 21) Day 42. statistically significant (p"S0.05) increase (maximum effect**+~** 100% at 28 days), with recovery occurring, at Day 42.



### - 8 -Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration Study Effect Concentration Occur

- 

### if so, Comments to controls (l1g/ L



### yt Occurred from Day Oto when? When Effect Occurred Not An approximate 30% applicable; decrease was No clear effect 20 Ilg/L; initial NA NA biomass only observed relative to 20.13 ± 0.58 IlgIL determined at controls that was not study statistically termination. significant (p. 2963). Macrophyte Not Magnitude ofeffect (Polygonum 200 Ilg/L; initial applicable; presented in article hydropiper) 98% decrease 195.50 ± 8.74 51 Not calculable biomass text (p. 2963). biomass (g wet Ilg/L determined at Effects statistically wt.) study significant (p<0.05, termination. Figure 2). Not Magnitude ofeffect 2,000 Ilg/L; initial applicable; presented in article 99% decrease 2,035 ± 51.54 51 Not calculable biomass text (p. 2963). Ilg/L determined at Effects statistically study significant (p<0.05, termination. Figure 2). Magnitude ofeffect estimated from 20 Ilg/L; Figure 4. Not Not No effect measured: 20.13 applicable Not applicable applicable Control and ± 0.58 Ilg/L treatment standard errorofmeans overlap Magnitude ofeffect Dissolved estimated from oxygen (mg/l) Figure 4.

- predusk Statistical measurements2 significance not 200 Ilg/L; reported, however, 60% decrease measured: 195.50 1 Not calculable Day 10 (see treatment and control
± 8.74 Ilg/L comments) standard error of means do not overlap. Recovery observed by day 10, however, treatment decreased relative to the control 

### - 9 -Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration Study Effect Concentration Occur

- 

### if so, Comments to controls (pglLyJ Occurred from Day Oto when? When Effect Occurred again starting day 20 and remained lower thro1Jgh the end of the experiment. Magnitude ofeffect estimated from Figure 4.
Statistical significance not reported, however, treatment and control 2,000 1Jg/L; standard error of Day 10 (see means do not 78% decrease measured: 2,035 I Not calculable comments) overlap. ± 51.54 1Jg/L Recovery observed by day 10, however, treatment decreased relative to the control again starting day 20 and remained lower through the end of the experiment. An approximate 20 1Jg/L and 200 3.4% increase was Gray tree frog No effect 1Jg/L; initial 20.13 Notreported Not calculable Notreported observed. This (Hyla ± 0.58 and 195.50 observation was not versicolor) ± 8.74 1Jg!L statistically survival significant at p<0.05. thro1Jgh An approximate 13% metamorphosis 2,000 1Jg/L; initial reduction in survival (%) No clear effect 2,035 ± 51.54 Not reported Not calculable Notreported was observed that 1Jg/ L was not statistically significant at p<0.05.
No effect 20 1Jg/L; initial NA NA NA



### -- Gray tree frog 20.13 ± 0.58 1Jg/L (Hyla 200 1Jg!L; initial versicolor) No effect 195.50 ± 8.74 NA NA NA
n larval period 1Jg/L duration (days) No effect 2,000 Ilg/L; initial NA NA NA


## --

- 10-I At 3 weeks post-treatment, atrazme concentratiOns mthe water had decreased 21%, 9% and 16% from mltIaI measured concentrations atthe 20, 200 and 2,000 Ilg/Ltreatmentrates, respectively; no additional quantitative results were presented. 2No effect observed at any treatment level for pre-dawn DO measurements whichrepresent changes due to

respiration. Pre-dusk DO measurements representthe net increase in DO during the day due to photosynthesis. Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration Study Effect Concentration Occur

- 

### if so, Comments to controls (pglLyJ Occurred from Day Oto when? When Effect Occurred 2,035 ± 51.54 Ilg/L
No effect 20 IlglL; initial Not reported Not calculable Notreported
-- 20.13 ± 0.58 IlgiL Gray tree frog (Hyla Magnitude ofeffect versicolor) 200 Ilg/L and calculated from
mass at 2,000 IlglL; initial Table 1. Effects metamorphosis 7% decrease 195.50 ± 8.74 Not reported Not calculable Notreported statistically (g) Ilg/L and 2,035 ± significant (p<O.OOI) 51.54 Ilg/L as compared to 0- and 20-llg/L groups. No effect 20 IlgIL; initial Magnitude ofeffect (+1% 20.13 ± 0.58 Ilg/L Notreported Not calculable Notreported calculated from Gray tree frog stimulation) Table 1. (Hyla Magnitude ofeffect versicolor) 200 IlgiL and calculated from length at 2,000 IlglL; initial Table 1. Effects metamorphosis 5% decrease 195.50 ± 8.74 Notreported Not calculable Not reported statistically (mm) IlgiL and 2,035 ± significant (p<O.OOI) 51.54 IlglL as compared to 0- and 20-llgiL groups.



### ...

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### Reviewer Comments: It

### is not stated whetherthe natural pond waterwas testedto establishthatitwas not contaminated withpollutants otherthan atrazine. Reportedly, water samples were collected from thenatural pond used as the water source for the microcosms to test for atrazine contamination; however, no results were provided. Atrazine was detected in untreated control microcosm water at 1.64 ± 0.09**~g/L** 

### atday O. This level ofatrazine contamination was consideredto not likely to have affectedthe study results. The source ofwheat straw introducedto each microcosm was notreported; this may have been anothervector ofcontamination.

The purity ofthe atrazine test compound was reported as technical, butnot further specified. Althoughthe study was conducted for 6weeks following atrazine application, atrazine concentrations were only determined at 0 and 21 days post-treatment.

No negative controls were utilized; only solvent (vehicle solution) controls were included.

The microcosms did notcontain a sedimentlayer. Atrazine does not adsorb strongly to soil, so this omissionis unlikely to have affectedthe biologically available fraction ofpesticide inthe water.
Multiple water quality parameters were not monitored, analyzed, and/or reported. Turbidity, alkalinity, total nitrogen, total phosphorus, conductivity, and particulate and dissolved organic carbon were not measured. Pre-dawn and pre-dusk water temperatures were reportedly monitored and analyzed, but no data were presented. Those results may be integral given that the microcosms were above ground plastic wading pools positioned on a bed ofgravel, and no shading was described. Likewise, effects of pool position on temperature were reportedly analyzed, butno results were presented.

The study authors suggested that the greater phytoplankton biomasses over the study duration in the 200 and 2,000**~g/L** 

### treated microcosms were likely a consequence of elimination of macrophytes. The more inhibited phytoplankton growth in the control and 20**~g/L** 

### treated microcosms may have been a consequence ofnutrient competition with surviving macrophytes, resulting in inhibitionvia allelopathy.

The mass ofphytoplankton introduced to each microcosm may have been too low to identify initial declines in population growth. Variability within (and across) treatment groups was another complicating factor, which may have precluded effective statistical analysis. The authors did notcite any tests orcorrections forhomogeneity ofvariance in theirmethods.

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### The study authors providedjustification for treatment rates based on literature values. The 20 flg/L 

### rate was selected as a no observed effect concentration (NOEC, Solomon 

### etai., 1996). The 200 flg/L 

### rate was selected as a concentration detected in static bodies ofwater that receive runofffrom atrazine treated fields (Kadoum 

### et ai., 1978). The 2,000 flg/L 

### rate was selected as a highly phytotoxic concentration considered unlikely to cause acute toxic effects in the frog larvae.

Because the experiment was not designed to distinguish between direct and indirect effects on frog larvae, all results are noted in this review. An assumption was made that any observed effects were mediated through the aquatic plant community (i.e., indirect effects), as the highest treatment level (2,000 flg/L) 

### had not been associated with acute toxic effects in tadpoles. The authors proposed a causal relationship between diminished DO and smaller size (eg., snout-vent length, mass) offrog larvae atmetamorphosis inthe highertreatments.

All frog larvae were obtained from a single lineage; this is inadequate ifnatural population-level inferences are to be made from the results.

The authors failed to report their primary method ofanalysis oflarval period duration, although they presented results. Based on their primary method, larval period duration was significantly longer for frogs inthe 2,000 flg/L 

### treatmentthan inthe 200 flg/L 

### treatment (5%, p=0.025). Lifetable analysis was also applied and indicated no significant effects ofatrazine concentration on this endpoint. No supporting calculations orresults were presented.

Removal offrog metamorphs from the microcosms prior to tail resorption introduces another variable that is not addressed. This may have confounded results for larval period duration and for size (snout-vent length, mass) atmetamorphosis.

A time lag must be considered when testing for indirect effects. For example, the authors suggest that younger frog larvae with undeveloped lungs may be more severely impacted by decreased DO associated with atrazine treatment. However, atrazine application in this study occurred when larvae were 11 

### days post-hatch, and the authors cite an average larval period duration ofapproximately 13 days. The study organisms may therefore have been insufficiently sensitive and/or housed in the microcosms for too short a duration to serve as an indicator of effect.

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### Description ofUse 

### in Document: Qualitative This study is sufficientto provide qualitative information for use inthe risk assessment process. Limitations with respectto experimental design and statistical analysis are noted limitations.



### Rationale for Use: The studyhas some utility for demonstrating that atrazine causedreduction inthe macrophyte community with corresponding changes in species composition leading to domination byphytoplankton at levels of200 Ilg/L and 2000 

### Ilg/L.

Limitations ofStudy: There were numerous reporting andmeasuring deficiencies. No effort was made to document whether otherchemicals were present in the treatment and/or control microcosms priorto study initiation, and analyses ofcontrol microcosms detected low levels of atrazine at Day 

### O. Atrazine concentrations were only determined at

## aand 21 days posttreatment, and the study was conducted for 6 weeks after application. Only solventcontrols were utilized; no negative controls were included. The microcosms consisted ofabove ground plastic wading pools positioned on a gravel bed. The microcosms containedno sedimentlayer. Microcosm waterwas notmonitored forturbidity, alkalinity, total nitrogen, total phosphorus, conductivity, or organic carbon. Watertemperature was monitored, butnotreported, andthose results may be integral given the microcosm system (above ground pool) utilized. Finally, unequal variance within and across treatments may have resulted in questionable statistical interpretation ofresults with regard to early phytoplanktonpopulation growth.


### Primary Reviewer: Lynne Binari, M.S., Subcontractorto Cambridge Environmental Inc. 

### Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division U.S. Environmental ProtectionAgency 

### Tertiary Reviewer: Brian Anderson, Biologist, Environmental Fate and Effects**Di~on** U.S. Environmental Protection Agency

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