### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOXRecord Number and Citation: Relyea, R. A. 2009. A cocktail ofcontaminants: how mixtures ofpesticides at low concentrations affect aquatic communities. Oecologia 159(2): 363-376. (MRID 48261129)


### 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 15,2009


### Purpose ofStudy: This study examines the effect offive insecticides (malathion, carbaryl, chlorpyrifos, diazinon, and endosulfan) and five herbicides (glyphosate, atrazine, acetochlor, metolachlor and 2,4-D) at low concentrations on outdoor mesocosms composed of zooplankton, phytoplankton, periphyton, and larval amphibians (gray tree frogs, 

### Hyla versicolor and leopard frogs, 

### Ranapipiens). The pesticides were studied alone, as a mixture ofthe five herbicides, as a mixture ofthe five insecticides, and as a mixture ofall ten herbicides and insecticides.


### Summary ofStudy Findings: The focus ofthe summary is onthe mesocosms treated with atrazine alone.



### Methods: The experiment was conducted atthe University ofPittsburgh's Pymatuning Laboratory ofEcology in northwestern Pennsylvania (USA). The experimentused a completely randomized design consisting of 15 treatments with4 replicates each for atotal of59 experimental tanks (the solvent control consisted ofonly 3 replicates). The treatments were as follows: a negative control (water), a vehicle control (ethanol), each ofthe five insecticides applied separately (malathion, carbaryl, chlorpyrifos, diazinon, and endosulfan), each ofthe five herbicides applied separately (glyphosate, atrazine,

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# 11 2087817acetochlor, metolachlor, and 2,4-D), a mixture ofall five herbicides, a mixture ofall five insecticides, and a mixture ofall ten pesticides. The mesocosms were 1,300-L cattle watering tanks. The tanks were filled (on May 15- 19,2006) with about 1000 L ofwell water (pH 8). On June 7th

, the tanks were supplemented with a mixture ofwater from nearby ponds which contained zooplankton, phytoplankton, and periphyton. The following day, eachtank was further supplemented with 300 g ofleaflitter (primarily 

### Quercus spp.) and 25 g ofcommercial rabbit chow (source ofalgal nutrients and additional surface for algal growth). On June 16th

, the periphyton samplers were addedto each tank in the form oftwo unglazed clay tiles (10 x 10 cm, oriented vertically). After 18 days (June 25th), the tadpoles were added. The tadpoles were collected for the experiment as newly oviposited egg masses and hatched in 200-L wading pools where they were fed rabbit chow 

### adlibitum until use inthe experiment. The time at which each species was oviposited differed (ten masses of leoJ?ard**fro~s,** collected on March 31 5t; 23 masses ofgray tree frogs, collected on May 14 and 17 ), thus the two species were ofdifferent ages when addedto the experiment. Added to each tank were 20 tadpoles ofeach species 

### (H versicolor, and

### R. pipiens) chosen from a mixture ofall egg masses (initial mass ± SE: gray tree frogs, 77 ± 4 mg; leopard frog, 134 ± 12 mg). The density oftadpoles in the experimental tanks was 91m2 for each species, which was within natural densities for the two species.

Two days aftertadpole addition, the tanks were exposed to atrazine (and other treatments) via a single dose oftechnical grade chemical (chemical purity, 98.1 %) at a nominal concentration of 10 !J.g/L (p.p.b.) using ethanol as the vehicle. Ethanol concentration was 0.003% inthe solvent control tanks and the tanks receiving a mixture ofall ten pesticides (ethanol concentration in the atrazine-only treatment was not stated). Control tanks without ethanol were also included. Atrazine was added to the tanks and the surface waterwas mixed with a 500-mL container. One hour later, water samples were collected midway in the water column; samples from the four replicate tanks were pooled and analyzed for atrazine by HPLC. The measured concentration ofatrazine was 6.4 !J.g/L (p.p.b.). The control tanks were not analyzed for any ofthe ten pesticides. The author states thatthe well water used for filling the tanks did nothave detectable concentrations ofany ofthe pesticides. However, it was not stated ifthe pond water (biota source) added to the tanks was analyzed for any ofthe pesticides.

The temperature, pH, and dissolved oxygen in eachtank were measured using a digital water meter on days 10 and 35 ofthe experiment.

On days 16 and 36, the zooplankton was sampled midway in the water column atthe four cardinal directions and the center ofthe tankusing a 0.2-L tube sampler. The five samples from eachtank were combined, filtered (62-!J.mNitex screen), and then preserved in 70% ethanol. The zooplankton was classified to species. Twelve taxonomic groups ofcladocerans and copepods were detected. The assemblage was dominated (91 %) by four species: two species ofcladocerans 

### (Daphniapulex and 

### Ceriodaphnia sp.)

2

### andtwo species ofcopepods (Skistodiaptomus oregonensis andLeptodiaptomus minutus). On days 16 and 35, phytoplanktonwas sampled via collecting 500 mL from the middle of the water column in eachtank. The samples were vacuum filtered through aWhatman GF/C filter. Chlorophyll 

### a concentrations were determinedby fluorometry afterthe filtered samples were prepared following the protocols ofArar 

### and Collins 1997 (including acidification).

On days 25 and 36, the periphyton was sampled via scrubbing one side ofthe unglazed clay tile into atub offiltered water. After filtration ofthe slurry (Whatman GF/C filters, pre-dried at 80°C and weighed) the filters were dried (80°C for 24 hours) andreweighed again to determine the biomass ofthe periphyton.
Metamorphs were removed from the tanks when they had at least one emerged forelimb (stage 42); the gray tree frogs emerged first due to their inherently shortertime to metamorphosis. Daily searches ofeachtankwere conducted starting day 21 (July 17) of the experiment when the first metamorph was observed through day 57 (August 22nd

, 

### last day ofexperiment). Afterremoval, the metamorphs were heldin one literlaboratory tubs containing moist sphagnum moss until tail resorptionwas complete (stage 46). The numberofdays from the beginning ofthe experimentto stage 46 was defined as time to metamorphosis. Each animal was euthanized in 2% MS-222, preserved, and weighed. The response variables forthe amphibians were percent survival, meantime to metamorphosis, and mean mass atmetamorphosis.

After most ofthe metamorphs ofboth species had emerged (determined laterto be 97.3% ofall live tadpoles), approximately 120 L ofwater was removed from each tank daily from day 50 to and including day 57 in orderto simulate pond drying (and accelerate metamorphosis). On day 57, the tanks were classified as "dry 

### ponds". The remaining amphibians were classified as dead, non-emergent (due to slow growth and development), or a successful metamorph (possessing at least one emerged forelimb; these were treated as described above).

The response variables were analyzed using multivariate ANOVA (MANOVA). The MANOVA was composed ofthe second measurements ofall abiotic conditions, cladoceran and copepod abundance, phytoplankton abundance (chlorophyll a), 

### periphytonbiomass, the survival ofgray tree frogs and leopard frogs, and size at and time to metamorphosis ofgray tree frogs. The metamorphosis data could notbe collectfor the leopard frogs intwo ofthe pesticide treatments due to nearly 100% mortality, so the leopard frog history responses were analyzed in a separate MANOVA. 

### Eachunivariate

.response variable was examined using either an ANOVA (forresponses that were measured atthe end ofthe experiment: tree frog mass, tree frog time to metamorphosis, tree frog survival and leopard frog survival) or arepeated-measures ANOVA (for responses measuredtwice during the experiment: temperature, dissolved oxygen, pH, the

3abundance ofcladocerans 

### (D. pulex and 

### Ceriodaphnia sp.), the abundance ofcopepods (S. 

### oregonesis and 1. 

### minutus), phytoplankton (chlorophyll 

### a) and periphyton biomass. The data was log-transformed when necessary. The zooplankton data was ranked priorto analysis since ithad heteroscedastic errors. The multivariate analyses controlledthe experiment-wise error rate at a = 0.05. To maximize the power ofthe analyses, posthoc comparisons were conducted using Fisher's LSD test which preserves the comparisonwise error rate at a = 0.05.

Results: The MANOVA on all final response variables (excluding the two life history traits of leopard frogs) revealed a significant effect ofthe treatments (Wilks' 

### A, F182,313 = 2.5, 

### P < 0.001); therefore, the ANOVA analyses were conduct for each response. These analyses were not specific to atrazine-only treatments and will not be discussed further.
Amphibians For leopard frog tadpoles 

### (R. pipiens), there was not a significant difference 

### (P> 0.05 using Fisher's LSD test) betweenthe atrazine treatment and the no-pesticide treatments (i.e. negative and ethanol control tanks) regarding survival to metamorphosis, mass at metamorphosis, and time to metamorphosis (Figure 1). For gray tree frog tadpoles (H 

### versicolor), there was not a significant difference betweenthe atrazine treatment and the no-pesticide treatments 

### (P> 0.05 using Fisher's LSD test) regarding survival to metamorphosis and time to metamorphosis (Figure 2). However, for mass at metamorphosis, gray tree frogs were larger in tanks treated with atrazine compared to those in the negative and ethanol control tanks (P = 0.045).

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Figure 1. Changes in the (a) survival, (b) mass at metamorphosis, and (c) time to metamorphosis of leopard frog tadpoles (Ranapipiens) in outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), lIVe separate insecticides (I) (carbaryl, malathion, chlorpyrifos, diazinon, and endosulfan), five separate herbicides (H) (acetochlor, metolachlor, glyphosate, 2,4-D, and atrazine), a mixture ofthe five insecticides, a mixture ofthe five herbicides, and a mixture ofall ten pesticides. In two ofthe treatments, there were no life history data (ND) due to a lackofsurviving animals. Data are mean ± 1 SE. Asterisks indicate treatments that are significantly differentfrom the no-pesticide treatment (P< 0.05 using Fisher's LSD test). Figure 1 from Relyea 2009.

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# W/**%:~~** Figure 2. Changes in the (a) survival, (b) mass at metamorphosis, and (c) time to metamorphosis of gray tree frog tadpoles (Hyla versicolor) in outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), five separate insecticides, five separate herbicides, and a mixture ofall ten pesticides. Data are mean ± 1 SE. Asterisks indicate treatments that are significantly different from the no-pesticide treatment (P< 0.05 using Fisher's LSD test). For abbreviations, see Figure 1. Figure 2 from Relyea 2009. 

### Zooplankton The abundance ofthe four species which dominated (91 %) the zooplankton was quantified: two species ofcladocerans 

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### Ceriodaphnia sp.) and two species ofcopepods (8. 

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### minutus). There was no effect from the atrazine treatment (P> 0.05 using Fisher's LSD test) relative to the control regarding the abundance ofthese species (Figure 3).

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Figure 3. Changes in the abundance ofcopepods [(a) Leptodiaptomus minutus and (b) Skistodiaptomus oregonensis] and c1adocerans [(c) Ceriodaphnia sp. and (d) Daphniapulex] in outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), five separate insecticides, five separate herbicides, and a mixture ofall ten pesticides. Data are mean ± 1 SE, averaged overtwo sample periods (day 16 and 36), on a log scale. Asterisks indicate treatments that are significantly different from the no-pesticide treatment (P< 0.05 using Fisher's LSD test). For abbreviations, see Figure 1. Figure 3 from Relyea 2009. 

### Phytoplankton andPeriphyton For chlorophyll 

### a (phytoplankton) measured on days 16 and 35, there was not a significant difference betweenthe atrazine treatment andthe no-pesticide treatments (P > 0.05 using Fisher's LSD test). Forperiphytonbiomass measured on days 25 and 36, there was not a significant difference betweenthe atrazine treatment andthe no-pesticide treatment (P> 0.05 using Fisher's LSD test). Phytoplankton and periphyton results are illustrated in Figure 4.



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Figure 4. The abundance of(a) phytoplankton (measured as chlorophyll a concentration) and (b) periphyton (measured as dry biomass on a clay tile) overtime in outdoor mesocosms exposed to no pesticides (control), solventonly (ethanol), five separate insecticides, five separate herbicides, and a mixture ofall ten pesticides. Data are mean ::I:: 1 SE on a log scale. Within each sample date, asterisks indicate treatments that are significantly different from the no-pesticide treatment (P< 0.05 using Fisher's LSD test). Forabbreviations, see Figure 1. Figure 4 from Relyea 2009.


### AbioticEffects TankwaterpH and dissolved oxygen were measured on days 10 and 35. There was not a significant difference between the atrazine treatment and the negative control 

### (P> 0.05 using Fisher's LSD test) for eitherpH or dissolved oxygen on either date 

### (Figure 5). The ethanol control differed from the negative control on day 10 for bothpH and dissolved oxygen butwas similarto the negative control on day 35.

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Figure 5. Changes in (a) pH and (b) dissolved oxygen overtime in outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), fIve separate insecticides, fIve separate herbicides, and a mixture ofall ten pesticides. Data are mean :I: 1 SE. Within each sample date, asterisks indicate treatments that are signifIcantly different from the no-pesticide treatment (P< 0.05 using Fisher's LSD test). For abbreviations, see Figure 1. Figure 5 from Relyea 2009.



### OverallResults Overall, based onthe results ofthe study, the study author concludedthat atrazine hadno significanteffect (P 

### > 0.05 using Fisher's LSD test) onthe biomass ofany ofthe trophic groups monitored inthis outdoormesocosm experiment. The only significanteffect of atrazine treatmentwhich was observed was the increase in mass at metamorphosis for gray tree frog tadpoles (P = 

### 0.045). The study authorreportedthatthe mechanism for this effectwas unclear. The overall effects ofthe controls and atrazine treatments on biomass oftrophic groups inthe food web are qualitatively illustrated in Figure 6.

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Figure 6. Changes in biomass oftrophic groups in the food web as a result ofexposure to (a) no pesticide (control), (b) solvent control (ethanol) and (i) atrazine. The size ofthe circle and font for each trophic group indicates the qualitative change in the biomass ofthattrophic group. R Basal resources for the two types ofalgae. Figure 6 from Relyea 2009.

10Data Summary Table:

Nommal concentratIOn was 10 IlgIL. Actual concentratIOn (6.4 IlgIL) was measured 1 hour after application. 2 Data analyzed was the average zooplankton abundance over two sampling dates (days 16 and 36). Time Nominal Weighted Magnitude 01 Atrazine Day 01 Average Did Atrazine Recovery Effect Effectrelative Concen- Study Effect Concentration Occur-if Comments to controls tration Occurred (llgIL/ IromDayOto so, when? When Effect

Occurred Chlorophyll aNo effect 6.4 IlgIL Days 16 and Not applicable Not P> 0.05 phytoplankton 35 applicable Biomass- Days 25 and Not P> 0.05 periphyton No effect 6.4llgIL 36 Notapplicable applicable Dissolved Days 10 and Not P> 0.05 oxygen No effect 6.4llgIL 35 Not applicable applicable Survival to Varied, metamorphosis No effect 6.4 11gIL depending Not applicable Not P> 0.05 -R. pipiens on tadpole applicable emergence Mass at Varied, metamorphosis No effect 6.4 11gIL depending Not applicable Not P> 0.05 -R.pipiens on tadpole applicable emergence Time to Varied, metamorphosis No effect 6.4 11gIL depending Not applicable Not P> 0.05 -R. pipiens on tadpole applicable emergence Survival to Varied, metamorphosis No effect 6.4 IlgIL depending Not applicable Not P> 0.05

- H versicolor on tadpole applicable emergence Mass at Varied, metamorphosis -23% increase 6.4llgIL depending Couldnotbe Couldnotbe P= 0.045

- H versicolor on tadpole determined. determined. emergence Time to Varied, metamorphosis No effect 6.4 11gIL depending Not applicable Not P> 0.05

- H versicolor on tadpole applicable emergence 1. minutus Days 16 and Not P >0.05 abundance No effect 6.4 IlgIL 362 Not applicable applicable S. oregonensis No effect 6.4 11gIL Days 16 and Not applicable Not P> 0.05 abundance 362 applicable Ceriodaphnia No effect 6.4 IlgIL Days 16 and Not applicable Not P> 0.05 sp. abundance 362 applicable D. pulex: No effect 6.4 IlgIL Days 16 and Not applicable Not P> 0.05 abundance 362 applicable
I

11Reviewer Comments: No measurements were made to determine ifatrazine and the othernine pesticides were present in the control tanks. The author states that the well water used for filling the tanks did not have detectable concentrations ofany ofthe pesticides used inthe study, although the data ortime at which it was analyzed was not reported. In addition, itwas not stated ifthere was chemical analysis for any ofthe pesticides in the pond waterwhich was added to the tanks as the source ofthe planktonic biota.

The authorreports thatthe concentration ofethanol was 0.003% inthe solvent control tanks and the tanks receiving a mixture ofall ten pesticides but does notreport ifthe concentration ofethanol in the atrazine-onlytreatment was also 0.003%.
Description ofUse in Document: Qualitative

Rationale for Use: The study contributes to the weight ofevidence regarding the potential for effects ofatrazine on aquatic plant communities.

Limitations ofStudy: The results have good applicability to mesocosms ornatural aquatic systems because the experimental mesocosms were outdoors during testing and contained phytoplankton, periphyton, zooplankton, and two species ofamphibians. Actual pesticide (atrazine) concentrations were not monitored throughoutthe experiment, although an initial measured value was reported. Control tanks were not analyzed for atrazine and the othernine pesticides tested. Additionally, it is unclearhow much ethanol was added to atrazine-only treatmenttanks.
Primary Reviewer: Lisa Muto, M.S., StaffScientist, Cambridge Environmental Inc



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

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

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