Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Relyea, R. A. 2009. A cocktail of contaminants: how mixtures of pesticides at low

concentrations affect aquatic communities. Oecologia 159(2): 363-376. (MRID

48261129)

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 (LaC).

Date of Review:

October 15,2009

Purpose of Study:

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, Rana pipiens). The pesticides were studied alone, as a

mixture ofthe five herbicides, as a mixture ofthe five insecticides, and as a mixture of all

ten herbicides and insecticides.

Summary of Study Findings:

The focus ofthe summary is on the mesocosms treated with atrazine alone.

Methods:

The experiment was conducted at the University of Pittsburgh's Pymatuning Laboratory

ofEcology in northwestern Pennsylvania (USA). The experiment used a completely

randomized design consisting of 15 treatments with 4 replicates each for a total of 59

experimental tanks (the solvent control consisted of only 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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1•••••••11

2087817
acetochlor, metolachlor, and 2,4-D), a mixture of all five herbicides, a mixture of all five

insecticides, and a mixture of all ten pesticides.

The mesocosms were 1,300-L cattle watering tanks. The tanks were filled (on May 15-

19,2006) with about 1000 L of well 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, each tank was further supplemented

with 300 g ofleaflitter (primarily Quercus spp.) and 25 g of commercial rabbit chow

(source of algal nutrients and additional surface for algal growth). On June 16th, the

periphyton samplers were added to 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 ad libitum until use in the

experiment. The time at which each species was oviposited differed (ten masses of

leoJ?ard fro~s, collected on March 31 5t; 23 masses of gray tree frogs, collected on May

14 and 17 ), thus the two species were of different ages when added to the experiment.

Added to each tank were 20 tadpoles of each species (H versicolor, and R. pipiens)

chosen from a mixture of all 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 after tadpole 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% in the solvent control tanks and the tanks receiving a mixture

of all 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 water was 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 of atrazine was

6.4 !J.g/L (p.p.b.). The control tanks were not analyzed for any ofthe ten pesticides. The

author states that the well water used for filling the tanks did not have detectable

concentrations of any 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 each tank 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 at the four

cardinal directions and the center ofthe tank using a 0.2-L tube sampler. The five

samples from each tank were combined, filtered (62-!J.m Nitex screen), and then

preserved in 70% ethanol. The zooplankton was classified to species. Twelve taxonomic

groups of cladocerans and copepods were detected. The assemblage was dominated

(91 %) by four species: two species of cladocerans (Daphnia pulex and Ceriodaphnia sp.)

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and two species of copepods (Skistodiaptomus oregonensis and Leptodiaptomus

minutus).

On days 16 and 35, phytoplankton was sampled via collecting 500 mL from the middle of

the water column in each tank. The samples were vacuum filtered through a Whatman

GF/C filter. Chlorophyll a concentrations were determined by fluorometry after the

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 a tub 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) and reweighed

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 shorter time to

metamorphosis. Daily searches of each tank were conducted starting day 21 (July 17) of

the experiment when the first metamorph was observed through day 57 (August 22nd, last

day of experiment). After removal, the metamorphs were held in one liter laboratory tubs

containing moist sphagnum moss until tail resorption was complete (stage 46). The

number of days from the beginning ofthe experiment to stage 46 was defined as time to

metamorphosis. Each animal was euthanized in 2% MS-222, preserved, and weighed.

The response variables for the amphibians were percent survival, mean time to

metamorphosis, and mean mass at metamorphosis.

After most ofthe metamorphs of both species had emerged (determined later to be 97.3%

of all live tadpoles), approximately 120 L ofwater was removed from each tank daily

from day 50 to and including day 57 in order to 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 of all abiotic conditions,

cladoceran and copepod abundance, phytoplankton abundance (chlorophyll a),

periphyton biomass, the survival of gray tree frogs and leopard frogs, and size at and time

to metamorphosis of gray tree frogs. The metamorphosis data could not be collect for the

leopard frogs in two ofthe pesticide treatments due to nearly 100% mortality, so the

leopard frog history responses were analyzed in a separate MANOVA. Each univariate

.response variable was examined using either an ANOVA (for responses that were

measured at the end ofthe experiment: tree frog mass, tree frog time to metamorphosis,

tree frog survival and leopard frog survival) or a repeated-measures ANOVA (for

responses measured twice during the experiment: temperature, dissolved oxygen, pH, the

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abundance of cladocerans (D. pulex and Ceriodaphnia sp.), the abundance of copepods

(S. oregonesis and 1. minutus), phytoplankton (chlorophyll a) and periphyton biomass.

The data was log-transformed when necessary. The zooplankton data was ranked prior to

analysis since it had heteroscedastic errors. The multivariate analyses controlled the

experiment-wise error rate at a = 0.05. To maximize the power ofthe analyses, post hoc

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) between the 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 between the 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).

4
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Figure 1. Changes in the (a) survival, (b) mass at metamorphosis, and (c) time to metamorphosis of

leopard frog tadpoles (Rana pipiens) 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 of all ten pesticides. In

two ofthe treatments, there were no life history data (ND) due to a lack ofsurviving animals. 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). Figure 1 from Relyea 2009.

5
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/q///~,.W/ J"J" ~J"~// %:~~ 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 of all 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 of cladocerans (D. pulex and Ceriodaphnia sp.) and two species

of copepods (8. oregonensis and 1. 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 of copepods [(a) Leptodiaptomus minutus and (b)

Skistodiaptomus oregonensis] and c1adocerans [(c) Ceriodaphnia sp. and (d) Daphnia pulex] in

outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), five separate

insecticides, five separate herbicides, and a mixture of all ten pesticides. Data are mean ± 1 SE,

averaged over two 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 and Periphyton

For chlorophyll a (phytoplankton) measured on days 16 and 35, there was not a

significant difference between the atrazine treatment and the no-pesticide treatments (P >

0.05 using Fisher's LSD test). For periphyton biomass measured on days 25 and 36,

there was not a significant difference between the atrazine treatment and the 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) over time in outdoor mesocosms exposed to no

pesticides (control), solvent only (ethanol), five separate insecticides, five separate herbicides, and a

mixture of all 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). For abbreviations, see Figure 1. Figure 4 from Relyea 2009.

Abiotic Effects

Tank water pH 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 either pH or dissolved oxygen on either date (Figure 5). The

ethanol control differed from the negative control on day 10 for both pH and dissolved

oxygen but was similar to the negative control on day 35.

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Figure 5. Changes in (a) pH and (b) dissolved oxygen over time in outdoor mesocosms exposed to no

pesticides (control), solvent only (ethanol), fIve separate insecticides, fIve separate herbicides, and a

mixture of all 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.

Overall Results

Overall, based on the results ofthe study, the study author concluded that atrazine had no

significant effect (P > 0.05 using Fisher's LSD test) on the biomass of any ofthe trophic

groups monitored in this outdoor mesocosm experiment. The only significant effect of

atrazine treatment which was observed was the increase in mass at metamorphosis for

gray tree frog tadpoles (P = 0.045). The study author reported that the mechanism for

this effect was unclear. The overall effects ofthe controls and atrazine treatments on

biomass oftrophic groups in the food web are qualitatively illustrated in Figure 6.

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(b)SoMInt oontrol (tIIhMD1)

(I) Atruine

Figure 6. Changes in biomass of trophic groups in the food web as a result of exposure to (a) no

pesticide (control), (b) solvent control (ethanol) and (i) atrazine. The size of the circle and font for

each trophic group indicates the qualitative change in the biomass of that trophic group. R Basal

resources for the two types of algae. Figure 6 from Relyea 2009.

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Data 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 Effect relative 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 Not applicable 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 11 gIL 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 Could not be Could not be 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

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Reviewer Comments:

No measurements were made to determine if atrazine and the other nine pesticides were

present in the control tanks. The author states that the well water used for filling the

tanks did not have detectable concentrations of any ofthe pesticides used in the study,

although the data or time at which it was analyzed was not reported. In addition, it was

not stated ifthere was chemical analysis for any ofthe pesticides in the pond water which

was added to the tanks as the source ofthe planktonic biota.

The author reports that the concentration of ethanol was 0.003% in the solvent control

tanks and the tanks receiving a mixture of all ten pesticides but does not report ifthe

concentration of ethanol in the atrazine-only treatment was also 0.003%.

Description of Use in Document: Qualitative

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

potential for effects of atrazine on aquatic plant communities.

Limitations of Study: The results have good applicability to mesocosms or natural

aquatic systems because the experimental mesocosms were outdoors during testing and

contained phytoplankton, periphyton, zooplankton, and two species of amphibians.

Actual pesticide (atrazine) concentrations were not monitored throughout the experiment,

although an initial measured value was reported. Control tanks were not analyzed for

atrazine and the other nine pesticides tested. Additionally, it is unclear how much ethanol

was added to atrazine-only treatment tanks.

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

..4 ' . Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 1!f;M / d

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA PJ~ lo /1 )/~ (

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