Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Rohr, J. R. and P. W. Crumrine. 2005. Effects of an herbicide and an insecticide on pond

community structure and processes. Ecological Applications. 15(4): 1135-1147. (MRID

48261130)

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 ofReview:

October 5, 2009

Purpose of Study:

This study examines the effect ofthe herbicide atrazine and the insecticide endosulfan

(tested independently) on constructed outdoor freshwater pond communities when the

presence of common community members (wood frog tadpoles, adult snails, and caged

dragonfly larvae) was varied.

Summary of Study 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 and reared in outdoor wading

pools (containing aged tap water and covered with 60% shade cloth). They were fed

rabbit chow ad libitum. Clutches were thoroughly mixed prior to experimentation. Adult

snails (PlanorbeUa trivolvis) were collected in early March 2003 from a pond at the

University ofKentucky's Ecological Research Facility. They were raised in pond 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 at the University of

Kentucky's Ecological Research Facility. Prior to the experiment, the dragonflies were

held individually in the laboratory and fed aquatic invertebrates and R. sylvatica tadpoles.

1

11111••111

2087816
The 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 of atrazine, and 10 1lg!L of

endosulfan), two tadpole treatments (presence or absence of lOR. sylvatica tadpoles,

Gosner stage 23-26), two snail treatments (presence or absence of lOP. trivolvis), and

two dragonfly treatments (presence or absence of one caged, late-instar A. junius). The

experimental densities ofthe community members were at the 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, transparent plastic cup (0.296 L capacity) secured to

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 at the University ofKentucky's

Ecological Research Facility. One March 31, 2003, each tub received 9.0 L of aged 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 of phytoplankton, zooplankton, and insect larvae/eggs. The tubs were

covered with 60% shade cloth to prevent predation and allowed to mature for two weeks

in order to establish a periphyton 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. For the solvent control, 246 ilL of acetone was

added to the appropriate tubs. For the atrazine treatment (25 Ilg/L), 27.8 ilL of atrazine

stock solution (8.984 mg atrazine per mL of acetone) 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 order to simulate a second run-off event. The maximum

nominal concentration of atrazine was 50 Ilg/L (i.e., for the second two week period of

the experiment). The authors do not state if a second dose of acetone was added to the

acetone control tubs. Atrazine concentration was not monitored during the experiment.

However, the study authors estimated that the nominal concentration was higher than 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 of each tub so that they were nonlethal to

tadpoles outside the cup but could be visually and chemically detected. Dragonflies were

2
fed two randomly chosen R. sylvatica tadpoles from the wading pool stocks three times

per week throughout the 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 off a microscope slide. Chlorophyll a was

measured using American Public Health Association standard protocols. Samples for

chlorophyll measurements were taken just prior to treatment (all tubs) and then from half

ofthe spatial blocks every other week throughout the 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 half of

the mesocosm), and the number oftadpoles moving during a 15-s interval. Metamorphs

(Gosner stage 42-46) were observed and removed only during the last week ofthe

experiment. The experiment was ended on May 12th for blocks 1 and 2 and on May 13th

for blocks 3 and 4. At experiment termination, surviving snails and tadpoles were

counted and weighed and snail egg masses were counted for each mesocosm. Surviving

tadpoles were euthanized with a 0.01% solution of benzocaine and preserved in 10%

neutral buffered formalin for later determination of Gosner developmental stage. Water

clarity was quantified by the study authors using a scale of 1 to 5. Chironomid larvae

(dipterans) ofthe genus Polypedilum (periphytivores and detritivores) and cladoceran

zooplankton ofthe genus Daphnia (primarily Daphnia pulex; phytoplanktivores) were

the most common invertebrates in the mesocosms. Their abundance was quantified on a

scale of 1 to 5. The study authors proposed that their introduction into the mesocosms

was via the pond water aliquots. The presence ofinsects and crustaceans in genera other

than Polypedilum and Daphnia were noted as the presence or absence of "other insects

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 and block

effects were included when significant. Three separate multivariate analyses ofvariance

(MANOVA) were conducted to analyze tadpole behavior (% hiding: % observed

avoiding cup, % observed moving), tadpole growth and development (mass/tadpole and

Gosner stage), and snail.growth and reproduction (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

per tadpole 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 of dragonfly survival. Repeated-measures ANOVA

3
was used to evaluate the effect of pesticide 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 of other insects and crustaceans and dragonfly

survival were evaluated using logistic regression for a binomial distribution (BR; with a

logit link). When the main effect ofthe pesticide was significant (P < 0.05) in ANOVAs,

the study authors performed a Turkey's honestly significant difference multiple

comparison test (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 Periphytic Algae

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 whether the 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

of dragonfly larvae which survived until the end ofthe experiment (atrazine, 12.5%;

endosulfan, 25%; solvent, 25%; BR, df = 2, 45, i = 1.08, P = 0.582) or the mean number

of days 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 not provide a statistical pairwise comparison between the atrazine treatment

and the control for either dragonfly survival or mean number of days alive.

4
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Figure 1. Effects of pesticide treatments (atrazine, endosulfan, solvent control) on (A) Polypedilum

(chironomid) larvae abundance, (B) Daphnia abundance, and (C) the percentage of mesocosms

containing insects or crustaceans other than Polypedilum and Daphnia. Bars represent means (± SE)

of 32 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.

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 <

0.001, 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, Figure 3).

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Figure 2. (A) Effects of pesticide (atrazine, endosulfan, solvent control) and dragonfly treatments

(presence and absence) on snail mass and (B) effects of pesticide and snail treatments (presence and

absence) on tadpole mass. Values represent means and SE of eight mesocosms, and probability

values are for the interaction between displayed treatments when controlling for significant

predictors [see Tables 1 and 3 of Rohr and Crumrine (2005) for predictors]. Figure 4 from Rohr and

Crumrine, 2005.

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Figure 3. Effects of pesticide 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) of 16 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 Tables 1 and 3 of Rohr and

Crumrine (2005) for predictors]. Figure 6 from Rohr and Crumrine, 2005.

Effects on Wood Frogs

The study authors detennined that the major temporal changes observed in wood frog

behavioral responses through time were primarily due to dragonfly mortality. Since there

were no significant treatment 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 not have a

7
significant effect on the number oftadpoles avoiding the cup compared to the control

(Tukey's hsd, P > 0.05, Figure 4). Atrazine treatment did not appear to affect tadpole

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, P = 0.067, Figure 3). Atrazine treatment did not have a

significant effect relative to the control on tadpole final mass (Tukey's hsd, P> 0.05,

Figure 2).

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Figure 4. Effects of pesticide treatments (atrazine, endosulfan, solvent control) on (A) the percentage

of tadpoles hiding (measured as the percentage of total individuals that were not observed) and (B)

the percentage of individuals observed that were avoiding the cup. Bars represent means (± SE) of 16

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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Figure 5. Interaction among pesticide (atrazine, endosulfan, solvent control), snail, and dragonfly

treatments (presence and absence) on the percentage of tadpoles 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 for Daphnia 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

ofEffect Atrazine Day ofStudy 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 of data mg/m2

);

15% 50 IlgIL collected over Could not be 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 not be be decrease (maximum)! determined. determined. OMR, p= score 0.004

Could not Figure 1

Daphnia No effect 50 IlgIL Unknown2 Could not be be abundance observed (maximum)! determined. determined. Tukey's hsd,

P> 0.05

9
Time Weighted

Magnitude Nominal Average Did Day ofStudy Atrazine

Effect ofEffect 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 not be (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 of data Unknown if

Could not difference Dragonfly 21% 50 ~g/L collected over Could not be be larvae lifespan decrease (maximum)! the four week determined. was

determined. significant study period. (no

statistical

comparison

provided),

SE values

suggest that

means may

not be

significantly

different.

10
Time Weighted

Magnitude Nominal Average Did Day ofStudy Atrazine

Effect ofEffect 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 not be 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 not be

per snail observed (maximum)! (study detennined. be Tukey's hsd, tenninationi detennined. p> 0.05

Figure 2

No effect

judged by

overlapping

Days 29/30 Could not

error bars

Tadpole final No effect 5OIlg!L Could not be for atrazine

observed (maximum)' (study detennined. be

mass treatment tennination)3 detennined. and control.

No

statistical

comparison

provided

Figure 3

Tadpole 5OIlg!L Days 29/30 Could not be 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 not be Could not

be Tukey's hsd, hiding hiding (maximum)' the four week detennined. detennined. P= 0.028 study period. (text)

Mean of data Could not Figure 4 Tadpoles 5OIlg!L collected over Could not be

avoiding cup

No effect (maximum)! the four week detennined. be

study period. detennined. P> 0.05

11
InItIal dose of atrazme was 25 l!g!L, (nommal) then after two 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 .

second two weeks was assumed by the study authors to have been a maximum of 50 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 half ofthe ponds on day 29 and the other half on day 30.

Time Weighted

Magnitude Nominal Average Did

ofEffect Atrazine Day ofStudy 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 of data Could not error bars

No effect 50 l!g!L collected over Could not be 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 Poisson regression

did not distinguish the individual effects of atrazine. In general, these analyses examined

the effects of "pesticide" treatment (atrazine, endosulfan, or solvent control) or the effects

of non-pesticide treatments (e.g. effects ofsnail absence or presence on chlorophyll a

with the "pesticide" treatments combined). The study authors presented the simple effect

of atrazine treatments for some but not all results, that is, a pairwise 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 that mesocosms 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 of atrazine (i. e., reduction of periphyton

chlorophyll a) resulted in indirect effects on snails, tadpoles, and Polypedilum

chironomid larvae.

12
Description of Use in Document: Qualitative

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

atrazine on aquatic plant communities.

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

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 not monitored throughout the

experiment. Although a nominal dose was reported, exposure was complicated because a

second dose of 25 ~g atrazine/L was made two weeks after the initial application of 25

~g!L and the study authors reported heavy rainfall (timing not reported) during the

experiment which would have diluted the pesticide in the mesocosms. Nominal

concentrations were verified in the stock solution and atrazine should have been stable

over a period oftwo weeks; therefore, the study authors made a conservative assumption

that 50 ~g atrazinelL was in each mesocosm It was not clear ifthe solvent control pond

received a second application of acetone after two weeks; effects of acetone would have

been underestimated if a second application was not made. There was no effort to

determine if atrazine or other contaminants were present in the source water for the

experiments or the control mesocosms. Although there was an acclimation period for the

periphyton, it does not appear that there was an acclimation period for the snails,

tadpoles, and dragonflies. In addition, it is unclear whether wood frog tadpoles, adult

snails, and dragonfly larvae may have been previously exposed to other contaminants

(including pesticides) prior to the experiment, given that no previous contaminant history

ofthe collection sites was provided.

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

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA '1'If;~/4

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA ~Sl.. lc (l3/\t

13