Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

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natural stressors in amphibian community mesocosms. Ecological Applications. 13(3):

829-841. (MRID 48261104)

Purpose of Review:

Determine 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 (LOC).

Date of Review: September 4, 2009

Purpose of Study:

This study examines the effects of atrazine and carbaryl (study design included individual

and combined exposure treatments) on phytoplankton and four amphibian species in

outdoor mesocosms. Tadpole density and photoperiod were also manipulated.

Summary of Study Findings:

The focus ofthe summary is on atrazine treatments (treatments including carbaryl are not

reported).

Methods:

Egg masses were collected for southern leopard frogs (Rana sphenocephala; 3 egg

masses) and spotted salamanders (Ambystoma maculatum; 21 egg masses) on March 14,

2000 and March 27,2000, respectively, from the Basket Wildlife Area in Boone County,

Ashland, Missouri. Egg masses were also collected for American toads (Bufo

americanas; 7 egg masses) and small-mouth salamanders (Ambystoma texanum; ca. 30

egg masses) on March 30,200 from the Forum Nature Area in Boone County, Columbia,

Missouri and April 9, 2001 from the Basket Wildlife Area, respectively. Eggs were

hatched in the laboratory at 23-25°C and held until all larvae were free-swimming.

Clutches were mixed within each species to homogenize genetic variation.

1 111••••111

2087802
The aquatic communities were prepared by filling polyethylene cattle tank ponds (1.85 m

in diameter, 1480 L volume) with 1000 L oftap water, 1 kg ofleaflitter from a mixed,

deciduous forest and plankton from natural ponds (500 mL of plankton per pond at six

different times) in mid-March 200 and 2001. The plankton populations were wellestablished prior to addition of larvae. The aquatic communities were maintained

outdoors during the experiments to simulate natural conditions. In order to protect the

test systems, the cattle pond tanks were located inside a fenced field and covered with

mesh lids during the experiments.

For Experiment I, the effects of competition, atrazine and carbaryl were studied by a fully

crossed design with three replicates (36 ponds) where initial anuran density was either

low (20 tadpoles/lOOO L) or high (60 tadpoles/lOOO L), carbaryl concentration was 0,3.5

or 7.0 mg/L, and atrazine concentration was 0 or 200 f.lg/L. Twelve spotted salamanders

were also in each pond. Carbaryl (liquid Sevin, 21.3% carbaryl) and atrazine (liquid

Aatrex, 40.8% atrazine) were added via mixing with 5 L of pond water and pouring

evenly over the pond surface without stirring. Uncontaminated pond water (5 L) was

added in the same manner to each control pond. The herbicide/insecticide treatment was

added between 1000 and 1100 hours Central Standard Time (pond water: pH 7.9 ± 0.01,

14.6 ± 0.06°C). Ponds with only carbaryl were sampled (triplicate 2-L aliquots from

each ofthe three ponds) at 1, 24, 48 and 96 hours post-treatment. Samples from each

pond were combined, and an aliquot (20 mL) was refrigerated for analysis. Ponds with

only atrazine were sampled (triplicate 2-L aliquots from each ofthe three ponds) at 1, 15

and 57 days post-treatment. Samples from each pond were combined, and an aliquot (l

L) was refrigerated for analysis. The half-lives were determined after HPLC analysis via

linear regression ofthe determined amounts: ca. 4.5 days for carbaryl and 34 days for

atrazine. After 56-58 days (39-41 days of exposure), the experiment was terminated.

Most ofthe tadpoles and salamander larvae had not reached metamorphosis. Mean body

mass, developmental stage, snout-vent length (SVL, salamander larvae only) and pond

survival was determined. Data were analyzed for effects of carbaryl, atrazine, density

(for tadpoles) and all interactions on mass, developmental stage and SVL using

multivariate analysis of covariance (MANCOVA) for ponds with both species.

Univariate analysis of covariance (ANCOVA) was used for determining which responses

were the strongest contributors to the multivariate effects. Survival was analyzed using

analysis of variance (ANOVA). ANOVA was also used to determine differences in the

control ponds with one or both species. To normalize data and stabilize variances, the

study authors angularly transformed all proportion data, log transformed mass and SVL,

and used rank procedure (PROC RANK) on developmental stage data before analyses.

Conservative pair-wise comparisons for significant main effects were performed using

Scheffe's multiple comparison tests.

For Experiment II, the effects of hydroperiod, atrazine, and carbaryl on amphibians

reared through metamorphosis were studied by a fully crossed design with four replicates

(32 ponds) where hydroperiod was either constant or dried with a movable standpipe,

carbaryl concentration was 0 or 5.0 mg/L, and atrazine concentration was 0 or 200 f.lg/L.

Twelve small-mouthed salamanders and 45 American toads were also in each pond;

2
initial anuran density was not variable since the results ofthe first experiment indicated

that competition between anurans and salamanders had no effect. Carbaryl (liquid Sevin,

22.5% carbaryl) and atrazine (liquid Aatrex, 40.8% atrazine) were added. The

herbicide/insecticide treatment was on day 8 (pond water: pH 7.7 ± 0.03, 13.3 ± 0.04°C).

Ponds with only carbaryl were sampled (triplicate 2-L aliquots from each ofthe three

ponds) at 1 and 48 hours post-treatment. Samples from each pond were combined, and

an aliquot (20 mL) was refrigerated for analysis. Ponds with only atrazine were sampled

(triplicate 2-L aliquots from each ofthe three ponds) at 1 day post-treatment. Samples .£'..~_.__ __1 ..1 __ • __ 1_.: ...J ..1 _.__1.: 4.- /1 T'\ .£'..•.: ._.L_..J .£'_~ 1 .:_

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Atrazine concentration was confirmed as 197 ug/L. After 88 days (80 days of exposure),

the experiment was terminated. Mean body mass, time to metamorphosis, and pond

survival was determined. Toad data were analyzed for effects of carbaryl, atrazine,

hydroperiod and all interactions on mass and time to metamorphosis using MANCOVA;

survival was the covariant. ANCOVA was used for determining which responses were

the strongest contributors to the multivariate effects. Survival was analyzed using

analysis of variance (ANOVA). Salamander data were analyzed for effects of carbaryl,

atrazine, hydroperiod, carbaryl by hydroperiod, and atrazine by hydroperiod interactions

on mass and time to metamorphosis using MANCOVA; survival was the covariant.

ANCOVA was used for determining which responses were the strongest contributors to

the multivariate effects. Prior to analysis, the following transformations were performed:

angular transformation (all proportion data) and log transformation (mass and time to

metamorphosis).

Chlorophyll content was measured by taking a l-L composite sample (from three 2-L

samples) from each pond. An aliquot (l00 mL) was filtered and incubated with 15 mL of

neutralized 90% acetone in the dark at 5°C for 24 hours. Chlorophyll was measured

using fluorometry. Water samples were taken before chemical application on day 15 and

then on days 22,29, and 42 (all post-application) for Experiment 1. For experiment II,

water samples were taken before chemical application on day 8 and then on day 50 (42

days of exposure). Data were analyzed for effects oftime, carbaryl, and atrazine for both

experiments in addition to density (Experiment I) or hydroperiod (Experiment II) using

ANCOVA.

Results:

Experiment I: effects ofcompetition, atrazine, and carbaryl on larval amphibians

Atrazine exposure at 200 ug/L did not influence the mass, SVL, developmental stage, or

survival ofspotted salamanders. There was a significant interaction of atrazine and

carbaryl on SVL that the authors attributed to atrazine acting as an ameliorating agent for

the carbaryl treatments. Similar observations occurred on body mass and developmental

stage, although the interaction was not significant (P>0.05). For leopard frogs, atrazine

exposure did not significantly influence survival. However, survival was significantly

influenced by an atrazine by population density interaction where atrazine exposure

decreased survival in the high-density ponds but not the low-density ponds relative to the

3
control ponds (treatments without atrazine). The multivariate responses of leopard frogs

were significantly affected by atrazine exposure; mass significantly decreased with

atrazine exposure compared to the controls (P=0.0052). Results ofthe competitive

controls indicated that no difference occurred between spotted salamanders reared alone

or with southern leopard frogs.

Experiment II: effects ofhydroperiod, atrazine, and carbaryl on amphibians reared

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Multivariate analyses on salamanders indicated that atrazine exposure and hydroperiod

(individually and their interaction) significantly affected mass and time to

metamorphosis. The effect of atrazine exposure was attributed to increasing time to

metamorphosis. Time and mass to metamorphosis were affected by the atrazinehydroperiod interaction, resulting in longer larval periods in constant hydroperiods and

smaller mass at metamorphosis in drying hydroperiods. For American toads, multivariate

responses were significantly affected by atrazine exposure. Atrazine exposure

significantly reduced toad mass at metamorphosis.

Chlorophyll content

In Experiment I, atrazine exposure had a significant effect on chlorophyll content. After

12 days of exposure, significantly lower chlorophyll content was observed in ponds with

atrazine exposure of200 f.lg/L (1.31 ± 0.48 f.lg/L) versus the control ponds (3.88 ± 0.48

f.lg/L; F1,24 = 15.65, P = 0.0006). Examination of Figure 1 suggests that the chlorophyll

content (3.88 f.lg/L) reported by the authors for control ponds included all ponds without

atrazine (i.e., some ponds contained carbaryl). The authors state that there was no

difference between treatments and controls on day 42 (after 25 days of exposure);

however, it was not stated if 'no difference' meant lack of a statistically significant

difference. However, chlorophyll content was higher than controls after 25 days of

exposure (day 42; See Figure 1 below).

In Experiment II, the effects of atrazine exposure on chlorophyll content were not

discussed, but Table 5 (summary in Table 1 below) ofthe study report indicates that

atrazine did not have significant effects on chlorophyll at the end ofthe study (P = 0.62).

However, chlorophyll content was only measured after 42 days of exposure in

experiment II. In experiment I, chlorophyll content had already recovered after 25 days

of exposure so it is reasonable tp assume that any effect in experiment II may have been

missed due to insufficient sampling. No figures were provided which illustrated the

chlorophyll content through Experiment II.

4
Table 1. Summary of repeated-measures ANOVA for effects of time and atrazine exposure on

chlorophyll content in pond water in Experiments I and II (from Table 5 in Boone and James, 2003).

Wilks'

Source of variation lambda df F P

Experiment I

Time 0.0351 3,22 201.69 0.0001

Time x atrazine 0.6858 3,22 3.36 0.0371

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Time 0.1974 1,24 97.59 0.0001

Time x atrazine 0.9895 1,24 0.25 0.6190

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Time from chemical dosing (d)

Figure 1. Pond chlorophyll measures for an atrazine-by-carbaryl interaction over time from

Experiment I. Numbers represent the concentration of carbaryl (in milligrams per liter) and atrazine

(in micrograms per liter), respectively, to which the ponds were originally exposed (Figure 10 in

Boone and James, 2003).

5
Data Summary Table:

Durmg expenment 1, atrazme was measured at day 1 (207 uglL) followmg exposure.

Time

Nominal Weighted

Magnitude Atrazine Average Did

Effect ofEffect Concen- Day ofStudy Atrazine Recovery Comments relative to tration Effect Occurred Concentration Occur-if

controls (pg/L) from Day Oto so, when?

When Effect

Occurred f"\ ___ ",,-.__ 1 1 ____ 1 _ .. _____

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higher than all

treatment levels at

Day 12 in

Experiment I

(Figure 1).

However, there

were no apparent

decreases in

chlorophyll in

other treatment

levels with atrazine

and chlorophyll in

the atrazine-only

treatment was

higher that the

control on Day 25.

Reduction in Not

chlorophyll None 2001 Not applicable Not applicable applicable There were no

content apparent effects in

Experiment 2

(Table 1) although

this analysis

appears to have

included treatments

containing both

atrazine and

carbaryl, thus is not

considered for the

summary table (see

reviewer

comments).

No experimental

variability was

available for this

effect. 1

Reviewer Comments:

The study was mainly an amphibian study in which chlorophyll content was measured in

order to examine effects of atrazine on amphibians via diminishing their food source, the

aquatic plant life. The study authors were looking for statistical trends and relationships,

and a limited amount ofthe data used in the analyses was reported.

6
The study authors reported that previous studies by other groups had shown that

phytoplankton was diminished by atrazine by 50-200 !lg/L although recovery of

phytoplankton was common.

It appears that most ofthe statistics presented did not look at the individual effects of

atrazine exposure, that is, in the absence of carbaryl exposure. Therefore, the atrazine

exposure results as presented in the article may be confounded by inclusion in the

analysis oftreatments containing atrazine and carbaryl and 'control ponds' without

atrazine but containing carbaryl. There was little raw data presented to tease out the

individual effects of atrazine, therefore the results should be interpreted with caution.

Background concentrations ofthe pesticides in neither the source water nor the controls

were reported.

The treatment was limited to one test concentration of atrazine (200 ug atrazine/L).

Atrazine was applied as part of a formulated product (Aatrex). It is unknown ifthere

were any effects due to other ingredients in the formulation.

There was an acclimation period but its duration is unclear. Natural phytoplankton

communities appear to have been added to each mesocosm twice. An initial 500 ml of

plankton was used to establish the ponds and acclimate. Then, the pesticides were mixed

with 5 L of pond water of unknown source and added to the mesocosms. It is unclear if

the pond water used for adding the chemical treatment had phytoplankton in it and ifso

how much. Based on the mean chlorophyll concentrations presented in Figure 1,

chlorophyll concentrations appear similar prior to and a few days after application. It

would be helpful to know more about the methodology to assess ifthe results may have

been confounded by addition of more phytoplankton with pesticide application.

Description of Use in Document: Qualitative

Rationale for Use: There was one endpoint relevant to aquatic plants measured in this

study (chlorophyll content). There was an observation ofreduced chlorophyll relative to

controls at Day 12; however, the reduction was somewhat slight and transient (effect

observed at Day 12, but not at the next measurement on Day 25). In addition, there was

no indication ofstatistical significance at Day 12 (i.e., if atrazine-only treatments were

statistically analyzed separately) and there was no indication of variability. Chlorophyll

content was higher than controls at Day 25. Therefore, this observation was not

considered to be treatment related.

7
Limitations of Study: Results have good applicability to mesocosms or natural aquatic

systems; however, the focus ofthe study was the effects of atrazine and carbaryl on the

amphibian community and not the effects on phytoplankton. Also, the study design, the

analysis, and the presentation and discussion ofresults ofthe study focused on interaction

of factors, as opposed to the single effect of atrazine. No statistics or information about

variation among replicates was provided regarding atrazine-only treatments. In addition,

lake water was used, and there was no indication of whether or not atrazine may have

been present in controls.

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

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA iflt0,,//1MA;/

Tertiary Reviewer: Brian Anderson, Biologist, EPA

8