Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

DeNovelles, Jr., F., W. D. Kettle, C. H. Fromm, M. F. Moffett, and S. L. Dewey. 1989. Use of

Experimental Ponds to Assess the Effects of a Pesticide on the Aquatic Environment.

Department of Systematics and Ecology, University of Kansas. Entomological Society of

America. Miscellaneous Publications No. 75: 41 - 56 (MRID 47543608).

Purpose of Review (DP Barcode or Litigation):

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:

November 24,2009

Summary of Study Findings

Purpose: This study examines the potential effects of atrazine to aquatic plants and animals in

experimental ponds (mesocosms).

Methods: Two studies with experimental ponds were conducted. The first study began in July

of 1979, lasted 136 days, and used six 0.04-ha (0.099 acres) experimental ponds filled to a depth

of2.1 meters (6.9 ft) representing 0.68 acre-feet of water. The second study began in June 1981,

lasted for 805 days, and utilized ten 0.04-ha experimental ponds filled to a depth of 2.1 meters.

All ponds were located at the University ofKansas Nelson Environmental Study Area near

Lawrence, KS.

Prior to any treatment with atrazine, all ponds were allowed an undisturbed year in order to

effectively establish a benthic plant and animal community. Then, the ponds were drained and

immediately refilled to a depth of 2.1 meters from an adjacent OA-ha pond maintained by well

water.

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II1IIIIIII

2087828
The ponds were stocked with 10 gizzard shad (Dorosoma cepedianum; 15.2 cm total length

(TL», 20 channel catfish (Ictalurus punctatus; 9.4 cm TL) and 50 bluegill sunfish (Lepomis

macrochirus; 5.1 cm TL) one week after filling and a few weeks before atrazine was added. The

same three species were used in both the l36-day and 80S-day studies. In addition, grass carp

(N=4 per pond) (Ctenopharyngodon idella; 18 - 23 cm) were added to the 10 ponds ofthe 805-

day study as a macrophyte grazer after the first year. Adult fish of all species were blocked from

the southwest comer of each pond (approx. 5% of the pond area) using a 2.5-cm mesh poultry

fence in order to separate the effects of atrazine on macrophytes from grazing by grass carp.

In the 136-day study, a commercial grade (formulated) atrazine (CO-OP Liquid Atrazine®; 41 %

active ingredient) was diluted in 20 liters of pond water and then dispensed through a hose

moved throughout the pond. In the 80S-day study, a suspension of reagent (technical) grade

atrazine (97% ai) was dispensed by adding suspending atrazine powder in 4-liter volume

portions into the ponds just below the surface ofthe water from a boat. Treatment solutions were

passed through fine screens so as to finely disperse particles for rapid dissolution.

The 136-day study consisted of two control ponds and two atrazine-treated ponds each at

nominal concentrations of 20llg/L and 500 Ilg/L. The 80S-day study started with two control

ponds, four ponds with 20 Ilg/L concentration (two formulated product, two technical grade),

three ponds with 100 Ilg/L concentration, (one formulated product, two technical grade), and one

pond with 200 Ilg/L concentration (formulated product and technical grade combined in a 1: 1

ratio) for the first year beginning in May of 1981. In the second 80S-day study, ponds were

retreated at the beginning of the second and third years. Beginning in the second year of the

study, formulated product was replaced with technical grade atrazine. The two ponds formerly

treated with formulated product at 20 Ilg/L were increased to 500 ~tg/L; the one pond formerly

treated with formulated product at 100 Ilg/L was increased to 200 Ilg/L, and the other pond

treated at 200 Ilg/L with a 1: 1 mix of formulated and technical grade was thereafter treated with

technical grade alone at 200 Ilg/L. The paper does not state what schedule was used for

'measuring atrazine concentrations; however, Figure 2 of the study suggests that samples may

have been collected at roughly 3-wk intervals.

Temperature, oxygen, and light penetration profiles, along with measurements of turbidity,

alkalinity, pH, and nutrient conditions for nitrogen and phosphorus were measured weekly one

month per pond prior to atrazine treatment then once or twice monthly through the following

Spring. Water quality samples were taken from water samples collected within 8 cm of the

pond floor from a walkway which extended one-third of the way toward the middle of each

pond.

Tolerance of phytoplankton to atrazine was determined in a laboratory by incubating water

samples with phytoplankton from all ponds for 24 hours with and without an additional 100 Ilg/L

of atrazine, while being gently rotated for mixing. Relative abundance of submersed

macrophytes was estimated using a modified version of the grapnel method where a rake

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attached to long pole is pulled along I m of the pond bottom and the contents are brought to the

shore for identification. Relative abundance of emergent macrophytes (cattails) was determined

by observing aerial photographs taken in August and September of 1982 and 1983. Values were

expressed as a percentage of shoreline covered. Browning of cattail blades was determined by

examining ten 25-cm quadrants within a cattail stand and counting the number of brown and

green blades.

Phytoplankton production, chlorophyl biomass and species abundance were determined from

water column samples.

Zooplankton was sampled with one vertical net haul taken at night using a IS7-llm mesh net

with a 29-cm opening. Insects were sampled every week from May through September in 1982

and 1983 using emergence traps. These values were expressed as total number captured per

sample. The 136-day study followed only the phytoplankton and zooplankton responses and did

not include detailed macrophyte and insect analysis whereas these parameters were measured in

the 80S-day study.

Fish and tadpole abundance was sampled by lowering the ponds to a depth of 0.75 meters. The

water was drained through a fine screen to ensure that all organisms were captured for sampling.

After this, each pond was completely drained to capture any remaining organisms.

Results

Water Quality

Alkalinity in the ponds ranged from 100 240 mg/L [as CaC03]; differences in alkalinity

between ponds was generally less than 60 mg/L. There were "some" declines in both dissolved

oxygen (DO) and pH over the first 2 weeks of atrazine addition in both the 136- and 80S-day

studies that the authors attributed to decreased photosynthesis. In the highest atrazine test

concentration (500 flg/L), DO is reported to have remained above 4.5 mg/L throughout the study,

and ponds did not differ by more than 1 pH unit with pH values ranging between 7 9 during

May - September. Water temperatures ranged from 20°C to 30°C during the May -September

sampling period. Turbidity was the only parameter that is reported to have been "highly"

correlated with atrazine concentration (r=0.82); turbidity increased with increasing atrazine

concentrations.
Atrazine Concentration

Initially measured atrazine concentrations were within 10% of nominal. By 6 months measured

concentrations had declined to 70% of nominal and by 12 months, atrazine had declined to 25%

of nominal in the 805~day study (Figure 1). A similar rate of decline was reported for atrazine

in the 136-day study.

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-'0.....0--6..... 5-0-;-'01.0-1.-7 5'-0--6-'-0o..J

Time (!lays)

Figure 1. Measured atrazine concentrations for the 805-d study. Values plotted as treatment means standard

error. Slashes along x axis indicate times of atrazine treatments. Figure from DeNoyelles et ai. 1989.

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Table 1. Summary of Effects of Atrazine in Aquatic Community.

Time

Weighted Did

Magnitude of Nominal Day ofStudy Average Recovery

Effect Effect relative to Atrazine Effect Atrazine Occur if Comments Concentration Concentration so, when controls (pg/L) Occurred fromDayOto (see

When Effect comment)?

Occurred

Recovery was not

specified at this

level, but assumed

to be 2::3 weeks,

given the lower

magnitude of effect

and recovery at the

50% (p$O.OS; higher

136-d and 80S-d 20 ~g/L :S3 weeks 16-20 ~g/L Yes concentrations.

studies) Range provided for

TWA (up to 3

weeks); further

detail was not

Phytoplankton provided and this

treatment was not biomass and graphically production represented in

('4C uptake) Figure 3, below.

(80S-d) 100 ~g/L 0 100 ~g/L Yes Recovery 2::3 weeks

Recovery 2::2

weeks; determined

~90% (80S-d) 200 ~g/L 0 200 ~glL Yes

from Figure 3 in

the study (and

below).

I

~90% (136-d i

I i 500 ~g/L 0 500 ~g/L IYes Recovery 2::3 weeks I and 80S-d)

L I I ._.._ ..

INot "po,"d 1 20 ~g/L Not reported Not calculable Not Reported for the

reported 136-d study; p

Change in ' ".."_.~._~-~ values for this

endpoint were not phytoplankton reported. species

composition Not reported 500 ~glL Not reported Not calculable Not There was a shift

reported away from

I

dominant green

I algae and I

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Time

Weighted Did

Magnitude of Nominal Day ofStudy Average Recovery

Effect Effect relative to Atrazine Effect· Atrazine Occur- if

Concentration Comments

controls Occurred Concentration so, when

(pg/L) from Day 0 to (see

When Effect comment)?

! Occurred

I .. _-_._--

dinoflagellates to

flagellated algae

and golden algae.

Tolerance was

evident.

I

I

""giL

I

reported Not calculable Magnitude of

effects estimated

! 75-80% reported

from Figure 7 in

the study document

(Figure 2 below)

and is relative to

pre-treatment

Emergent abundances;

macrophyte No includes areas with

cover (i.e.,

and without grass

Typha) carp.

~90% 5OOIlg/L Not reported Not calculable

Results are from

the 80S-d study and

effects were

statistically

significant at

p:SO.OS.

50% - no grass Magnitude of

carp 20 f.1g/L N. •"f-'UI ,"'U Not calculable effects estimated

carp

from Figure 7 in

the study document

and is relative to

carp

pre-treatment

Submerged 100 JIg/L Not reported ""I.." l"h1.. abundances (Figure

macrophyte 100% - grass No 2 below).

cover carp

I

(with or I Results are from

grass 200llg/L Nat reported Not calculable the 80S-d study and

carp) effects were

statistically

100% (with or Not I Not calculable I

significant at

without grass

In<i1 n.:;

!
Effect

Magnitude of

Effect relative to

controls

i

Nominal

Atrazine

Concentration

(llgIL)

Day ofStudy

Effect

Occurred

Time

Weighted

Average

Atrazine

Concentration

from Day 0 to

When Effect

Occurred

Did

Recovery

Occur- if

so, when

(fee

comment)?

Comments

Balded llues are those data

Biological Data

I

spe'~lrlied·1 in the "Cosm Data Points and Exposure PIVIIIC~" spreadsheet.

There was a statistically significant (p<0.05) decrease In photosynthesis measured In

phytoplankton in ponds treated with atrazine. According to the author, these plants are directly

affected by atrazine, which inhibits the electron transport in photosystem II by inactivating

electron transport carrier proteins upon occupying a specific binding site in the protein structure

ofthe chloroplast. Potentially, this reduction in photosynthesis could result in a decrease in total

biomass ofthe aquatic system.

According to the study authors, ponds treated with 20 jlg/L did not develop communities of

phytoplankton with tolerance to higher concentrations of atrazine as evidenced by initial declines

in biomass in ]982 when 500 jlg/L was added to two ponds with concentrations of 20 jlg/L).

Phytoplankton declined in biomass and production at all concentrations during the first three

weeks of exposure in both studies. Phytoplankton declines of 50% occurred at 20 jlg/L while

there was near complete cessation of carbon intake and biomass declines to 10% of control pond

levels in higher (500 ug/L) atrazine treatment concentrations. However, after that three week

period, the biomass and production recovered, and returned to the levels ofthe control pond at all

treatment concentrations. It was determined that all persisting phytoplankton were tolerant of or

resistant to the herbicide.

In the 136-day study, changes in species composition were recorded with a shift away from the

originally dominant green algae (Chlorophyceae) and dinoflagellates (Dinophyceae) to

flagel1ated algae (Cryptophyceae) and golden algae (Chrysophyceae). According to the authors,

the ponds receiving atrazine at all concentrations became dominated by organisms resistant to or

tolerant to atrazine exposure. Phytoplankton from ponds treated with 100 J.lg/L returned to

control pond levels of carbon intake within 3 weeks and did not exhibit a decline with the next

treatment of atrazine the following year. However, the pond treated with 20 J.lg/L exhibited an

initial decline following the second year oftreatment when atrazine concentrations were raised to

500 jlg/L. According to the study authors, the decline in carbon uptake following addition ofthe

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500 Ilg/L dose indicates that tolerant communities were not present in the pond initially treated

with 20 Ilg/L atrazine.

Phytoplankton collected over the 80S-day study were exposed to an additional 100 /lg/L of

atrazine and incubated for 24 hours. Phytoplankton from all atrazine-treated ponds exhibited less

reduction in carbon uptake than control pond phytoplankton. According to the study authors,

this result provides further evidence of the shift toward more tolerant species of phytoplankton

exposed to atrazine.

The authors concluded that direct toxicity of atrazine appears to be minimal to animals for the

concentrations tested in the mesocosms from this study. There were "some declines" in numbers

of grazing zooplankton following the initial declines in phytoplankton biomass in the 136-d

study, but the zooplankton returned to control pond levels within three weeks; these declines

were attributed to food availability and not to the direct effect of atrazine. However, because of

the direct effects on photosynthetic organisms, there may be indirect effects on animals that

consume these organisms.

The biomass of channel catfish at the end of the 80S-day study was unaffected by atrazine

treatments. Grass carp were lost from one control pond and one 500 /lg/L treatment after the

second year of the study. Grass carp biomass declined at > 100 Ilg/L; according to the author,

grazing by grass carp had a greater effect on macrophyte abundance with increasing atrazine

concentration.

Percentage of cattail cover, submersed macrophyte relative abundance, tadpole final biomass,

emergent insect numbers, grass carp and bluegill final biomass were significantly affected

(p<O.OS) by the atrazine treatment (Figure 1). These measurement endpoints showed significant

(p<0.10) negative linear trends with increasing atrazine concentrations. Only cattails in the area

where grass carp were present were not affected. Declines in biomass of submersed macrophytes

were most evident at atrazine concentrations 2100 Jlg/L.

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lOll

!l1l6"~fi'H:O "~C;f!OP"YT£S

-. wlih ",UfUrp

... -0'. n,tr ,.tttl U,~

o

.,

r

.+,

\

\

\

\ """"

\-- '~-- ... -- "" ... ., .. .._,,-........-_-i!tlOG

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4

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a 0

laa

Figure 2. Depiction of percentage of cattail cover, submersed macrophyte relative abundance, tadpole final

biomass, emverging insect numbers, grass carp final biomass and bluegill final biomass in ponds treated with

0,20,100,200 and 500 f.1g atrazine/L in 80S-day study. Figure from DeNoyelles et al. 1989.

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--_...._--_..._--- -----------------------
control _

n

Ii "'.

II f l\ J f Ii f I r . I il ,4 I 11/f\ II 1, \i I f V i

\1

control _

100 IJg/L......

198\

control _

100 ug/L"'.'

200 ..""x

1982

control -

500Ug/L ......

1982

o 10 20 30 o 10 20 0 10 20 30

DAYS

o 10 20 30

Fig. 3. Cllrbcm-14 uptake by sumt}le, of phytoplankton communities li'om mntrol (solid line) and atrH:t.ine lrented (doHcJ

line) ponds for the first 30 tI of exposure in the 136-d study lIlI(1 the first 25 lind 35 d of expOlmre in years 1 and 2,

respectively, of the 805-d study. rn 1982, the 100- and 200·fJ.W1ltt>r pllnds shown here r<.'Crlved their second year's dOSt'.

lind 500 fLg/liter wns lidded to tWIl previolls 20'fLwliter ponds, CllrbOlHlptllkc is plotted as lhe ll1j;>an (;-loI counts per

minute (cpm) from four samples from each pond.

Declines in tadpole biomass, primarily bullfrogs (Rana catesbiana) were evident by the end of

the 80S-day study even at the lowest atrazine concentration of 20 I-lg/L; however, the report does

not state the extent ofthe decline.

Reviewer Comments

The area surrounding the study sites is not completely characterized in terms of previous

chemical treatments. However, a companion study by DeNoyelles and Kettle (1980) indicates

that the ponds received no watershed runoff and, therefore, could not be affected by local

herbicide runoff.

It is stated that prior to atrazine addition, the experimental ponds were drained and then

immediately refilled with water from an adjacent OA-ha pond maintained by well water. It is not

stated whether this water was tested to ensure that it was not contaminated with any pollutants.

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If this were the case, it could potentially lead to false conclusions. No analyses were conducted

to either qualitatively or quantitatively describe whether atrazine or other pesticides/chemicals

were present in the control or treatment ponds prior to study initiation. The study does not

mention whether any atrazine degradates were measured.

Ponds were allowed to equilibrate/acclimate for an entire year prior to use; however, the ponds

were drained just prior to initiating the study. It is unclear what was accomplished during the

acclimation period since draining the ponds would likely have disrupted the aquatic community

in spite of the authors' belief that it "probably does not alter the already established benthic plant

and animal communities". The methods section does not state how much time was allowed for

the aquatic community to re-establish itself after draining; however, the text implies that 3 weeks

may have elapsed. If the ponds were to be drained prior to the study, then information could

have been collected on vertebrate, invertebrate, and aquatic plant communities and this

information could have in turn been used to compare recovery data.

In the first study, atrazine was already dissolved in the concentrated stock solution; however, in

the second study, atrazine powder was suspended and not dissolved. It is uncertain whether the

atrazine in the second study ever went into solution. Although the suspension was passed

through a fine-mesh screen to ensure that the compound was dissolved, it is possible that the

screen acted as a filter that prevented larger suspended particles from entering the pond

ingredient. According to the authors, measured concentrations differed by less than 10% of

nominal [at study initiation] and were 70 and 25% of nominal after 6 and 12 months,

respectively.

In the second year ofthe multi-year study, the researchers stopped using formulated product and

relied on technical grade atrazine. Nominal treatment concentrations of the one pond treated

with formulated product at 100 JlglL were increased to 200 Jlg/L with technical grade, while the

two ponds at nominal treatments of20 Jlg/L with formulated product were increased to 500 JlglL

with technical grade in the second and third years of the study. Presumably then, the study

consisted of two ponds each at 20, 100, 200, and 500 Jlg/L for the last two years of the study

with all of the ponds treated with technical grade atrazine. Thus, test chemical and exposure

concentrations for three ofthe test ponds changed in the middle ofthe study.

With only two replicates at each treatment, the study has a low power to detect any treatment

effects should they occur.

Based on the caption to Figure 2 ofthe paper, a "small amount" of atrazine was required to bring

the treatment ponds up to the nominal concentrations in the third year ofthe study. This implies

that each of the ponds was treated to raise the treatment concentrations back to nominal rather

than retreating each the ponds at the original rate. Thus, it is uncertain how much atrazine was

added to each ofthe ponds in the second and third years ofthe study.

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It is unclear from the longer second study whether ponds were retreated using the same method

for dispersing the chemical as that used in the first year.

The basis for the stocking rates of fish used in the study is not discussed in the paper. Rather, the

reader is referred to an earlier publication by DeNoyelles.

The methods used for measuring phytoplankton productivity, chlorophyll biomass and species

abundance are not described in the paper. The frequency ofthese analyses is not discussed.

Considering that ponds were in close proximity to one another and were all filled with wellderived water from adjacent ponds, there was a relatively large range in alkalinity (100 - 240

mg/L). It is unclear why these differences in alkalinity existed; however, such differences could

impact primary productivity and confound interpretation of the study results since differences in

productivity could influence dissolved oxygen, pH and turbidity.

Although the authors report that dissolved oxygen (DO) concentrations did not drop below 4.5

mg/L, this measurement was for surface water where diffusion would have been greatest. More

reliable measures of DO would have been at several depths in the water column.

Phytoplankton in treated ponds recovered to levels that were comparable to controls after three

weeks, and the authors hypothesize that the recovery was due to the growth of algae that were

tolerant to atrazine given that atrazine levels had not declined substantially in the treated ponds.

In the shorter 136-d study, changes in species composition were recorded with a shift from

originally dominant Chlorophyceae (green algae) and Dinophyceae (dinoflagellates) to

Cryptophyceae (flagellated algae) and Chrysophyceae (golden algae). However, in the 805-d

study, no data were provided on the species of phytoplankton present and whether any species

was displaced by another. Replacement of one species by another could have marked effects on

aquatic communities where foraging species may not be as resilient to such changes in their diet.

It is mentioned in the study that tadpoles were collected trom the pond; however, no data on the

potential effects of atrazine on the tadpoles are provided; it is only stated that atrazine had a

minimal direct effect on animals, and that reduction in tadpole biomass is likely due to loss of

macrophyte refuges as feeding sites.

Due to loss of grass carp in one of the control ponds and one of the 500 ~g/L treatment ponds,

the level of grazing by these herbivores was disproportionate across the study. According to the

authors, the fish were lost due to predation. This suggests that the study environment was not

particularly stable.

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There is very little that can be concluded with absolute certainty from this study. While it is

good in theory, there are far too many inconsistencies in the methods and procedures. Many of

the processes are not explained, and it remains uncertain how reliable measurements were

obtained. Performing experiments based on mesocosms is clearly important to determine the

effects of pesticides on organisms, but in order to glean any significant information a study will

have to be more tightly controlled. This is further evidenced by the fact that bullfrogs had

moved into the ponds and were one ofthe treatment effects noted in the study.

Description of Use in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use:

Exposure was not sufficiently characterized, and baseline aquatic community assembledges were

not documented prior to study initiation. The study has some utility for demonstrating that

aquatic communities were not permanently impaired at a functional level, i.e., aquatic systems

recovered, in terms of abundance; however, there may have been effects on species diversity as

plant species more tolerant to atrazine reportedly displaced less tolerant species.

Limitations of Study: No effort was made to document whether other chemicals (including

atrazine) were present in the treatment and/or control ponds prior to study initiation. Presampling was not conducted to determine whether aquatic communities were similar to one

another prior to study initiation. The study switched test chemical from formulated product to

technical grade atrazine after the first year and also switched exposure concentrations for several

of the ponds, i. e, two of the lowest exposures at 20 !1g/L were increased to 500 !1g/L while one

pond formerly at 100 !1g/L was increased to 200 !1g/L. Atrazine degradate concentrations were

not reported. Phytoplankton and zooplankton were not identified at the species level in order to

determine whether more tolerant species had displaced less tolerant species. Large grazing

animals (grass carp) were unequally distributed across the studies due to predation; thus, there

was likely differential grazing in the study ponds. Additionally, bullfrogs were immigrated into

the ponds during the course of the study, and were not likely evenly distributed across

treatments; thus, predation by the bullfrogs would have been different across treatments.

Controls exhibited wide variability and given that there were only two controls for each of the

two studies, the ability ofthe study to detect treatment effects is limited.

Primary Reviewer: Joseph Gandolfi, Intern Biologist, Environmental Fate and Effects

Division, U. S. Environmental Protection Agency.

Secondary Reviewers:

Thomas Steeger, Ph.D., Senior Biologist, Environmental. F.~te and Effect~ Division, U:..§. I

Environmental Protection Agency. ~]~J../J...-. <4 t_~ '.--- &6/11 v·

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Anita Pease, M.S., Senior Biologist, Environmental Fate and Effects Division, U.S.

Environmental Protection Agency. ~'7UX / /3/1 \

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