Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Hoagland, K. D., R. W. Drenner, J. D. Smith, and D. R. Cross. 1993. Freshwater community

responses to mixtures of agricultural pesticides: effects of atrazine and bifenthrin.

Environmental Toxicology and Chemistry. 12: 627-637. (MRlD 48261118)

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 3, 2009

Purpose of Study:

This study examines the potential effects of atrazine and bifenthrin on aquatic plants and animals

in experimental tanks (mesocosms).

Summary of Study Findings:

This review focuses on treatments effects of atrazine.

Methods:

The study was conducted in two phases. Phase 1 (addition oflow dose applications) was

initiated May 22, 1990, and lasted 14 days, and phase 2 (addition of high dose applications to the

same tanks used in phase 1) was initiated June 12, 1990, and lasted 14 days (community effects

reported for day 7 only). Both phases utilized six experimental tanks as controls and atrazineonly treatments (12 other tanks had treatments ofbifenthrin only or bifenthrin plus atrazine),

each a 5,500-L fiberglass tank (1.8-m diameter, 2.2-m height), located at the Texas Christian

University (Fort Worth, Texas) outdoor mesocosm facility. The tanks were filled (water depth,

volume not provided), on May 6, 1990, with water containing natural phytoplankton

assemblages from a pond (0.7 ha) located adjacent to the mesocosm facility and were

continuously mixed throughout the study using an airlift mixer system (pond was sampled 'near

- 1 -

11111••111

2087857
the shore', no details on how pond water was distributed to the tanks). The tanks were stocked

with bluegill sunfish (Lepomis macrochirus, 10 fish per tank) electrofished from the same pond,

and supplemented with zooplankton collected from a local reservoir (not further detailed).

Throughout the study, all ponds were amended every other day with 10% ofinitial ambient total

phosphorus and total nitrogen to prevent nutrient depletion. Tanks were allowed to acclimate

for two weeks before adding pesticide.

For both phases, atrazine was applied via soil slurry to simulate storm run-off. Formulated

atrazine (Hi-Yield, 40.8% a.i. w/v) was combined with 150 mL deionized water and 0.5 L of

sandy loam soil (58.3% sand, 19.6% silt, 22.1% clay, 2.17% organic matter) then, just prior to

treatment, was slurried with 125 mL of pond water. The treated slurry was evenly distributed

across the surface water in the tanks via the airlift mixing system, with turbidity appearing

uniform within 10 minutes. Untreated slurried soil (no pesticide additions) was added to control

tanks in the same manner.

A factorial design was used with treatments containing three levels of atrazine and three levels of

bifenthrin resulting in nine treatment combinations. Phase 1 consisted of duplicate control tanks

and duplicate atrazine-treated tanks at nominal concentrations of 0 f.!g/L, 20 f.!g/L and 200 f.!g/L.

For phase 2, the duplicate tanks treated during phase 1 at 0 f.!g/L, 20 f.!g/L and 200 f.!g/L were

increased to 250 f.!g/L and 1,500 f.!g/L, respectively. Bifenthrin was added at nominal

concentrations of 0, 20, and 200 ng/L (phase 1) and 0,20, and 1600 ng/L (phase 2). With this

design, there were four atrazine-only treatments (20, 200, 250, and 1500 f.!g/L).

Atrazine concentrations were measured at 1 hour and 12 days after the initial application (phase

1), and at 1 hour and 14 days after the second application (phase 2). Water samples were taken

just below the surface near the tank center. Atrazine was extracted (method not detailed) and

analyzed by gas chromatography equipped with a 63N electron-capture detector. The limit of

detection was 5 f.!g/L.

Tanks were sampled for water quality, phytoplankton, and zooplankton two days prior to

pesticide treatm,ent (phase 1), seven and 14 days post-treatment in phase 1, and seven days posttreatment in phase 2. Tanks were sampled for water chemistry (dissolved oxygen, total

phosphorus, total nitrogen, pH and turbidity) by common techniques. Primary productivity was

measured using the light:dark bottle, dissolved oxygen method (sampling technique unspecified).

Algal chlorophyll was determined using a chloroform-methanol extraction method (sampling

technique unspecified). Water temperature and fish mortality were monitored daily.

Zooplankton samples (2 L) were collected from the surface at three locations in each tank (6 L

total per tank), filtered (54-f.!m mesh Nitex netting) and the filtrate preserved with 5% sucroseformalin (w/v). Organisms were identified to genus and counted using a Wildco counting wheel

and Wild M5 dissecting microscope.

- 2 -
Phytoplankton samples (250 mL) were collected, without filtration, just below the surface near

the tank center and preserved with 0.5% Lugol's iodine. For community monitoring, subsamples (4 mL) were settled overnight, then ten random fields were counted using a Wild M-40

inverted microscope.

Standing stock of plankton community components (nano-, micro- and macroplankton) were

assessed via size fractionation by passing the sample through filters of decreasing porosity (200,

20 and 2 IJ.m), with the resultant material retained on the filters analyzed for total phosphorus by

acid persulfate hydrolysis and the orthophosphate malachite green method.

Response variables were analyzed using analysis of variance (ANOVA) on individual sample

dates with SYSTAT v4.0. Treatment effects were analyzed on a date-by-date basis to determine

when significant effects occurred. A probability value (p) ::SO. 1 level ofsignificance was selected

to reduce the probability iftype II errors (accepting a false null hypothesis, i.e., no effect).

Results:

All statistical tests presented in this section are based on ANOVA results as provided by the

study authors. These statistics do not distinguish between the atrazine treatment levels.

Additionally, the statistical analysis was based on all atrazine treated tanks, including tanks

treated with both atrazine and bifenthrin. Statements about differences between atrazine-only

treatments and the control (data from only one control was presented by the authors - the second

was dropped due to bifenthrin contamination) are based on estimates by the reviewer from the

study figures and have no statistical basis (i.e., no statistical tests were presented regarding

differences between atrazine-only treatments and the control; furthermore, except for

measurements of atrazine, no information about variation between treatment replicates was

presented).

The reviewer concurs with the study authors' statement that community changes observed during

phase 2 were the combined result of effects from phase 1 plus the subsequent increased treatment

levels of atrazine.

At the 20 and 200 IJ.g/L applications rates (phase 1), measured atrazine concentrations in the

atrazine-only tanks were 48-85% and 55-95% of nominal, respectively, at 1 hour, and 35-105%

and 80-85% of nominal, respectively, at 12 days (Table 1). At the 250 and 1,5001J.g/L

applications rates (phase 2), measured atrazine concentrations were 148-196% and 120-180% of

nominal, respectively, at 1 hour, and 144-200% and 73-93% of nominal, respectively, at day 14

(after the start of phase 2). In the control tanks (no atrazine or bifenthrin added), measured

atrazine concentrations were <5 IJ.g/L (n = 2) at 1 hour after the initial application (phase 1), but

were not monitored thereafter. Note that community and water quality measurements are plotted

for only control tank #2 in Figures 1,2,3, and 4.

- 3 -
Turbidity was reduced at day 7 during both phases, but in the higher atrazine levels only

(atrazine-only treatments). Total phosphorus was reduced at day 7 for both the high and low

atrazine conditions (atrazine-only treatments), but this effect was only observed in phase 2

(Figure 2). Total nitrogen was significantly increased at all intervals (ANOVA), but the

increases may have been due to the nitrogen present in atrazine. Water pH was 7.8-8.0 in the

treated tanks at all intervals, but increased in the control tank from pH 7.9 at days 7 and 14 of

phase 1 to almost pH 9 at day 7 of phase 2 (Figure 2).

Chlorophyll was significantly affected by the effects of atrazine during both phases (ANOVA:

phase 1, p=0.061 and phase 2, p=O.OOl); the study authors concluded that "recovery" had

occurred after 14 days in phase 1 based on ANOVA results. During phase 1, effects of atrazineonly treatments on chlorophyll were not evident while reductions were evident for treatments

with both atrazine and bifenthrin (Figure 1). During phase 2, chlorophyll was reduced relative

to the control in the atrazine-only treatments. Atrazine had no significant effect on primary

productivity or green colonies during phase 1, but both were significantly (ANOVA: p=O.OOl)

affected at 7 days following treatment in phase 2. In phase 1, atrazine-only treatments showed

an apparent reduction in primary productivity relative to the control (Figure 1). In phase 2

atrazine-on1y treatments, primary productivity and green colonies were reduced at low (250

Ilg/L) and high applications (1500 Ilg/L) in excess of 80%, relative to controls (Figure 1). There

were no significant effects (ANOVA) to diatoms or green unicells at any interval. In atrazineonly treatments, there was an apparent decrease in green unicell density 14 days following

treatment in phase 1 (Figure 1).

Particulate phosphorus (2-20 11m fraction) was significantly (ANOVA: p=0.022) affected by

atrazine during phase 1. Phosphorus concentrations in the atrazine-only treatments were greater

than or equal to the control (Figure 3). During phase 2, significant atrazine effects were evident

again (ANOVA: p=0.069), with> 60% reductions in particulate phosphorus (2-20 11m) relative

to the control (Figure 3). The 20-200 11m fraction was significantly (ANOVA: p=0.054)

affected during phase 1 (14 days following treatment), but not during phase 2 (ANOVA:

p=0.112). Phosphorus concentrations in the atrazine-only treatments were less than the control

(Figure 3: phase 1, day 14; phase 2, day 7). There were no significant effects on the >200 11m

fraction following treatment at during phase 1, but there were significant (ANOVA: p=O.OOl)

effects during phase 2. Phosphorus concentrations in the atrazine-only treatments were less

than the control (Figure 3: phase 1, day 7; phase 2, day 7).

There were significant atrazine treatment effects (ANOVA) on zooplankton densities (Bosmina,

copepod nauplii, and rotifers) with the exception of cyclopoid copepodids (Figure 4). In phase 1

atrazine-on1y treatments, rotifer densities in atrazine-only treatments were lower than the control

(14 days following treatment), copepod naup1ii densities were lower in the high-atrazine

treatment (200 Ilg/L) at all intervals, while Bosmina and cyclopoid copepodids densities appear

similar to the control (Figure 4). No fish mortality was reported in the atrazine-only treated

tanks.

-4-
PHASE 1 PHASE 2

Day 7 Day 14

PRIMARY PRODUCTIVITY ... 0.28., ., % 0.875 0.158

0.08b0.718 boO'258

l\oI • 0.581 0.28'"

o 0.0'" ........... : .".. '..

Day 7

CHLOROPHYLL

.... 8°bO.278 0.081

~"'O ~"lIot 0.188

&20 - ......

0'18'"

'.. 0.531

_~ 0.52'"

~--

0'008

0.001

;:;•• 0.030

1 1\ 0 .... 35

l\ 0.523

! \ 0 .... 88

! \

I

L O.001

0.001

0.001 •...'.

']. 0.831

0'255

0 ....80

... ::--

DIATOMS

8"'} 0.173

25-

0

.

702

...~~ 0.880

1 ••'

.' IX

W

...

.... GREEN COLONIES ;

Q.

::L~::~: 0 .... 87 Ll~I." 0.725~:~:: O.

5 U ..,4'

>C

L

O 3

0 08

0.371

N L H

0 ....85

0.803

0.328

.. ,"If. 1c \

:)X'" \

". N L H

BIFENTHRIN

GREEN UNICELLS

8~0'855 0.558

... 0 .... 82

2 ••••.

N L H

IX

W

m

::::E

:;:)

z

........... No Atrazine

--- Low Atrazlne

• High Atrazine

Figure 1. Mean responses of primary productivity, chlorophyll and phytoplankton to atrazine and bifenthrin

additions; atrazine only results above "N" on x-axis. Probability values from ANOVA of bifenthrin (top),

atrazine (middle) and their interaction effects (bottom) are given to the right of each graph, for days 7 and 14

of phase 1 and day 7 of phase 2. Initial concentrations/densities are indicated by an arrow on y-axis. N = no

bifenthrin; L = low bifenthrin; H = high bifenthrin. Figure from Hoagland et al. 1993.

- 5 -
PHASE 1 PHASE 2

Day 7 Day 14 Day 7

TOTAL PHOSPHORUS

0.010

0.346

0.315

0.036

0.055

....... 0.814

ti·..fi

0.833

0.929

0.351

10

....

....

t1l

:10 6

TOTAL NITROGEN

0.150

0.080

0.531

0.014

0.008

0.615 ............

.....+ .....

0.018

0.001

0.387

pH

9.0l 0.261 1 0.206 l .... 0.453

8.0 ..+ ..... 0.211 0.105 0.001

% •••••• • Q, 0.500 0.738 0.017

7.9 ..,

.....-.

4

::)

IZ 2

TURBIDITY

0.001

0.077

0.041

N L H

0.001

0.004

0.012

N L H

BIFENTHRIN

N L H

0.001

0.277

0.610

............ No Atrazine

- ....- Low Atrazine

• High Atrazlne

Figure 2. Mean responses of Iimnological parameters to atrazine and bifenthrin additions; atrazine only

results above "N" on x-axis. Probability values from ANOVA of bifenthrin (top), atrazine (middle) and their

interaction effects (bottom) are given to the right of each graph, for days 7 and 14 of phase 1 and day 7 of

phase 2. Initial concentrations/densities are indicated by an arrow on y-axis. N = no bifenthrin; L = low

bifenthrin; H = high bifenthrin. Figure from Hoagland et al. 1993.

- 6 -
PHASE 1 PHASE 2

Day 7 Day 14 Day 7

PHOSPHORUS (>200 pm)

0.007 0.002 \ 0.001

4 0.895 0.812 0.001

-J

..... 0.583 0.001 0.184

Q 2

PHOSPHORUS (20-200 pm)

0.038 •• 0.232 0.001

7 0.395 .::.~ 0.054 0.112 -J

..... 5 0.376 0.608 0.052 Q .....•

'

3 ••••

PHOSPHORUS (2-20 pm)

-J 10 .....

Q

5

N L H

0.311

0.022

0.151

"10 0.114

, 0.885 tt:4" 0.911

••••

N L H

BIFENTHRIN

"1>~'.'.

::x

N L H

0.051

0.089

0.287

.....+ No Atrazine

- - Low Atrazlne

• High Atrazine

Figure 3. Mean responses of particulate phosphorus size fractions to atrazine and bifenthrin additions;

atrazine only results above "N" on x-axis. Probability values from ANOVA of bifenthrin (top), atrazine

(middle) and their interaction effects (bottom) are given to the right of each graph, for days 7 and 14 of phase

1 and day 7 of phase 2. Initial concentrations/densities are indicated by an arrow on y-axis. N = no

bifenthrin; L =low bifenthrin; H = high bifenthrin. Figure from Hoagland et al. 1993.

- 7 -
PHASE 1 PHASE 2

Day 7 Day 14 Day 7

BOSMINA

1 0.001 0.001 , 0.001

0.952 0.002 \ 0.001

0.5 0.451 0.018 0.001

\

.... '\

CYCLOPOID COPEPODIDS

0.2 0.001 0.014 0.441

a: 0.917 0.524 0.238

w 0.1 0.946 0.706 •.....•.... ... 0.679

-J ...... cz:

W

Q.

t')

0 COPEPOD NAUPLII ,..

0.001 t=--. 0.137 0.020 2 ,

a: 0.010 '........ "

0.005 ..'\.. 0.330

w 0.123 ...... 0.955 •• 0.703 ID 1 ,\

'\ ;:) 1 z

0.078

0.013

0.380

N L H

0.488

0.032

0.415

........

.~. ".:4 ..'

N L H

BIFENTHRIN

0.004

0.015

0.083

N L H

.....+ No Atrazlne

- - Low Atrazlne

• High Atrazlne

5

10

Fig. 4. Mean responses of zooplankton to atrazine and bifenthrin additions. Probability values from ANOYA of

bifenthrin (top) and atrazine (middle) effects and their interaction effects (bottom) are given to the right of each graph.

for days 7 and 14 of phase 1and day 7 of phase 2. Off-scale initial densities are indicated by an upward-pointing arrow. N = no bifenthrin; L = low bifenthrin; H = high bifenthrin.

- 8 -
Table 1. Atrazine concentrations in experimental mesocosms (adapted from Table 1 ofstudy). Atrazine-only

(Le. no bifenthrin) treatment tanks are presented.

Atrazine (J,12:1L)

Phase 1 Phase 2

Tank Hour Day Mean- MeanTreatment No. 1b, c 12 measured Hour 1b Day 14 measured

Control (no

atrazine or

bifenthrin) 1e <5 NA --- <5 NA --- 2 <5 NA --- <5 NA ---

Low

atrazine3

13 17 21 19 490 500 495

11 9.5 6.9 8.2 370 360 365

AVG: 14 AVG: 430

High

atrazine 3

15 110 170 140 2700 1100 1900

7 190 160 175 1800 1400 1600

AVG: 158 AVG: 1750

a Low tanks treated at nommal concentratIOns of20 Ilg/L (phase 1) and and 250 IlgIL (phase 2). HIgh tanks treated

at nominal concentrations of200 IlglL (phase 1) and 1,500 Ilg/L (phase 2).

b Hour 1 is post-treatment of pesticide.

C The initial date of application appears to have been May 22, 1990, and not May 25, 1990, as presented in the article

table (pp. 628-629).

d Table reported 10 days post treatment, reviewer assumed day 12 (see reviewer comments)

e For this tank, community data were not presented nor used for analysis due to contamination with bifenthrin.

NA= Not analyzed

- 9 -
Data Summary Table: Data are summarized for phase 1 experiments. Phase two experiments

are not included because community changes observed during phase 2 were the combined result

of effects from phase 1 plus the subsequent increased treatment levels of atrazine.

Time Weighted

Magnitude of Nominal Day ofStudy Average Atrazine Did

Effect Effect relative to Atrazine Effect Concentration Recovery Comments

controlsI Concentration Occurred from Day Oto Occur-if

(pglLl When Effect so, when?

Occurretf

Magnitude of

effects estimated

from Figure 1.

No statistical tests

~40% (decrease) 20 11giL 7 14 Ilg/L Day 14 or raw data

showing

experimental

variability were

available for this

effect.

Primary

productivity Magnitude of

effects estimated

from Figure 1.

No statistical tests

-40% (decrease) 2OOllg/L 7 158 Ilg/L No or raw data

showing

experimental

variability were

available for this

effect.

-19% (increase) 20llgiL 7 141lg/L Not Magnitude of

applicable effects estimated

from Figure I.

No statistical tests

or raw data

showing

experimental

Chlorophyll variability were

1581lg/L Not available for this

-19% (decrease) 2OOIlg!L 7 applicable effect.

This is considered

not likely a

treatment related

effect given the

increase at low

atrazine

- 10 -
Time Weighted

Magnitude of Nominal Day ofStudy Average Atrazine Did

Atrazine Concentration Recovery Effect Effect relative to Concentration Effect from Day Oto Occur-if Comments

controli (pglLl Occurred When Effect so, when?

Occurretf

concentrations and

equal decrease at

high atrazine

concentrations.

~220%

20 11 giL 7 141lgIL Not Magnitude of

(increase) applicable effects estimated

from Figure 1.

No statistical tests

Diatoms or raw data

(density) ~I50% Not showing

(increase) 200llgiL 7 158 Ilg/L applicable experimental

variability were

available for this

effect.

No observed 2Ollg/L Not Not applicable Not Magnitude of

effect applicable applicable effects estimated

from Figure 1.

No statistical tests

Green colonies or raw data

(density) No observed Not Not showing

effect 2OOllg/L applicable Not applicable applicable experimental

variability were

available for this

effect.

~50% (decrease) 20 11gIL 14 14 11 giL Not reported Magnitude of

effects estimated

from Figure 1.

No statistical tests

Green unicells or raw data

(density) ~75% (decrease) 200llgiL 14 158 IlgIL Not reported showing

experimental

variability were

available for this

effect.

Phosphorus ~45% (decrease) 20llgiL 7 14 IlgiL 14 Magnitude of

(standing effects estimated

stock of>200 from Figure 3.

11m size- No statistical tests fraction of ~20% (decrease) 2OOllg/L 7 1581lg/L 14

or raw data plankton showing community) 4

experimental

- 11 -
Time Weighted

Magnitude of Nominal Day ofStudy Average Atrazine Did

Effect Effect relative to Atrazine Effect Concentration Recovery Comments

controlsl Concentration Occurred from Day Oto Occur-if

(llgIL/ When Effect so, when?

Occurretf

variability were

available for this

effect.

-20% (decrease) 20 jlg/L 14 14 jlg/L Not reported Magnitude of

Phosphorus effects estimated

(standing from Figure 3.

stock of20-

200 jlm size- No statistics or

fraction of ~50% (decrease) 200 jlg/L 14 158 jlglL Not reported experimental

plankton variability data was

community) 4 available for this

effect.

~15% (increase) 20 jlg/L 7 14 jlg/L Not Magnitude of

Phosphorus applicable effects estimated

(standing from Figure 3.

stock of2-20

jlm size- No statistics or

fraction of Not experimental

plankton ~38% (increase) 200 jlg/L 7 158 jlg/L applicable variability data was

community)4 available for this

effect.

No effect 20 jlg/L Not Not applicable Not Magnitude of

Bosmina

observed applicable applicable effects estimated

(density) from Figure 4.

No effect 200 jlglL Not Not applicable Not

observed applicable applicable

No effect 20 jlg/L Not Not applicable Not

Cyclopoid observed applicable applicable Magnitude of

copepodids effects estimated

(density) No effect 200 jlglL Not Not applicable Not from Figure 4.

observed applicable applicable

13% (increase) 20 jlglL 7 Not applicable Not Magnitude of

applicable effects estimated

from Figure 4.

Copepod

nauplii No statistics or

(density) experimental ~57% (decrease) 200 jlglL 14 158 jlglL Not reported variability data was

available for this

effect.

Rotifers -38% (decrease) 20 jlg/L 14 14 jlg/L Not reported Magnitude of

- 12 -
All effects reported m table are for atrazme-only expenmental treatments (l.e. WIthout addItIOn ofbifenthnn)

2 Tanks treated at nominal concentrations of20 Ilg/L and 200 1lg!L for Phase 1, and at 250 1lg!L and 1,500 Ilg/L for

Phase 2.

3 Average concentration from day 0 (1 hour post-treatment) to day 12 (day 12 assumed by reviewer - see reviewer

comments).

4 Plankton community comprised of phytoplankton and zooplankton.

Time Weighted

Magnitude of Nominal Day ofStudy Average Atrazine Did

Effect Effect relative to Atrazine Effect Concentration Recovery Comments

controls} Concentration Occurred from Day Oto Occur-if

(flgIL/ When Effect so, when?

Occurretf

(density) Not reported effects estimated

from Figure 4.

No statistics or ~50% (decrease) 200llg/L 14 158 Ilg/L experimental

variability data was

available for this

effect.

No results reported

for atrazine-only

treatments, and the

None reported at authors did not

Bluegill any atrazine- report that there

mortality only treatment was mortality for

level these treatments, so

it is assumed that

no mortality

occurred.

. .

- 13 -
Reviewer Comments:

This study also investigated the individual effects of bifenthrin and the combined effects of

atrazine plus bifenthrin on the aquatic community.

The study authors provide sound statistical analysis; however, the results of those analyses are of

limited use. This is because the results of ANOVA did not distinguish between atrazine

treatment levels. Furthermore, the analyses also did not distinguish between treatments that

contained atrazine-only and those that contained atrazine plus bifenthrin, that is, atrazine effects

as reported in the study were based on all treatments that contained atrazine including those that

contained bifenthrin as well. All statistical tests reported in the results section of this review are

based on ANOVA as provided by the study authors. Atrazine-only effects summarized in this

review (data summary table and results section) are estimates based on data in study figures of

atrazine-only treatments and have no statistical basis regarding differences between treatments

and the control. Additionally, except for measurements of atrazine, no information was provided

about replicate variation of community variables (e.g. chlorophyll).

The experimental tanks were filled with water from a natural pond (0.7 ha). It is not stated

whether the natural pond water was tested to establish that it was not contaminated with any

pollutants. Ifthis were the case, it could potentially lead to false conclusions. Furthermore,

zooplankton were obtained from a different water source (a nearby reservoir). It is unclear if

water from the reservoir was added to the tanks and, ifso, if it was contaminated with pesticides.

Determinations for atrazine in the control or treatment tanks prior to study initiation were not

provided. The control tanks were analyzed for atrazine at 1 hour after initial application but

were not monitored thereafter during phase I or phase 2. The authors report the atrazine

concentration in the control ponds was <5 ~g/L, which is the limit of detection ofthe method

they used to measure atrazine. The limit of detection is a fairly high concentration and was 25%

ofthe lowest concentration tested (20 ~g/L). Bifenthrin contamination in one ofthe control

tanks was sufficiently high enough for the study authors to exclude it from analysis. Given all

this, there is little evidence to discount the potential for atrazine contamination, particularly

relative to the lowest treatment concentration.

It is unclear how many days after the initial pesticide application that atrazine was measured

(phase I). The authors report that it was both 10 and 12 days throughout the paper. Based on the

actual dates provided, the reviewer assumed that the measurement was made 12 days after the

first application.

The tanks were allowed to equilibrate for 16 days prior to treatment, and there was no indication

that the tanks contained sediment other than the sediment slurry used as the test compound

vehicle. The tank systems were also continually mixed throughout the study.

- 14 -
Tank water was not monitored hardness. Temperature (daily) and dissolved oxygen (weekly)

were reportedly monitored; however, no results were provided.

Meteorological conditions were not monitored. Atrazine concentrations were only determined at

two intervals following each application. Methods used to extract atrazine from the water

samples were not described. Ponds were sampled for water parameters, phytoplankton and

zooplankton only at two intervals after the initial application (phase 1) and at a single interval

after the second application (phase 2) .

Phytoplankton were not identified at the species level to assess any changes in species

composition. As the study authors noted, any effects which occurred in phase 2 of the study

were a product of both the residual phase 1 effects and increased atrazine treatment levels, and,

therefore, are of limited value. Only one control tank was used for analysis (and presented in

figures) as the second control was excluded due to contamination with bifenthrin.

Description of Use in Document: Invalid

Rationale for Use:

Data from only one control pond was presented due to bifenthrin contamination in the second

replicate, thus there is increased uncertainty about the apparent effects due to atrazine based on

differences between the control tank and the treatment tanks. The study authors provide sound

statistical analysis; however, the results are of limited use. This is because the results of

ANOVA did not distinguish between atrazine treatment levels. Furthermore, the analyses also

did not distinguish between treatments that contained atrazine only and those that containe~

atrazine plus bifenthrin. Atrazine effects reported by the study authors were based on all

treatments that contained atrazine, including those that contained bifenthrin as well. Therefore,

there is no statistical basis for the reviewer to establish atrazine-only treatment effects

(additionally, the variation between replicates is unknown).

Limitations of Study: There were numerous deficiencies in the measuring and reporting of

experimental conditions. No effort was made to document whether other chemicals (including

atrazine) were present in the treatment and/or control tanks prior to study initiation (i.e.,

experimental addition of pesticides). The tanks were equilibrated for 16 days prior to treatment,

and, apparently, contained no sediment layer to buffer the system. It is not clear how similar the

tank communities were to one another prior to pesticide application - only a mean value is

reported for each parameter measured (e.g. chlorophyll). Meteorological conditions were not

monitored. Additionally, it would have been useful to identify plankton at the species level to

assess any changes in species composition. The study had a factorial design that included

treatments containing both atrazine and bifenthrin. The statistical analysis performed was sound

but did not examine the simple effects of atrazine-only treatments. Furthermore, the statistics

provided did not distinguish between atrazine treatment levels. Therefore the statistics provided

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were oflimited use. Data from only one control pond was presented due to contamination in the

second replicate, thus there is even greater uncertainty about the apparent treatment effects due

to atrazine based on differences between the control tank and the treatment tanks. Community

changes observed during phase 2 were the combined result of effects from phase I plus the

subsequent increased treatment levels of atrazine, thus effects due to phase 2 treatments cannot

be isolated.

Primary Reviewer: Lynne Binari, M.S., subcontractor to Cambridge Environmental Inc.

" .~

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA '/!t{iW J:"1-1,M;I v .

Tertiary Reviewer: Brian Anderson, Biologist, EPA

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