Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Diana, S. G., W. J. Resetarits, Jr., D. 1. Schaeffer, K. B. Beckmen and V. R. Beasley. 2000.

Effects of atrazine on amphibian growth and survival in artificial aquatic communities.

Environmental Toxicology and Chemistry. 19 (12): 2961-2967. (MRID 48261107)

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: October 5, 2009

Summary of Study Findings

Purpose of Study:

This study examines the effects of atrazine on a simple aquatic community including aquatic

plants and the larval gray tree frog (Hyla versicolor) in experimental outdoor microcosms. The

focus ofthis review is on treatment-related effects to the aquatic plant community, althollgh

possible indirect effects on Hyla versicolor are noted.

Methods:

Plastic wading pools were used to construct sixteen outdoor experimental ponds (microcosms) in

mid-May 1996 at the experimental pond facility ofthe Illinois Natural History Survey (Urbana,

Illinois). Each microcosm measured 1.22-m diameter; depth was not reported. The microcosms

were arranged in a 4 x 4 Latin square design on a bed of gravel. Each was filled with 90 L water

from a nearby pond, not further described, and amended with 150 g dry wheat straw (unreported

source) as a slow-release nutrient source. The microcosms were covered with fiberglass screen

(3.2-mm mesh) to retain added amphibians and exclude predators. Clear, greenhouse,

polyethylene film covers (6-llm thickness) were suspended 43 cm above the microcosms to

exclude rainwater, with one cover shielding two microcosms. The microcosms were allowed to

acclimate 5 weeks prior to treatment. Water lost through evaporation was replaced twice weekly

with deionized (DI) water.

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Iflllllllll

2087824
Study organisms were obtained from nearby natural locations, not further described. Eighty

stems ofthe aquatic macrophyte marsh-pepper knotweed (Polygonum hydropiper) were

collected, potted, and distributed five plants per microcosm. Captive-born Hyla versicolor larvae

were initially maintained in pond water indoors. 40, 9-day-old larvae (5 days post-hatch) were

then randomly assigned to each ofthe outdoor microcosms. Larvae were 15 days old (11 days

post-hatch) at the time of atrazine application.

Atrazine stock solution was prepared through drop-wise addition oftechnical grade atrazine

(purity unspecified) in acetone (5.002 mg/ml) to DI water, with continuous stirring. The final

atrazine concentration ofthe premix was 30.01 mg/L. A vehicle control solution was similarly

prepared by adding an equal volume of acetone to OI water. Both the test and vehicle control

solutions were purged with air overnight to remove acetone through volatilization. Each

microcosm was treated with 6 L of an appropriate combination of aqueous atrazine test solution

and/or vehicle control solution to yield nominal atrazine treatment rates of 0, 20, 200 and 2,000

Jlg/L. Four replicates were included per treatment. Solutions were evenly sprinkled over the

water surface using a watering can, after which the water was briefly mixed.

Three series of water quality measurements were taken to analyze general water quality

parameters, atrazine concentrations, and chlorophyll a. Dissolved oxygen (DO), temperature,

and pH measurements were recorded in situ at pre-dawn and pre-dusk, daily, from Dayll -1

through Day 10. Measurements were continued every 3 days thereafter through Day 51. Water

samples (unreported volume) for atrazine analysis were collected at Day 0 and 3 weeks posttreatment. Concentrations were quantified using the Ohmicron Atrazine RaPID Assay

(Ohmicron Environmental Diagnostics), with intra- and inter-assay coefficients of variance of

3.91% and 1.24%, respectively. Additional water samples (1 L) were collected for chlorophyll a

analysis prior to treatment and at 7, 14, 21, 28 and 42 days post-treatment (Figure 1).

Chlorophyll a was extracted from the water with acetone and quantified by measuring

absorbance at 630,647,664 and 740 nm with a split-beam spectrophotometer (Lambda 3,

Perkin-Elmer) with quartz cuvettes (1-cm path length). Intra- and inter-assay coefficients of

variance were 1.2% and 1.58%, respectively. Extraction recovery efficiency was 89.29 ± 2.77%

(USEPA Method 446.0, EPA/600/R-97/072).

Periphyton that had adhered to duplicate glass microscope slides suspended in the microcosm

water were similarly analyzed for chlorophyll a on a weekly basis. To determined macrophyte

biomass, plants were cut at the soil line, blotted dry, and immediately weighed (wet weight) as

microcosms were drained at study termination. During the study, metamorphosing frog larvae

were collected nightly from microcosm sidewalls and maintained in a laboratory at 26.7 ± laC

until completion ofmetamorphosis (tail resorption). Following tail resorption, the frogs were

sacrificed by immersion in a 0.1 % aqueous solution of 3-amino benzoic acid ethyl ester. Snoutvent length (to nearest 0.05 mm), body mass (to nearest 0.001 g) and date of metamorphosis

I Day 0 = Date of atrazine application (treatment)

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completion were recorded. Survival through metamorphosis was calculated as the percentage of

frogs that completed metamorphosis, once no additional larvae could be found in the

microcosms.

Statistical tests were conducted using SYSTAT v 7.0 software. Repeated measures analysis of

variance (ANOVA) was used to evaluate potential effects of pool position on temperature.

Phytoplankton chlorophyll a concentrations, DO, pH, and temperature were also analyzed by

repeated-measures ANOVA. One-way ANOVA was used to analyze response values for

macrophyte biomass and arcsin-transformed survival values. Frog larval duration was analyzed

by life-table analysis and a second method (unreported, presumably ANOVA).

For all analyses, a probability value (P) of 0.05 was selected to detect significant differences in

responses. When ANOVA detected an overall difference between treatment groups, responses

were compared using the Tukey HSD multiple comparison test and linear contrasts. Leastsignificant differences were determined for repeated-measures ANOVA, using a 95% confidence

level, to compare responses at the same interval as well as responses between consecutive

intervals.

One control microcosm was excluded from data analysis because it experienced an early and

sustained zooplankton bloom, resulting in a clearing ofthe phytoplankton, which did not recover.

Adherent periphyton chlorophyll a was excluded because it was "almost uniformly" less than the

limit of detection.

Results:

At Day 0, measured atrazine concentrations in the water were 1.64 ± 0.09 Ilg/L, 20.13 ± 0.58

Ilg/L, 195.50 ± 8.74 Ilg/L and 2,035.83 ± 51.54 Ilg/L for the 0, 20, 200 and 2,000 Ilg/L treatment

rates, respectively. At 3 weeks post-treatment, atrazine concentrations in the 20, 200 and 2,000

Ilg/L treated microcosms had decreased 21 %, 9% and 16%, respectively, from initial

concentrations..

Based on ANOVA, phytoplankton chlorophyll a changed significantly over the study duration

within the treatment groups (p<O.OOI) and differed significantly between treatment groups

(p=O.OI, Figure 1). There were no clear treatment-related changes in chlorophyll a at Day 7.

Chlorophyll a levels at 200 Ilg/L were higher than controls, and chlorophyll levels at 20 Ilg/L

and 2000 Ilg/L were lower than controls. Chlorophyll a levels ranged from approximately 5 to

10 Ilg/L to 40 Ilg/L at Day 7. By Day 14, a more consistent pattern was observed. Chlorophyll a

levels at 200 Ilg/L and 2000 Ilg/L atrazine were approximately 2- to 4-fold higher than controls

until the end of study. The highest chlorophyll a levels were observed in the 2000 Ilg/L

treatment group from Day 7 to Day 21, and in the 200 Ilg/L on Day 28 and thereafter.

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The overall effect of atrazine on the macrophyte Polygonum hydropiper was highly

significant (p<O.OOl) with biomass decreased 30%, 98% and 99% in the 20, 200 and 2,000 Ilg/L

treated microcosms, respectively, as compared to controls at study termination.

No data or analysis results were provided with respect to temperature. Figure 4 shows

effects on dissolved oxygen (DO). Decreasing concentrations of DO levels occurred in pre-dusk

water with increasing atrazine concentrations, as compared to controls, with significant

differences between treatment groups (p<O.OOI), as well as in the change of DO over time

(p<O.OOl). The same trends were seen in pre-dawn DO levels (p=0.007 and p=0.002,

respectively). In 20 Ilg/L treated microcosms, pre-dusk DO was decreased 10-20% at 5-7 days,

as compared to controls, then recovered at 10 days and remained near control levels thereafter.

At the 200 and 2,000 Ilg/L treatment rates, pre-dusk DO was decreased ~50% and 75%,

respectively, at 1-7 days, as compared to controls. DO recovered to control levels by 14 days,

then again decreased to ~20-40% and ~40-50%, respectively, from 30 days to study termination.

There were significant differences in pre-dawn and pre-dusk pH levels between treatment

groups, as well as change in pH over time (both p<O.OOI), and although there were significant

decreases in pH in the 200 and 2,000 Ilg/L treated microcosms, as compared to controls, pH

levels generally remained between approximately 7 to 9.

At the 200 and 2,000 Ilg/L treatment rates, frogs at metamorphosis were significantly shorter

(5%) with lower body mass (7%), as compared to frogs in the control and 20 Ilg/L treated

microcosms (p<O.OOI,Table 1, attached Worksheet). Results of analyses differed with respect to

larval period duration. Life table analysis indicated no significant differences across treatments

(calculations not reported). However, in the 2,000 Ilg/L treatment, larval period duration was

significantly longer than in the 200 Ilg/L treatment (+5%, p=0.025, method unreported). No

statistically significant differences in survival were seen across treatments, although survival in

the 2000 Ilg/L treatment was notably lower at 70.7% ±4.9 (attached Worksheet). For all

treatment groups, mean snout-vent length and mass initially decreased at 2-3 weeks posttreatment and increased at 3-5 weeks. The increase continued at 5-6 weeks in control and 20

Ilg/L treated microcosms, but decreases occurred in the 200 and 2,000 Ilg/L treated microcosms

(Figure 3).

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160

140 •

120

100 , -,

"

i • lU 80 •

>. , 60 -a /_1--.. - .c I

-'. 0-

0 t '/ 40 -,' '. ,-

.,;....... ..

E 20 • .s= . -"-. ~.-._::' ". -. :

U . - 0

-20 ... ,,-- _... ---.-....----

0 10 20

+>-'- •

.. -'----- '.:- '- -:-

.-- ..

T---------

30

• o~~

• - 20 I'QIl

.. 2OO...wt

... ~ooo I'9"l

._,

40 so

Days from atrazine addition

Figure 1. Mean phytoplankton chlorophyll a concentrations (± standard error) in water; n = 4 for each

atrazine treatment level, n = 3 for controls. Figure 1 from Diana et al. 2000.

-_.- ."._.. --- -----_··-------1,

I

I

f

' ____ po, ".WJ _____•_____•

--_ .. _.

30

A

- 25 j 0)

.......,

.s:::. - 20 0) , 'Q) 1

15 I

1 -Q)

10 -1

5

0

B 8

o 20 200

Atrazine (jJ.g/L)

2000

Figure 2. Mean macrophyte biomass (± standard error), "A" and "B" indicate that groups differed

significantly (p<0.05); n = 4 for each atrazine treatment level, n =3 for controls. Figure 2 from Diana et al.

2000.

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f l

[ ; 0 and 20 ~glL 'I

-11- 200 and 2000 ~g/L r

I

2.0 J

!

I

1.9

I

--- E

(.)

-.c 1.8 -O'l

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C1l I

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0.7 {

I

I

0.6

--- O'l

'-'

III

III 0.5

ro

0.4

0.3 J

1 2 3 4 5 6

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7

Weeks from atrazine addition

Figure 3. Mean (± standard error) snout-vent length and mass at metamorphosis for gray tree frog (Hyla

versicolor); statistically indistinguishable groups are combined, n = 7 for pooled 0- and 20-ltglL treated

groups and n = 8 for pooled 200- and 2,000-ltg/L treated groups. For 0- and 20-ltg/L treated groups at 2

weeks, frogs had emerged from only one microcosm; consequently, no standard error is presented. Figure 5

from Diana et al. 2000.

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Table 1. Frog survival through metamorphosis, larval duration, mass and length at metamorphosis (mean ±

st. dev., n = 97,134,135 and 112 individual for 0-, 20-, 200- and 2,000-l-lg/L treatment rates, respectively;

from Table 1 ofstudy).

Atrazine Microcosm Larval period duration

(Ilg/L) replicates Percent survivall (days)

0 3 80.8 ± 5.6 13.38 ± 0.59

20 4 83.1 ± 7.9 13.55 ± 0.59

200 4 84.4 ± 4.1 12.39 ± 0.76

2,000 4 70.7 ± 4.9 13.99 ± 0.693

1 Differences were not statistically significant at p<0.05.

2 Significantly (p<O.OO 1) less than the 0- and 20-llg/L treatment groups.

3 Significantly (p=0.025) greater than the 200-llg/L treatment group.

~'~"I

I

o·f.... j

I

.- . -~l.'.d

......

'.......... ".. _....,., .. ""1""

Fig. 4. Mean (± standard error of mean [SED (a) Predusk and (b)

predawn dissolved oxygen (DO) concentrations as a function of atrazine

concentrations and time N =3 for the control group and 4

for each atrazine-treated group.

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Mass at

metamorphosis

(g)

0.482 ± 0.026

0.484 ± 0.021

0.449 ± 0.0192

0.448 ± 0.021 2

Length at

metamorphosis

(mm)

19.00 ± 0.28

18.9 ± 0.20

18.0 ± 0.202

18.1 ± 0.202
d Daat Summary TahIe: Ph illOpJan t l kton, macropJhryites and amp,hObOI tans In out oor microcosms

Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pgIL/ Occurred from Day Oto when?

When Effect

Occurred

The study authors

reported that "no

significant

differences in

phytoplankton

20 /lg/L; initial chlorophyll a

No clear effect NA NA NA concentration were 20.13 ± 0.58 /lg/L detected between

control and 20 /lg/L

treatment groups at

any time." The

reviewers agree with

this conclusion.

Magnitude of effect

estimated from

Not determined Figure 1.

Approximately 200 /lg/L; initial (measured No; effect Chlorophyll a levels

a 3-fold concentrations was present increased at all Phytoplankton Stimulation 195.50 ± 8.74 14

were only at the end of intervals as compared chlorophyll a above controls /lg/L reported at Day the study. to controls. (/lg/L) oand Day 21) Maximum effect was

statistically

significant (p"S0.05).

Magnitude of effect

estimated from

Figure 1.

Initial decrease was

Not determined Yes, observed that was not

Approx.. 2- (measured chlorophyll a statistically

fold increase 2,000 /lg/L; initial concentrations levels were significant, p value

relative to 2,035 ± 51.54 14

were only similar to

not reported),

controls. /lg/L reported at Day controls by followed by

oand Day 21) Day 42. statistically

significant (p"S0.05)

increase (maximum

effect +~ 100% at 28

days), with recovery

occurring, at Day 42.

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Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (l1g/Lyt Occurred from Day Oto when?

When Effect

Occurred

Not An approximate 30%

applicable; decrease was

No clear effect 20 Ilg/L; initial NA NA biomass only observed relative to

20.13 ± 0.58 IlgIL determined at controls that was not

study statistically

termination. significant (p. 2963).

Macrophyte Not Magnitude of effect

(Polygonum 200 Ilg/L; initial applicable; presented in article

hydropiper) 98% decrease 195.50 ± 8.74 51 Not calculable biomass text (p. 2963).

biomass (g wet Ilg/L determined at Effects statistically

wt.) study significant (p<0.05,

termination. Figure 2).

Not Magnitude of effect

2,000 Ilg/L; initial applicable; presented in article

99% decrease 2,035 ± 51.54 51 Not calculable biomass text (p. 2963).

Ilg/L determined at Effects statistically

study significant (p<0.05,

termination. Figure 2).

Magnitude of effect

estimated from

20 Ilg/L; Figure 4.

Not Not No effect measured: 20.13 applicable Not applicable applicable Control and

± 0.58 Ilg/L treatment standard

error of means

overlap

Magnitude of effect

Dissolved

estimated from

oxygen (mg/l) Figure 4.

- predusk Statistical

measurements2

significance not

200 Ilg/L; reported, however,

60% decrease measured: 195.50 1 Not calculable Day 10 (see treatment and control

± 8.74 Ilg/L comments) standard error of

means do not

overlap.

Recovery observed

by day 10, however,

treatment decreased

relative to the control

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Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pglLyJ Occurred from Day Oto when?

When Effect

Occurred

again starting day 20

and remained lower

thro1Jgh the end of

the experiment.

Magnitude of effect

estimated from

Figure 4.

Statistical

significance not

reported, however,

treatment and control

2,000 1Jg/L; standard error of

Day 10 (see means do not

78% decrease measured: 2,035 I Not calculable comments) overlap. ± 51.54 1Jg/L

Recovery observed

by day 10, however,

treatment decreased

relative to the control

again starting day 20

and remained lower

through the end of

the experiment.

An approximate

20 1Jg/L and 200 3.4% increase was

Gray tree frog No effect 1Jg/L; initial 20.13 Not reported Not calculable Not reported observed. This

(Hyla ± 0.58 and 195.50 observation was not

versicolor) ± 8.74 1Jg!L statistically

survival significant at p<0.05.

thro1Jgh An approximate 13%

metamorphosis 2,000 1Jg/L; initial reduction in survival

(%) No clear effect 2,035 ± 51.54 Not reported Not calculable Not reported was observed that

1Jg/L was not statistically

significant at p<0.05.

No effect 20 1Jg/L; initial NA NA NA -- Gray tree frog 20.13 ± 0.58 1Jg/L

(Hyla 200 1Jg!L; initial versicolor) No effect 195.50 ± 8.74 NA NA NA n

larval period 1Jg/L duration (days)

No effect 2,000 Ilg/L; initial NA NA NA --

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I At 3 weeks post-treatment, atrazme concentratiOns m the water had decreased 21%, 9% and 16% from mltIaI

measured concentrations at the 20, 200 and 2,000 Ilg/L treatment rates, respectively; no additional quantitative

results were presented.

2 No effect observed at any treatment level for pre-dawn DO measurements which represent changes due to

respiration. Pre-dusk DO measurements represent the net increase in DO during the day due to photosynthesis.

Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pglLyJ Occurred from Day Oto when?

When Effect

Occurred

2,035 ± 51.54

Ilg/L

No effect 20 IlglL; initial Not reported Not calculable Not reported -- 20.13 ± 0.58 IlgiL

Gray tree frog

(Hyla Magnitude of effect

versicolor) 200 Ilg/L and calculated from

mass at 2,000 IlglL; initial Table 1. Effects

metamorphosis 7% decrease 195.50 ± 8.74 Not reported Not calculable Not reported statistically

(g) Ilg/L and 2,035 ± significant (p<O.OO I)

51.54 Ilg/L as compared to 0-

and 20-llg/L groups.

No effect 20 IlgIL; initial Magnitude of effect

(+1% 20.13 ± 0.58 Ilg/L Not reported Not calculable Not reported calculated from

Gray tree frog stimulation) Table 1.

(Hyla Magnitude of effect

versicolor) 200 IlgiL and calculated from

length at 2,000 IlglL; initial Table 1. Effects

metamorphosis 5% decrease 195.50 ± 8.74 Not reported Not calculable Not reported statistically (mm) IlgiL and 2,035 ± significant (p<O.OOI)

51.54 IlglL as compared to 0-

and 20-llgiL groups.

...

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Reviewer Comments:

It is not stated whether the natural pond water was tested to establish that it was not

contaminated with pollutants other than atrazine. Reportedly, water samples were collected from

the natural pond used as the water source for the microcosms to test for atrazine contamination;

however, no results were provided. Atrazine was detected in untreated control microcosm water

at 1.64 ± 0.09 ~g/L at day O. This level of atrazine contamination was considered to not likely to

have affected the study results. The source of wheat straw introduced to each microcosm was

not reported; this may have been another vector of contamination.

The purity ofthe atrazine test compound was reported as technical, but not further specified.

Although the study was conducted for 6 weeks following atrazine application, atrazine

concentrations were only determined at 0 and 21 days post-treatment.

No negative controls were utilized; only solvent (vehicle solution) controls were included.

The microcosms did not contain a sediment layer. Atrazine does not adsorb strongly to soil, so

this omission is unlikely to have affected the biologically available fraction of pesticide in the

water.

Multiple water quality parameters were not monitored, analyzed, and/or reported. Turbidity,

alkalinity, total nitrogen, total phosphorus, conductivity, and particulate and dissolved organic

carbon were not measured. Pre-dawn and pre-dusk water temperatures were reportedly

monitored and analyzed, but no data were presented. Those results may be integral given that

the microcosms were above ground plastic wading pools positioned on a bed of gravel, and no

shading was described. Likewise, effects of pool position on temperature were reportedly

analyzed, but no results were presented.

The study authors suggested that the greater phytoplankton biomasses over the study duration

in the 200 and 2,000 ~g/L treated microcosms were likely a consequence of elimination of

macrophytes. The more inhibited phytoplankton growth in the control and 20 ~g/L treated

microcosms may have been a consequence of nutrient competition with surviving macrophytes,

resulting in inhibition via allelopathy.

The mass of phytoplankton introduced to each microcosm may have been too low to identify

initial declines in population growth. Variability within (and across) treatment groups was

another complicating factor, which may have precluded effective statistical analysis. The

authors did not cite any tests or corrections for homogeneity of variance in their methods.

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The study authors provided justification for treatment rates based on literature values. The 20

flg/L rate was selected as a no observed effect concentration (NOEC, Solomon et ai., 1996). The

200 flg/L rate was selected as a concentration detected in static bodies of water that receive runoff from atrazine treated fields (Kadoum et ai., 1978). The 2,000 flg/L rate was selected as a

highly phytotoxic concentration considered unlikely to cause acute toxic effects in the frog

larvae.

Because the experiment was not designed to distinguish between direct and indirect effects on

frog larvae, all results are noted in this review. An assumption was made that any observed

effects were mediated through the aquatic plant community (i.e., indirect effects), as the highest

treatment level (2,000 flg/L) had not been associated with acute toxic effects in tadpoles. The

authors proposed a causal relationship between diminished DO and smaller size (eg., snout-vent

length, mass) offrog larvae at metamorphosis in the higher treatments.

All frog larvae were obtained from a single lineage; this is inadequate if natural population-level

inferences are to be made from the results.

The authors failed to report their primary method of analysis of larval period duration, although

they presented results. Based on their primary method, larval period duration was significantly

longer for frogs in the 2,000 flg/L treatment than in the 200 flg/L treatment (5%, p=0.025). Lifetable analysis was also applied and indicated no significant effects of atrazine concentration on

this endpoint. No supporting calculations or results were presented.

Removal of frog metamorphs from the microcosms prior to tail resorption introduces another

variable that is not addressed. This may have confounded results for larval period duration and

for size (snout-vent length, mass) at metamorphosis.

A time lag must be considered when testing for indirect effects. For example, the authors

suggest that younger frog larvae with undeveloped lungs may be more severely impacted by

decreased DO associated with atrazine treatment. However, atrazine application in this study

occurred when larvae were 11 days post-hatch, and the authors cite an average larval period

duration of approximately 13 days. The study organisms may therefore have been insufficiently

sensitive and/or housed in the microcosms for too short a duration to serve as an indicator of

effect.

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Description of Use in Document: Qualitative

This study is sufficient to provide qualitative information for use in the risk assessment

process. Limitations with respect to experimental design and statistical analysis are noted

limitations.

Rationale for Use: The study has some utility for demonstrating that atrazine caused reduction

in the macrophyte community with corresponding changes in species composition leading to

domination by phytoplankton at levels of 200 Ilg/L and 2000 Ilg/L.

Limitations of Study: There were numerous reporting and measuring deficiencies. No effort

was made to document whether other chemicals were present in the treatment and/or control

microcosms prior to study initiation, and analyses of control microcosms detected low levels of

atrazine at Day O. Atrazine concentrations were only determined at aand 21 days posttreatment, and the study was conducted for 6 weeks after application. Only solvent controls

were utilized; no negative controls were included. The microcosms consisted of above ground

plastic wading pools positioned on a gravel bed. The microcosms contained no sediment layer.

Microcosm water was not monitored for turbidity, alkalinity, total nitrogen, total phosphorus,

conductivity, or organic carbon. Water temperature was monitored, but not reported, and those

results may be integral given the microcosm system (above ground pool) utilized. Finally,

unequal variance within and across treatments may have resulted in questionable statistical

interpretation ofresults with regard to early phytoplankton population growth.

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

Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division

U.S. Environmental Protection Agency

Tertiary Reviewer: Brian Anderson, Biologist, Environmental Fate and Effects Di~on

U.S. Environmental Protection Agency ~n c / ~._

! ~~

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