Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Stay, E.F., A. Katko, C.M. Rohm, M.A. Fix, and D.P. Larsen. 1989. The effects of

atrazine on microcosms developed from four natural plankton communities. Arch.

Environ. Contam. Toxico!. 18:866-875. (tY/(1 0 Y1-~i.(.3 ':,-/ S- )

Purpose of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this

study were also used to parameterize the Comprehensive Aquatic System Model (CASM)

and establish the model-associated LOC for atrazine.

Date of Review:

November 14,2008

Purpose of Study:

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

Leffler microcosms.

Summary of Study Findings:

Methods:

Microcosms were prepared according to the protocol of Leffler (1984), The use ofselfselected, generic aquatic microcosmsfor pollution effects assessment, in the book

"Concepts in marine pollution measurement." Microcosms are l-L beakers filled with

950 mL Taub and Dollar (TD) media (see Stay, et aI., 1985 review) and 50 mL of a stock

culture developed from a natural aquatic community. Each week during the experiment,

each system was reinoculated with 50 mL ofthe same culture. The microcosms initially

stabilized for 6 weeks and then predominant organisms were identified. Each microcosm

was then assigned randomly to a treatment or control group and atrazine was added. The

day of atrazine addition was Day 0 and the experiment ended on Day 42. During the 42

days ofthe experiment, the microcosms were incubated at 20±1°C under a 12 hour

light/12 hour dark cycle with light at 5400 lux.

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Stock cultures were prepared from four natural ponds and lakes using identical

techniques and acclimation to the lab environment in 37 L aquaria for at least 2 months.

Contemporaneous samples were taken in the springtime (year not stated) from Waverly

Lake (WL; an urban lake in Albany, Oregon), Freeway Lake (FL; a lake in an

agricultural watershed 2 km southeast of Albany), Triangle Lake (TL; a rural mountain

lake in a forested watershed in the Coast Range, 45 km west of Albany), and Peavy

Arboretum Pond (PA; a lake in an agricultural watershed 7 km northeast of Corvallis,

Oregon). WL and FL are the most productive lakes and PA is the least productive and

most turbid. (No other information was provided on lakes, water quality, sampling

locations within the water bodies, background levels of atrazine, or prior atrazine

exposure.)

Reagent grade atrazine was mixed with TD media to prepare stock solutions before each

experiment. Five replicate microcosms were used at each nominal treatment level: 0

(control),20, 100,200,500,1,000, and 5,000 f.lg/L. Since large volumes of water were

required for atrazine analysis, Day 0 concentrations of atrazine were determined from

replicate surrogate microcosms (1 L ofTD media). On Day 42, when experiments were

terminated, water from the experimental microcosms was analyzed for atrazine. See

Table 1 for a summary ofthe average and 95% confidence interval Day 0 concentrations,

and the average percent remaining on Day 42 over the four experiments. Atrazine was

analyzed by extraction from 100 mL water samples with 20 mL pesticide-grade ethyl

acetate, concentration of extract, and analysis by gas/liquid chromatography. Average

extraction recovery was 104% (determined with internal spikes) and average precision

was 6% relative standard deviation (determined with duplicates).

The response variables measured were dissolved oxygen concentration (DO), pH, redox

potential (Eh), heterotrophic activity (HA), net primary productivity (Pn), dark

respiration (Rni), and Pn/Rni ratio. Measurements were made on Days 1,2,3,5,7, 10,

14,21,28,35, and 42. Before sample collection or measurement, microcosms were well

mixed with a magnetic stir bar. All measurements and samples were taken 5 cm below

the surface at the center ofthe microcosm. DO, pH, and Eh measurements followed

standard methods (USEPA, 1979, Methods for chemical analyses ofwater and wastes;

and APHA, 1980, Standard methodsfor the examination ofwater and wastewater).

Precisions were determined for each sampling series by making 5 replicate measurements

in biota-free TD media near oxygen saturation at 20°C. Average precision for DO

measurements was 6% relative standard deviation. Average Eh measurement precision

was 5% with a platinum redox electrode calibrated with 192 and 258 poised solutions.

Average pH measurement precision was 0.23% with an Orion Model 801 A ion meter

and Ross electrode (accuracy defined using pH 7 and pH 10 buffers).

Heterotrophic activity, defined as the fraction of glucose decomposed to CO2, was

measured according to Kadota (1972), Techniques for the assessment ofmicrobial

production and decomposition infresh waters, IBP Handbook No. 23. 14C-labeled

glucose (0.1 f.lCi) was added to a 10 mL sample, and the sample was then incubated at

2
20°C for four hours, acidified, and the CO2 purged through a C02 trap. Trapped 14C02

was counted by liquid scintillation. Net primary productivity and dark respiration were

estimated by the amount of DO change during the light and dark periods, respectively.

Estimates were corrected for oxygen exchange with the atmosphere by the procedures of

Stay, et al. (1985). Pn/Rni ratio was calculated directly from Pn and Rni.

Statistical analyses on the response variables were performed to determine differences

between treatments and controls on each measurement day. After determining

homogeneity ofreplicate variances with a folded F test, a linear contrast t test with either

pooled variance or non-pooled variance was conducted, the former requiring no

significant differences among replicate variances at p-value < 0.05.

Results:

The study lists predominant taxonomic groups in the lake communities and in the

microcosms on Day 0 of each experiment in a table (not provided in this review). Lake

communities from the four lakes were generally different, with only the Melosira genus

predominate in all four. After lab acclimation, the stock cultures were more similar to

one another than to the original communities. On Day 0, Scenedesmus, Oedogonium,

Anabaena, Hyallella, Cyclops, Lecane, and Philodina were predominate genera in all

four microcosm communities. The acclimation process changed the basic structure ofthe

community from one dominated by diatoms to one dominated by green and blue-green

algae.

The response variables had different sensitivities to atrazine treatment. HA levels in

treatment microcosms were not generally significantly different from controls (p<0.05).

Generally the percentage of added glucose converted to C02 was less than 15%. Eh was

highly variable and results were not assessed. DO levels were sensitive to atrazine, but in

a manner controlled by primary production and respiration, so results are not provided.

Rni was relatively insensitive to atrazine treatment and microcosms exposed to 500 Ilg/L

or less generally were not significantly different from controls (p<0.05).

Pn, Pn/Rni, and pH were the variables with the most response to treatment (see Figure 1,

Figure 2, and Figure 3). The 1,000 and 5,000 Ilg/L treatments showed similar effects as

the 500 Ilg/L treatment, and so are not shown in the figures. Responses to atrazine were

generally greater in the microcosms derived from WL and FL than in those from TL and

PA.

Pn values for microcosms with atrazine treatments of 100 Ilg/L or more and developed

from lakes WL, FL, and TL were significantly different from their respective controls

over the first 14 days (p<0.05; see Figure 1). There was generally no recovery in these

microcosms over the 42-day experiment. Pn values for microcosms made from PA

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communities were consistently less than controls, but differences were only significant at

atrazine treatments of 200 Ilg/L or above (p<0.05).

Pn/Rni ratios were significantly less than controls (p<0.05) at atrazine treatments of 100

Ilg/L or more during the first 14 days. Responses were more consistent among the four

experiments (WL, FL, TL, and PA) than the responses for Pn. Pn/Rni ratios all recovered

by the end ofthe 42 day experiment. See Figure 2.

The pH values were the most sensitive response variables to atrazine treatment (Figure

3). All treatment microcosms exposed to 100 Ilg/L or more had significantly lower pH

than controls (p<0.05), and microcosms exposed to 200 Ilg/L or more generally did not

recover during the 42 day experiment.

Table 1. Average concentration (l1g/L) and 95% confidence interval of atrazine added on Day 0, and

average percent atrazine remaining on Day 42, of all microcosm experiments.

Nominal 95% Confidence Day 42

concentration N Day 0 Mean Interval % Remaining

20 II 17.7 16.1 - 18.7 76

100 6 99.9 90.6 - 109.2 69

200 13 204.2 190.3-218.1 71

500 15 491.7 465.7-517.7 76

1000 7 961.4 859.0-1063.8 75

5000 12 4929.2 4742.3 - 5116.1 80

4
40 50

- Conlrol

D. 20 IJg/l

0100 IJg/l

o 200IJg/1

0500 IJg/l

20 30

Day

10

- Control >.

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0100 IJg/J § -2 o 200IJg/I a:

0500 IJg/l OJ -4 z 40 50 0

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Figure 1. Mean net primary productivity (Pn) of Leffler microcosms exposed to atrazine. Source

communities used to develop these microcosms were from Waverly Lake (WL), Freeway Lake (FL),

Peavy Arboretum Pond (PA), and Triangle Lake (TL). Points ofsignificant (p<0.05) linear contrast

from control are shown with solid/darkened symbols. 1,000 and 5,000 !!g/L treatments are not

shown, because they were similar to 500 !!glL treatments.

5
FREEWAY LAKE

5.-------------~ WAVERLY LAKE 5,-------------~

4 4

3

-I

-Control

620lJg/1

0100 ug/l

0200 lJg/I

o 500lJg/1

c

a::

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0200 lJg/I

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Day

40 50 o 10 20 30

Day

40 50

5,,----------------,

PEAVY ARBORETUM POND TRIANGLE LAKE 5,--------------~

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-Control

620 lJg/1

o 100~/l

0200 lJg/1

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o /0 20 30

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40 50 o /0 20 30

Day

40 50

Figure 2. Mean net primary productivity/respiration (Pn/Rni) ratio of Leffler microcosms exposed

to atrazine. Source communities used to develop these microcosms were from Waverly Lake (WL),

Freeway Lake (FL), Peavy Arboretum Pond (PA), and Triangle Lake (TL). Points ofsignificant

(p<0.05) linear contrast from control are shown with solid/darkened symbols. 1,000 and 5,000 Ilg/L

treatments are not shown, because they were similar to 500 Ilg/L treatments.

6
WAVERLY LAKE II FREEWAY LAKE II

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--+

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r r a. a.

7 -Control -Control

/>201-19 /1 (). 20119 /1

6 01001-19/1 6 o 100119/1

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50 10 20 30 40 50 50 10 20 30 40 50

Day Day

PEAVY ARBORETUM POND

II TRIANGLE LAKE

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5 0'--~--"-o10~-J---=20'-:c--'--~30"----'--"""""':4""0"---"'----7"0

Figure 3. Mean pH of Leffler microcosms exposed to atrazine. Source communities used to develop

these microcosms were from Waverly Lake (WL), Freeway Lake (FL), Peavy Arboretum Pond (PA),

and Triangle Lake (TL). Points ofsignificant (p<0.05) linear contrast from control are shown with

solid/darkened symbols. 1,000 and 5,000 Ilg/L treatments are not shown, because they were similar

to 500 Ilg/L treatments.

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Data Summary Table:

Effect Magnitude of Nominal Day ofStudy Time Did Comments

Effect relative Atrazine Effect Weighted Recovery

to controls Concentration Occurred Average Occur- if

(/lg/L) Atrazine so, when

Concentration (see

pom Day 0 to comment) ?

When Effect

Occurred

No effect 20 !lg/L Day 0 17.7 !lg/L N/A Values

estimated from

Decrease in 20% 100 !lg/L Day 0 99.9 !lg/L No graph for

pH Waverly Lake.

30% 200 !lg/L Day 0 204.2 !lg/L No Differences

30% 500 !lg/L Day 0 491.7 !lg/L No significant at

p<0.05.

No effect 20 !lg/L NA NA NA Values

Decrease in estimated from

primary 50% 100 !lg/L Day 0 99.9 !lg/L No graph for

production Waverly Lake.

80% 200 !lg/L Day 0 204.2 ~lg/L No Differences (Pn) significant at 120%

(negative) 500 !lg/L Day 0 491.7 !lg/L No p<0.05.

Change in

dissolved

oxygen and Variables are too closely related to Pn and dark respiration to be considered separate effects Pn/dark

respiration

ratio

No effect 20 !lg/L N/A NC N/A Study text

reports

Change in No effect 100 !lg/L N/A NC N/A (p<0.05)

dark No effect 200 !lg/L N/A NC N/A insignificant . . I respIratIOn differences at

NR 500 !lg/L NR NC NR treatments

<500 llg/L

No effect 20 !lg/L N/A NC N/A Study text

Change in No effect 100 )lg/L N/A NC N/A reports that

differences heterotrophic No effect 200 )lg/L N/A NC N/A were "usually activit/ not significant" No effect 500 !lg/L N/A NC N/A (p<0.05)

Note: NR = not reported; N/A = not applIcable; NC = not calculable

No time-weighted averages needed to be calculated because effects occurred on Day O.

Mean atrazine concentrations on Day 0 from Table 1 are used as TWA concentrations.

I Ibid.

2 Ibid.

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Reviewer Comments:

The study was well controlled and clearly presented, although some reporting

deficiencies were noted. For example, it would have been helpful in terms of interpreting

the between-lake differences ifmore information about the initial community samples

had been provided. Background or prior exposure to atrazine in the natural lakes was not

analyzed, and the precise timing and location ofthe sampling was not provided. Some

results (heterotrophic activity, dark respiration) were not fully described, leading to

incompleteness in the Data Summary Table.

One ofthe goals ofthe Leffler microcosm approach is to remove differences in the

natural communities by acclimating them to lab conditions. It is then expected that the

source ofthe initial community sample is reduced so that the experiment may be easily

replicated. This assumption was shown to be not completely realistic, as some betweenlake differences were clear in the four experiments, despite the fact that the dominate

genera became very similar after lab acclimation. For this reason, it would have been

useful to know more about the conditions ofthe source samples. Overall, though, the

applicability ofthe Leffler microcosms to natural aquatic systems seems much better than

that ofthe Taub microcosms of Stay, et ai. (1985), because the community is derived

from natural sources and is at near-steady state when the experiment commences.

The authors describe their results in the context ofthe deNoyelles and seminatural

mesocosm experiments as a different "level of biological organization" than the full

ecosystems ofthe latter. The microcosms cannot capture effects on macrophytes or

synergistic responses due to fish grazing, though they provide more complexity than

single-species toxicity studies. The authors assert that the lowest treatment concentration

at which treatment effects are observed in microcosm experiments probably constitutes a

lower bound on the concentration likely to cause effects in experimental ponds.

However, they also state that one cannot necessarily predict the ecological response at

one level of biological organization from observations at another.

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

Rationale for Use: Study limitations do not invalidate results. The study adds to the

weight of evidence regarding potential effects of atrazine to aquatic plant communities.

Limitations of Study: This was a well-controlled and well-conducted study, but it has

limitations. The presentation ofthe results was incomplete. Although Leffler

microcosms appear more applicable to natural aquatic systems than Taub microcosms,

they are still highly simplified representations of actual ecosystems. The biotic

community changed significantly after lab acclimation, so that experimental results

9
should not be considered directly representative ofresponse of a natural community,

despite the natural source ofthe samples.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Brian Anderson, U.S. EPA

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