Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Lampert, W., W. Fleckner, E. Pott, U. Schober, and K.U. Storkel. 1989. Herbicide

effects on planktonic systems of different complexity. Hydrobiologia. 188/189:415-424.

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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 19,2008

Purpose of Study:

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

laboratory microcosms and in experimental enclosures in a natural lake.

Summary of Study Findings:

Methods: A set of experiments was conducted ranging from direct toxicity tests of

atrazine on Daphnia to full community assessments that allowed for interpretation of

indirect effects. Methods and results presented here will focus primarily on the tests that

allowed for at least a two-step food chain.

Atrazine used in all experiments carne from Ciba Geigy, Basel (it is not stated whether it

was reagent grade or in a commercial product formulation). A solvent was not used. The

authors state, "All chemical and biological analyses were carried out according to routine

protocols" (the source ofthe protocols used is not stated). Particulate organic carbon

(POC) was measured using an infrared carbon analyzer described in a 1981 German

journal article (Krambeck et aI., GIT Fachz. Labor. 25).

Laboratory tests were carried out with an obligately pathenogenetic strain ofDaphnia

pulicaria Forbes and with D. magna Straus, whose stocks had been kept for several years

at 20°C and continuous dim light. The food for the daphnids in all experiments was

III1IIIIII

2087866
Scenedusmus acutus Meyen green algae, grown in CHU 12 media in chemostats

(described in a 1976 article by the lead author). 0.45-flm membrane filtered lake water

from either Lake Constance or a pond in the Frankfurt Botanical Gardens was the test

water, kept at 20°C for all experiments. Acute lethality tests of atrazine on daphnids

were conducted, as well as sublethal tests for effects on growth and reproduction and on

feeding inhibition.

A two-step "food chain" system in the laboratory assessed the effects of atrazine on algae

and Daphnia. See Figure 1 for a diagram ofthe set-up. Scenedesmus was grown in two

different chemostats, one with and one without atrazine, and the algae samples were

diluted with filtered pond water (with or without atrazine) before addition to the

microcosms containing D. magna females. The experiment began with 10 neonate D.

magna females in each microcosm (2-L glass beaker). Four replicate microcosms were

created at each treatment regime: atrazine in algae chemostat only; atrazine in dilution

water only; atrazine in both algae chemostat and dilution water; and no atrazine exposure

(controls). Experiments were run for atrazine concentrations of 0.3, 0.1,0.05 (two

experiments), and 0.01 mg/L (300,100,50, and 10 flg/L). Each experiment ran for 3

weeks, and afterwards the number of animals per beaker, dry weight ofthe population,

and the number of eggs were measured. (Atrazine concentrations were apparently not

monitored.)

Enclosure experiments with natural communities were carried out in plastic bags

mounted in an aluminum frame floating in Schohsee, a moderately eutrophic lake in

northern Germany (then West Germany). The lake has a small catchment area where no

com or treated crops are raised, and so no prior exposure to atrazine was expected to

affect the community responses. The enclosures had about 1 m diameter and were about

2.7 m long, and held 1.7 m

3 ofwater. Experiments were carried out in different seasons

and with different initial atrazine conccntrations (0.1, 1, 10, and 100 flg/L). In each

experiment, four bags were filled with lake water that had been filtered through a 100 flm

mesh to screen out large zooplankton, and the bags were then inoculated with equal

amounts of zooplankton collected with a plankton net. Prior experiments had shown that

all four bags developed "very similar" communities when left untreated (prior

experiments not further described). Two bags received atrazine, and two remain as

controls. Experiments lasted "typically" "no longer than 3 weeks" because ofthe growth

ofperiphyton on the bags (exact durations ofthe experiments not stated). SamplesJor

chemical and biological analysis, including zooplankton sampled by vertical 1OO-flm net

haul, were taken "at regular intervals" (about every few days; see results figures).

Atrazine concentrations were measured by gas chromatography (a 1989 dissertation from

Univ. Kiel is cited but no additional details are provided).

Results: Methods and results presented here will focus primarily on the tests that allowcd

for at least a two-step food chain. Direct atrazine toxicity tests found that Daphnia has a

48-hr LDso of about 10 mg/L (10,000 flg/L), with sublethal effects observed at about I

2
order of magnitude lower atrazine concentrations. At 2 mg/L (2,000 /lg/L) atrazine,

Daphnia had significantly (p<0.05; test not stated) reduced reproduction and growth.

Significant (p-value < 0.0 1) reductions in final daphnid biomass in the two-step

laboratory food chain system were found at 0.3 and 0.1 mg/L (300 and 100 /lg/L)

atrazine, and at 0.05 mg/L (50 /lg/L) atrazine in one ofthe two experiments at this

concentration (p-value < 0.01 for combined effect, p-value < 0.05 for algae effect only).

These reductions were seen only in the treatments where algae were affected by atrazine

treatment. An apparent contradictory effect occurred, where microcosms with Daphnia

being directly but not indirectly exposed to atrazine (i.e. atrazine was in the dilution

water, but not in the algae growth chamber) actually had increased biomass relative to

controls. The authors explain this effect as being due to initial but subtle relative

decreases in the development ofthe directly-exposed daphnid population, which allowed

these populations to avoid the Daphnia bloom (and subsequent crash) that affects the

control populations; they state that the duration ofthe experiments should have been

either shorter or much longer in order to avoid this artifact. See Figure 2 for a figure

showing the results ofthe laboratory food chain experiments.

In the enclosure experiments, similar results were found for all atrazine concentrations

between 1 /lg/L and 100 /lg/L. Atrazine was persistent in these experiments, with day 18

concentrations being within 90% to 95% of initial values. In these experiments, primary

production and chlorophyll a decreased in the treatments after about 7 days, and as algae

died, POC grew and oxygen decreased. (See Figure 3 and Figure 4.) Concurrently,

zooplankton abundance declined and composition changed, with cladocerans declining

more than copepods (see Figure 5). (Figures are for the 1 /lg/L atrazine experiment only;

the authors state that 10 /lg/L and 100 /lg/L atrazine experiments "were qualitatively

identical to those at 1 /lg/L, but were even stronger" and do not provide figures or data.)

The authors also note that bacterial and blue-green filaments occurred at the time of

oxygen decline, and attribute the increase in particulate organic carbon to bacterial

production and detritus formation. They also attribute the excessive cladoceran decline

relative to copepods to loss oftheir algal food base and increased abundance of flakes

and filaments that interfere with their food collection, whereas the copepods still had

abundant small zooplankton prey.

Tests with 0.1 /lg/L atrazine were performed twice in 1982, once in June/July and once in

September, and once in August and September 1984. In the latter experiment, the

authors state that no differences between treatments and controls were seen, so results are

not presented. The 1982 experiments have results shown for the Warm (W; June/July)

and Cold (C; September) experiments. The Cold experiment, bags were left in the lake

after experiment termination for another 17 days and sampled again on Day 42. Atrazine

in the treatment bags was no longer detectable after 10 days. In the 0.1 Ilg/L atrazine

experiments, algal biomass (chlorophyll a) did not differ between treatments and controls

in the Warm study, but transient relative decreases with treatment were noted in the Cold

study that ultimately led to a large spike (algal bloom) noted on Day 42 (see Figure 7).

3
Primary production (photosynthetic rate) was more sensitive to treatment in both the

Warm and Cold experiments, but like biomass it appeared to recover by the end ofthe

experiments. Oxygen was reduced in the treatments and did not fully recover, except

partially after 42 days in the Cold experiment (see Figure 8). (The authors state

"recovery ofthe oxygen concentration occurred after two and three weeks, respectively";

it is not clear what "respectively" refers to, and the statement seems in contradiction with

the figure provided.) Daphnia was nearly eliminated in the treatment bags, but was seen

to rebound after 42 days in the Cold experiment (see Figure 9).

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Figure 1. Schematic of the "food chain" laboratory experimental set-up. s: chemostat containing

Scenedesmus; M: algal nutrient medium; W: filtered pond water; PP: peristaltic pump; PI: piston

pump; MP: multichannel peristaltic pump; 0: overflow. Asterisk denotes presence of atrazine.

Treatments labeled by letters. C: controls; W: water effect; A: algae effect; T: combined effect.

4
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Concentration of atrazlne (mg/U

Figure 2. Biomass of Daphnia populations in the "food chain" laboratory experiments after 3 weeks

with different atrazine concentrations. Letters denote treatments. C: controls; W: water effect; A:

algae effect; T: combined effect. Vertical bars show the standard deviation (n=4). Asterisk indicates

that the mean is significantly different from the control mean according to Tukey's test. Large

asterisk: p-value < 0.01; small asterisk: p-value < 0.05.

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Figure 3. Enclosure experiment with 1 J.tg/L atrazine. Time series of chlorophyll a and particulate

organic carbon (POC). Open circles: control bags; closed/solid circles: treatment bags.

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Figure 4. Enclosure experiment with 1 Ilg/L atrazine. Time series of oxygen saturation. Open

circles: control bags; closed/solid circles: treatment bags.

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Figure 5. Enclosure experiment with 1 /-lgiL atrazine. Time series of zooplankton. NAU: nauplii;

DA: Daphnia; CY: Cyclops; BO: Bosmina. Open circles: control bags; closed/solid circles: treatment

bags.

6
Figure 6. Enclosure experiment with 0.1 /-lg/L atrazine. W: Warm experiment; C: Cold experiment.

Time series of chlorophyll a. Open circles: control bags; closed/solid circles: treatment bags.

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Figure 7. Enclosure experiment with 0.1 /-lglL atrazine. W: Warm experiment; C: Cold experiment.

Time series of primary production. Open circles: control bags; closed/solid circles: treatment bags.

7
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Figure 8. Enclosure experiment with 0.1 /-lg/L atrazine. W: Warm experiment; C: Cold experiment.

Time series of oxygen saturation. Open circles: control bags; closed/solid circles: treatment bags.

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Figure 9. Enclosure experiment with 0.1 /-lg/L atrazine. W: Warm experiment; C: Cold experiment.

Time series of Daphnia abundance. Open circles: control bags; closed/solid circles: treatment bags.

8
Data Summary Table:

Effect Magnitude Nominal Day of Time Weighted Did Comments

ofEfJect Atrazine Study Average Atrazine Recovery

relative to Concentra EfJect Concentration Occur - if

controls tion Occurred from Day 0 to so, when

(flgIL) When EfJect (see

Occurred comment)?

>95% 300 Ilg/L 300 Ilg/L Results of laboratory "food Decline in chain" experiments with Daphnia Day 21 combined algae and water biomass 90% 100 Ilg/L (end of 100 Ilg/L No (end of effects. P-values for effects are resulting from study) study) all <0.01. Magnitudes effects on food

(algae). 60% 50 Ilg/L 50 Ilg/L

estimated from Fig.2; the leftmost 0.05 mg/L plot is used.

No effect 10 Ilg/L 10 Ilg/L

NR 100 Ilg/L NR not calculable NR

Decline in NR 10 Ilg/L NR not calculable NR Results of enclosure

Daphnia experiments. Magnitudes

biomassI >90% I Ilg/L Day II 0.97 Ilg/L No estimated from Figs. 5 and 9

>90% 0.1 Ilg/L Day 18 0.03 Ilg/L No ("C").

Decline in NR 100 Ilg/L NR not calculable NR Results of enclosure

NR 10 f,lg/L NR not calculable NR experiments. Magnitudes primary est.imated from Fig. 7 ("C"). production NR 1 J.1g/L NR not calculable NR

(photosynthet Effect at 0.1 ug/L was not

ic rate) 60% 0.1 J.1g/L Day 18 0.03 J.1gIL Yes - Day considered to be clearly related

25 to atrazine treatment.

NR 100 Ilg/L NR not calculable NR

NR 10 Ilg/L NR not calculable NR Results of enclosure

Decline in 50% 1 J.1g/L Day 11 0.97 J.1g/L No experiments. Magnitudes

chlorophyll a estimated from Figs. 3 and 6

No clear 0.1 J.1g/L NA NA NA ("C").

effect

NR 100 f,lg/L NR not calculable NR Results of enclosure experiments.

NR 10 f,lg/L NR not calculable NR Results presented in Figs. 4 and 8

("C"). There is uncertainty in the

50% 1 J.1g/L Day 11 0.97 J.1gIL No results at 0.1 ug/L because the

oxygen levels may have been

affected by daphnid mortality and

Decline in corresponding decomposition.

oxygen However, there was an apparent

saturation2 decrease in photosynthesis at

40% 0.1 J.1g/L Day 18 0.03 J.1g/L No (partial approximately the time of

at Day 42) observed reductions in oxygen

levels. Therefore, these data

suggest that atrazine may have

impacted photosynthesis and

corresponding oxygen levels at 0.1

ug/L.

Note: NR=Not Reported; NA = Not applIcable; Balded values are those data pomts specIfied m the "Cosm

Data Points and Exposure Profiles" spreadsheet.

I Ibid. Note that three other zooplankton declines appear in Fig. 5; only Daphnia is included here, because

it is the only zooplankton that was also examined at 0.1 Ilg/L, and it has the most extreme responses of all

zooplankton studied.

2 Ibid.

9
Since the laboratory "food chain" experiments used controlled continuous inputs of

treated water and growth medium, it was assumed that no significant declines in atrazine

concentration occurred over time. For the enclosure experiments, time weighted

averages were calculated based on the information in the study text: I) 90% ofthe

atrazine from the 1 /lg/L treatment bags remained on Day 18; and 2) No atrazine could be

detected in the 0.1 /lg/L treatment bags on Day 10. The detection limit was not reported;

therefore, a conservative assumption of 0 ug/L was used to calculate time-weighted

average concentrations at time points when no atrazine was detected. Chemographs were

computed assuming a linear decline in atrazine concentration over the time period of the

experiment, and time-weighted averages were then simple cumulative average

concentrations. Calculations are shown below.

0.01 ug/L1day 0.005556 ug/L1day

0.1 ug/L 1 ug/L

Cone Day TWA Cone Day TWA

0.10 0 0.10 1.00 0 1.00

0.09 1 0.10 0.99 1 1.00

0.08 2 0.09 0.99 2 0.99

0.07 3 0.09 0.98 3 0.99

0.06 4 0.08 0.98 4 0.99

0.05 5 0.08 0.97 5 0.99

0.04 6 0.07 0.97 6 0.98

0.03 7 0.07 0.96 7 0.98

0.02 8 0.06 0.96 8 0.98

0.01 9 0.06 0.95 9 0.98

0 10 0.05 0.94 10 0.97

0 11 0.05 0.94 11 0.97

0 12 0.04 0.93 12 0.97

0 13 0.04 0.93 13 0.96

0 14 0.04 0.92 14 0.96

0 15 0.03 0.92 15 0.96

0 16 0.03 0.91 16 0.96

0 17 0.03 0.91 17 0.95

0 18 0.03 0.90 18 0.95

Reviewer Comments:

Interpretation ofthis study was hampered by inadequate descriptions ofthe protocols and

methods used for analysis, as well as incomplete presentation ofthe results ofthe

enclosure experiments. The lack offull descriptions ofthe protocols is particularly

problematic because this study took place in Germany, and so differences with standard

American procedures may affect understanding ofthe results. Exact dates and durations

ofthe enclosure experiments are not provided. The data summary table is incomplete

because results for 10 /lg/L and 100 /lg/L atrazine experiments were not presented. The

fact that wann versus cold experiments were conducted at 0.1 /lg/L atrazine levels but not

10
for other concentrations also confuses interpretation ofthe results, as inter-treatment level

comparisons may not be appropriate.

The laboratory "food chain" experiment barely qualifies as an ecological study, since

only one zooplankton species and one algal species were utilized. Indeed, the authors use

this experiment as an example ofthe effects that can be observed at a level only slightly

more complex than a direct toxicity bioassay. Results ofthe laboratory experiment thus

cannot be generalized to natural ecosystems; however, the laboratory conditions allowed

a reasonable level ofreplication (four replicates at each treatment) and well controlled

conditions.

The enclosure experiments appeared well controlled, but only two replicates were used at

each treatment level, so no statistical tests could be applied. Additionally, the bag

enclosures may have generated artifacts in the observations due to inhibited mixing and

inability of communities to recolonize, which limit their applicability to natural ponds.

Overall, the study is successful in its goal ofshowing that indirect effects may be more

important than direct effects in a complex community, but the experiments do not provide

a realistic simulation of environmental conditions.

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

Rationale for Use: The utility ofthis study is limited by the deficiencies described in

this review, and its results should be interpreted with caution. However, the study does

contribute to the weight of evidence regarding potential effects of atrazine to aquatic

communities.

Limitations of Study: The highly simplified, artificial structure ofthe laboratory "food

chain" experiments limits their applicability to real-world aquatic systems. Results for

higher-concentration enclosure experiments were not provided, and the use of "warm" VS.

"cold" lowest-concentration enclosure experiments interferes with comparison of effects

at different treatment levels. Finally, the study does not describe or provide sources for

nearly all analytical methods and sampling protocols used.

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

Secondary Reviewer: Brian Anderson, U.S. EPA #~_

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