### 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. 

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Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study were also used to parameterizethe Comprehensive Aquatic System Model (CASM) and establish the model-associated LOC for atrazine.



### Date ofReview: November 14,2008


### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in Leffler microcosms.


### Summary ofStudy Findings:

Methods: Microcosms were prepared according to the protocol ofLeffler (1984), 

### The use ofselfselected, generic aquatic microcosmsforpollution effects assessment, inthe book "Concepts in marine pollution measurement." Microcosms are l-L beakers filled with 950 mL Taub and Dollar (TD) media (see Stay, 

### etaI., 1985 review) and 50 mL ofa stock culture developed from a natural aquatic community. Each weekduring the experiment, each system was reinoculated with 50 mL ofthe same culture. The microcosms initially stabilized for 6 weeks andthen predominant organisms were identified. Each microcosm was then assignedrandomly to atreatment orcontrol group and atrazine was added. The day ofatrazine addition was Day 0 andthe experiment ended on Day 42. During the 42 days ofthe experiment, the microcosms were incubated at 20±1°C under a 12hour light/12 hour dark cycle with light at 5400 lux.



# 111111••11 2087835Stock 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 (yearnot stated) from Waverly Lake (WL; an urban lake in Albany, Oregon), Freeway Lake (FL; a lake in an agricultural watershed 2 km southeast ofAlbany), Triangle Lake (TL; a rural mountain lake in a forested watershed in the Coast Range, 45 km west ofAlbany), and Peavy Arboretum Pond (PA; a lake in an agricultural watershed 7 km northeast ofCorvallis, Oregon). WL and FL are the most productive lakes and PA is the least productive and mostturbid. (No other information was provided on lakes, water quality, sampling locations within the waterbodies, background levels ofatrazine, orprior 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 ofwater were required for atrazine analysis, Day 0 concentrations ofatrazine 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 percentremaining on Day 42 overthe four experiments. Atrazine was analyzed by extraction from 100 mL water samples with 20 mL pesticide-grade ethyl acetate, concentration ofextract, 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), netprimary 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 stirbar. 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, 

### Methodsfor chemicalanalyses of waterandwastes; and APHA, 1980, 

### Standardmethodsfor the examination of waterandwastewater). 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 measurementprecision 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 ofglucose decomposed to CO2, was measured according to Kadota (1972), 

### Techniquesfor the assessmentof microbial production anddecomposition infresh waters, IBP HandbookNo. 23. 14C-labeled glucose (0.1 f.lCi) was added to a 10 mL sample, and the sample was then incubated at

220°C for four hours, acidified, and the CO2purgedthrough a C02 trap. Trapped 14C02 was counted by liquid scintillation. Netprimary productivity and darkrespiration were estimatedby the amount ofDO change during the light and darkperiods, respectively. Estimates were corrected for oxygen exchange with the atmosphere by the procedures of Stay, 

### etal. (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 contrastt test with either pooled variance ornon-pooled variance was conducted, the former requiring no significant differences among replicate variances atp-value < 0.05.



### Results: The study lists predominanttaxonomic groups in the lake communities and in the microcosms on Day 0 ofeach experiment in atable (notprovided 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 similarto one anotherthan 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 ofadded 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 notprovided. 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 similareffects 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 ormore and developed from lakes WL, FL, and TL were significantly different from theirrespective controls overthe first 14 days (p<0.05; see Figure 1). There was generally no recovery in these microcosms overthe 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 of200 Ilg/L orabove (p<0.05).

Pn/Rni ratios were significantly less than controls (p<0.05) at atrazine treatments of 100 Ilg/L ormore 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 ormore had significantly lowerpH 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 ofatrazine added on Day 0, and average percent atrazine remaining on Day 42, ofall 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



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Figure 1. Mean netprimary productivity (Pn) ofLeffler microcosms exposed to atrazine. Source communities used to develop these microcosms were from Waverly Lake (WL), FreewayLake (FL), Peavy Arboretum Pond (PA), and Triangle Lake (TL). Points ofsignificant (p<0.05) linearcontrast from control are shown with solid/darkened symbols. 1,000 and 5,000 !!g/Ltreatments are not shown, because they were similarto 500 !!glLtreatments.

5FREEWAY LAKE**5.-------------~** WAVERLY LAKE**5,-------------~**
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Figure 2. Mean net primary productivity/respiration (Pn/Rni) ratio ofLeffler microcosms exposed to atrazine. Source communities used to develop these microcosms were from Waverly Lake (WL), Freeway Lake (FL), Peavy Arboretum Pond (PA), and TriangleLake (TL). Points ofsignificant (p<0.05) linearcontrastfrom control are shown with solid/darkened symbols. 1,000 and 5,000 Ilg/ L treatments are not shown, becausethey were similarto 500 Ilg/L treatments.



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Figure 3. Mean pH ofLeffler 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 contrastfrom 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 of Study Time Did Comments Effectrelative 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 darkrespiration 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 differences at respIratIOn 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 = notreported; N/A = not applIcable; NC = not calculable



### No time-weighted averages neededto be calculatedbecause effects occurred on Day O. Mean atrazine concentrations on Day 0 from Table 1 are used as TWA concentrations.

I Ibid. 2Ibid.

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### Reviewer Comments: The study was well controlled and clearly presented, although some reporting deficiencies were noted. For example, it wouldhave been helpful in terms ofinterpreting the between-lake differences ifmore information aboutthe initial community samples had been provided. Background orpriorexposure to atrazine in the natural lakes was not analyzed, and the precise timing and location ofthe sampling was notprovided. Some results (heterotrophic activity, darkrespiration) were not fully described, leading to incompleteness in the Data Summary Table.

One ofthe goals ofthe Lefflermicrocosm approach is to remove differences in the natural communities by acclimating them to lab conditions. It 

### is then expectedthatthe source ofthe initial community sample is reduced so thatthe experimentmay be easily replicated. This assumption was shown to be not completelyrealistic, as some betweenlake differences were clear in the four experiments, despite the fact thatthe dominate generabecame very similar after lab acclimation. Forthis reason, itwouldhave been useful to know more aboutthe conditions ofthe source samples. Overall, though, the applicability ofthe Leffler microcosms to natural aquatic systems seems much betterthan that ofthe Taub microcosms ofStay, 

### etai. (1985), becausethe community is derived from natural sources and is atnear-steady state when the experimentcommences.

The authors describe theirresults in the contextofthe deNoyelles and seminatural mesocosm experiments as adifferent "level ofbiological 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 assertthatthe lowesttreatment concentration atwhich treatment effects are observed in microcosm experiments probably constitutes a lowerbound on the concentration likely to cause effects in experimental ponds. However, they also state that one cannotnecessarily predictthe ecological response at one level ofbiological organization from observations at another.
Description ofUse in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: Study limitations do not invalidate results. The study adds to the weightofevidence regarding potential effects ofatrazine to aquatic plant communities.

Limitations ofStudy: This was awell-controlled and well-conducted study, but ithas limitations. The presentation ofthe results was incomplete. Although Leffler microcosms appearmore applicable to natural aquatic systems than Taub microcosms, they are still highly simplifiedrepresentations ofactual ecosystems. The biotic community changed significantly after lab acclimation, so that experimental results

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### should not be considered directly representative ofresponse ofanatural community, despite the natural source ofthe samples.


### Primary Reviewer: SaraHendrix, M.S., StaffScientist, Cambridge Environmental Inc


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

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