### Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Brockway, D.L., P.D. 

### Smith, and F.E. Stancil. IYM. 

### Fate and effects ofatrazine on small aquatic microcosms. Bull. Environ. Contam. Toxicol. 32:345-353. (MRID 47543602).


### Purpose ofReview: Determine potential effects ofatrazine 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 ofReview: December 1l, 2008


### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals (net plankton plusAufWuchs) 

### in both static and continuous-flow microcosms.


### Summary ofStudy Findings:

Methods: Two sets ofexperiments were conducted: one in continuous-flow aquaria, and one in static beakers. Atrazine of98.2% purity was obtained from the Reference Standards Repository, USEPA, Research Triangle Park, North Carolina. Analyses used high performance liquid chromatography, a 2% water-methanol mobilephase, a Whatman Partisil ODS-2 column, and a variable wavelength UV-VIS detectorat222 nm.

Continuous-flow experiments Continuous-flow experiments occurred in 12 seamless glass aquaria, 20 em x 

### 30 em x 

### 18 em, in atemperature-controlledroom at 22°C. Light sources were daylight-balanced high intensity fluorescent lamps with intensity 6500±500 lux, on a 12 hours on, 12 hours offphotoperiod. Microcosms were initialized with a 0.5-cm layer ofpond sediments, 7 L ofpond water, and "an inoculum" (size/concentration not described) ofnetplankton plus AufWuchs 

### from a mesotrophic, softwater, 2 hapond (location, land use, etc. 

### not described). Microcosms also received weekly additional inocula for the duration ofthe 

# 1811.11111 2087801

### 49-weekexperiment. Freeman's standardreference waterwas addedthrough aglass and stainless steel system at arate of0.6 mL/min, giving an 8.1 day retention time in the microcosms. Microcosms were continuously mixed by chain-driven steel stirrers at 50 rpm.

Twelve microcosms were organized into 4 sets of3 containers, each set on a separate nutrient feed system. After allowing 39 weeks for microcosms to develop, atrazine
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### systems for the fourrespective sets. The microcosms were not spiked, so the concentration increased gradually. For acclimation tests, concentrated spikes were added to each microcosm directly. Forresilience tests, 50% ofthe nutrientmedium was removed and the tankrefilled with uncontaminated nutrientmedium on two successive days, and the normal nutrient inputwas switchedto uncontaminated medium.



### Treatments were as follows: Treatment 1: 5 ug/L Treatment 2: 50 ug/L (Day 0to 55) 

### +0 ug/L (removal ofatrazine from the system at Day 56) 

### + 50 ug/L (Day 63) Treatment 3: 0.5 ug/L (Days 0 to 28) 

### + 50 ug/L (Day 29) 

### + 100 ug/L (Day 35) 

### +0ug/L (removal ofatrazine from the system at Day 50) 

### + 100 ug/L (Day 63)

Six parameters were monitored in the continuous-flow experiments. Dissolved oxygen (DO) and pH were measuredbefore and afterthe 12-hourlightperiod, using an Orbisphere DO meter and an Extech Model 609 digital pH meter. Total dissolved phosphorus, nitrate, nitrite, and ammonia concentrations were determined weekly, on the same day and atthe same time (1 

### hour after lights-on), using aTechnicon Auto Analyzer II (following USEPA, 1979, 

### Methodsfor chemicalanalysis of waterandwastes).

Static Experiments Static experiments used l-L glass beakers containing 600 mL ofthe nutrientmedium. The beakers were inoculated with 2-g aliquots (wetweight) ofmixed biota from I-yearold laboratory flow systems established for stock purposes. The inoculum was dominated by 

### Spirogyra sp., and also included 

### Oedogonium, Microcystitis, Apthanothece, Scenedesmus, other algal species, flagellate protozoans, and nematodes. Temperature, lighting, and DO and pH monitoring proceeded as in the continuous-flow experiments. Fourreplicates each at atrazine treatment levels of0 (control), 50, 500, and 5,000 )lg/L were tested for 12 days in the static experiments, with one set ofreplicates sacrificed on days 1 and 7. A I-replicate abiotic control was also included. A second experimentused 3 replicates at each atrazine treatmentlevels of0 (control), 0.5, 5, and 100 )lg/L and lasted for 7 days.

2

### Results: Measured atrazine concentrations were close to nominal and stable in the continuousflow systems (Figure 1), with the static tests showing slightly more variability (results not described).

In the static experiments, net DO production during the day was similarto controls in the atrazine treatment levels of0.5 and 5 Ilg/L, decreased by 25-30% in the 50 Ilg/L treatments, decreased 40-50% in the 100 Ilg/L treatments, decreased 90% in the 500 Ilg/L treatments, and went to zero ornegative production in the 5,000 Ilg/L treatments (see Figure 2). Within-treatment variations were very low (coefficients ofvariation orCYs were typically below 7%). (No significance testing results were provided.) Biotic community composition was similarin all treatments, and appeared healthy at and below atrazine treatments of500 Ilg/L, although after 12 days at 5,000 Ilg/L the algae were totally bleached (viable rotifers and nematodes were still present).

In the continuous-flow experiments, during the pretreatmentmicrocosm development phase, net DO production during the day increased from about 4 mg/L in week 1 to 11 mg/L during week 14, leveled offuntil week 24, and then slowly declined until the end of the pretreatment phase. After atrazine treatment commenced, oxygen production (as % ofcontrol production) fluctuated in response to various treatments, spike, and dilutions (see Figure 3). The 5 Ilg/L constanttreatment appearedto eventually recover (around day 50 oftreatment) with respectto the net diurnal DO production, whereas the more complex treatment regimes showed clear drops immediately after atrazine spikes were added and sudden rises immediately after dilutions occurred, apparently regardless of previous exposures. Within-treatment CYs were very good, typicallybelow 19%, though controls varied by ± 10% during a period ofseveral days, so treatment effects were not judged as qualitatively significant (no statistical significancetestresults provided).

Ofthe otherchemical parameters measured, only dissolved nitrate changed significantly after atrazine treatment. Nitrate concentrations increased significantly when Treatment 3 was spiked to 100 Ilg/L (Student's t-test, p-value < 0.01). Dilutions were similarly marked by decreases in nitrate (no statistics provided). See Figure 4. The authors calculated that the nitrate decrease was what could be expected from the decrease in nitrate utilization caused by inhibition ofphotosynthesis, as estimated from DO changes.
Microscopic observations ofbiotathroughoutthe experiments showed similar composition through all times and treatment levels. Bluegreen algae dominated, green algae and diatoms were also abundant, and copepods, ostracods, and annelids were also present.

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Figure 3. Netdiurnal dissolved oxygen production, as percentage ofcontrols, during the continuous-flow experiments. Treatment 1: 5 I!g/L; Treatment 2: 50, then 0, then 50 I!g/L; Treatment3: 0.5, then 50, then 100, then 0, then 100I!g/L. Timing ofTreatment3 spikes given by Arrows 1,2, and 5; of Treatment3 dilution given by Arrow 3. Timing ofTreatment 2 dilution given by Arrow 4 and Treatment2 spike given by Arrow 5. Each pointis the mean of3 replicates.

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Figure 4. Dissolved nitrate concentrations as mg NIL before and after atrazine treatment. Treatment 1: 5 Ilg/L; Treatment 2: 50, then 0, then 50/-lg/L; Treatment3: 0.5, then 50, then 100, then 0, then 100/-lg/L. Timing ofTreatment3 spikes given by Arrows 1,2, and 5; ofTreatment3 dilution given by Arrow 3. Timing ofTreatment 2dilution given by Arrow 4 and Treatment 2 spike given by Arrow 5. Each point is the mean of3 replicates.

6

### Data Summary Table: Note that all endpoints appear in Attachment 2, butwith misleading Test Cone, Results, PlantGroup, and Recovery information. Effect Magnitude Nominal Day of Time Did Recovery Comments ofEffect Atrazine Study Weighted Occur

- ifso, relative to Concentration Effect Average when (see controls (llg/L) Occurred Atrazine comment)? Concentration from Day 0to When Effect Occurred
no effect 0.5 J-lg/L NA NA/variable NA Decrease in Continuous-flow 20% 5J-lg/L Day 30 3.8 J-lg/L Yes

- Day 48 experiments with net diurnal Partial by Day time-varying dissolved 30% 50 J-lg/L Day 30 33.5 J-lg/L 55 treatments. oxygen Day] Magnitudes estimated production No (>]5 days 40% ]00 J-lg/L after spike 11.4 J-lg/L after spike) from Fig. 3. treatment
no effect 0.5 J-lg/L NA NA/variable NA

no effect 5J-lg/L NA NA/variable NA Continuous-flow Increase in 50% 50 J-lg/L Day] 8(?) 23.4 J-lg/L No (>40 days) experiments with dissolved time-varying nitrate 65% treatments. concentration (different Day I(?) No (>15 days Magnitudes estimated from 100 J-lg/L after spike I ].4 J-lg/L after spike) from Fig. 4. control at treatment p=O.Ol)
no effect 0.5 J-lg/L NA 0.5 J-lg/L NA Decrease in no effect 5J-lg/L NA 5J-lg/L NA net diurnal 25% 50 J-lg/L Day] 50 J-lg/L No (>12 days) Static experiments. dissolved Magnitudes estimated oxygen 40% 100 ).1g/L Day I ]00 J-lg/L No (>7 days) from Fig. 2. production 90% 500 J-lg/L Day] 500 J-lg/L No (>12 days) >100% 5,000 J-lg/L Day 1 5,000 ).1g/L No (>12 days) Note: NA=Not Applicable



### No data on atrazine concentrations for the static experiments were provided, so forthose experiments, time-weighted average atrazine concentration was assumed to be nominal. Forthe continuous-flow experiments, atrazine concentrations (as shown in Figure 1) slowly increased overthe first 20 days ofthe experiment. Additionally, calculation of time-weighted averages was complicated by the time-varying treatmentregimes for Treatment 2 (50 Ilg/L, then 0 Ilg/L, then 50 Ilg/L) and Treatment 3 (0.5 Ilg/L, then 50 Ilg/L, then 100 Ilg/L, then 0 Ilg/L, then 100 Ilg/L). Chemographs were determined from a combination ofFigure 1, assuming concentrations equal to nominal (assuming nominal spike ordilution concentrations) afterDay 20, and the timing ofspikes and dilutions (i.e. from Figure 3 arrows), and then running averages were computed. For simplicity, linear increases between datapoints on Figure 1 and zero concentrations on Day 0 were assumed for creation ofchemographs, and it was assumed thatthe nominal 0.5 Ilg/L

7treatment (Treatment 3) followed the pattern ofTreatment 1 forthe first 20 days, with concentrations at l/lOth the level, since these datawere not provided in Figure 1. Timeweighted averages are computed for all time elapsed since Day 0 ofthe treatment phase. The authors found the latterwas abetter determinerofthe effects they saw. See the linked Excel spreadsheetbelow. (It continues beyond the end ofthe page to coverthe whole 75-day interval.)

8Treatment 1 Treatment 2 Treatment 3 Day Cone. TWA Cone. TWA Cone. TWA 0 0.0 0.0 0.0 0.0 0.0 0.0 1 0.4 0.2 1.4 0.7 0.0 0.0 2 0.8 0.4 2.8 1.4 0.1 0.0 3 1.1 0.6 4.1 2.1 0.1 0.1 4 1.5 0.8 5.5 2.8 0.2 0.1 5 2.0 1.0 10.3 4.0 0.2 0.1 6 2.5 1.2 15.2 5.6 0.3 0.1 7 3.0 i.4 20.0 7.4 0.3 O.-i 8 3.3 1.6 22.5 9.1 0.3 0.2 9 3.5 1.8 25.0 10.7 0.4 0.2 10 3.8 2.0 27.5 12.2 0.4 0.2 11 4.0 2.1 30.0 13.7 0.4 0.2 12 4.2 2.3 33.3 15.2 0.4 0.2 13 4.3 2.4 36.7 16.7 0.4 0.2 14 4.5 2.6 40.0 18.3 0.5 0.3 15 4.6 2.7 40.8 19.7 0.5 0.3 16 4.8 2.8 41.6 21.0 0.5 0.3 17 4.9 2.9 42.4 22.2 0.5 0.3 18 5.0 3.1 45.0 23.4 0.5 0.3 19 5.0 3.2 46.7 24.5 0.5 0.3 20 5.0 3.2 48.3 25.7 0.5 0.3 21 5.0 3.3 50.0 26.8 0.5 0.3 22 5.0 3.4 50.0 27.8 0.5 0.3 23 5.0 3.5 50.0 28.7 0.5 0.3 24 5.0 3.5 50.0 29.6 0.5 0.4 25 5.0 3.6 50.0 30.3 0.5 0.4 26 5.0 3.6 50.0 31.1 0.5 0.4 27 5.0 3.7 50.0 31.8 0.5 0.4 28 5.0 3.7 50.0 32.4 0.5 0.4 29 5.0 3.8 50.0 33.0 50.0 2.0 30 5.0 3.8 50.0 33.5 50.0 3.6 31 5.0 3.8 50.0 34.0 50.0 5.0 32 5

..0 3.9 50.0 34.5 50.0 6.4 33 5.0 3.9 50.0 35.0 50.0 7.7 34 5.0 3.9 50.0 35.4 50.0 8.9 35 5.0 4.0 50.0 35.8 100.0 11.4 36 5.0 4.0 50.0 36.2 100.0 13.8 37 5.0 4.0 50.0 36.6 100.0 16.1 38 5.0 4.1 50.0 36.9 100.0 18.2 39 5.0 4.1 50.0 37.2 100.0 20.3 40 5.0 4.1 50.0 37.5 100.0 22.2 41 5.0 4.1 50.0 37.8 100.0 24.1 42 5.0 4.1 50.0 38.1 100.0 25.8 43 5.0 4.2 50.0 38.4 100.0 27.5 44 5.0 4.2 50.0 38.6 100.0 29.1 45 5.0 4.2 50.0 38.9 100.0 30.7 46 5.0 4.2 50.0 39.1 100.0 32.1 47 5.0 4.2 50.0 39.4 100.0 33.6 48 5.0 4.2 50.0 39.6 100.0 34.9 49 5.0 4.3 50.0 39.8 100.0 36.2 50 5.0 4.3 50.0 40.0 0.0 35.5 51 5.0 4.3 50.0 40.2 0.0 34.8 52 5.0 4.3 50.0 9 40.4 0.0 34.2 53 5.0 4.3 50.0 40.5 0.0 33.5 54 5.0 4.3 50.0 40.7 0.0 32.9

### Reviewer Comments: The continuous-flow study data were difficultto interpret within the framework oftimeweighted averages, days to recovery, 

### etc., due to the time-varying nature oftwo ofthe three treatment regimes. Additionally, statistical significance tests were not conducted forthe vast majority ofthe variables measured, so despite the reasonably good replication it is not clearhow significant are most ofthe results.

Microcosms, with simplifiedbiota communities, were the subject ofstudy. The source pond for the continuous-flowbiota was notwell described. Biota consisted only ofdrift (net) plankton and

### Aufwuchs that could grow from an inoculum. Microcosms were given a reasonable (nearly 40 week) amount oftime to acclimate to the laboratory and grow to steady-state, however, they remained simple (consisting ofmainly algae with some small animals). Re-introduction ofbiota during weekly inoculum additions may have unduly affected results as well.


### Description ofUse in Document (QUAL, QUAN, INV): QUAL



### Rationale for Use: Study contributes to weight ofevidence regarding potential atrazine effects to aquatic communities.



### Limitations ofStudy: Data Summary Table mustbe interpreted with care due to timevarying nature ofcontinuous-flow atrazine treatments. Statistical tests were not performed for most data, andraw data are notprovided. Microcosms, though the biota were natural in origin, were very artificial, and results may not be applicable to more complex natural ecosystems.


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


### Secondary Reviewer: Brian Anderson

10