### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: DeLorenzo, M. E., 

### J. Lauth, P. L. Pennington, G. 1. Scott and P. E. Ross. 1999. Atrazine effects on the microbial food web in tidal creekmesocosms. Aquatic Toxicology. 46: 241-251.


### Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plantcommunities and associated determination ofa Level ofConcern (LOC). 

### Date ofReview: 9/9/09


### Summary ofStudy Findings
Purpose ofStudy: This study examines the potential effects ofatrazine on phytoplankton and zooplankton in experimental estuarine mesocosms.



### Methods: To simulate non-point source atrazine exposure in an estuarine environment, three compartmentalizedmesocosm systems were setup inside a greenhouse and assigned one ofthree treatmentlevels. Eachmesocosm system consisted ofthree chambers (Units A, B and C), connected viaa "sub-tidal" channel. Each chambercontained a low, middle, and high zoneto simulate tidal creekconditions. Tidal flow ofan unspecifiedrate and volume and a salinity gradient of7-25% were simulated. Each system included a single exposureportal, connectedto the subtidal channel, in UnitA (Figure 1 below). Slope, dimensions, and total volume ofthe mesocosms were notreported. Uncharacterized sediment, cordgrass (Spartina alterniflora) and glasswort (Salicornia virginica) plants, oysters (Crassostrea virginica), mud snails (Nassarius vibex), grass shrimp (Paleomonetespugio), fiddler crabs (Uca sp.) and mummichog fish (Fundulus heteroclitus) were collected from areference marsh located onthe western branch of Leadenwah Creek, Wadmalaw Island, South Carolina, andtransferred to the mesocosmunits. The acclimation interval forthepreparedmesocosms priorto treatment was notreported, but was aminimum of3 days.

Three days priorto treatment, artificial substrates [polyurethane foam units (PFUs), each4 x 5 x 6 cm3] were positioned inthe sub-tidal channel ofeach mesocosm systemto colonize

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1•••••••

# 11 2087812microorganisms. Attached weights held the artificial substrates belowthe water level at "low tide". Atrazine (purity notreported) in acetone at 2.013 mg/mL was addedto 56.6 L offreshwater in UnitA ofone mesocosm at40 /lg/L andto UnitA ofa secondmesocosm at 160 /lgiL. The third mesocosm system was maintained as a solvent control. All three mesocosms received an equal volume ofacetone (test solution vehicle) with a final concentration of0.079 mLiL. The atrazine was introduced into UnitA as an uncharacterizedpulsed exposure at "ebb tide" and allowedto mix through Units Band C viathe subtidal channel withthe incoming "hightide.". This application method was designed to simulate arun-offeventwhereby apesticide is introduced into atidal creek system, diluted by incoming tides, and transported away from the point of entry.

Water samples (unreported volume) were collected for atrazine analysis immediately after treatment (Time = 0) and at48 hours posttreatment. Additional 10 mL water samples were collectedinto abeaker from the sub-tidal channel ofeachmesocosm at 2 (48 hours), 5, and 9 days posttreatmentfor chlorophyll 

### a analysis. Dissolved oxygen measurements were taken at 1 (24 hours), 2 (48 hours), and 9 days posttreatment.

Artificial substrates (PFUs) were collected from eachmesocosm immediately priorto atrazine treatment and at24 and 48 hours posttreatment. Additionally, clean PFUs were placed in each mesocosm24 hours aftertreatment, then collected after 8 days ofexposure (9 days posttreatment). Microorganisms were collectedby gently squeezingthe PFU contents**(~60** mL) into individual, sterile sample cups.

Atrazine. Water samples were loaded onto solid-phase C18 (SEP) cartridges pre-conditioned withmethanol (two columnvolumes) followed by distilled, deionized water (one column volume). Afterloading, the cartridge was rinsed with water (one columnvolume), driedby drawing airthroughthe cartridge**(~5** minutes), then wrapped in aluminum foil and storedfrozen (-20°C, interval not specified). Atrazine was eluted withethyl acetate (two column volumes) and the resulting eluate concentrated**(~0.5** mL) viaTurboVap. The concentratedresidues were diluted with isooctane**(~10** mL), re-concentrated**(~0.5** mL), broughtto volume (l mL) with isooctane, then transferred to GC vials, capped and stored frozen (interval not specified) until analysis. Atrazine was quantified by GC underthe following conditions: J&W Scientific DB-5 5% pheny1:95% methy1-po1ysiloxane column (0.25 mm x 0.25 mm, 30 m length), injection volume 2 IlL, sp1itless mode, average linearvelocity 33 cm/s, carrier gas helium, nitrogenphosphorus detection (NPD), atrazine reference standard at 120 ng/mL; limit ofdetection was notreported.
Phototrophic carbon assimilation. An aliquot (5 mL) ofcollectedmicroorganisms (from PFUs) were treated with [14C]sodium bicarbonate (NaH14C03), at 1 /lCi/mL, then maintained at in situ light and temperature regimes (notfurther detailed) in an incubatorfor 24 hours. Following incubation, the samples were filtered (0.45-/lm, cellulose, nitrate membrane), thenthe filters 

### - 2 -were rinsed with filtered (0.2-jlm) seawater, placed in scintillation vials with 10% HCl (1 mL) and allowedto sit in darkness overnight. Afterwhich, scintillation fluid was added, the samples were equilibrated for 4 hours, then analyzed for total radioactivity using LSC.
Chlorophyll a. 10 mL water or 3 mL microorganism samples were filtered (glass fiber, Type GF/F), then the filters were placed in vials with 1 mL saturated magnesium carbonate (MgC03 ) solution and stored frozen (-2°C, unspecified interval) until analysis. Priorto analysis, samples were thawed, treated with acetone (9 mL), shaken, refrigerated overnight, shaken and again refrigerated overnight, then brought to room temperature and analyzed using a fluorometer (Sequoia-TurnerModel 450) with excitation (NB440) and emission (SC665) filters.
Phototrophic (algal) bio-volume. Microorganism samples (5 mL) were preserved in 2% buffered formalin (final concentration), then stored (interval not specified) at 4°C in darkness until analysis. Aliquots (40 jlL) ofdiluted formalin preserved samples were analyzed for algal cells by counting at 500 magnification. Algae were identified to genus where possible, and the number ofcells, average cell shape, and size were noted for eachtaxon. Phototrophic biovolumes (jlm3) were estimated for eachtaxon using cell sizes and simple geometric shapes, then multiplied bythe total number ofcells pertaxon in each sample.

Bacterial abundance was determined viaepifluorescence microscopy. Formalinpreserved samples (described above) were treated with chemical dispersant (Na3 PPi, 0.25 mL), sonicated, diluted with filtered (0.2-jlm) seawater, then stained with acridine orange (100 mg/L, final concentration) for 3 minutes. The stained samples were filtered (O.2-jlm blackmembrane), then bacterial counts were conducted under oil immersion at 1,250 magnificationusing a Whipple disc and Zeiss fluorescence microscope equipped with a superhigh-pressure mercury lamp with a blue light filter (470-490 nm). Bacterial density was calculated using the following equation: Bacterial density (cells/mL) 

### = (NxAf)/(dx Ag ), where N

### =average number ofcells counted, Af= the effective area ofthe filter (mm\Ag

### =area ofthe counting grid (mm2), and d

### =dilution factor.

Ciliates and flagellates (heterotrophic). Microorganism samples (5 mL) were preservedwith 0.5 mL Lugol's solution [acetic acid:Lugol's (50 mL:200 mL)], then diluted samples were counted at 500 magnification using a Sedgewick-Rafter slide (1 mL). A minimum of200 organisms/sample were counted, with a standard error of<1 0%. Non-pigmented ciliates and flagellates were sorted into size classes of<20 and >20 jlm. Taxonomy was not determined.
Response values from three PFUs were averaged for eachtreatment (0, 40 and 160 jlg/L), with statistics calculated using MINITAB software. Pair-wise comparisons betweentreatments of each endpointmeasured (chlorophyll, bacterial abundance, etc.) were done by one-way analysis ofvariance (ANOVA) for eachtime point (0,24 and 48 hours and 9 days).

- 3 -Results: At the low treatment rate (40 Ilg/L), measured atrazine concentrations were 121%, 22.7% and 0.6% ofnominal in Units A (entry point), B and C, respectively, at time O. At 48 hours, levels had reached 59.2%, 9.7% and 10.3% of nominal, respectively. Mean atrazine levels in the low treatments decreased by 45.3% from 19.3 Ilg/L to 10.5 Ilg/L in 48 hours (Table 1 below). At the high application rate (160 Ilg/L), measured atrazine concentrations were 157%, 0.4% and <0.1% ofnominal in Units A, Band C, respectively, at time O. At 48 hours, they had reached 15.2%, 13.0% and 3.5% of nominal, respectively. Mean atrazine levels in the high treatments decreased by 79.8% from 84.0 Ilg/L to 16.9 Ilg/L in 48 hours. In the untreated (solvent) control mesocosm, atrazine was detected in the water at 0.097 Ilg/L at time 0 and at 0.233 Ilg/L at48 hours. Detection limits were notreported.
One-way ANOVA detected no significant effects of atrazine treatment on dissolved oxygen (DO) All phototrophic parameters measured (chlorophyll a in water, chlorophyll a in organisms, phototrophic assimilation and phototrophic bio-volume) were significantly decreased (p=S0.001 at maximum effect) at both treatment levels (attached Worksheet), although carbon assimilation was not measured beyond 48 hours. Chlorophyll a in the water and organisms were decreased (p=S0.002) 77-79% and 46-47%, respectively, at 24 hours for both treatmentrates, as comparedto the control mesocosm, and remained significantly (p<O.OO1) reduced for the remainder ofthe study (attached Worksheet). Phototrophic assimilation was decreased (p=0.032) 39% and 62% at the 40 Ilg/L and 160 Ilg/L rates, respectively, at 24 hours and was decreased (p<O.OOl) 72% and 83%, respectively, at 48 hours, but was not monitored thereafter. Phototrophic bio-volume was decreased 54-56% (p=O.OOI) at 24 hours for both treatment rates and remained significantly (p=S0.001) reduced for the remainder ofthe study.
Twenty-eight algal taxawere identified from the colonized artificial substrates, with the majority (twenty) ofthe identified taxa diatoms (Chrysophyta, Table 2 below). Atrazine exposure caused noticeable changes in algal community composition with total taxa decreasing from twenty-eight to twenty in the atrazine treated mesocosms. Total algal bio-volume was decreased 54-56% at 24 hours for bothtreatmentrates, as comparedto the control, and was decreased 54% and 73% at the 40 Ilg/L and 160 Ilg/L rates, respectively, at 9 days; ANOVA analyses were not reported (Table 3, below). Diatom, dinoflagellate, chlorophyte and cryptophyte bio-volume decreased in atrazine treated mesocosms; however, cyanophyte bio-volume increased. The study authors reported there were decreases in numbers ofChlamydomonas and Cryptomonas, with increases in a small CymbeUa sp. and many coccoid blue-green algae; quantitative results were not presented. Additionally, diatom Cylindrotheca sp. had fewer, smaller and more curved cells in atrazine treated mesocosms.

Bacterial abundance was significantly decreased (p=O.OOI) at 48 hours for both treatment rates, as compared to the control, but there were no significant differences at study termination (see attached Worksheet). For protozoa, there were no significant effects on numbers of smaller ciliates «20 Ilm) in atrazine treated mesocosms, as compared to the control, but larger ciliates (>20 Ilm) were significantly increased at 48 hours (p=0.010) and 9 days (p=0.003) for both 

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### treatmentrates. Smallerflagellates «20 /lm) 

### were significantly increased (p=0.022-0.023) at 24 and 48 hours in atrazine treated mesocosms (both rates) and remained elevated (p=0.055) at 9 days. However, larger flagellates (>20 /lm) 

### were significantly decreased (p=0.013-0.021) at 48 hours and 9 days for bothtreatmentrates. 

### Unit A 

### Unit 8 

### Unit C

( llig.h Marsh)

( Mid Marsh)

( Low MarSh)
( Channel ) [ rIig.h Marsh)

( Mid Marsh)

( Ltlw MarSh)

[ Channel} ( ,lig.h Marsh

( Mid Marsh

( Low Maflih 

# ( Dilution

Water

Toxicant Delivery Saltwater Reservoir

Figure 1. Mesocosm unit design simulating an estuarine (tidal creek) ecosystem (Lauth etai., 1996). Figure from DeLorenzo etai. 1999.

Table 1. Atrazine concentrations in experimental tidal mesocosms (from attached Workshee Atrazine (I-Ig/L) Mesocosm Unie Measured Nominal % Decrease l Time 0 48 hours Control 0.097 0.223 A 40 48.544 23.681 B 9.072 3.861 C 0.243 4.101 Average 19.286 10.548 45.31 A 160 251.202 24.382 B 0.609 20.725 C 0.103 5.677 Average 83.971 16.928 79.84 t).

1 Decrease m average atrazme concentratIOn at 48 hours posttreatment.

- 5 -Table 2. Algal taxa identified in experimental mesocosms (from colonized artificial substrates) over the course ofthe study; grouped as to phylum (Prescott, 1978, from Table 2 ofstudy). Taxon Chlorophyta**.r;.~lq'!!:x4?lr}?,!q~?P:m** Chyrsophyta Amphiprora sp. Asterionella sp. Bacillaria sp. Biddulphia sp. Chaetoceros sp. Coscinodiscus sp. Cylindrotheca sp. Cymbella sp. Dinobryon sp. Diploneis sp. Fragilaria sp. Gomphonema sp. Licmophora sp. Melosira sp. Navicula sp. Nitzschia sp. Pleurosigma sp. Rhizosolenia sp. Sellaphora sp. Stauroneis Cryptophyta

............**mr;.1)!Pt?lr}?'!q~?P:m** . Cyanophyta Cylindrical blue-green Filamentous blue-green Oscillatoria sp.

............**.§X'!~c.~?c.?c.c.1!~~P:mm** Euglenophyta**§1![JI~'!C:?P:m** Pyrrhophyta Gymnodinium sp.



### - 6 -Table 3. Change in algal community composition; expressed as percent decrease in average bio-volume as able 3 ofstudy). compared to control (i.e., magnitude ofeffect), except where noted as an increase (+) (from T 40 JlglL Treatment 160 Jlg/L Treatment Phytoplankton 24 h 48 h 9 d1 24 h 48 h 9 d1 Diatoms 49.53 47.02 53.81 45.62 44.21 73.85 Dinoflagellates 89.27 88.58 100.00 91.07 96.43 100.00 Chlorophytes 20.00 78.15 82.05 47.90 83.19 100.00 Cryptophytes 76.74 72.09 36.46 88.37 100.00 89.99 Cyanophytes 64.67 +29.29 +52.49 31.33 +94.70 +87.02 Total algae 56.49 56.43 53.95 54.09 53.61 73.02 Artificial substrates placed III mesocosms at 24 hours posttreatment and removed after 8 days ofexposure (9 days posttreatment).



### T hi D t 

### S

aa ummary a e: Time Weighted Nominal Average Did Magnitude of Effect Atrazine Day of Study Atrazine Recovery Effect Relative to Controls Concentration EffectFirst Concentration Occur- Comments (p.gIL/ Detected from Day Oto If So, When Effect When? Occurred Chlorophyll a 77%*--v 40 JlglL 2 in water p**<O.OOl (mglL) 79%*--v 160 JlglL 2 Chlorophyll a 47%*--v 40 Jlg/L 1 Magnitude ofeffects in organisms 46%*--v p=0.002 calculated (mglL) 160 JlglL 1 from results Not calculable No Phototrophic 39%*--v 40 JlglL 1 in Table 1 assimilation p=0.032 ofstudy (mg C/L*h) 62%*--v 160 Jlg/L 1 (attached Worksheet). Phototrophic 56%*--v 40 Jlg/L 1 bio-volume p=O.OOl (Jlm3 x 105) 54%*--v 160 Jlg/L 1 Diatoms 50%--v 54%--v 40 JlglL 1 9 (average bio46%--v 74%--v Magnitude volume) 160 JlglL 1 9 ofeffects 89%--v IOO%--v from Table Dinoflagellates 40 JlglL 1 9 3 above; (average bio91%--v IOO%--v Not calculable statistical volume) 160 JlglL 1 9 No significance Chlorophytes 20%--v 82%--v 40 Jlg/L 1 9 not (average bio- reported. volume) 48%--v 100%--v 160 Jlg/L 1 9 Day 9 values (on Cryptophytes 77%--v 36%--v 40 Jlg/L 1 9 the right) - 7 -Time Weighted Nominal Average Did Magnitude of Effect Atrazine Day of Study Atrazine Recovery Effect Relative to Controls Concentration EffectFirst Concentration Occur- Comments (pgIL/ Detected from Day Oto If So, When Effect When? Occurred (average bio- 88%-.It 90%-.It 160 Ilg/L 1 9 are from volume) substrates introduced 65%-.It 52%t 40 11giL 1 9 Yes to the Cyanophytes (Day 2) mesocosms (average bio- at 24h and volume) 31%-.It 87%t 160 Ilg/L 1 9 Yes (Day 2) are rounded from two 56%-.It 54%-.It 40llgIL 1 9 decimal places. Total algae (average bio- No volume) 54%-.It 73%-.It 160llg/L 1 9

1 Forthe nommal treatment levels of40 and 160 IJ.g/L , mean (n=3) atrazme concentratiOns mthe water column

decreased from 19.3 and 84.0 IJ.glL attime 0 to 10.5 and 16.9 IJ.g/L, respectively, at48 hours. No further measurements were taken. 

### * Reportedto be statistically significant; level ofstatistical significance was notreported. 

### ** Probability (p) values were notreported separately for treatment levels. Values could not be reproduced because

only means were presented.

Reviewer Comments: 1. The purity and source ofthe atrazine testcompoundused were notreported.

2. The tidal mesocosms were preparedusing sediment, plants and animals obtained from a natural reference marsh. Itis not stated whetherthe natural marsh site was testedto establish whetheritwas contaminated withpollutants. Atrazine was detected inthe water ofthe untreated control mesocosm at 0.097**~g/L** attime 0 and 0.233**~g/L** at 48 hours, suggesting thatthe levels ofatrazine detected in the treated mesocosms attime 0 could be due eitherto the atrazine treatment orto priorcontamination. The detection limits for atrazine analysis were notreported.

3. No negative controls were included; only solventcontrols were used.

4. Atrazine concentrations were only determined at two intervals (24 and 48 hours) following application.

- 8 -5. The study authors noted that chlorophyll 

### a and total biovolume in the control mesocosm steadily increased overthe 9-day study, indicating thatthe microorganism communities (colonization) had not achieved equilibrium with the surrounding waterpriorto treatment.

6. Nine-day measurements were only reported for substrates introduced 24 hours after dosing with atrazine.

7. Reportedly, previous experiments (Lauth 

### etai., 1996) using tidal mesocosms determined that pesticide concentrations reached equilibrium in the water ofeach unit within 24 hours (two tidal exchanges). As the study authors noted, applied atrazine had not reached equilibrium betweenthe three units inthe treated mesocosms after48 hours inthis study.

8. The water of the tidal mesocosms was not monitored for pH, temperature, turbidity, alkalinity, total nitrogen, total phosphorus, conductivity, or particulate and dissolved organic carbon.

9. The mesocosm sediment was not analyzed for pesticide content, particle SIze, cation exchange capacity, organic content, andpH priorto treatment.

10. Dimensions, total volume, and slope (to create low/middle/high zones) ofthe mesocosm units were notreported.

11. Pairwise comparisons (one-way ANOVA) were reportedly used to compare each treatment endpointto the control. However, only one setofp-values is reported (see Table 1).

12. The study authors reported that the diversity of the phytoplankton taxa colonized in the control mesocosm of this study was similar to that observed. in a reference tidal creek (DeLorenzo, unpublished data), butno supporting datawere presented.

13. The initial microbial communities were dominated by diatoms. The relative abundance of diatoms decreased throughout the study in the atrazine-treated units. The total number of taxa identified also decreased from 28 to 20. The authors assertthatthe effects on substrates introduced at 24 hours and examined at 9 days indicate decreased colonization ability in the presence of atrazine. However, it is unknown whether the source microbial assemblages were comparable across treatments atthe time these substrates were introduced.

14. Overall, phytoplankton and larger flagellates decreased in abundance and productivity, while bacteria, larger ciliates and smaller flagellates increased in abundance. However, dissolved 

### - 9 -oxygen (DO) levels in the atrazine-treated units was similar overall to DO in the control units, which the authors suggest might indicate (l) a compensatory phototrophic response or (2) that metabolic rates ofthe heterotrophic organisms may have decreased with decreased oxygenproduction.

15. The study authors noted that atrazine had a significant effect on the microbial food web of the experimental tidal mesocosms by influencing interactions betweenphytoplankton (algae), bacteria, and grazers (heterotrophic protozoa). This may have occurred through direct effects (phytoplankton), by influencing competitive interactions (bacteria), and/or by impacting the heterotrophic food source food source (grazers).

16. The study authors also proposed that the trend toward smaller microorganism cell size in the atrazine treated mesocosms has potential adaptive significance: It may allow the alga to sustain its nutritional needs while photosynthesis is impaired, and/or it might reduce adsorption ofthe pesticide via a decreasing cell surface area:volume ratio.
Description ofUse in Document (QUAL, QUAN, INV): This study is consideredInvalid (INV) for use inthe riskassessmentprocess.

Rationale for Use: No quantitative exposure-response relationships could be inferred, as the system failed to reach equilibrium during the study period andthe treatments lacked sufficientreplicates.

Limitations ofStudy: Numerous deficiencies existed withrespectto the experimental design and implementation ofthis study. No effortwas made to document whether other chemicals were presentinthe treatment and/or control mesocosms priorto study initiation, and analyses of the control mesocosm indicate the systems mayhave been contaminated with atrazine priorto treatment. The acclimation interval was insufficientto establish equilibrium-phase colonization ofmicroorganisms priorto treatment. Mesocosm sedimentwas not characterized, andthe water was not monitored for pH, temperature, turbidity, alkalinity, total nitrogen, total phosphorus, conductivity, ororganic carbon. Purity ofthe test compound was notreported. Frequency and intensity ofatrazine pulses was not described. An acetone solventcontrol (0.079 mLlL) was used, butno separate negative control was established. Atrazine concentrations were only determined attwo intervals following application, and equilibrium ofthe appliedtest substance between the three units ofeachtreated mesocosm had notbeen achievedby the second sampling interval at48 hours post-treatment.

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-Primary Reviewer: Lynne Binari, M.S., subcontractorto Cambridge Environmental Inc.
Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division U.S. Environmental Protection Agency



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