### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Carder, j .P. and KD. Hoagland. 1998. Combinedeffects ofalachior and atrazine on benthic algal communities in artificial streams. Environ. Toxicol. Chern. 17(7): 1415- 1420. 

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



### Date ofReview: December 12, 2008


### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants (epipelic algae) in experimental streams.


### Summary ofStudy Findings:

Methods: The experiment was a duplicated, 3 x 3 factorial design testing effects ofzero (control), low, and high levels, ofatrazine, alachlor, and acombination ofboth, in 18 artificial streams. Details ofalachlor-related methods that are not essential to understanding the overall. design orthe atrazine methods will notbe reported.

Stream water and benthic substrate were collected from Wahoo Creek, 1.5 km southeast ofWahoo, Nebraska, on March 25, 1993. Wahoo Creekis a third-order, sedimentdominated stream draining primarily agricultural land, and is subjectto majorrunoff events during the growing season. Analysis ofWahoo Creek water atthe onsetofthe experiment showed background atrazine concentrations of2.9**~g/L.** Waterwas collected via centrifugal pump from the stream surface and was transported to the University of Nebraska Laboratory Stream Facility in a 3,800 L polyethylene tank. Sediments (epipelon) were collected from the upper 2 cm ofbenthic substrate using agarden trowel.

1 ••••

# 1.111 2087871Samples were placed in a bucket, homogenized by stirring with the trowel, and transported back to the laboratory.
Eighteen recirculating artificial streams were constructed from 114 L, oval Rubbermaid® plastic livestocktubs (Rubbermaid, Wooster, Ohio), with a lengthwise wooden divider cut 25 cm from each end to allow for the passage ofwater, and lined with two layers of4- mil (1.016 mm) clearplastic. The authors cite a 1997 alachlorpaper for aphotograph of

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.....44111..;:) 11.1 ,1 UBU-VBI 1/3 increments to ensure homogeneity among the streams' water, and all were filled to within 10 cm ofthe top ofeach tub (about 86 Lofwater in each). The tubs were arranged on three parallel tables, with an electric variable-speed motor attached to the end ofeach table controlling a belt-driven shaft abovethe streams, driving a paddle wheel dipping approximately 3 cm into the water surface ofeach stream to produce the current. The electric motors were adjusted daily to achieve similar velocities in the streams (no details on how similarity was determined). At four different times in the experiment (timing not described), a Marsh-McBimey currentmeter(Marsh-McBimey, Frederick, Maryland) was used to determine the water velocity in the center ofeach channel, at a depth ofone-halfthe water column. In the sending segment, velocities were 0.05-0.1 mis, and in the returning segment, they were 0.01-0.05 m/s. A 1,000 W metalarc lamp was the light source (the authors parenthetically reference**"~600** Ilmollm2/s" and it is unclear what this refers to, as it is not in any standard illuminance or irradiance units), and operated on a 12 hour light, 12 hourdarkphotoperiod. The ambient room temperature was controlled (no details provided). Approximately 1-2 Lofwater was lost from each stream daily due to evaporation, so stream water from the collection tank was used to maintain water levels. The stream water was mixed in circulating holding streams for at least 24 hours before being added. The initial batch ofwaterwas insufficient, so additional stream waterfrom Wahoo Creek was collected "on several otheroccasions" (no details orbackground atrazine levels on these collections provided).

Three unglazed masonry tiles (20 cm x 20 cm x I cm) were placed on the bottom ofthe return channel ofeach stream to shape the liner andprovide a uniform surface for the epipelon sampling units. Sampling units were 60 mm x 

### IS mmplastic Petri dish bottoms filled with Wahoo Creekbenthic sediments. Units were initially placed immediately after water was added to the artificial streams, four on each tile (12 in each stream). Epipelon notused in the initial setup was stored in a circulating holding stream, and these sampling units were used to replace the sampling units that were removed for sampling. The replacement was only to ensure that stream flow aroundthe units was not affected by sampling unitremoval- replacement units were not used for measurements. All streams were allowed 6 weeks to acclimate to lab conditions before the 4 week experiment commenced.

Four I L solutions ofboth high and low concentrations ofalachlor and atrazine were developed (methods or solvents not described) so that 100 mL ofsolution would create the desired test concentrations in the 86 L streams. Atrazine was a commercial product

2(Farmland Industries, Kansas City, Missouri; 40.8% active ingredient). Atrazine test concentrations were nominally 12.0jlg/L ("low") and 150.0jlg/L ("high"). The 3 x 3 factorial design cross-classified control, low, and high alachlortreatments with control, low, and high atrazine treatments, with duplication. Treatments were randomly assigned to the 18 streams (randomization method not described). Treatments (l00 mL solutions) were added to the appropriate streams immediately aftercollecting the initial algal samples on May 5, 1993.

Herbicide analyses were conducted on water samples taken from each stream 24 hours afterherbicide addition and atthe end ofthe experiment. Samples were frozen until they were analyzed using Ohmicron RaPID Assay® (Newton, Pennsylvania) immunoassay kits for the detection ofatrazine (coefficient ofvariation was <5% according to the manufacturer). The ability ofthe immunoassay kit to accurately determine herbicide concentration was also verified using gas chromatography on several randomly selected samples.

On each sampling day priorto algal sampling, pH and stream temperature measurements were taken using aFisher Scientific Accumet® portable pH meter (model 1002; Fisher Scientific, Pittsburgh, Pennsylvania), dissolved oxygen (DO) concentrations were measured using a Yellow Springs Instruments portable DO meter and probe (model 57; YSI, Yellow Springs, Ohio), and conductivity was measured using aportable Orion® conductivity/total dissolved solids (TDS) meter (model 124; Orion, Boston, Massachusetts). Additionally, water samples from "fourrandomly selected streams" (treatments ofselected streams not described) were taken immediately before the experiment, on May4, 1993 and immediately after it ended, on June 3, 1993, for nutrient analysis. Nutrients analyzed were total nitrogen, using the alkalinepotassiumpersulfate digestion (D'Eliaet aI, 1976, 

### Limnol Oceanogr22) and ultraviolet absorption (American Public Health Association's 1985 standards) methods, and total phosphorus, using the ascorbic acid method (Lind, 1985,

### Handbookof Common Methods inLimnology).

For sampling ofepipelon, all paddle wheels were stopped and arandomly selected sampling unit was removed and areplacement unitput in immediately (as previously described). In all cases, this occurred between 0900 and 1030, the time ofday when motile epipelic species would be expectedto have migrated towards the surface for light absorption. The removed sampling unit was subsampled with a sharpened 1 em copper tube in arandom (randomization not described) location, and the upper 2 mm ofepipelon from the inside ofthe tube was drawn offusing O.S-cm tygon tubing attached to a vacuum pressure system. The subsample was drawn directly into a graduated 120 mL wide-mouth glassjar and 40 mL of6:3:1 (water:ethanol:formalin) was used to rinse the vacuum system and preserve the sample. Sampling occurred before herbicide addition (May 5, 1993), 24 hours afterherbicide addition, and 1, 2, and4 weeks afterherbicide addition.



### 3Algal samples were analyzed for cell density (results not described), cell biovolume, and relative abundance ofthe six dominanttaxa (meaning those comprising >5% by biovolume in the initial samples). Samples were homogenized by gently swirling the samplejar, a 0.10 mL aliquot was pipetted into a Palmercounting cell and scanned at 400x using an Olympus BH-2 compound microscope (Olympus, Tokyo, Japan), and then algae were counted until 300 viable (chloroplast intact) cells had been quantified, or 100 fields ofview had been observed. Data were analyzed using analysis ofvariance (ANOYA) with repeated measures, performed with SAS Version 6.0 software, with statistical significance inferred for p-values:::; 0.05.

Results: To the extentpossible, only atrazine results will be described. However, for some results, either all streams or "randomly selected" streams were sampled; these results include streams treated with alachlor in the factorial design.

Average atrazine concentrations in all streams were relatively close to nominal (see

Table 1), though they started out higherthan nominal due to existing background levels ofatrazine in the stream water. The high atrazine treatment streams decreased in atrazine concentration by 13% overthe four-week experiment. The low atrazine treatment streams actually increased in concentration by nearly 15% overthe course ofthe experiment, which probably reflects changes in background contamination (as evidenced by increase in "control" concentrations in

Table 1). The authors state thatphysical and chemical waterquality measurements did nothave any obvious trends or differences among the experimental streams (however, there appeared to be some changes in nutrient levels and conductivities). Over all streams, means (and standard deviations) were: dissolved oxygen, 9.0 (0.3) mg/L; pH, 8.8 (0.1); and temperature, 1704 (004) °C. Conductivities over all streams were 0.89 (0.04) mS/cm initially and lAO (0.1) mS/cm atthe end ofthe experiment. Nutrient concentrations in four randomly selected streams changed somewhat overthe course of the experiment: total nitrogen increased from 1,007.5 (172.5) Ilg/L to 1,571.9 (82.8) Ilg/L, while total phosphorus declined from 117.0 (10.0) Ilg/L to 81.8 (7.7) Ilg/L (these results are not interpreted in the paper,justprovided in atable it is difficultto interpret them withoutknowing which streams were sampled).

Effects ofboth atrazine and alachlor on algal biovolume are shown in Figure 1. No significantdifferences were found between pretreatment streams and control, orbetween streams at 24 hours after addition. Time-independent analyses showed significant atrazine effects atboth high and low concentrations at week 1 (both with p=0.0005) and week4 (both with p=O.OOOI), with high concentration treatment effects atweek 2 (p=0.0064). No significantdifference between high and low treatments was found at week 1, but there was a significant difference at week4 (p=0.0006). Time-dependent analyses found thatboth treatment levels induced significantchanges in algal biovolume throughout the experiment (p=0.0083), butthe difference between high and low treatment levels was not significant (p=0.3629).

4Relative abundances ofdominant algae werenot significantly affected by atrazine, although two were affected by alachlor. The six dominant algae taxa were: Fragilaria capucina (Chrysophyta, diatoms) Fragilaria spp. (diatoms) Cylindrospermum majus (Cyanophyta, blue-green algae) GyroSlgma eXln1lum ldiatom) Rhopalodiagibba (diatom) Navicula spp. (diatom)

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Figure 1. Percentage ofmean control algal biovoJume for each treatment combination on each ofthe five sampling dates. ("LO"=Jow, "HI"=high, "al"=alachlor, "at"=atrazine; for atrazine-only data, see the third and fourth bars from the left on each subplot). 

### t indicates a significant (p-value 0.05) time-dependent effect based on repeated measures analysis; 

### * indicates a significant (p-value 0.05) time-independent effect based on ANOVA ofthe individual sampling period; and 'I' indicates a significant interaction.

Table 1. Measured atrazine concentrations in the artificial streams. Initial concentrations were taken 24 hours after atrazine addition, and final concentrations were taken 4weeks later upon termination ofthe experiment. Means (and standard deviations) over all streams are shown.



### 6Data Summary Table: Effect Magnitude of Nominal Day of Time Did Comments Effect Atrazine Study Effect Weighted Recovery Relative to Concentration Occurred Average Occur- if Controls**(~g1L)** Atrazine so, when Concentration (see from Day 0to comment)? When O",-,Ui l"U**~50%** Decrease in (different 12**~g1L** Day 7 16.4 Ilg/L No (>28 from control days) benthic at p={) ()O{) 1. ) Magnitudes from algal community**~60%** Fig. 1. biovolume (difTerent ISO 7 145.21lglL No (>28 from control at n=O OO!)1) i

Time weighted averages are averages ofthe initial and final means given in

Table 1.

Reviewer Comments: This study had a factorial design, including duplication, although the alachlorresults are not ofinterest from the perspective ofthis review. Most sampling and analytical methods were reasonably well described and either followed standards orwere cross-validated.

The only real endpointpresented by the study was changes in epipelon biovolume. Water quality measurements that may have been affected by treatment, like dissolved oxygen and nitrogen levels, were nottaken in enough streams at enough sampling times for properexamination.

The main complication ofthe study was the significant and apparently time-varying background levels ofatrazine in the stream water. Given thatthe average "control" atrazine concentration started out at 2.9 Ilg/L and increasedto 6.4 IlglL by the end ofthe experiment, and the "low" treatmentconcentrations also increased somewhat, there was clearly significantbackground atrazine. Itwould have been informative to learn what kind ofrunoffevents, herbicide applications, etc., were occurring in the area around the times ofstream watercollection. The factthatthe epipelic communities clearly had prior exposure to atrazine was not discussed, and could confound the results somewhat, as the community may have some pre-developed atrazine tolerance. In addition. the "controls" were not contaminant-free, so some effects may appear weaker relative to the "controls" than they would ifcomparedto truly contaminant-free controls.

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

### INV

Rationale for Use: The study was limited because ofthe presence ofatrazine in the controls at levels thatmay influence the endpoints evaluated in this study.



### Limitations ofStudy: Background water and "controis;; had 2-7 

### llg/L concentrations of atrazine, so effects "relativeto controls" are misleading. Only benthic algal communities werepresent in the experimental streams, so the ecological dynamics were highly simplified and may notbe applicable to natural streams.


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


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

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