Open Literature Review Summary

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

ECOTOX Record Number and Citation: Pratt, 

### l.R., N.J. Bowers, B.R. Niederlehrer, andJ. Cairns, Jr. 1988. Effects ofatrazine on freshwater microbial communities. Arch. Environ. Contam. Toxicol. 17:449-457.



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### l(SS"il.-f) Purpose ofReview: Determinepotential effects ofatrazine on an aquatic community. Endpoints from this study were also usedto parameterizethe Comprehensive Aquatic SystemModel (CASM) and establish the model-associated LOC for atrazine.

Date ofReview: December 10, 2008

Purpose ofStudy: This study examines thepotential effects ofatrazine to aquatic microbial communities in artificial streams (microcosms).

Summary ofStudy Findings:

Methods: 21 microcosms were constructed inhigh-densitypolyurethane tubs (35 cm x 28 cm x 15 cm), each with an inlettube at one end andthree drain holes atthe otherend. The inlet tube delivered amixture ofdiluent andtoxicantwith a flow rate giving approximately 7 turnovers ofthe 7 Ltestvolume perday. Three replicates were used ateach treatment level: diluent control, solvent(methanol) control, and five concentrations ofatrazine. Diluentwater, consisting oftap water (source: New River, Virginia; hardness -70 ppm CaC03, alkalinity -45 ppm CaC03, pH -8.4) dechlorinatedby activated charcoal filtration, flowed from a head box into a flow splitterand 21 mixing chambers. Each mixing chamberwas also fed by atoxicantreservoircontaining appropriate concentrations ofatrazine, diluent, and solventfor each microcosm. Each microcosm
was then fed by the test solution flowing outofits mixing chamber. (No diagram provided.) Lighting was from Vita Litebulbs (CRI>90, Duro Test Corp.) located -30 cm above the microcosms (intensity -5000 lux). Lights were setto a 16 hours light, 8 hours darkphotoperiod. Watertemperature was uncontrolled andranged from 13.5°C to 15°C.

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# 11111111111 2087818Atrazine added to the microcosms was technical grade (98.6%, FL851205, Ciba-Geigy), while analytical standards were made from analytical grade atrazine (98.8%, Code #S85- 0653-3, Ciba-Geigy), Primary stockwas made by dissolving 200.84 mg ofatrazine in 100 ml ofmethanol and diluting in distilled waterto I L. Secondary stocks were made from primary stockby diluting with diluentwater. Bothprimary and secondary stocks
were made on day 0 and again on day 10. Nominal atrazine concentrations in the microcosms were 3, 10, 30, 100, and 300 Ilg/L. The solventcontrol test solutions were made with methanol at aconcentration of0.015% in diluentwater.

Atrazine concentrations in the microcosms were measured on days 0, 10, and 21. For these measurements, approximately 500 mL oftest solution were collected from each microcosm and filtered through a 0.45-llm GA-6 Metricel membrane filter (Gelman). Atrazine was extracted from 250 mg offiltered waterusing C18 SPE columns (IT. Baker) and eluted from the columns with 2.5 mL ofHPLC grade acetonitrile (ACN) for a 100-fold increase in concentration. These samples were then stored in glass vials with Teflon caps at4°C for analysis the nextday. Standards were extractedthe same way for each analysis. Samples were analyzed with ahigh performance liquid chromatograph and Beckman Ultrasphere ODS columns (5 Ilm mesh, 4.6 mm ID x 250 mm). A 67:33 water:ACN solution was used to elute the atrazine at 1.5 mLimin. Absorbance at 220 nm was used to monitorpeakheight, and concentration was calculated from standards. Precision (based on replicate measures) had acoefficient ofvariation of2.04%, and average recovery was 104.1%. The minimum detectable level ofatrazine with the technique was 0.5 Ilg/L.

Testmicrobial communities were collected on polyurethane foam substrates (6.0 cm x 5.0 cm x 4.0 cm) in Pandapas Pond, Montgomery County, Virginia. (Pond, watershed, surrounding land use, etc. not described.) Communities included at least 150-200 species ofbacteria, protozoa, algae, fungi, and small metazoans. The substrates were placed in the pond and collected 14 days later(no further description ofdate or location of sampling). In each microcosm, two polyurethane foam substrates were placed as species sources, and five barren substrates ("islands") were also placedto provide colonizable habitat. (Spatial arrangementnot described.) One ofthe island substrates was removed from each microcosm after 3, 7, 14, and 21 days and examined forprotozoan species richness. Three reference substrates were examinedpriorto the start ofthe testto deteITI;line initial species richness, and atthe end ofthe experiment, the species sources were examinedto evaluate survival.

Each substrate was sampledby placing it in arandomly numbered sterile Whirl-Pak® bag, squeezing to remove as much ofthe contents as possible, and discarding the substrate. Samples were allowedto settle and then "repeatedly" subsampled and examined at200-400x for identification ofprotozoato the lowestpossible taxonomic unit (usually species, sometimes genus). (Time allowed for settling and number/size of

2subsamples notprovided.) The total number oftaxawas recorded andthen matchedwith the original sample using the random ID number.

Waterquality measurements (temperature, pH, dissolved oxygen, and conductivity) were taken in each microcosm once perweek, aboutone hour afterthe lights came on and before substrate collection. Additionally, waterhardness and alkalinity were measured weekly in the diluent control, solventcontrol, andthe highesttestconcentration. Water quality measurements followed the protocol ofUSEPA, Methodsfor the Chemical Analysis ofWaterand Wastewater, 1985. On Day 21, several non-taxonomic variables
were evaluated. Biomass was estimated as protein concentration. Samples were concentratedby centrifugation (1500 rpm for 10 minutes), protein was extracted with 0.5 N NaOH, and then protein concentrations in the extract was measured following the method ofBradford (1976),A rapidandsensitive methodforthe quantitation of microgram quantities of protein, utilizing theprinciple of protein-dye binding, Anal Biochem 72. Aliquots ofeach sample were fixed to pH<2 with trace pure HN03 for analysis ofcalcium, potassium, and magnesium (again following USEPA, 1985). Metals
were analyzedby flame atomic absorption spectrophotometry on aPerkin-Elmermodel 603 spectrophotometer. Chlorophyll 

### a was extracted in 90% acetone overnight and concentrations detenninedby the trichromatic method (following the American Public Health Association's 1985 standards).

For statistical analysis ofprotozoan colonization ofinitially barren substrates, data was fitted to the MacArthur-Wilson equilibrium model, given by: 

### S 

## =Seq(1

- e -Gr ) where Sis the number ofspecies attime t, Seq is the equilibrium number ofspecies, and Gis the colonization rate. Parameters Seq and Gwere estimated using nonlinearleast squares regression, andthen the curves for differenttreatmentconcentrations were compared using dummy variable analysis (factors chosen for dummy variables are not described). Foreach sampling day available, species numbers as well as non-taxonomic variables were compared using analysis ofvariance (ANaYA) (exactprocedure not described). When ANaYA found significant differences atp<0.05, multiple comparisons were made using Fisher's LSD. No-observable-effects concentrations and lowest-observable-effects concentrations were also defined, butwill notbe described in the results because they are trivial.

Results: Atrazine levels remained stable during the 21-day study period, though concentrations
were slightly differentthan nominal (see Table 1). Waterchemistry (pH, temperature, hardness, alkalinity, conductivity) remained in the nonnal range for dechlorinatedtap water.

3Nonlinearregression was adequate to describe protozoan colonization ofthe polyurethane foam substrates. Results showed some significanttreatmenteffects, but treatment as well as time effects were not consistent (see Figure 1). Low atrazine treatmentmicrocosms «30 Ilg/L nominal concentrations) experienced increased species numbers on days 14 and 21. The two highest atrazine treatments (l00 and 300 Ilg/L nominal concentrations) showed initial decreases, butby day 21 the species number decrease was only significantatthe highesttreatment level. Colonization curves were significantly different among treatments (F 12,70 = 11.02, p<O.OOOI; it is not clearto what testthese statistics refer). Seq was significantly greaterat <30 Ilg/L nominal atrazine concentrations and significantly lowerat 300 Ilg/L nominal. Gwas significantly lowerat all treatment levels except atthe highesttest concentration (300 Ilg/L nominal). See Table 2. Atthe end ofthe experiment, there were no differences in survival rates on the
source substrates.

Non-taxonomic measurements taken on Day 21 showed some significanteffects. Protein and chlorophyll alevels were significantly increased in the two lowestatrazine treatments (3 and 10 Ilg/L nominal), butwas significantly decreased in the highest treatments (300 Ilg/L nominal). Midmorning dissolved oxygen, an indicatorofprimary productivity, was significantly decreased in treatments with 30 Ilg/L nominal concentrations orhigher. Forthe metals, magnesium and calcium also were significantly decreased in treatments with 30 Ilg/L nominal concentrations orhigher, while potassium
was only reduced in the highesttreatments(300 Ilg/L nominal). See Table 3 andTable 4 forthe summary data.

Table 1. Measured atrazine concentrations in microcosms. Means and standard deviations (SD) are shown. (study table 1)

Nominal Measured atrazine concentration (gg/L) concentration Day 0 Day 10 Day 21 Time Mean (gg/L) Mean SO Mean SO Mean SO Mean SO Control <0.5 <0.5 <0.5 <0.5 Solvent Control <0.5 <0.5 <0.5 <0.5 3 3 0.11 3.6 0.33 3.1 0 3.2 0.34 10 9.2 0.23 11.3 0.58 9.6 1.1 10 1.15 30 29.7 2.31 33.3 1.34 33.1 4.82 32 3.26 100 102 8.02 102 10.4 126 14.4 110 15.6 300 324 8.02 336 17 350 14.4 337 16.4

4Table 2. Effects ofatrazine on estimated MacArthur-Wilson colonization parameters: equilibrium species number (Se,/) and colonization rate (G). Parametervalues are based on nonlinear regression fits to the protozoan species numberdata. Asymptotic standard errors (SE) are also provided. Values significantly different from control are marked with (*). (study table 2)

Nominal concentration (gg/L) Seq Seq SE G GSE Control 33.2 2.34 0.349 0.104 SolventControl 34.4 1.92 0.309 0.068



## .. 42.4 * 2.65 0.167 * 0.03 j 10 52.6 * 5.06 0.122 * 0.028 30 61.7 * 12.26 0.081 * 0.031 100 34.8 3.05 0.138* 0.031 300 24.1 * 1.18 0.266 0.048

Table 3. Non-taxonomic measures: data from Day 21, means and standard deviations (SD). Values significantly different from control according to one-way ANOVA are marked with (*). (study table 3, part 1 of2)

Nominal Dissolved oxygen concentration Protein (mg/L) Chlorophyll (ug/L) (mgL!J (gg/L) Mean SD Mean SD Mean SD Control 35.7 7.4 1326 491.8 9.23 0.6 SolventControl 31.9 8 732 241.5 8.67 0.81 3 49.7 * 11 2247.3 * 711.3 8.9 0.1 10 57.2 * 20.6 2356 * 1330.8 7.47 1.63 30 47 5.3 1037 216.1 6* 1.08 100 42.7 2.28 538.7 50.4 6.3 * 1.55 300 21.2 * 5.1 88.3 * 124.1 6.73 * 0.15

Table 4. Non-taxonomic measures: data from Day 21, means and standard deviations (SD). Values significantly different from control according to one-way ANOVA are marked with (*). (study table 3, part 2of2)

Nominal concentration Potassium (mg/L) Magnesium (mg/L) Calcium (mg/L) (gg/L) Mean SD Mean SD Mean SD Control 2.04 0.44 3.82 0.36 24.1 2.19 SolventControl 2.04 0.54 3.69 0.49 23 0.29 3 1.73 0.1 4.17 0.45 23 0.29 10 2.56 0.9 4.17 0.45 21.8 0.82 30 2.17 0.52 3.15 * 0.06 20.2 * 0.29 100 1.66 0.11 3.45* 0.2 22.7 * 2.5 300 1.14 * 0.19 3 * 0.11 20 * 0

53 7 14 Day. 21

3.2 ug/L 110 

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Figure 1. Colonization ofpolyurethane foam substrates by protozoa overtime. Curves were fitted using the MacArthur-Wilson equilibrium model. Points marked with asterisks are significantly differentfrom controls. Average (not nominal) concentration for each treatment level is provided in the upper left cornerofeach subplot.

6Data Summary Table: Effect Magnitude of Nominal Dayof Study Time Did Comments Effect Atrazine Effect Weighted Recovery relative to Concentration Occurred Average Occur- 

### if controls (pg/L) (measurements Atrazine so. when onlyreported Concentration (see forDay21) from Day 0to comment)? When Effect Occurred No stat. sig. adverse 3 I!g/L NA 3.2 1!g!L NA Magnitudes effect No stat. sig. estimated Numberof adverse 10 I!g/L NA 10 I!g/L NA from Fig. 1. protozoa effect (*) indicates species in No stat. sig. significant difference. new adverse 30 I!g/L NA 32 I!g/L NA Increases colonies effect No stat. sig. were observed at 3 adverse 100 I!g/L NA 110 I!g/L NA to 30 ug/L. effect 15% * 300 lJ,g/L Day 21 337 lJ,g/L NA No stat. sig. NA adverse 3 I!g/L 3.2 I!g/L NA Magnitudes effect calculated No stat. sig. NA from table 3. adverse 10 I!g/L 10 1!g!L NA (*) indicates Protein effect significant biomass of No stat. sig. NA difference. protozoa adverse 30 I!g/L 32 I!g/L NA Significant effect increases No stat. sig. NA were adverse 100 I!g/L 110 I!g/L NA observed at 3 effect and 10 ug/L. 40% * 300 lJ,g/L Day 21 337 lJ,g/L NA No stat. sig. NA Magnitudes adverse 3 I!g/L 3.2 I!g/L NA calculated effect from table 3. No stat. sig. NA (*) indicates adverse 10 I!g/L 10 I!g/L NA significant Chlorophyll effect difference.
a ofalgal No stat. sig. NA Significant protozoa adverse 30 I!g/L 32 I!g/L NA increases effect were 60% 100 lJ,g!L Day 21 110 ug/l NA observed at the lowest 90%* 300 I!g/L Day21 337 I!g/L NA two test levels. Primary No stat. sig. Magnitudes productivity adverse 3 I!g/L NA 3.2 I!g/L NA calculated (mid- effect from table 3.

7Effect Magnitude of Nominal Day of Study Time Did Comments Effect Atrazine Effect Weighted Recovery relative to Concentration Occurred Average Occur- 

### if controls (pg/L) (measurements Atrazine so, when only reported Concentration (see for Day21) from Day 0 to comment)? When Effect Occurred morning No stat. sig. (*) indicates dissolved adverse 10 !!g/L NA 10 !!g/L NA significant oxygen) effect difference. 35% * 30 !!g/L Dav21 32 ug/L NA 35% * 100 )..lg/L Day 21 110 )..lg/L NA 30% * 300 ug/L Day 21 337 )..lg/L NA No stat. sig. NA adverse 3 !!g/L 3.2 !!g/L NA effect No stat. sig. NA Magnitudes adverse 10 !!g/L 10 !!g/L NA calculated effect from table 3. Potassium No stat. sig. NA (*) indicates adverse 30 !!g/L 32 !!g/L NA significant effect No stat. sig. NA difference.
adverse 100 !!g/L 110!!g/L NA effect 45% * 300 )..lg/L Day 21 337 ug/L NA No stat. sig. NA adverse 3 !!g/L 3.2 !!g/L NA Magnitudes effect No stat. sig. NA calculated from table 3. Magnesium adverse 10 !!g/L 10 !!g/L NA (*) indicates effect 20% * 30 ug/L Dav21 32 ug/L NA significant
10% * 100 )..lg/L Day 21 110 )..lg/L NA difference.
20% * 300 )..lg/L Day 21 337 Ug/L NA No stat. sig. NA adverse 3 !!g/L 3.2 !!g/L NA effect Magnitudes No stat. sig. NA calculated adverse 10 !!g/L 10 !!g/L NA from table 3. Calcium effect (*) indicates 15% * 30 !!g/L Day 21 32 !!g/L NA significant difference. 5% 100 !!g/L Day 21 110 !!g/L NA 15% * 300 !!g!L Day 21 337 )..lg/L NA Note: NA=Not AvaIlable (effect observed on last day ofexpenment)

Time weighted averages are taken directly from study table 1 (time means).

Reviewer Comments:

8This was awell-controlled microcosm study with reasonable replication, five levels of treatment, and both diluent and solventcontrols. Methods are generally well described and followed published standards, although more detail on the statistical methods and exactly when they were applied would have been useful. Also, the conditions ofthe pond from where the protozoacolonies were collectedwere not described; background exposure to atrazine may therefore be possible.

Protozoacame from apond, buttheir colonization in a running water scenario was examined. The incoming water was dechlorinated tap water, devoid ofthe nutrients of natural water, and the substrate was likewise unnatural. The ecosystem under study consisted entirely ofprotozoa (Aufwuchs) thathad attached to polyurethane substrates in the pond. By not including largerplants and animals, the ecosystem structure was greatly simplified from that ofanatural ecosystem, and therefore the effects noted may notbe applicable to a more complete ecosystem structure.

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

Rationale for Use: Study contributes to the weight ofevidence ofpotential effects of atrazineto aquatic ecosystems.

Limitations ofStudy: This microcosm study had goodreplication and methods. However, the focus was protozoacolonies on artificial polyurethane substrates, watered by dechlorinated tap water. These conditions are highly simplified from a natural ecosystem structure, and so results may notbe easily generalized.

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

Secondary Reviewer: Brian Anderson, U.S. EPA

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