### Open Literature Review Summary

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



### ECOTOX Record Number and Citation: Hamala, 

### l.A. and H.P. Kollig. 1985. The effects ofatrazine on periphyton communities in controlled laboratory ecosystems. Chemosphere. 14:1391-1408 (MRID 47543615).



### Purpose ofReview: Determine potential effects ofatrazine on aquatic community. Endpoints from this study contributeto the weight-of-evidenceregarding effects ofatrazine on aquatic plant communities and associated determination ofaLevel ofConcern (LOC).



### Date ofReview: November 24,2009



### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants (periphyton community) in experimental laboratory flow-through chambers (microcosms).



### Summary ofStudyFindings:

Methods: The experimentwas conducted atthe EPA's Aquatic Ecosystem Simulator(AEcoS), a laboratory chamber systembuiltin a 19.5 m long, 0.46 m wide, 0.51 m deep, "U"-shaped flume. The flume was divided into 4 independentflow channels (A, B, C, and D), each further subdivided into four 250-L microcosms (1, 2, 3, and4), which were setup in flow sequence so thatthe effluent from an upstream microcosm passed overaweir into the next downstream microcosm. The flume was made ofPlexiglass and lined with 4-mil Teflon film. Uniform mixing in each microcosm was achieved with longitudinally suspended paddlewheels. Deionized waterwas gravity fed into the upstream microcosm at a flow of250 L/day. Watertemperature was maintained at 15°C. Aphotoperiod of12 hours light, 12 hours dark was maintained with fluorescent lights at 8608 lux. The microcosms AI, Bl, Cl, and Dl were used for initial nutrient introduction (viaperistaltic pumps) and additional nutrients were addedto A2, B2, C2, and D2. See Table 1 for nutrient concentrations. The second set ofmicrocosms (A2, B2, C2, D2) were the set sampled for the experiment, two as controls andtwo as treatments at a single treatment concentration. 

# '1.1.11111 2087840

### Periphyton communities were established in the microcosms by inoculating and then cross-inoculating each with composite tow and littoral sediment samples from local northeast Georgiaponds that were notexpectedto have background atrazine exposure (no further information on pond sources provided). Teflon-coveredplastic strips (10 x 

### 30 cm) were suspended along the sides ofeach microcosm for 3 different 14-day colonizations, with sampling on days 44, 59, and 80 after inoculation. Microcosms were allowedto be colonized andreach steady state for 43 days, by which time dissolved oxygen diurnal cycles hadbecome similar, with concentrations from 7-13 mg/L, indicating functional stability.

Atrazine was added to the microcosms A2 and D2 on the 44th 

### day afterperiphyton inoculation. Firsteach microcosm was "loaded" to aconcentration of 100 Ilg/L, andthen this concentration was maintainedwith aconstant-head flask and dripper input system calibratedto deliver 2mUmin. The atrazine spiking solution was preparedby dissolving technical grade atrazine (96.5% active ingredient, Ciba-Geigy Corporation) in methanol in avolumetric flask. Aportion ofthis stock solution was addedto an Erlenmeyerflask, the methanol was evaporated with dry nitrogen gas, and distilled waterwas added and stirred on amagnetic stirringplate for 24 hours. Samples were takenperiodically and analyzedto confirm complete dissolution ofthe atrazine. Atrazine inputto the treatment microcosms was stopped on the 59th 

### day afterperiphyton inoculation (Day 14 after addition), andthe microcosms were allowedto recover for 21 days (until Day 35 after addition). (Note that although the article numbers days beginning from periphyton inoculation, the review will countDay 0 as the day ofatrazine addition.) Triplicate water samples were taken for atrazine analysis every 3 days with methylenechloride using an ultrasonic Politron apparatus, and analyzedusing aTracor 900-78S HPLC equipped with Partisil ODS-2 column and water-methanol mobile phase. Atrazine concentrations during the exposure period averaged 89.6 ± 

### 3.3 Ilg/L in microcosm A2 and 96.9 Ilg/L ± 

### 6.0 Ilg/L in microcosm D2.

Perophyton strips were sampledon Day -1, 14, and 35 afteratrazine addition (one day beforetreatmentbegan, atthe endofthe treatmentperiod, and at end ofa 21-day recovery period), in each case after 14-day colonizationperiods. Aknown strip areawas scraped into abeaker, the sample was broughtup to aknown volume with distilled water andblended athigh speed for 5 seconds, and aliquots ofthe resultant slurry were dispensed for algal enumeration, chlorophyll 

### a content analysis, and determination of ash-free dry weight (AFDW).

Foridentification andenumeration, algal samples were preserved with diluted acid Lugol's solution and examined with aZeiss invertoscope 0 

### microscope with brightfield andphase contrastcapabilities. Enumeration, excluding diatoms, was based on the inverted microscope method ofUtermohl. Density (excluding diatoms) was estimated from counting at least400 algal units at 640x, 

### units being unicells, coenobia, palmellas, colonies, orfilaments. For diatoms, permanent slides were preparedby "cleaning" the

2sample using Van derWerff(1958)'s organic digestion procedure, drying a diluted aliquot on a round coverslip, and mounting the coverslip onto a microscope slide with Hyrax medium. Diatom density was then estimatedby counting at least400 diatom valves at 1000x. A total of33 algal taxawere identified in the experiment (see Table 2). Algal species diversity (H') was determined with the Shannon-Weinerequation, and changes in algal community structure were evaluated with the SIMI index, which has a maximum of 1 when species composition and average relative densities are identical and 

# owhen they are completely different. (The study cites sources for these measures, but does not define them within the article.)

Algal biomass as chlorophyll 

### a was analyzed with a TurnerDesign Modell0 fluorometer and corrected for phaeophytin 

### a. Samples were extracted with 90% acetone within two hours ofcollection using a B. Braun cell homogenizer. Total community biomass as AFDW was measured according to the American Public Health Association's 1981 standard methods.

Dissolved oxygen (DO) in the microcosms was measured hourly using an Orbisphere Model 2708 monitor interfaced with a computerized system. Community metabolism
measures (net and gross productivity, nighttime and total respiration) were calculated from the hourly DO changes with a computerized modification ofOdum's (1956) mass balance equation for open, flowing water systems (no further description provided).

A nested analysis ofvariance was used to test significance ofdifferences between controls and treatments on each periphyton sampling day in the following variables: total algal density, algal division density, chlorophyll a, and AFDW. All algal densities were transformed by 10glO(X+1) priorto statistical analysis (the purpose ofthis transformation is not explained, normality tests ofraw and transformed data are not mentioned, and specific ANOVA techniques are not described). Community metabolism results were averaged within replicate treatments and plotted overtime without any statistical analysis.



### Results: Bythe end ofthe pretreatmentperiod (43 days afterinoculation), all colonizable surfaces in the microcosms were covered with a dense mat ofperiphyton. Plankton were not a significantpart ofsystem biomass due to the flow-through nature ofthe microcosms (this
was confirmed by unpublished data). Microinvertebrates in the periphyton mat were sparse except for rotifers and nematodes. Periphyton communities were dominated by (average relative abundances follow in parentheses) Chlorophyta (71 %), Bacillariophyta (19%), Cryptophyta (3%), and Cyanophyta (2%). See Figure 1. Dominant species were 

### Chlamydomonas spp. (27%), 

### Stigeoclonium lubricum holdfast cells (23%), 

### Characium ambiguum (7%), 

### Chlorella sp. (7%), 

### Fragillaria crotonensis (17%), 

### Cryptomonas ovata (3%), and 

### Chroococcus minor (2%). Community biomass (chlorophyll 

### a and AFDW) before treatment was within the range observed during previous AEcoS experiments, and

3algal diversity estimates indicated arelatively complex, mature periphyton community (see Table 3 pretreatmentvalues). No significantdifferences existedbetween control and treatment microcosms priorto atrazine addition (see Table 3 and Figure 2).

After 14 days ofatrazine treatment atnominal levels of 100 !Jg/L, community metabolism was profoundly disrupted relative to controls. Netproductivity decreased within hours ofatrazine addition and continued to decrease throughoutthe exposure period, falling to negative values by Day 10 after addition. The effect on respiration was much weakerthan the effect on productivity, with no consistent change in nighttime respiration during the first week after atrazine addition. During the second week, transient increases in nighttime respiration of8-34% over controls were noted, which the authors attribute to a delayed increase in heterotrophic activity. The P/Rratio decreased to 0.63 immediately after atrazine addition and continuedto decline until atrazine inputs ceased, with a minimum of0.29 on Day 13 after addition. See Figure 2 for time-series plots ofnetproductivity, dark period respiration, and P/R.

Community structure changes on periphyton strips that were colonized during the treatment period were observed, including decreased algal density and diversity, altered species composition, andreducedbiomass accumulation in treatments relative to controls. Total algal and Chlorophyta average density were significantly different from controls (p

## :s 0.05), decreasing to 23% and 15% ofcontrol values respectively. See Table 3. Green algal species with large average density decreases included

### Ankistrodesmus faleatus (11 % ofcontrol value), 

### Chlamydomonas spp. (16%), 

### Cosmarium reinsehii (10%), Scenedesmus dimorphus (1%), 

### Straurastrum manfeldtii (10%), and

### Stigeoclonium lubricum holdfast cells (15%). See Table 4. The Bacillariophyta and Cryptophytawere minor components ofthe periphyton community in both controls and treatments (see Figure 1). Substantial decreases in density were observed for these divisions, however, comparedto control values (see Table 3). 

### Chroocoecus minor, a small unicellular Cyanophyte, increased to 224% ofcontrol values (Table 4) and accounted for the large increase in Cyanophytarelative abundance in treatment microcosms (Figure 1). As Table 4 shows, individual algal species varied considerably in theirresponse to atrazine level of 100 !Jg/L for 14 days; while mostdeclined in cell density, sometimes dramatically so, a few species increased. The authors note thatthese effects were perhaps characteristic of natural assemblages and couldnotnecessarily have been predicted from single species bioassays. The algal diversity also decreased with treatmentrelative to controls: H' was 2.35 in treatments and 2.77 in controls, and total number ofspecies was 18 in treatments and 24 in controls. SIMI (based on all observedtaxa) was 0.64 in treatmentrelative to controls (indicating dissimilarity), although only 3.3% ofthe total relative abundance in control microcosms were not observed in the treatment microcosms. The authors interpretthis as meaning that atrazine eliminated some ofthe rarer algal species, and changedthe relative abundances ofthe dominant species. See Table 3 for summary data.

Chlorophyll a and AFDW decreased to 8% and 52% ofcontrol values, respectively, during the treatment, but sample variation among the two replicates was high and so the

4

### differences were found to be insignificant. However, since chlorophyll 

### a was apparently
more affected by atrazine treatmentthan AFDW, the authors state that eithermuch ofthe AFDW biomass was nonalgal (note thatnematodes and rotifers were presentin the periphyton mats), orelse the per-cell chlorophyll a content declined due to treatment. In eithercase, although the results are not statistically significant, the authors concludethat atrazine didreduce the overall algal growth rate.

During the 21-day recovery period, in which no atrazine inputs were appliedto the treatment microcosms (complete washout occurred within 4 days in the sediment-free microcosms), daily netproductivityrecovered steadily until itwas similarto control values onDay 31 after atrazine addition. In nighttime respiration, there was atransient decrease (82.5-86% ofcontrol values on Days 15-16 after atrazine addition) which came backto within 90% ofcontrol values afterDay 16. P/R

### ratio recovered similarlyto net productivity, achieving avalue of1.04 on Day 31. See Figure 2. The authors suggest that the fairly rapid recovery in these parameters indicate that atrazine's primary effect is algistatic and not algicidal in nature.

In contrastto the community metabolism measures, periphyton community structure measures showed little to partial recovery during the recovery period. Significant(p

## :s 0.05) differences were still found inthe total algal average density (25% ofcontrols) and total Chlorophyta average density (25% ofcontrols). See Table 3. Some green algal species increased as percentages ofcontrol abundances compared to the treatmentperiod (Characium ambiguum, Cosmarium reinschii, Stigeoclonium lubricum), while others remained low (Ankistrodesmusfalcatus, Scenedesmus dimorphus, Staurastrum manfeldii). See Table 4. The total average density ofBacillariophytaincreased in treatment microcosms to 74% ofcontrol values, due primarilyto a large increase in Nitzschiapalea, which comprised 96% oftotal Bacillariophyta(Table 4). Total average density ofCryptophytaremained low in treatment microcosms (8% ofcontrol values) while that ofCyanophytadecreasedto percentage ofcontrol values seen in the pretreatmentperiod (calculated from Table 3 data). Algal diversity increased substantiallyto become similarto orhigherthan control andpretreatmentvalues (Table 3), butthe SIMI value remained low at 0.60, indicating that community composition remained dissimilar from controls. The authors note that it seems some ofthe rare species were able to reestablish themselves, butthe relative abundances ofthe dominant species remained substantially different.

Chlorophyll a 

### and AFDW community biomass estimates in treatmentmicrocosms were 25% and 33%, respectively, ofthe control values. In comparison with the treatment period, chlorophyll 

### a increased and AFDW decreased (Table 3). The authors interpret the results to indicate that chlorophyll a 

### per algal cell increased during the recovery period, although total algal density remained lower.



### 5

### At one point, the authors tangentially describe unpublished data showing that, after a full recovery post-treatment, the periphyton communities did not appearto develop atrazine resistance when again exposed to 14 days of 100 /lg/L atrazine.

Table 1. Elemental concentrations ofnutrient input to microcosms AI, 81, Ct, and D1.

Macronutrient m /L Micronutrient P 0.01 B Si 5.88 Mn HC03 75 Zn Mg 2.67 Fe K 3.02 Co NOrN 0.083 Cu Ca 9.65 Mo Na 37.96 S04 13.91 Cl 38.08 EDTA Additional inputs to A2, B2, C2, and D2: NOrN 0.747 P 0.09 C 4 /L 30.8 100 14.9 31.4 0.33 0.0035

2.75

440

Table 2. Algal species identified during the experiment.

Cyanophyta Chroococcus minor(Kuetz.) Naegeli Oscillatoria subbrevis Schmidle
BacilJariophyta Achnanthes minutissima Kutz. Cymbella minuta Hilse ex. Rabh. Gomphonema gracile Ehr. emend. V.H. Gomphonemaparvulum (Kutz.) Fragilaria erotonensis Kitton Navicula minima Grun. Nitzschiapalea (Kutz.) W. Sm. Synedra delicatissima W. Sm. Synedra socia WalJace Synedra ulna (Nitz.) Ehr.
Cryptophyta Cryptomonas ovata Ehr. Chlorophyta Ankistrodesmusfalcatus (Corda) Ralfs Chaetosphaeridium globosum (Nordst.) Klebahn Characium ambiguum Hem1ann Chlamydomonas spp. Chlorella sp. Coelastrum mieroporum Naegeli Cosmarium meneghiniiBreb Cosmarium reinschii Cosmarium undulatum Mougeotia sp. Oedogonium sp. Scenedesmus abundans (Kirch) Chodat Scenedesmus bijuga (Turp.) Lagerheim Scenedesmus denticulatus Lagerheim Scenedesmus dimorphus (Turp.) Kuetzing Spirogyra sp. Staurastrum manfeldtii Staurastrumparadoxum Meyen Stigeoclonium lubricum (Dillw.) Kuetzing

6Ulothrixzonata (Weberand Mohr) Kuetzing

Table 3. Periphyton community structure estimates after each ·atrazine exposure period (pretreatment, treatment, recovery). Mean values (n=6, 2 replicate microcosms, 3 replicate water samples from each) and standard deviations (SD) are shown. Values in bold are significantly differentthan control values (p 0.05).

Pretreatment Treatment Recovery Mean SD Mean SD Mean SD AlgalDensity (unitslcm2 ) TotalAlgae Control 8.76E+05 1.09E+05 4.83E+06 1.01E+06 8.13E+06 2.45E+06 Treatment 8.84E+05 1.55E+05 1.01E+06 2.19E+05 2.03E+06 7.06E+05 Chlorophyta Control 7.25E+05 6.70E+04 4.19E+06 8.00E+05 5.74E+06 5.39E+05 Treatment 6.13E+05 2.24E+04 6.16E+05 1.46E+05 1.46E+06 5.56E+05
Bacillariophyta Control 1.16E+05 7.07E+04 9.31E+04 6.59E+04 3.78E+05 3.93E+05 Treatment 2.l7E+05 6.24E+04 1.81E+04 6.72E+03 2.84E+05 1.44E+05 Cyanophyta Control 1.40E+04 l.79E+04 2.00E+05 I.72E+05 1.73E+05 6.75E+04 Treatment l.76E+04 2.77E+04 4.32E+05 6.55E+04 1.44E+05 2.48E+04 Cryptophyta Control 2.06E+04 9.06E+03 3.46E+05 3.IOE+05 1.85E+06 1.93E+06 Treatment 3.64E+04 1.04E+04 3.04E+04 2.32E+04 1.41E+05 7.17E+04 SIMI(similarity) Trt. vs. Crl. 0.88 0.64 0.6

H' (diversity) Control 3.18 2.77 2.88 Treatment 3.26 2.35 3.26 Total #Species Control 22 24 25 Treatment 21 18 22 Chl. a (flglcm2 ) Control 3.22 0.86 3.89 1.55 3.92 1.33 Treatment 3.89 1.05 0.3 0.04 0.98 0.47 AFDW(mglcm2 ) Control 0.3 0.03 0.27 0.08 0.33 0.13 Treatment 0.27 0.04 0.14 0.08 0.11 0.03

7Table 4. Average algal species densities, as percentages ofcontrol values, for dominantspecies on periphyton strips after each atrazine exposure period. * denotes thatthe species was not observed in eithercontrol ortreatment microcosms. Blank values for Synedra ulna appear in the original article.

Pretreatment Treatment Recovery
126 224 90

132 11 16 93 0 118 117 16 38 56 44 78 97 0 

### * 131 10 46 133 1 3 86 10 17 268 53 110 56 15 76

60 29 128 203 34 7 122 25 110 164 87 32 Species Bluegreen Chroococcus minor Green

Ankistrodesmusfalcatus Characium ambiguum Chlamydomonas spp. Chlamydomonas spp. (palmella stage) Chlorella sp. Cosmarium reinschii Scenedesmus dimorphus Staurastrum manfeldtii Stigeoclonium lubricum S. lubricum holdfast cells Diatoms Achnanthes minutissima Fragilaria crotonensis Nitzschiapalea Synedra socia Synedra ulna Cryptophyta Cryptomonas ovata

8 177 9 8Pre- Treatment Treatment Recovery "'- 

### o t':' 80 

# "-

60

40

20

o 

### c 'I 

## c 

## c T

Figure 1. Average relative abundance ofalgal divisions (% oftotal) in Control (C) and Treatment (T) microcosms before, during, and 21 days after atrazine treatment at 100 IlglLfor 14 days.

9(b)

'I ,., I /\' ,I 

# f

150.0 

### I I I ar-j I J I I I I I (i', i , iIi I ' I 36 40 '" 48 52 56 60 64 68 72 76 80 Time (days) 300.0
o
:.:: 1-0 275.0 ""J:**.~** C\l Co

.... 250.0 CIl""'... ll.l_ 0::""'... N Z25.0 "t:IO 

### .8 CIl 1i> 200.0 Cl. c 

### S :t 175.0 to Cl (a)

I**I-~"** '\,.,

....J

36 4Q '" 48 52 56 60 64 68 72 76 80 Time (days) o 

### S 0.0 :t -50.0

-100.0

400.0 350.0 1-0 >,J: 300.0



## .... ':;N**250.0~** 

## ......... 

## t:.::- 200.0 :::l""'... "t:I N 150.0 0 0 1-0 100.0 c.. CIl 50.0



## .... Cl) Z

1.50 (c) -'- Control lLicrocosms

--- Treatment Kicrocosm.s

0.75

0.50 Fia=e 2. Average Net Productivity (2&). Dark Period Respiration (2b). and PjR Ratio1 (2.:) in Control and Treatment lLicrocO:mls.

I Gross Productivity (GP) 1) P R = . I TotAl Respiration (TR) where GP

- Net Productivity + Dark Period Respiration and TR = 2 (Dark Period Respiration)**'-~** 1III I I I... f L f" \,.'\,J v

'_, I 

# t '---''Jf

0.25 1.25

o 1.00



# -- .... Il:l 

### c:: c:: ""'... c..

0.00 

### I i 

### Ti-j i I I I I I

# ",riO, iIi I I I ' I 36 40 '" 48 52 56 60 64 68 72 76 80 Time (days)

Figure 2. See caption in lower right subplot area. Atrazine input began on "Day 45" (called Day 0in the review) and ended 14 days later.

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### Data Summary Table: E.ffect Magnitude of Nominal Day of Study Time Did Comments Effect relative Atrazine Effect Weighted Recovery to controls Concentration Occurred Average Occur

- if (j1g/L) Atrazine so, when Concentration (see from Day 0 to comment)?

When Effect Occurred Not during From Fig. 2 Decrease in treatment, and textp.
net 100% butfull 1398. community (became 100/-lg/L Day 6 93.3 J..lg/L recovery Recovery productivity negative) after -16 days treatment postended. treatment. Decrease in Day 14 Partial at total (effect end of21- number of 25% 100/-lg/L measured at 93.3 J..lg/L day From Table end of 3. periphyton treatment recovery species period) period

Decrease in Day 14 biomass of (effect periphyton 50% 100/-lg/L measured at 93.3 J..lg/L No FromTable end of 3. (ash-free treatment dry weight) period) Change in 35% Day 14 algal species (different (effect From Fig. 1, composition Chlorophyta 100/-lg/L measured at 93.3 J..lg/L No significance (decrease in cell density end of from Table Chlorophyta with p < treatment 3. abundance) 0.05) period) Day 14 Decrease in 75% (effect total algal (different 100 J..lg/L measured at 93.3 J..lg/L No From Table end of 3. density with p < 0.05) treatment period)
Decrease in Day 14 Yes, at periphyton (effect end of21- measured at From Table algal 15% 100 J..lg/L end of 93.3 J..lg/L day 3. diversity treatment recovery (H') period) period Balded values are those datapomts speCIfied mthe "Cosm Data Pomts and Exposure Profiles" spreadsheet.



### Time weighted averages are the average of89.6 Ilg/L and 96.9 Ilg/L, cited on p.I394 as the average measured atrazine concentrations in the two microcosms overthe treatment period. Since application was continuous and no further concentration datawere provided, the time weighted average on Day 6 was assumed equal to that ofDay 14.

11Reviewer Comments: This was a well-controlled laboratory microcosm study with periphyton. Results were presented clearly and relatively completely. Although the laboratory system was not very realistic, it provides areasonable test bed for the limited question ofdirect effects of atrazine onthe periphyton community. Since the microcosms are not complete with drift plankton and othercommunities that would be found in a natural aquatic ecosystem, indirect ecosystem-level effects cannot occurin this experiment. The experimental design was further limited in that it used only two replicates and one treatment level, the atrazine treatment lasted only two weeks, and most measurements (besides DO for community metabolism) were taken infrequently.

Some aspects ofthe methodology couldhave been further described. The natural source ofthe inoculantperiphyton was not described in detail. The equations used for community metabolism were not defined, norwas their implementation on the computer system described; likewise, the community diversity and similarity measures were not defined within the article. The explanation ofthe statistical analyses was vague and left unclearthe interpretation ofthe "significantdifferences" that were found, although with only two replicates, itwould have been difficultto detect significant differences in any case.

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

Rationale for Use: The study demonstrates that atrazine has direct and indirect effects
on periphyton community structure at constantexposure concentrations of 100 Ilg/L. Communityproductivity was immediately and severely inhibited during the 14-d exposure period; however, the recovery was fairly rapid. Changes in community structure were evidenced by decreased algal density and diversity, altered species composition, and reduced rates ofbiomass accumulation. Little to no recovery in community structure was observed during the 21-day recovery period.

Limitations ofStudy: Only two replicates and one test concentration were used in this study, community composition and biomass measurements were infrequent (there was only one "treatment" measurement day), and the laboratory microcosm design precluded any ecosystem-level effects. In addition, it is unclearwhetherthe source ofthe periphyton communities taken from the northeast Georgiaponds had been exposedto background levels ofatrazine and/or otherpesticides However, direct effects of continuous 100 Ilg/L atrazine on the periphyton community metabolism and algal cell density were obvious.

Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc

12

### Secondary Reviewer: Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. Environmental Protection Agency.

## ~!l~/11

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