### Open Literature Review Summary

ChemicalName: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Gustavson, K. 

### and Wangberg, S. 1995. Tolerance induction and succession in microalgae communities exposed to copper and atrazine. Aquat. Toxico!. 32:283-302 (MRID 47543614).
Purpose ofReview: Determine the potential effects ofatrazine on an aquatic community. Endpoints from this study contributeto the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associateddetermination ofa Level ofConcern (LOC).

Date ofReview: November 24, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine to microalgae in experimental lake enclosures. Specifically, the objectives ofthe study wereto follow the succession of species and tolerance ofexposed phytoplankton communities and compare tolerance levels in phytoplankton and periphyton communities.
Summary ofStudy Findings:

Methods: This study also examined effects ofcopper, and toleranceto zinc and diuron. Methods and results described in this study review will be limitedto thoserelevant for the atrazine investigations.

The experiments tookplace over20 days, August 8-29 (the yearis not stated). Enclosures were created out ofclosed-bottom, 3.5 mdeep, 1.5 m diameter clearnontoxic polyethylene cylinders with 0.1 mm thick walls, each containing about 6 m3 

### oflake water and attached to apontoon bridge in Lake Bure, Denmark. LakeBure is a mesotrophic clear lake that is not fed by wastewaterrunoff. No further information about the size ofLakeBure orthe distance ofthe enclosures from shore was provided. Six enclosures received atrazine at 3 treatment concentrations (2 replicates at each treatment level) of0.0046 /lM (1.0 /lg/L), 

### 0.046 /lM (9.9 /lg/L), 

### and 0.092 /lM (19.8 /lglL). Three


# 1111•••111 2087841

### enclosures were controls. The atrazine (appears to bereagentgrade: Pesternal, Riedel-de Haen, MW == 

### 215.6 g/mol) was dissolved in distilled water to 10 ppm with ultrasound and no organic solvents. This solution was added slowly to the enclosures using a soft silicone tube to distribute it evenly with depth. The positions oftreatment and control enclosures on the pontoon were random.

Water samples were taken from the enclosures and the lake with a 3-m-Iong rigid tube sampler ofvolume 2.3 

### L. Macrozooplankton were removed during the sampling by a 140 J.1m filter. Periphyton was sampled on submerged 1.5-cm2

### -areaglass discs in the enclosures at a depth of0.5 m.

Tolerance measurement ofphytoplankton and periphyton (in terms ofJ4C uptake after additions ofatrazine) was begun within 0.5 hours after sampling, in a mobile laboratory. lO-mL scintillation vials, containing 9.9 mL lake/enclosure waterand 0.1 mL ofatrazine test solution, wereprepared; for periphyton, each vial contained a 1.5 cm2 

### glass disc and 9.9 mL filtered (GF/C) lake water. The atrazinetest solutions were prepared in distilled waterto achieve a geometric series of15 nominal vial concentrations ranging from 0.00082 J.1M to 2.8 J.1M atrazine (about 0.2 J.1g/L to 603.9 J.1g/L), one single replicate for each test concentration and five control replicates. A 50 J.1L (1 

### J.1Ci) aliquot of14C_ bicarbonate

# e 4

### CAgency, Denmark) was addedto each vial. After apreincubation period ofI hour, the vials were incubated for 2 hours in clear acrylic holders, in situ, at a depth giving irradiance ofabout 300 J.1E/m2

### /s corresponding to light-saturatedphotosynthesis. The non-fixed 14C was removed after acidification with hydrochloric acid. The biotic fixed 14C was counted on a Beckmann Liquid scintillation spectrometerLS 1801 (the authors say that abiotic 14C 

### was always less than 1%

- no details on how this was determined were provided). Tolerance was determinedby examining the dose-response curve and determiningthe ECso 

### value via log-linearinterpolation after normalizing the 14C fixation to the mean ofthe 5 controls.
Phytoplankton biomass in water samples was measured as chlorophyll a, afterextraction in ethanol for 12 hours and spectrophotometry analysis. Phytoplankton community composition and succession were determined by counting cells in Lugol-fixed samples using an inverted microscope. Species composition was evaluated using the S0rensen similarity index (the 1948 paper is cited, butthe index is not defined in the text), and via detrended canonical correspondence analysis (DCCA) using the software package CANOCO (TerBraak, 1988) after transforming the cell counts to square roots to increase the influence ofthe less abundant species. Particulate organic carbon (PaC) andnitrogen (PaN) were measured by dry combustion ofparticles retained on precombusted GF/C filters in an Elemental Analyzer. Standards were runroutinely after every four samples. Surface quantum flux densities were measured with a LI-COR spherical quantum sensor (model notprovided). Nutrients were determined "according to conventional standard methods" (no reference provided), and pH was measured in situ (no details provided).

2Atrazine was analyzed in threereplicate 100 mL water samples taken from each enclosure on different days. Samples were liquid/liquid extracted twice using 100 mL of dichlormethane, and the extracts then reduced to dryness in arotary evaporator, dissolved in 1 mL dichlormethane, and injected in the HPLC (Waters model 510 and a Waters UVdetector440) using a Novapak C-I8 HPLC column with dichlormethane as the mobile phase (1 mLlmin). Concentrations ofatrazine were determined using peakintegration and standard curves constructed the same day as the analysis, and extraction blanks were run routinely after each sample. The detection limit was 0.0001 IJ,M, and replicate measurements had a2% coefficientofvariation.

Results: Only results relevant to atrazine will be described. It should be noted, however, thatthe study focuses on the copperresults, because more effects were seen with copper than with atrazine.

Atrazine concentrations in the enclosures remained stable overthe course ofthe experiment, ranging from 0.005 to 0.003 IJ,M (0.6 to 1.1 IJ,g/L) for the low (0.0046 IJ,M or 1.0 Jlg/Lnominal) enclosures, from 0.046 to 0.069 IJ,M (9.9 to 14.9 IJ,g/L) forthe medium (0.046 IJ,M or 9.9 IJ,g/L nominal) enclosures, and 0.082 to 0.111 IJ,M (17.7 to 23.9 IJ,g/L) for the high (0.092 11M or 19.8 IJ,g/L nominal) enclosures. (No further details are provided.)

Atrazine tolerance in the phytoplankton communities was relativelyconstantthroughout the experiment, and additions ofatrazine to the enclosures did not induce atrazine tolerance (no data provided) (see Figure 1 for comparison ofatrazine tolerance in lake vs. control enclosure phytoplankton communities

- they werevery similar). Likewise, when the tolerance ofperiphyton was measured 22 days after atrazine addition to the enclosures, there were no changes in periphyton community tolerance based on prior atrazine exposure, orcontrols vs. lake samples. The ECsovalues for atrazinewere very similar in periphyton and phytoplankton (see Table 1).

Atthe beginning ofthe experiment, chlorophyll concentrations in the enclosures were about 10 llg chl-a/L. The addition ofatrazine caused a small increase in the chlorophyll concentrations after 2 weeks (no data or statistics provided). pH in the enclosures increased as the experiment continued due to reduced watermovement in the enclosures, which resulted in reduced uptake ofcarbon dioxide from the atmosphere (seeFigure 2).

POC/PON ratios became as low as 6.6 in the atrazine enclosures, whereas the control enclosures had ratios 01'7.5 to 8.4 (no further details ordataare provided). The chlorophyll/POC ratio was about 30% higher in the high concentration atrazine enclosures (no further details ordata are provided). Nutrient concentrations remained similar over all atrazine enclosures and the lake, and constantovertime (no further details or data are provided).The number ofrecorded phytoplankton species was similar in all enclosures. In the control enclosures, a succession was seen with increased numbers of

### Anabaena ef seheremtiev, Closterium aeutum, and 

### Chrysoehromulinaparva, and similartrends but with stronger magnitudes were observed in the atrazine enclosures (no magnitudes or statistics are provided). See Table 2 and Table 3 for species distribution data. The communities from the atrazine enclosures, control enclosures, and the lake all had Sorensen index between 

### 0,593 and 

### 0.957 (no further details or data are provided). DCCA ordination could explain only 60% atmost ofthe total variation in species composition, but found that medium and high atrazine concentration enclosures had succession in the same direction as the controls (see Figure 3).



### Cladoeera and copepods were more abundant in the enclosures than in the lake due to lack ofpredation, and enclosures with atrazine addition had largernumbers of

### Cladocera, though the mean length ofthe zooplankton was not affected by atrazine level. There was no noticeable decrease in zooplankton density across atrazine enclosures and controls. (No further details ordata are provided.)



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## nazin. concentf,tion (JlMJ Figure 1. Atrazine tolerance ofphytopJankton communities from Jake samples (solid line) and control enclosures (dotted line), Jines representing the mean and error bars the standard deviations over all samples and all days ofmeasurement (13 data points tota)). Vertical dashed lines represent nominal atrazine concentrations in the enclosures.

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### 8.5



### 8.0 12 18 

### Date in August 24 

### 30

Figure 2. pH values in the enclosures. Solid squares 

### =Lake; open circles with errorbars = control, low and medium atrazine treatment, and low copper treatment enclosures (mean and range); triangle with dotted error bars high atrazine treatment enclosures (mean and range). Stars are high copperconcentration, and diamonds are notdefined in the study.

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### 1Figure 3. Ordination ofa nCCA ofphytoplankton community composition data. Relevant symbols are the open circles (controls); solid squares (lake); triangles (medium atrazine treatments); and inverted triangles (high atrazine treatments). All are clustered in the upper left corner ofthe plot.

Table 1. ECso values for phytoplankton and periphyton communities. Low atrazine treatments have a nominal concentration of1.0 /!g/L, medium treatments have 9.9 /!g/L, and high treatments have 19.8/!g/L. (from study Table 2) Phytoplankton (29**Aug~** M atrazine atrazine Periphyton (30 August) M atrazine atrazine

LowAtrazine Lake Control 0.27 58.2 0.24 ± 0.Q35 a 51.8 ± 7.5 a 0.26 ± 0.021 b 56.1 ± 4.5 b MediumAtrazine 0.23 ± 0.008 b 49.6 ± 1.7 b 0.27 0.0025 b 58.2 ± 0.5 b mean from mean with n

### =3
b mean and deviation from mean ofduplicates 0.24 0.25 0.35 0.15 0.3 51.8 53.9 75.5 32.4 64.7

Table 2. Number ofspecies in different systematic groups on August 28. It is assumed that the last column heading on study Table 3 is a typo, and should read "A_high(a),**A~high(b)".** (from study Table 3)

Systematic class Lake Controls Cyanophyceae II 8 10 10 10 10 I 1 Bacillariophyceae 7 5 5 4 5 6 6 Eug1enophyceae 0 0 1 0 0 0 0 Chlorophyceae 18 17 22 22 22 18 22 Dinophyceae 2 2 2 1 I I 1 Cryptophyceae 3 3 3 3 3 3 3 Chrysophyceae 2 3 2 1 1 2 2 Prymnesiophyceae 2 2 2 2 2 2 2 Numberof 46 40 47 43 44 42 47

Table 3. Cell numbers on August 28 for algal species that in some cases had more than 1000 cells/mL. 

### "+" notation is not defined in the study, but reproduced here for completeness. It is assumed that the last column heading on study table 4 is a typo, and should read**"A~high(a),** A_high(b)", and that the second 28 Aug. heading should read "C_l, C_2". (from study Table4)

Systematic class Lake Controls Anabaena cfscheremetievi + 10,040 30,981 38,174 59,714 Lyngbya limnetica + 291 74 163 378 Monoraphidium cf + 8 + minutumlcirernale Closterium cfacutum 565 423 914 1,747 672 1,396 A1ougeofia sp. 1,692 660 689 409 538 511

6Rhodomonas minuta 906 83 130 5 44 172 Katablepharis sp. 302 + 630 398 135 216 378 Chrysochromulinaparva 4,047 2,966 5,298 8,904 1,434 8,555 7,698 906 540 867 741 184 288 I 65



### Data Summary Table: E.lfect Magnitude Nominal Day of Study Time Weighted Did Comments ofEjJect Atrazine EjJect Average Recovery relative to Concentration Occurred Atrazine Occur- 

### if controls (f.Jg/L) Concentration so, when from Day 0 to (see

I When EjJect comment)

# ~t;;.'t·nkto**~o e~ecl_**  1.0 .glL Occurred ?**~~-** not 0.9 IlgiL not a applicable Study states periphyton

. ff t

. 9 9 IL not 12.4 Ilg/L not lack ofeffect. community no e ec
. I1g 

## . applicable No data a tolerance to no effect 19.811g/L not 20.81lg/L not provided. atrazine applicable applicable

-- Smail increase 

# I Increase in NRJ"small" NR Day 14 Not calculable NR after 2 weeks

chlorophyll a stated; no data.
,..----**---~- .~~**  ..**_-~~ ~~-** **~------~~~**

Decrease Study reports ratio of only: 6.6=low particulate**·~15-20%** NR NR Not calculable NR value for organic treatments, vs. carbon to p.o. 

# -t1 7.5 to 8.4

### = nitTogen control**range~_** Increase in chlorophyll to No further data particulate 30% 119.8 jlg/L NR 20.8 11giL NR or details organic reported.

I carbon ratio 

# I- I not no effect 1.0 IlgiL a Ikable 0.91lg/L applicable Study states 

# (;:00'

no effect 9.9 jJ.g/L 

### I not 12.4 Ilg/L not lackofeffect.

concentrations applicable applicable No data

no effect 19.8 jJ.g/L not 20.81lg/L not provided. applicable applicable Enhancement Qualitative ofenclosurecaused statement successional NR NR NR Not calculable NR based on observation changes in andDCCA phytoplankton 

# ;*

O'd;",tion**~om~llnity** 

### I

-**~~----~** Increase in No further data Cladocera NR NR NR Not calculable NR ordetails
09 IL -_. not--1

# ~~~d~:~~t~ Mean hmnthc effect 1.0 not

. Ilg applicable 

# I lack ofeffect. density of not not No data zooplankton effect 9.9 12.4 IlglL**applicabl~rovided.** 

# _J

7Note: NR=Not Reported Balded values are those datapoints specified inthe "Cosm Data Points and Exposure Prames" spreadsheet.

Since no time series ofatrazine concentrations were provided, butthe study stated that atrazine concentrations did not change much over time, the midpoint ofthe range of concentrations measured ateach treatment level was used as the time-weighted average. The minimum and maximum measured concentrations, in !lM, were cited in the study text. Calculation oftime-weighted averages involved simply converting to units of !lg/L and taking the average ofthe minimum and maximum. See below, and the linked Excel spreadsheet.

11

### M J,lg/L midpoint of range::: TWA atrazine atrazine (ug/L) range - low 0.005 1.1 0.9 0.003 0.6rangemed 0.069 14.9 12.4 0.046 9.9rangehigh 0.111 23.9 20.8 0.082 17.7



### Reviewer Comments: Many factors limitthe usefulness ofthis study for the purposes of the atrazine literature review. Most obviously, since the authors observed much more substantive impacts with copper addition, they focused primarily onthe copperresults at the expense ofafull presentation ofthe atrazine results.

The basic study design was limited byhaving only two controls at each treatment level, and all treatment concentrations were low. The study lasted only a few weeks, andthe enclosures had strongly altered physical, chemical, and biological conditions (i.e. mixing with depth, pH, and predation) which limitthe generality ofthe results to naturallentic systems.

ManY'details regarding both methods and results were not provided. The year ofthe experimentis not stated, only the month and days. Very little information is provided about the site and the general setup, including the lake size and surrounding land uses, the distance ofthe enclosures and lake samples from the shore, and the relative locations of treatment and control enclosures. It is not statedwhen orhow abiotic 14C was determined in the 14C uptake measurementprotocol. There is no information on how nutrient levels orpH were determined. Atrazine concentrations appearto be exhaustively measured; however, the results are not shown, and only minimum and maximum values at each

8

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# l treatment level are provided. Many results, both with and withoutreportedeffects, are not described adequately (that is, with the use ofdata tables orgraphs). These include atrazine tolerance with prior atrazine exposure; increases in chlorophyll 

### a and the chlorophyll/POC ratio; decrease in the paC/paNratio; lack ofeffects on nutrient concentrations and zooplankton; and time-dependentphytoplankton community composition (i.e. successional changes). The Data Summary Table has many missing entries due to the lack ofcompletereporting onthese results.

In terms ofpresentation, there were otherproblems with the study. The DCCA (detrended canonical correspondence analysis) was notuseful it 

### was poorly described and was able to account for only 60% ofthe total species abundance variability in the first two components. Multiple figures and tables were not fully explained, that is, symbols were presented in the tables for which no definition was available in the study text or in the caption, and additionally some apparenttypos in the tables interfered with interpretation.
Description ofUse in Document(QUAL, QUAN, INV): Qualitative

Rationale for Use: The results ofthe study show that atrazine caused no effectto phytoplankton andperiphyton community tolerance at concentrations::: 20 !!g/L.

Limitations ofStudy: The study is limited by only two replicates at each treatmentlevel, very low atrazine treatment concentrations (20 !!g/L orless) studied for a shortperiodof time (3 weeks), and physical/chemical/biological artifacts due to the Closed-bottom 6 m3 

### water volume enclosures. In addition, no effort was made to document whether atrazine orotherchemicals were present in lake water orenclosure controls. Moreover, very little information aboutthe atrazine effects (orlackthereof) was provided in the article. Other limitations associated with this study are described in the Reviewer's Comments.
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc
Secondary Reviewer: Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. Environmental Protection Agency.