### Open Literature Review Summary Chemical Name: Atrazine 

### CAS No: 1912-24-9
ECOTOX Record Number and Citation: Knauert, S., U. Dawo, J. Hollender, U. Hommen, and K. Knauer. (2009). Eflects of Photosystem II Inhibitors and Their Mixture on Freshwater Phytoplankton Succession 

### in Outdoor Mesocosms. Environmental Toxicology and Chemistry. 

### 28(4): 836-845. (MRID 

### 48261120) 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 plant commUnities and associated determination ofa Level ofConcern (LaC).


### Date ofReview: October 

### 2, 2009 Purpose ofStudy: This study examines the individual and combined effects ofthree photosystem II inhibitors (atrazine, isoproturon, and diuron) on the community structure offreshwater phytoplankton studied in outdoor mesocosms. 

### Summary ofStudy Findings: This review focuses on atrazine-only experimental treatments.


### Methods: The data in this study appear to have been collected from the same seven-month mesocosm experiment as reported in Knauert 

### etal., 

### 2008. The experimentdates and site were the same and the measured atrazine concentration data reported in Knauert 

### etal., 

### 2008 was the same as reported in this study. In the prior study, the analysis focused on the inhibition ofphytoplankton photosynthesis whereas this study focused on effects to phytoplankton community structure.
Atrazine 

### (99% purity) was studied alone and as a mixture with diuron 

### (98.4% purity) and isoproturon (analytical standard). Diuron and isoproturon were studied alone, as well. For the single compound treatments, the 

### 30% hazardous concentrations (HC30) were
1 

# 11111••111 2087800selected based on species sensitivity distribution (SSD) curves from laboratory experimental gro\\-1:h ECso data on a broadrange ofphototrophic organisms (described in more detail in Knauert 

### etal., 2008). The HC30 for atrazine was 70**~g/L.** The test site was the Syngenta Crop Protection AG located in CH-8260 Stein, Switzerland (47° 33'10" N, 7° 57'47"E, 300 m a.s.l.). The test site composed of28 highdensity polyethylene tanks (3 m diameter, 

### ca. 15 cm soil-sediment layer, 

### ca. 130 cm 'va1-"'-**",~1.'m'" ~/"**  If),.,...3 .,,,,1

.. ,.,...,,,\ <"J..:",J..**<.,~-",** J..
..**_:~;t :~**  +J..",**~_",** ..**~;t** +",**~+",J..:I:~",** +J..", v\,**"'"'~** \..IVlU. lU.L, \,u. V U.J. VV1U-l11\.1),> VY1.11'--'U. YY\.IL\,.; UUJ.J.\....u I.U UJ.\..t 5J.VUlJ,U l.V .;,t.Q.U1I1L\..' Ul"'-' temperature. Phytoplankton, zooplankton, macroinvertebrates and other organisms were introduced into the mesocosms via water and sediment from a nearby man-made supply pond 

### (ca. 500 m3 volume, 4 m depth), as well as by aerial colonization during the year. Mesocosms were dominated by 

### Elodea canadensis (Hydrocharitaceae) populations, which were planted in the sediment. During March to April 2006, water was circulated among the mesocosms and the supply pond after which water circulation stopped and the mesocosms were considered isolated. Priorto exposure, mesocosms were randomly assigned to the different treatments. Each treatment and the control were replicated in three ponds. The exposure was initiated on May 3, 2006 (day 0) after a six-weekpre-exposure period. Herbicide concentrations and effects on photosynthesis were investigated over a seven month period from April to October 2006 with a five-week constant exposure period (day 0-34) and a five-month post-exposure period. Atrazine was dissolved in methanol; the final concentration of methanol in the ponds was < 0.002%. Methanol was not added to the control ponds. During the constant exposure period, atrazine was maintained within ± 20% ofthe target concentration. Atrazine (as well as diuron and isoproturon) was applied to the water surface using a commercial sprayer. After application, the water column was mixed with a polyethylene tube. Separate sampling equipment was used for each treatment to avoid cross-contamination.
For chemical analysis and biological sampling, depth-integrated water samples were taken from the four quadrants ofeach mesocosm using a polyethylene tube (120 cm long, 4.5 cm diameter). Samples were taken close to the sediment, to avoid contamination due to sedimentperturbation (it was not stated whatthe authors meant by 'contamination', i.e., the sediment itselfor something in the sediment). Samples for biological analysis were separated into phytoplankton and zooplankton with an Apstein plankton net (25 cm diameter,**55~m** mesh size). Phytoplankton samples (100 mL) were taken from each mesocosm, stained and preserved with approximately 2 mL ofLugol-iodine solution; the samples were stored in the dark for taxonomic determination. Sampling intervals for chemical analysis ofthe mesocosm water were on the day offirst application, twice weekly during the constant-exposure period, weekly from day 34 to 68 ofthe postexposure period and biweekly from day 68 to 173 ofthe post-exposure period. Water samples were stored frozen at4°C until analysis. Biological sampling ofthe**ph~ytoplankton** was performed weekly from days -8 to 54 and then biweekly from days 54 to 173.

2

### For atrazine analysis, the water samples were filtered (cellulose acetate, 0.45 Ilm). Eighteen (18 mL) aliquots were adjusted to pH 4 with acetate buffer, and radiolabelled atrazine was added as an internal standard. Analysis was performed with online solidphase extraction-liquid chromatography-tandem mass spectrometry. The water sample was enriched using a strataX column (20 mm length, 2.1 mm Ld.) eluted with methanol using an online system. Separation and detection ofatrazine was performed using HPLC withthe following conditions: Xbridge C18 column (2 mm Ld. x 55 mm), room temperature, flow rate 200 ilL/min, gradient mobile phase consisting ofwater with 0.1 % formic acid and methanol with 0.1% formic acid, and MS-MS detection (selected reaction mode with positive electrospray ionization). The indicative transitions for atrazine were described in Knauert 

### etai., 2008. The limits ofquantification were 3 nglL. Quantification was performed using the internal standard. The intraday precision ofthe method used for thepesticides applied in this study was 1-3%, and the recovery ofthe enrichment step was 92-115% (spiking level, 100 ng/L).
For the taxonomic determination, aliquots ofthe fixed phytoplankton samples were taken; cells were allowed to settle for at least 24 hours in a sedimentation chamber. Quantitative evaluation was done according to the method ofUtermohl as described by Schwoerbel using an inverted microscope (400x magnification). Taxawereidentified at the species level or to the lowest possible taxonomic level. Cell counts were expressed as individuals/mL for each taxon.
The dissipation ofatrazine in the post-exposure period (day 34-173) was described by calculation ofthe half-life (tv,) 

### using first-order kinetics. Phytoplankton data were summarized by determining total number ofalgal cells (total abundance), absolute and relative abundances ofsingle algal groups and species, and the number ofdifferent taxa found per sample. On the taxon level, mean abundances pertreatment were tested for significant differences using Duncan's multiple 

### t test. The Shannon index, which considers species number and distribution ofindividuals across species, was used as a measure for diversity ofthe phytoplankton community in a sample. Community data were further analyzed using ordinationtechniques, such as PCA (Principal Component Analysis) and PRC (Principal Response Curves; the constrained PCA based on redundancy analysis), performed using CANOCO 4.5. The ordination diagram ofPCA was created for each sampling data and indicated the dissimilarity ofthe community in these samples by the distance between the two sample scores. The ordination diagram of PRC displays the deviation ofthe community structure ofthe treated ponds from that of the controls overtime. Only log-transformed abundances were used for the ordinations.



### 3

### Results: 

### Chemical (Atrazine) Analysis In orderto maintain the target 

### HC30 (70 Jlg atrazine/L) in the constant exposure period, atrazine was reapplied one time at day 20. Time-weighted average atrazine concentrations in the constant exposure period were maintained within ± 20% ofthe target 

### HC3

### o. The primary reviewer estimated a range of69-84 IlgiL for days 0-28 based on 

### Figure 1 (see 

### Table 1 and 

### Figure 1 for details). In the,post-exposure period (days 

### 34-**17~\** thP**h~lf** ..llfp ofQt,.Q71np**~'''>"** I'Ql1'1l1Qtp,i Q" 1/17 r!"""" (,.'- = /1 0')\ h"""pr! An f'i,.d_A,.,i",,. -A. # '"")'

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..........._ ...**~~** w_... __....**_~_~** _u ... ...., t --Ju \..1.- ....,./_j ....,_u v ................. u?o-

_ .... _ ......... kinetics. The atrazine concentration declined to 

### 30 

### Ilg/L (reviewer-estimated) at the end ofthe experiment (day 173; 42.4% ± 2.4% ofthe 

### HC30, n 3; mean standard deviation calculated by the study authors). In the controls, the atrazine concentrations ranged from 6 to 28 ng/L throughout the study. The results ofthe atrazine concentration measurements (in 

### Jlg/L) are reported in 

### Table 1; the results ofthe herbicide concentration measurements for atrazine, diuron, isoproturon and the herbicide mixture are illustrated in 

### Figure 1.

4n Ica es va ue a er reapPlIea Ion 0 a razme I Time post first treatment (days) Atrazine single treatment (JigIL) 

## I
0 1 84 

### Y 7 1 101 76 141 74 171 70 21 1 69 (77)* 28 1 77 I 352 74

i 42t 70 49k 562 I i 702 i 842 i Table 1. Mean atrazine concentrations in the water phase overtime for atrazine single treatment. All mean values, including time, were estimated by the primary reviewer from Figure 1. Asterisk i d' tIft 

### r f f t

1 Constant exposure 2 Post-exposure period.



### 5

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# • • 0.0 0 20 40 60 80 100 120 140 160 180 200 0 0 20 40 60 80 100 120 140 160 180 200 Days pre- and postfirst application Days pre- and postfirst application Figure I. Actual measured herbicide concentrations in ""gIL for the different treatments: Atrazine (A), isoproturon (B), diuron (C), atrazine in the mixture (D), isoproturon in the mixture (E), and diuron in the mixture (F). Dashed lines indicate the target concentration ± 20%. Each data point represents the mean ± standard deviation ofthree replicate ponds. Arrows indicate reapplications of the herbicides.



### Totalabundance Prior to application (day -8), total abundance in all mesocosms was similar (one way ANOVA,p> 0.05). During the five-week constant exposure, mean total abundance reached approximately 6000 individuals/mL in the control on day 26. The total abundance in the atrazine treatment was reduced approximately three-fold to 2500 individuals/mL at day 26 (statistical significance not stated). From day 40 to study termination, atrazine treatments were not significantly different from the control. The study authors report that significant differences occurred on day 110 (one-way ANOVA followed by Duncan's multiple t 

### test,p > 0.05); however, they do not specify ifthe

6

### significant difference was for the atrazine-only treatment. The total abundance results for control and various herbicide treatments are illustrated in 

### Figure 2.



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# o 20 40 60 80 100 120 140 160 180 Time (days 

# post application) Figure 2. Total abundance (individuals/ml) determined in the control (*), atrazine (0), diuron (e), isoproturon (0), and mixture (.) treatment over the entire course ofthe experiment. Dashed lines indicate the minimum and maximum values ofthe control community. The v.ertical dashed line indicates the day ofthe first application (day 0)

.



### . Community structure andsuccession On day 5, control phytoplankton communities were dominated mainly by Chrysophyceae and Cryptophyceae (each accounting for 40% oftotal abundance). At days 19-26, the cryptophytes comprised up to 80% oftotal abundance, whereas chrysophytes had been reduced to only 10%. At the end ofJune, the abundance ofthe chlorophytes increased, yielding more than 80% oftotal phytoplankton because ofa bloom ofChoriocystis 

### cf. minor 

### and Monoraphidium circinate. 

### During August (days 82-110), Chrysophyceae and Cyanophyceae were the dominant algae, comprising 80% ofthe phytoplankton. During September and October (days 140-173), the Chlorophyceae, dominated by Oocystis naegelii (ca. 

### 3,000 individuals/mL), made up 60% oftotal phytoplankton. Before application (day

- 8) and a few days after application (day 5), the species compositions ofthe atrazine-treated communities were not significantly different (p > 

### 0.05) from those ofthe control communities. On every sampling date from days 12 to 173 (study termination), statistically significant differences in species composition were observed between the control and the atrazine-treated mesocosms (one-way ANOVA



### 7

### followed by Duncan's multiple t 

### test, p 

### < 0.05); these comparisons were based on sample scores from the first axis ofthe principal component analysis. Atrazine treated communities showed differences in major algal classes from day 12 to 173. For instance, Cryptophyceae abundance in atrazine treated communities was lower than in controls from day 19 to 40. Differences were also apparent at the species level. For example, the abundances ofChroomonas acuta, the prasinophyte Nephroselmis cf. olivaceae (data not shown) andAnkyrajudayi were also reduced in the atrazine treatment comparedto the control (generally starting on day 12). During the post-treatment period (days 34-173),
""""1 1

. 1 ; 1 4 1 • .. 1 1 .",..... • • \.."IllUrupny\;cat: werc lIlt: InU:Sl UUIIllnaIll algal gruup 1Il lilC alHlzmt: lrcalIIlCIll, l;urnpn:smg 

### 40 to 90% oftotal abundance. The bloom ofCyanophyceae and Chrysophyceae observed in the control ponds during August was nearly completely suppressed in the atrazine-treated pond because ofthe sensitivity ofChroococcus sp. (days 82-140) and Chromulina cf. obconica (day 110) toward atrazine. In contrast, growth ofChoriocystis cf. minor was significantly enhanced in the atrazine treatment compared with the control (days 82-110). The relative abundance ofmajor algal classes for control and various herbicide treatments are illustrated in Figure 3. The abundances ofselected species for the control and various herbicide treatments are illustrated in Figure 4. The PCA results for control and various herbicide treatments are illustrated in Figure 5.



### 8

### Control

-8 5 12 19 26 40 54 82 110 140 173 Time (daYli postapplication) 

### Diuron

-8 5 12 19 26 40 54 82 110 140 173 11me (daYli postapplication) 

### Atrazine

-8 5 12 19 26 40 54 82 110140 173 • Chrysophyceae 

### lS.'S1 Bacillariophyceae 

### CJ Cyanophyceae 

### mD Cryptophyceae 

## Iaa ZygnenuHophyceae 

### b.cl Prasiiioptiyceae 

# m Chlorophyceae



### Isoproturon

-8 5 12 19 26 40 54 82 110140 173 Time (days postapplication) 

### Mixture

.. 5 12 19 26 40 54 82 110140 173 Time (days post application) lime (days postapplication) Figure 3. Relative abundance ofmajor algal classes in the control and the various treatments.

9Cryptomonas erosa fitovata

Chromulina of. obconica

Oocystis naegeli

..... Control -_. Control minimum

--. Control maximum -e- Atmzlne

... DiI.ron -It- lsoproturon

- Mixt...-e 

## o**~"~1001~1~1"1~** Time (days postapplcatlon) Ankyrajudayi Chroomonasaeula

ChroOCOCCU8 spec.

Chodocystis cf. minor



## o**~"~1001~1~1"1~** Time (days postapplication) 105 10" 103 102 10 1 10 104


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### 10· 103 102 101 10°
Figure 4. Absolute abundances ofthe two cryptophytes, Chroomonas acuta and Cryptomonas erosa et ovata; the cyanophyte Chroococcus sp.; the chrysophyte Chromulina

### cr. obconica; and the three chlorophytes, Choriocystis cf. minor, Oocystis naegelii, andAnkyrajudayi in the different treatments. Data points localized on the x axis indicate that the abundance ofthe respective species was below the detection limit ofmicroscopic analysis on these sampling days. The vertical dashed line indicates the day ofthe first application (day 0).

10

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### -1.0 Figure 5. Plots ofprincipal component analysis sample scores for the phytoplankton data from three different sampling days. *
- control; 0- atrazine; • - diuron; 0

- isoproturon; _ - herbicide mixture.



### In contrast to the PCA, which coordinates the data for each sampling date separately and with apriori infonnation on the treatment ofthe samples, the PRC attempts to explain the data by considering time and treatment. Therefore, the PRC does not correspond exactly to the sample scores ofthe first PCA axis. According to the study authors, the PRC indicates the communities in atrazine treatments could recover within three to five weeks during the post-treatment period (this seems to conflictwith the results ofPCA which
11show no recovery ofatrazine treated communities at study termination). The PRC also indicates, however, that later on, depending on the substance, the community structure also differed from the controls: some species, especially 

### Chroomonas acuta, reached higher abundances in the atrazine mesocosms compared with controls. For some algae, such as the green alga

### Scenedesmus ecornis, an opposite pattern is predicted by the PRC: an increase during the treatmentperiod, followed by lower abundances in the atrazine mesocosms than in controls during the post-treatment period. The PRC results for control and various herbicide treatments are illustrated in 

### Figure 6. 

### It is not clearhow +1..0.**n~,o.n.~~** .....**~"r",,;r":h+n** _ ... rJol"'<

.....**_+~rl ~_**  v.:_....**_~** I:. _,.,.1

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1.0
0.5
-0 u -0.5
-1.00 00**001001~1~100100** Time (days post**applicBtion~** Chroomonas Bcura ChOliocystis cf. minor NeDhroseltJiis cf. oIlvacea MonOOJphidium circinsle Nitzschia lea

Ankyra PS:
. CryptomonBs C uIa**~jN8tOri8** tfNUJis Chromulina cf. oboonica C06Iastrom mictOporum**C~=$ ~vatfans**  OszillBtanBlacustris Chroococcusspec. ScenedeSfflus ecomis -4 -2 0 2 4 6 8 10 Species weight Figure 6. Principal response curves (PRCs) representing a significant part ofvariance in the phytoplankton community data set (p
- 0.002). Time (x axis) and treatment could explain 36% and 27%, respectively, ofthe total variance. The PRC (y axis) captured 16%. The y axis represents the difference in community structure between the atrazine (0), diuron (e), isoproturon (0), and mixture (.) treatments and the control expressed as regression coefficients (Cdt) ofthe PRC model. Taxa with species weights ofless than' - 1 orgreaterthan 1 are presented on the right. The species weight can be interpreted as the affinity ofthe taxon to the PRe.


### Number of differenttaxa andShannon diversity Before application (day

- 8), the average number ofdifferent taxa in the ponds was 30 ± 3. A few days after application (day 5), 23 ± 6 different species were in the control. During constant exposure, the number oftaxa in the atrazine treatments were comparable to those ofthe control (one-way ANOVA, p 

### > 0.05), with an exception on day 5 (38 different species, reviewer-estimated from Figure 7, unclear ifthis time point was significantly different from the control). During the post-treatment period, the number of species in the atrazine treatments continued to be comparable to those ofthe control (oneway ANOVA,p 

### > 0.05). Before application (day

- 8) and few days after application (day 5), the Shannon indices ofthe control and the atrazine-treated communities were similar (one-way ANOVA, p 

### > 0.05). However, in the following three exposure weeks, the Shannon index ofthe

12

### atrazine-treated communities remained constant, while those ofthe control decreased by a factor oftwo (indicating an unbalanced distribution ofthe organism among the various taxonomic groups in the control

- the secondary reviewerbelieves the lower Shannon index also reflects the decreased species richness shown on the same dates); it is unclear ifthe atrazine treatments and controls were significantly different. During the post treatmentperiod, in general, no significant differences in the Shannon index were observed (one-way ANOVA,p

### > 0.05), however, atrazine treatments were higherthan controls on days 110, 140, and 173 and the authors do not specify whetherthese were significant differences. The species number data and Shannon index results for control and various herbicide treatments are illustrated in 

### Figure 7.

13

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## Time (days post application) Figure 7. Diversity ofcontrol and exposed phytoplankton communities. Total number ofalgal species (A) and Shannon index (B) as determined for the control (*), atrazine (0), diuron (e), isoproturon (0), and mixture (.) treatment are presented. Dashed lines indicate the minimum and maximum values ofthe control community. The vertical dashed line indicates the day ofthe first application (day 0). Overall, the study authors concludedthat atrazine treatments substantially influenced phytoplankton community structure due to the sensitivity ofa few algal species for endpoints including abundance, diversity and species composition. However, the majority ofthe taxonomically identified algal species in the mesocosm were unaffected by atrazine (datanot reported).

14

### Data Summary Table:


## . Time Nominal Weighted Average Did Magnitude of Atrazine Day of Atrazine Recovery Effect Effect relative Concen- Study Effect Occur 

### if Comments Concentration to controls tration Occurred fromDayOto so, when? (pgIL) When Effect Occurred i bstimate of magnitude based on Figure 2. It is unclear if effect is statistically significant. Abundance in 50% reduction atrazine Total (transient**77~glLl,2** No treatment was abundance effect

- see 84**~glL** Day 19 greater than (phytoplankton) comments) control on day 5, then equal to control on day 12. Given the variability in the dataset overtime the reviewer considered this a transient effect. Statistically significant (p < 0.05): comparison based on sample scores on first axis of principal Community component structure (based Not able to 84**~gIL** Day 12 78**~glLJ** No analysis. on species determine composition) Effects on individual species and algal groups

I i ! 

### I**~·15\.Ll** ""



### 15I Time Nominal Weighted Magnitude of Atrazine Day of Average Did Effect Effect relative Concen- Study Effect Atrazine Recovery Comments to controls tration Occurred Concentration Occur-if (pgIL) from Day Oto so, when? When Effect

Occurred Estimated from Figure 7. It is unclear if this was a statistically significant difference between control and treatment. The reviewer discounts the Community increased structure**~50%higher** 84 f..lg/L Day 5 81 f..lg/L 1 No species (species number as a number) treatment related effect. Species richness in the atrazine treatment community substantially increased (25%) from t= 

## oto t=5 (see reviewer notes i for further discussion). Magnitude of effect Community Unclear estimated from given Figure 7. It is structure**~30%higher** 84 f..lglL Day 12 78 f..lglLJ temporal unclear ifthis (Shannon diversity index) variability was a in data. statistically significant difference. 1 Based on the reviewer-estimated values reported ill Table l. 2 Value based on the atrazine measurement prior to adjustment. 

### The study authors calculated the half-life ofatrazine as 107 days.

16

### Reviewer Comments: Atrazine was present in the control ponds (6 to 28 ng/l) throughout the study period. Although statistical analysis was performed, the results were not always clearregarding specific comparisons (e.g. atrazine treatments on specific dates). This is because the authors generally made statements regarding trends in the data and typically did not mention orpresent statistical results for individual comparisons. Statements about statistical signiticance in this review are based on statements made by the study authors.
The PRe results were difficult to interpret as presented.
The study authors reported that the control ponds were not dosed with methanol because the methanol final concentration ofthe testponds was less than 0.002%, and the study authors believedthat no effects to the aquatic community would be observed at that methanol concentration.
A previous study (Knauert et aI., 2008) investigatedthe inhibition ofphotosynthesis by a mixture ofthese three photosystem II inhibitors versus the single herbicides inthe same seven-month mesocosm experiment which was described in this study.
It is unclear why the average species richness increased from t=O to t=5 inthe atrazine treatmentponds. This effect is unlikely to be treatmentrelated (i.e. atrazine treatment causing speciation). One explanation for the increase in species richness in the atrazine treatmentponds is that the species were initially present, but undetected. Ifthis is the case, it is likely that there was a high degree ofnon-treatment related variability in the dataset. Furthermore, the variability in the temporal trends throughout the experiment (fluctuations in increasing/decreasing richness) suggests that there was a high degree of error in sampling and species counting. Another explanation is that the species were introduced between t=O and t=5 through aerial colonization, contamination, or some other means. Species richness ofeach replicate mesocosm was not provided; therefore, it cannotbe determined ifall orjust a single replicate showed this increase. Species richness data from each ofthe replicates would be useful in evaluating the importance of the apparent increase in the number ofspecies. 

### Description ofUse 

### in Document: Qualitative


### Rationale for Use: Mesocosm experiments contribute to the weight ofevidence regarding effects ofatrazine on aquatic plant communities.

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### Limitations ofStudy: The results have good applicability to mesocosms or natural aquatic systems given that the experiments were performed in the field. However, atrazine was present in the control ponds (6 to 28 ng/l) throughout the study period. While this level ofatrazine has not been shown to cause adverse effects to aquatic plants based on the available data, it is unclear ifbackground levels ofatrazine in the control ponds may have altered the phytoplankton community structure to more tolerant species in all ponds prior to the addition ofatrazine. Water chemistry ofthe microcosms was not adequately monitored either priorto or following treatment. See the Reviewer's Comments for further limitations associated with this study. 

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

### Secondary Reviewer: Michael Lowit, Ph.D., Ecologist,

# EPA,ftiU**/~** Tertiary Reviewer: Anita Pease. M.S., Senior Biologist, EPA 

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