Open Literature Review Summary

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

ECOTOX Record Number and Citation: Seguin, F., F. Le Bihan, C. Leboulanger, and A. Berard. 2002. A risk assessment ofpollution: induction ofatrazine tolerance in phytoplankton communities in freshwater outdoormesocosms, using chlorophyll fluorescence as an endpoint. Water Research. 36(13): 3227-3236. (MRID 48261133)
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 8, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine on phytoplankton in experimental ponds (mesocosms) over 25 days, including short-termtoxicity tests on the mesocosm communities to assess induction ofatrazine tolerance over the course ofthe mesocosm experiment.
Summary ofStudy Findings

Methods: The experimenttookplace during spring 2000. Mesocosms consisted of4 outdoorcirculartanks atthe INRA (InstitutNational de laRecherche Agronomique, Rennes, France) experimental platform. The tanks were 3.2 m in diameter, 1.2 m high, held approximately 5000 L, and were sunk about 0.4 m into the ground to keep temperatures more stable. The mesocosms were created by first filling the tanks to 7 cm with natural sediment from a local uncontaminatedpond, then filling them to a depth of0.7 m with natural pond water from a nearby site (not stated whetherthe site is the same as the sediment collection pond, orwhether sediment and/orpond water were sampled for atrazine contamination). The natural pond water was used as the source for phytoplankton. Zooplankton added to the mesocosms were sampled from "several local ponds" (not described further) using a net with a mesh size of63 Jlrn. Aquatic macrophytes
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# 1111.1.111 2087862(Glyceria maxima) were also added. Seguin et al (2001b, also reviewed) is cited for more details aboutthe mesocosm setup. Mesocosms had 3 weeks to develop before treatment. Two mesocosms were treated with 30**~g/L** atrazine and two were left as controls. Brand, grade, and application method ofatrazine were not described, and it is not stated whether a solventwas used.

Dissolved oxygen and pH were monitored in the rnesocosms daily (early morning). Water temperature was measured continuously in situ, and surface irradiationmeasurements were taken from a nearby meteorological station (exact location/distance from mesocosms not stated). Phytoplankton was sampledusing ahomemade sampler consisting ofa ?O-cm long Plexiglass tube with atotal volume of2.5 L. Samples were pooled from four differentpoints in each experimental unit (mesocosm). Chlorophyll a was determined once a week in each mesocosm. One liter water samples from eachmesocosm were filtered through a Whatman GFIC filter, then chlorophyll 

### a was extracted using 90% acetone for 24h at 4°C and measured according to SCOR-UNESCO (1966). Phytoplankton dry weight was measured after filtering samples (unknown volume) through 1**~m** Nucleopore filters and drying at 105°C to a constantweight. Cells and filaments were counted in preserved samples at 400x using the Utermohl method. Phytoplankton analysis was done on days -1 (the day before atrazine treatment), 2, 4,9, 11, 16, 22, and 25 aftertreatment.

Short-termphytoplankton community tolerance tests (effects ofatrazine on fluorescence) were conducted three times a week on water samples from eachmesocosm. Samples (volume not reported) were placed in 20 mL Corning glass tubes and 10**~L** ofatrazine-acetone stock solution was addedto eachtube. Concentrations ranged from 1 to 10,000 nM atrazine (0.22

- 2160**~g/L).** Only some ofthe intermediate concentrations were provided (Figure 7). Based onthe log scale in Figure 7, it appears that a dilution series of101\0.5 may have been used, without a treatment level at 0.68**~g/L.** The final acetone concentration was 0.05% intreatments and controls. Six replicates were used as solvent controls, and three replicates were used at each atrazine concentration. After incubating tubes under 115**~E/m2** 

### Is ofcontinuous light at 10±2°C for 1 hour, followed by a 30 min darkperiod, the in vivo fluorescence was measured using a Turnerfluorometer (model 111).

Shannon-Weaverdiversity (H') was calculated for the mean species densities ofthe two replicates for the treatment and control communities. Bray-Curtis dissimilarity indices (BCl) for differences between treatment and control communities were calculated for each sampling day. BCl was calculated from phytoplankton cell densities. The Mann-Whitney test was used to detect significant differences between C-C (within-control) BCls and A-C (control vs. treatment) BCls. A principal component analysis was conducted on the total densities ofdominant species inthe treatment and control communities using the ADE software package.

Forthe short-termtolerance tests, EC125 values were calculated. EC125 was defined as the atrazine concentrationthat increased fluorescence by 25% over controls. It was notpossible to - 2 -accurately determine the more common EC50 measure because the maximal range of fluorescence increase could not be predicted. Dose-response curves were compared betweenthe mesocosm treatment levels using the Wilcoxon signedrank test after log-transforming the values to fulfill normality assumptions. Correlation analysis was used to compare short-term sensitivities to experimental factors (water quality and biological characteristics ofthe mesocosms). The correlations were determined by calculating a Pearson correlationmatrix using ADE software.

Results: Initial pH and DO were the same in all four mesocosms, but starting about 3 days after atrazine treatment, the two treatmentmesocosms had lowerpH and DO than controls (Figure 1). Atrazine treatments and controls showed less difference in DO about 12 days aftertreatment, but the difference increased again toward the end ofthe experiment The exact dates in Figure 1 are unclearbecause the x-axis labels do notmatch the scale. In general, nutrient levels were similar betweentreatments and controls (Table 1).
Chlorophyll a and dry weight ofphytoplankton samples in the treatmentmesocosms were lower than in controls nine days after contamination (4/11), and the difference became more pronounced with time (Table 1 and Figure 2). Additionally, changes inphytoplankton community structure with atrazine treatmentwere visible starting 11 days after contamination; Cyanobacteriawere more abundant in the controls relative to the treatments through the end of the experiment and Chlorophyceae were more abundant in treatments relative to controls through day 22 (there was a small difference on day 25) (Figure 3). This change in community structure was also reflected in the principle component analysis (Figure 4) that showed an increasing separation oftreatments and controls about 12 days aftertreatment (4/14) on factor two (i.e., axis 2, which explained 16% ofthe total variance). Bray-Curtis indices (BCIs) between controls and treatments showed a decreasing trend starting nine days aftertreatment, and were significantly different from within-control BCIs from day 9 on as well (Figure 5, Mann-Whitney test, "90%", the reviewer assumes that "90%" represents 1 - p-value, with the resultantp-value = 0.1). The study authors state thatthe results, taken together, indicate a simplification ofthe communities in boththe control and treated mesocosms which they attributed to the phenomenon of confinement. This is perhaps most apparent from the lower species diversity by the end ofthe experiment (Table 1).

Short-term community tolerance tests showed similar dose response curves for all mesocosms priorto atrazine treatmentofthe mesocosms. The dose response curves oftreated and control mesocosms beganto diverge one day aftertreatment ofthe mesocosm. According to the study authors, the dose-response curves ofthe treated mesocosms were significantly lower (Wilcoxon signed-ranktest, p-value not stated) than those ofthe controls starting on April 5 throughthe end ofthe experiment (Figure 7: dose-response curves only shown for d-l, d+1, and d+24). Additionally, the treatmentmesocosm EC125 values were significantlyhigherthan those of controls (Wilcoxon signed-ranktest, no p-value provided) and varied with time (Figure 6). It is not clear ifthe significant differences reported bythe study authors were for each date or for the 

### - 3 -entire study period. The tolerance ofthe atrazine-treated communities peaked (highest ECl25) on day 3 aftertreatment and there was a sharp decline intolerance ofboth treatment and control communities on day 15 aftertreatment (Figure 6).

Short-term atrazine sensitivity (i.e. EC125) ofthe control mesocosm communities was significantly correlated with dry weight biomass, Shannon diversity index, 

### 02 concentration, nutrient concentration (orthophosphates, ammonium), and temperature (Table 2). For atrazine treated mesocosm communities, EC125 was significantly correlated with temperature and orthophosphate. Over all ofthe tolerance tests, the ratio between control and treatment sensitivity (EC 125) was shifted from the 1: 1 line (shifted to a higher tolerance in the atrazinetreated mesocosm communities) (Figure 8). The study authors interpretthese results as showing that environmental conditions during the experimenthad a strong influence on community tolerance, butthatthere was a stronger effect from prior atrazine exposure at 30**~g/L;** they point to the decreased correlations oftolerance with other factors in the atrazine-treated mesocosm communities as opposedto control communities (Table 2).

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Figure 1. Water quality changes overtime in the four mesocosms. "Days to contamination" means days since treatment (d-l = day before treatment). Squares = control mesocosms, circles = atrazine treatment mesocosms (30 J.1g/L). Labels on x-axis do not seem to correspond to the scale, making it difficult to determine exact dates ofdata plotted on x-axis (study figure 1)

- 5 -Table 1. Water quality and phytoplankton community characteristics ofthe mesocosms during the experiment. Values are averages over 2 replicates (control or30 !!g/L atrazine). H' = Shannon-Weaver diversity index. (study table 1)
Date Temp. (0C) NH4+ N02- N03- PO/ Chi a Dry weight H' (mg NIL) (mg NIL) (mg NIL) (mg P/L) (!!g/L) (mg/L) (bits/individual) Controls 4/2 9.30 2.14 0.00 7.20 0.02 13.66 4.93 0.93 4/4 8.26 1.30 0.06 4.20 0.01 25.42 5.90 0.89 4/6 7.30 8.60 0.00 0.93 4/9 10.08 7.60 0.03 0.91 4/11 7.20 0.78 0.15 5.60 0.02 24.42 6.32 0.91 4/13 8.84 7.40 0.02 4/16 7.62 7.66 0.88 4/18 9.50 8.76 4/20 10.38 0.09 0.17 9.60 0.00 30.11 8.80 4/25 12.26 9.40 0.81 4/27 12.40 0.02 0.15 7.00 0.00 28.63 9.04 0.67 Atrazine Treatment 4/2 9.30 2.18 0.00 7.00 0.01 13.93 4.89 0.91 4/4 8.26 1.35 0.06 4.25 0.01 23.09 5.35 0.91 4/6 7.30 10.75 0.01 0.93 4/9 10.08 6.25 0.03 0.86 4/11 7.20 1.20 0.12 5.50 0.02 19.07 4.40 0.92 4/13 8.84 7.50 0.01 4/16 7.62 5.70 0.9 4/18 9.50 5.70 4/20 10.38 0.18 0.14 7.75 0.00 20.86 5.93 4/25 12.26 4.05 0.87 4/27 12.40 0.49 0.16 6.75 0.00 12.23 2.93 0.76

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Figure 2. Growth ofphytoplankton in the four mesocosms expressed as chlorophyll a. "Days to contamination" means days since treatment (d-l =day before treatment). Squares 

### =control mesocosms, circles = atrazine treatment mesocosms (30 J.1g/L). (study figure 2)



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Figure 3. Class-level taxonomic composition (as population density) ofalgal communities in mesocosms (C = control, A =atrazine treatment) over time. "Days to contamination" means days since treatment (d-l =day before treatment). (study figure 3)

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Figure 4. Principal component analysis ofphytoplankton communities at various sampling times during the experiment. Figure A: Label XIJ defined as: X (A for atrazine treatment, C for control); I (sample number, 1 for 4/3, 2 for 4/5, 3 for 417, 4 for 4/12, 5 for 4/14, 6 for 4/19, 7 for 4/25, 8 for 4/28); J (replicate, 1 or2). Figure B: Correlation circle ofexplanatory variables (corresponding to the PCA ordination shown in Figure A). 1: Chlamydomonas sp.; 2: Chlorella vulgaris; 3: Scenedesmus quadricuada; 4: Dictyosphaeriumpulchellum; 5: Crucigenia apiculata; 6: Crucigenia quadrata; 7: Synedra acus; 8: Nitzschia acicularis; 9: Pediastrum duplex; 10: Oocystis naeglii; 11: Scenedesmus sp.; 12: Tetraedron minimum; 13: Chodatella quadriseta; 14: Scenedesmus acuminatus; 15: Monoraphidium minitum; 16: Hyaloraphidium contortum; 17: Scenedesmus sp.; 18:Xanthopyceae; 19: Scenedesmus spinosus; 20: Cryptomonas ovata; 21: Closterium acutum; 22: Nitzschia sp.; 23:Scenedesmus sp.; 24: Rhodomonasminuta; 25: Actinastrumhantschii; 26: Navicula sp.; 27: Pseudoanabaena sp.; 28: Crucigeniafenestrata; 29: Oscillatoria limnetica; 30: Pseudoanabaena sp. (study figure 4) - 8 -1 0.9 0.8 

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### days to contamination Figure 5. Bray-Curtis indices ofthe phytoplankton communities over time, as calculated for the density of replicate communities between treatment and controls. Difference between treatmentvs. control BCI (A-C) and within-control BCI (C-C) was tested for statistical significance using the Mann-Whitney test ("90%" assumed to mean p-value = 0.10) and days with significant differences are marked with an asterisk (*). "Days to contamination" means days since treatment (d-l = day before treatment). (study figure 5)



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Figure 6. Calculated (from fluorescence measurements) EC125 values for the four mesocosms overtime. "Days to contamination" means days since treatment (d-l =day before treatment). Squares =control mesocosms, circles = atrazine treatment mesocosms (30 ,.g/L). (study figure 7)

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Figure 7. Short-term effects ofatrazine on phytoplankton community fluorescence, using phytoplankton community samples from the 4 mesocosms. Tests were conducted the day before mesocosm atrazine treatment (4/3), the day after treatment (4/5), and at the end ofthe experiment (4/28). Fluorescence is expressed as percentage ofcontrol fluorescence. Squares =control mesocosms, circles = atrazine treatment mesocosms (30 Jlg/L). (study figure 6)

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-Table 2. Correlation analysis results between short-term atrazine sensitivity (log ECI2S) and different environmental parameters and biological characteristics ofthe mesocosms. NS =Not Significant. (study table 2) Tee) NH/ 

### po/- O2 Chi a DryWeight H' Mean (controls)

r -0.7021 0.8713 0.6830 0.6814 -0.7243 -0.9334 0.9123

n 11 5 8 11 5 8 8 P 0.01 0.05 0.05 0.05 NS 0.001 0.001 Mean (atrazine-treated)

r -0.6006 0.5011 0.6518 0.2532 0.0411 -0.1628 0.4530

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Figure 8. Correlations between atrazine sensitivities (log ECI2S) in control vs. treatment mesocosm communities. Means are plotted. (study figure 8)

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-Data Summary Table
Time Weighted Did Magnitude of Nominal Day of Study Average Recovery Effect Effectrelative Atrazine Effect Atrazine Occur- if Concentration Concentration so, when Comments to controls Occurred (f.lg/L) from Day Oto (see When Effect comment)? Occurred
Effectmagnitude
Decrease in calculated from review Table 1 chlorophyll a 22% 30/lg/L Day 9 Unknown (no No mean values (also concentration measurements) see Figure 2).
No significance tests provided. Effect magnitude Decrease in dry calculated from weight of 30% 30/lg/L Day 9 Unknown (no No review Table 1 phytoplankton measurements) mean values. No significance tests provided. Change in phytoplankton community No change 30/lg!L Not diversity Not applicable Not applicable See review Table (Shannon applicable 1. diversity index)

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-Time Weighted Did Magnitude of Nominal Day of Study Average Recovery Atrazine Atrazine Occur-if Effect Effectrelative Concentration Effect Concentration so, when Comments to controls (pg/L) Occurred from Day Oto (see When Effect comment)? Occurred
Effect magnitude estimated from review Figure 3. No significance tests provided. Abundance in Lower abundance**~50%** 30 J.lglL Day 11 Unknown (no No control increased ofcyanobacteria measurements) relative to the treatment group to day 16 then on, day 22 the treatment group abundance started to decrease relative to the control. Change in abundance of Not Seereview Figure Cryptophyceae, No effect 30 J.lg/L Not applicable Not applicable Bacillariophyceae, applicable 3. Xanthophyceae
Effectmagnitude Increase in estimated from abundance of**~100%** 30 J.lg/L Day 11 Unknown (no Yes -Day review Figure 3. measurements) 25 Chlorophyceae No significance testprovided.
See review figure Change in 5. phytoplankton Effects were community Unknown (no statistically composition (BCI within-contro1 30 J.lglL Day 9 Unknown (no No significant ("90%" between control & BCls provided) measurements) according to the treatment, vs. BCI study authors). ofwithin- The reviewer controls) assumed the pvalue = 0.10. Change in Unknown Unknown (no See review Figure phytoplankton (cannot 30 J.lglL Day 12 measurements) No 4A. Axis 2 community quantify) accounts for 16%

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-Time Weighted Did Magnitude of Nominal Day of Study Average Recovery Atrazine Atrazine Occur-if Effect Effectrelative Concentration Effect Concentration so, when Comments to controls (pg/L) Occurred from Day Oto (see When Effect comment)? Occurred composition ofthe total (divergence of variance. treatments and controls on axis 2 ofprincipal No significance component testprovided. analysis based on species densities)
See review figure 1. No significance test provided. Decreased Unknown (no dissolved oxygen -20% 30llgIL**~Day3** measurements) Day 12 Days ofeffects and (% saturation) recovery are approximate because scale on Figure 1 is difficult to interpret as presented.

Not Effects estimated**~5%** 0.221lg/L 1.5 hours Not applicable applicable from Figure 7A (short-term effects Decreased ofatrazine on efficiency of mesocosm photosynthesis communities prior (increased to long-term fluorescence exposure to yield) atrazine). 2.15 Ilg/L 1.5 hours Not applicable Not**~5%** applicable No significance testprovided

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-Time Weighted Did Nominal Average Recovery Magnitude of Atrazine Day of Study Atrazine Occur-if Comments Effect Effectrelative Concentration Effect Concentration so, when to controls Occurred from Day Oto (see (pglL) When Effect comment)? Occurred

Dose for three other atrazine treatments was not defmed. Unknown (no Not**~20%** 21.5/lg/L 1.5 hours measurements) applicable Effects at 0.22 and 2.15 /lg/L are not considered different from the control.
Although in some cases the magnitude of effects are greater for communities sampled later Unknown (no Not during the**~50%** 215/lg!L 1.5 hours measurements) applicable mesocosm experiments (Figure 7B and 7C), those results may have been affectedby changing conditions (community and water quality) over the experiment. In addition, shortUnknown (no Not term exposure for**~70%** 2156/lg/L 1.5 hours measurements) applicable atrazine-treated mesocosm communities is uncertain (see reviewer comments)

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-Reviewer Comments: The study was presented relatively clearly with several limitations similarto those ofrelated studies by this lead author (also reviewed). The descriptions ofthe sources and sampling methods for sediment, water, andplankton are sparse, and itis unclearwhether all came from uncontaminated natural waterbodies. Control mesocosms were not measured for atrazine contamination. Therefore, background exposure to atrazine inthe mesocosms cannotbe ruled out. No information is provided on what type orbrand ofatrazine was used orhow itwas applied. The study authors do notreport using a solvent for the mesocosm experiments. According to the primary reviewer, ifa solvent was not used, then the atrazine may nothave dissolved enough for the actual initial concentrations in the mesocosms to approximate the nominal concentrations. Since actual atrazine concentrations in the mesocosms were never measured, and the application method is not described, it is unclear ifnominal concentrations adequately estimate the actual atrazine concentration atthe beginning ofthe experiment.
Only 2 replicates were used and only 1 treatment level was employed (30 Ilg/L). Forthe shortterm tests, 8 concentrations were tested, three ofwhich the authors did notprovide the exact concentration.

No information was provided on how nutrients were measured in the mesocosms.

Some endpoints are difficultto interpret due to the data analysis. The Bray-Curtis index was used, butits implementation was notwell-described, which may leadto some misinterpretation. Since BCl is never defined with an equation, it is unknown exactly which version ofitis being used, overwhat set ofspecies ortaxonomic groups it is being defined, and so on. The Principal ComponentAnalysis, while qualitatively interesting, is limited in usefulness. The only component (Axis 2) showing visible changes with treatment explains only 16% ofthe variance, and the first two components together account for less than halfthe variance. Finally, as with other studies by these authors whichhave been reviewed, p-values are generally notprovided when statistical tests are applied, and many endpoints have no statistical analyses performed at all. The reviewer assumed ap-value 

### = 0.1 for the Mann-Whitney test (regarding this test, the study authors state "90%" with no context ofwhatthis means).
Exposure concentrations are uncertain for the short-term toxicity testing on communities from mesocosms that were pre-exposedto atrazine (nominal 

### = 30 Ilg/L). Forthese tests, water samples were used from the atrazine treated mesocosms which contained not only the phytoplanktonbut also the nominal amount ofatrazine added to the mesocosms atthe beginning ofthe experiment (30 Ilg/L). Therefore, the actual concentrations would have been higherthan the nominal values reported for the short-term experiments. Moreover, the nominal concentration ofatrazine in the atrazine-treated mesocosms was greaterthan three ofthe nominal concentrations addedto the samples for the short-term tests. Finally, this is also an indication thatthe actual exposure for the shortterm experiments would nothave been the same for communities from atrazine-treated mesocosms and those from control mesocosms. Short-term

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-toxicity datafrom the atrazine-treated mesocosm communities was not considered for the data summary table due to the uncertainty surrounding exposure concentrations.

Some ofthe dates provided inthe tables and figures do notmatch statements made by the study authors in the text. For example, the chlorophyll analysis seems to suggest that 4/3 was the date ofatrazine application in the mesocosms, whereas the caption offigure 7 implies the date was 4/4.

Overtime, there was increasing short-term sensitivity (i.e. EC125) ofthe control mesocosm communities to atrazine. Results from correlation analysis suggestthat mesocosm characteristics which changed overtime (i.e. community composition and environmental conditions) likely accounted for the increased sensitivity inthe control mesocosm communities overtime. Although fewer mesocosm characteristics were significantly correlated with EC125 in the atrazine-treated mesocosms, the temporal change in atrazine sensitivity oftreated mesocosms communities may have been aproduct ofatrazine exposure in combination with environmental and biological factors as well. It is uncertainto what degree factors otherthan atrazine affected fluorescence.


### Description ofUse in Document: Qualitative



### Rationale for Use: The study contributes to the weightofevidence regarding effects ofatrazine on aquatic plant communities.



### Limitations ofStudy: The study is limitedby a single atrazine test concentration (mesocosms), lowreplication (mesocosms), lack ofinformation about atrazine treatment methods and actual concentrations ofatrazine overtime, lack ofclear statistical results (i.e. no p-values) for most endpoints, and the potential for background atrazine exposure. Fluorescence tests were shortterm (l.5 hours ofexposure) andperformed on communities eitherpre-exposed ornotpreexposedto atrazine. Actual exposure in the short-term tests is uncertain; confounded by the addition ofatrazine from waterused in the treated mesocosms. 

### Primary Reviewer: SaraH. Hendrix, M.S., Subcontracting Scientist, Cambridge Environmental Inc. 

### /'A/J ' 

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

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Tertiary Reviewer: AnitaPease, M.S., SeniorBiologist, EPA

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