### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Knauert, S., B. Escher, H. Singer, J. Hollender, and K. Knauer. (2008). Mixture toxicity ofthree photosystem II inhibitors (atrazine, isoproturon, and diuron) toward photosynthesis offreshwater phytoplankton studied in outdoor mesocosms. Environmental Science & Technology. 42(17): 6424-6430. (MRID 48261119)


### 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 (LOC).



### Date ofReview: October 2, 2009


### Purpose ofStudy: This study examines the individual and combined effects of three photosystem II inhibitors (atrazine, isoproturon, and diuron) on their inhibition ofphotosynthesis of freshwater phytoplankton studied in outdoormesocosms.


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



### Methods: 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. Forthe single compound treatments, the 30% hazardous concentrations (HC30) were selected, based on species sensitivity distribution (SSD) curves. The SSD curves were generated based on ECso growth data from laboratory experiments investigating a broad range ofphototrophic organisms. The HC30 was defined as the hazardous concentration for 30% ofthe species. The HC30 for atrazine was defined as 70 f.lg/L. Forthe mixture, 1/3 ofthe HC30 was used for each herbicide.

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# 11.1.11111 2087852

### For short-term dose-response relationship testing (to determine ifchosen HC30 resulted in similar effects on photosynthetic activity ofmesocosmphytoplankton), untreated phytoplankton was collected from the mesocosms and concentrated (5-fold) by centrifugation. Algal samples of3 mL were dosed with atrazine at 12.5,25,50 and 100 f.lg/L (separate samples were dosed with diuron and isoproturon). Controls were dosed with the same amount ofmethanol (0.1 %) and the herbicide-exposed samples. Exposure conditions were illumination at 40 f.lmol m-2 S-1 

### at 25°C and 100 rpm for 3 hours. Methods for photosynthesis measurements are described below.

Formesocosm experiments, 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 was described in full detail in Knauer et at. 

### (2005). No details were provided about the biota in the mesocosms. 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 (day 34-173). During the constant exposure period, atrazine was maintained within ± 

### 20% ofthe target concentration. Atrazine was dissolved in methanol for a final solvent concentration of<0.002%. The reviewer inferred from the results that 70 f.lg atrazine/L was the nominal concentration used inthe mesocosm experiments. The atrazine (as well as diuron and isoproturon) application was performed via spray drift on the water surface. After application, the water column was mixed with a polyethylene tube. Separate sampling equipment was used for each treatmentto avoid cross-contamination.

For chemical analysis and biological sampling, depth-integrated water samples were taken from the four quadrants ofeach mesocosm using apolyethylene tube (120 cm long, 4.5 cm diameter). Samples were taken close to the sedimentto 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, 55f.lm 

### mesh size). Phytoplankton samples 

## « 55f.lm) 

### were 5-fold concentrated inthe laboratory (a 50 ml sample from each pond was centrifuged at 3000 rpm for 5 minutes atroom temperature). The algal pelletwas resuspended in 10 mL ofthe pond water (40 ml ofthe supernatantwas removed and discarded). 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. Samples were stored frozen at 4°C until analysis. Biological sampling ofthe phytoplankton was performed weekly from days -8 to 54 and then biweekly from days 54 to 173.

For atrazine analysis, the water samples were filtered (cellulose acetate, 0.45 f.lm). 

### Eighteen (18) mL aliquots were adjusted to pH 4 with acetate buffer, and radiolabelled

2atrazine was added as an internal standard. Analysis was performed with online solidphase extraction-liquid chromatography-tandem mass spectrometry. The water sample was emiched usinga strataX column eluted with methanol using an online system. Separation and detection ofatrazine was performed using HPLC withthe following conditions: Xbridge C18 column (2 mm i.d. x 50 mm), room temperature, flow rate 200 J.!Llmin, 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 monitored transitions for atrazine and internal standards were m/z 216 ----+ 174 for the parent and m/z 188 ----+ 146, 104 for desethylatrazine. Quantification ofdesethylatrazine was performed using the internal standard ofatrazine. The limits ofquantification were 3 ng/L. Desethylatrazine was monitored starting seven weeks after the first application until the end ofthe study.

The concentrated samples for effects on photosynthetic activity ofphytoplankton (from short-term and mesocosm toxicity testing) were analyzed by a Toxy-PAM fluorometer using the saturating pulse method. I mL ofthe concentrated algal suspension sample was transferredto a quartz glass cuvette and analyzed. The PSII quantum yield was determined according to the following equation: Y= (F m'

- F) I (F m '), where Fis the momentary fluorescence yield measured betweenthe pulses and Fm' is the maximum fluorescence yield induced by a saturationpulse leading to full inhibition ofenergy conversion at PSI! reaction centers. Afterthe arithmetic mean of

### Yfor the three replicated ponds was determined, the inhibition ofphotosynthetic activity (1) was calculated using the following equation: 1(%) =:' [1

- (Yapplication I Ycontroz)] X 100%. Standard deviations were estimated according to Gaussian errorpropagation. Fluorescence data from the short-termtests were analyzed using linearregression with GraphPad Prism 4.0, generating the equation I (c) 

### = m log (c) 

### + n, where I is the inhibition ofphotosynthetic activity (%), c is the atrazine concentration, m is the slope parameter and n is the y-axis-intercept. Forthe constant-exposure period (day 0-34) of the mesocosm experiment, the time-weighted average effects on the photosynthesis of phytoplankton were calculated as mean ± standard deviation ofall measuring datapoints for each treatment. The dissipation ofatrazine in the post-exposure period (day 34-173) was monitored via calculation ofthe half-life (tv,) using first-order kinetics.

Relative herbicide concentrations in the single herbicide treatments were expressed as toxic units (TU i ) which were calculated using the following equation: TUj = (ci)/(HC30i), where Ci was the measured concentration ofthe herbicide 

### iin the water column and the HC30i was the 30% hazardous effect concentration ofthe respective herbicide i obtained from SSD curves. The study authorpresented the results in TVunits. The reviewer used the previously described formula to convert from TVto J.!g atrazine/L.

3Results: 

### Herbicide (Atrazine) Concentrations in the Water Column In orderto maintainthe target 

### HC30 in the constant exposure period, atrazine was reapplied one time at day 20. Atrazine concentrations in the constant exposure period were maintained within ± 20% ofthe target 

### HC30. The study authors reported that atrazine ranged from of0.95 to 1.22 TU for days 0-34 (see Figure 1 and estimates from primary reviewer in Table 1 for details). Inthe postexposure period (days 34-173), the half-life ofatrazine was calculated as 107 days (r2 

### = 0.92) based on first-order kinetics. The atrazine concentration was 1.07 TU at day 35 (reviewer-estimated) and declined to 0.42 ± 0.02 TU (n = 3; calculated by the study authors) at the end ofthe experiment (day 173). Atrazine degradationproducts desethylatrazine and desisopropylatrazine were less than 11% and 6% respectively ofthe target concentration ofthe parent. Estimates ofTU measurements and the inhibition ofphotosynthetic activity for atrazine are reported in Table 1 and are based on results illustrated in Figure 1.

4Table 1. Mean relative herbicide concentrations in the water phase and inhibition ofphotosynthetic activity ofphytoplankton over time for atrazine single treatment. All mean values, including time, were estimated by the primary reviewer from Figure 1. Asterisk indicates value after reapplication ofatrazine Time post first treatment Atrazine single treatment (days) Atrazine (TV) Inhibition ofphotosynthetic activity (%)

01 1.2 60 3 1 1.18 50 

### i 1.15 40 10 1 1.1 35 141 1.08 40 

### Ii 1.05 NA 21 1 1.0 (1.1)* 50 28 1 1.1 35 352 1.07 50 422 1.05 35 492 1.07 30 562 0.95 35 702 0.8 35 842 0.82 30 982 0.75 25 1122 0.74 30 1262 0.7 30 1402 0.65 30 1542 NA 25 1732 0.42 25 I Constant exposure perIod. 2 Post-exposure period.

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### I Figure 1. Relative herbicide concentrations in the water phase (expressed as TV) (.) and inhibition of photosynthetic activity ofphytoplankton (0) over time for the single treatments atrazine (A), isoproturon (B), diuron (C), and the mixture treatment (D). In general, data points represent arithmetic means ± standard deviations. TVi and TVmixwere calculated according to eq.s 5 and 6. Standard deviations were estimated according to Gaussian error propagation. Dashed lines indicate target concentrations ± 20%. Arrows mark reapplications ofthe herbicides. Inhibition of photosynthetic activity ofphytoplankton that was not significantly different from control level is indicated by an asterisk (two-tailed, unpaired t test,p > 0.05).

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### Effects on Photosynthesis -LaboratoryShort-Term Toxicity Tests In short-term laboratory toxicity tests onthe phytoplankton sampled from untreated mesocosms, atrazine-only treatments of 12.5,25,50 and 100 Ilg/L caused 4-14%, 18- 28%,38-48% and 46-58% inhibition ofphotosynthetic activity, respectively (inhibition ranges estimated by the primary reviewer based on Figure 2). The HC30 ofatrazine (70 Ilg atrazine/L) caused 43.0 ± 7.6% inhibition ofphotosynthesis. It is unclear what the datapoints plotted in Figure 2 represent (i.e., subsamples ofmesocosms, laboratory replicates, etc.)



### • Atrazine • Isoproturon • Diuron 80



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Figure 2. Inhibition ofphotosynthetic activity (%) ofphytoplankton due to the three single herbicides atrazine (_), isoproturon (T), and diuron (e) determined in short-term laboratory experiments. Experimental data (_,T ,e) and statistical fit (-) according to a linear regression are shown. Logarithmic herbicide concentrations were calculated from concentrations values expressed in IlglL. Dashed lines indicate the effect levels that correspond to the HC30 ofthe three herbicides. 

### Effects on Photosynthesis

- Mesocosm Experiment Priorto application (day -8), photosynthetic activity ofphytoplankton in all mesocosms was comparable (Y

### = 0.457 ± 0.008, n 

### = 15). The yield values ofthe control mesocosms increased in spring (days 0-68; 

### Y= 0.602 ± 0.020, n = 3) due to higher photosynthetic activity and were constant during the summer (days 68-173; 

### Y= 0.571 ± 0.034, n 

### = 24). At day 2 (2 days after application), atrazine caused a statistically significant (p < 0.05) inhibition ofphotosynthetic activity ofphytoplankton (51.4 ± 10.8% with 1.15 ± 0.07 TU, n = 3) (Figures 1 and 3). Overthe constant exposure period (days 0-34), atrazine concentrations ranged 0.95 to 1.22 TU, with a time-weighted average of 1.09 ± 0.07 TU (n = 30). For atrazine, the average inhibition ofphotosynthetic activity during the constant exposure period was 45.6 ± 9.3 (n = 7). The mean effects induced by the HC30 ofatrazine in the mesocosm experimentwere also similarto the effects ofthe HC30 of

7atrazine from the shortterm laboratory toxicity tests (one way ANOVA,p> 0.05), although the phytoplankton exposed in the field tended to be more strongly effected. The study authors proposed that the stronger inhibition for the phytoplankton in the field was caused by a longer exposure time. During the entire mesocosm experiment (days 0-173), a lineartrend between declining effects on photosynthesis and decreasing atrazine concentrations was observed (see 

### Figure 4). At the end ofthe experiment (day 173), atrazine treatments continued to show statistically significantreductions of photosynthetic activity compared to the controls.

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.. FIGURE 3. Herbicide-induced inhibition ofphotosynthetic activity (%) determined on day 2 after application. Each bar represents mean and standard deviation ofthe three replicated mesocosms. Statistical analysis ofthe data (one way ANOVA; P > 0.05) revealed no significant differences among the single bars. Corresponding water herbicide concentrations (in TV) detected on day 2 were added.

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Figure 4. Concentration-response relationship between herbicide concentrations (expressed as TV) and inhibition ofphotosynthetic activity ofphytoplankton (in %) for atrazine (_), isoproturon (T), diuron (e), and the mixture (0). Measuring data from the entire mesocosm experiment are presented.
In regards to the observed inhibition ofphotosynthetic activity by atrazine overtime, the study authors concluded that the decline in inhibition was related to the decrease in atrazine water concentrations andthat community tolerance likely played a minorrole due to the fact that 0.4 TU ofatrazine at experimentend (day 173) resulted in a 25% inhibition ofphotosynthetic activity.

9Data Summary Table: Time Nominal Weighted Average Did Magnitude of Atrazine Day of Atrazine Recovery Effect Effectrelative Concen- Study Effect Occur-if Comments Concentration to controls tration Occurred from Day Oto so, when? (pgIL) When Effect Occurred Not Short-term 4-14% 12.5 f..lglL 3 hours Not applicable calculated toxicity Not applicable Not laboratory test. 18-28% 25 f..lg/L 3 hours calculated Not Effect range 38-48% 50 f..lg/L 3 hours Not applicable calculated estimated by the reviewer based on Figure 2. Photosynthetic Variation activity among control inhibition replicates (short-term unknown. No laboratory tests) Not statistical 46-58% 100 f..lg/L 3 hours Not applicable calculated testing of treatment compared to control. Type ofreplication unknown. High solvent concentration.

10Time Nominal Weighted Magnitude of Atrazine Day of Average Did Atrazine Recovery Effect Effectrelative Concen- Study Effect Concentration Occur- if Comments to controls tration Occurred (pg/L) from Day Oto so, when? When Effect

Occurred Mesocosm experiment (Figures 1 and 3). Statistically significant (p < 0.05)
TV= toxic units. TWA concentration was based on the reviewer estimate for Photosynthetic initial TV (1.2 activity 1.2 TV 1.18 TV (82 TV) and the inhibition (long- 51.4 ± 10.8% (84 /lg/L)l Day 2 /lg/L) I No reported TVat term day 2 (1.15 mesocosms) TV).
The timeweighted average inhibition over the entire exposure period (34 days) was 45.6 ± 9.3% at an average atrazine concentration of 1.09 ± 0.07 (TV)
1 ConcentratIOn calculated by reviewerusmg equatIOn proVidedby study author. TV= c/HC30 where c IS the measured concentration ofatrazine in the water column and HC30 is the 30% hazardous effect concentration ofatrazine (70 /lg/L as reported by the study authors). c (/lg/L) = TVx 70 /lg/L.


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

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### Reviewer Comments: The study authors used toxicity units (TU) in the place ofstandard concentration units (such as Ilg/L) in the majority ofthe report. This unit ofmeasure was used in orderto relate the different herbicides to one another. The reviewer used a formula provided by the study authors to calculate concentrations ofatrazine as Ilg/L.

The study authors reported that otherprevious literature studies concurred with the halflife findings offreshwater microcosm studies ofother laboratories.

The subsequent study report, Knauert 

### et. ai., 2009, was an expansion ofthis study in which effects onphytoplankton community structure were investigated. It appears that the information included in Knauert 

### et. al., 2008 (this study) and that presented in Knauert 

### et. al., 2009 were both collected from the same seven-month mesocosm experiment. The experiment dates and experimental site were the same. The measured atrazine concentration datareported in the 2009 study for the mesocosm experiment was the same as the value reported in the 2008 study, exceptthat it was reported in units of Ilg/L.

.Furthermore, the calculated half-life ofatrazine was the same in both studies.

The authors do not provide information about background concentrations ofatrazine in the controls; however, in the 2009 study the authors report that atrazine was present in the control ponds (6 to 28 ng/L) throughout the study period.

The concentration ofsolvent used in the short-termtoxicity tests (0.1 %) was higher than that used in the mesocosm experiments «0.002%). Methanol was included in the controls for the short-termtests. Forthe mesocosm experiments, the authors do not state whether methanol controls were used; however, in the 2009 study, the authors report that the mesocosm control ponds did not containmethanol.


### Description ofUse 

### in Document: Qualitative (except results from short-termtoxicity tests, which are invalid).



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



### Limitations ofStudy: The results have applicability to mesocosms ornatural aquatic systems since experiments were performed inthe field, as well as the laboratory. The study authors used toxicity units (TU) inthe place ofstandard concentration units (such as Ilg/L) in the majority ofthe report; therefore, the reviewer assumed that the conversion to Ilg atrazine/L could be made using a formula provided by the study authors. The methanol solvent concentration in the short-termtoxicity testing of0.1 % (1 mIlL)

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### exceeded the criteria for study acceptance «0.5 mlll, static-renewal testing); therefore, the toxic effects ofatrazine may have been confoundedby the addition ofsolvent. In addition, the type ofreplication used inthe short-termtoxicity testing was unclear. With respect to the mesocosm studies, water chemistry was not adequately monitored either priorto or following treatment. The study authors did notreport ifatrazine or other contaminants were present inthe controls; however, inthe 2009 follow-up study, the authors reported that atrazine was detected in the control samples (6-28 ng/L), based on the same mesocosm experimental dates.
Primary Reviewer: 

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

### Michael Lowit, Ph.D., Ecologist, EPA

# l~t:;J 

# "V

Tertiary Reviewer: 

### Anita Pease, M.S., Senior Biologist, EPA 

# MtJ.t (l3/ \

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