### Open Literature Review Summary

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

ECOTOX Record Number and Citation: Dorigo, U. and C. Leboulanger (2001). A pulse-amplitude modulated fluorescence-based method for assessing the effects ofphotosystem II herbicides on freshwater periphyton. Journal ofApplied Phycology. 13(6): 509-515. (MRID 48261108).

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 ofaLevel ofConcern (LOC).

Date ofReview: 9/4/09

Purpose ofStudy: This study examines the use ofPulse-Amplitude Modulated (PAM) fluorometry to
measure 

### in vivo chlorophyll 

### a fluorescence in orderto assess the short-termtoxicity effects ofatrazine and isoproturon on freshwater periphytoninrivers.

Summary ofStudy Findings: Datawere presented onboth isoproturon and atrazine. This review focuses onthe effects ofatrazine.

Methods: The study site was the River Ozanne which is located inthe center ofFrance. The river drained an area ofintensive colzacrops and, therefore, contained agricultural runoff composedpartly ofherbicides, mainly atrazine and isoproturon. The river was sampled five times during one season from May 2000 to January 2001. River samples were collected from established stations chosen from up to downstream (Oz#6, Oz#4, Oz#3.1 and Oz#1.1). The water was characterized by an institutional monitoring ofthe Ozanne watershed which measured herbicide contamination. Atrazine was measured using gaschromatography coupled with mass spectrometry. Atrazine and isoproturon were simultaneously measured using an ELISA kit. Water samples were collected along with each periphyton sampling to determine background concentrations ofeachherbicide.

1 

# / ••1.1.111 2087823

### Forperiphyton sampling, approximately 120 small glass discs (1.5 cm2 

### area) were glued onto aPlexiglas plate (18 x 22 cm, 4 mmthickness) using aquarium silicon sealant. After roughening the upper surface ofthe glass discs, the Plexiglas plate was adhered to concrete blocks which was placed into the running part ofthe river and exposed to sunlight (width varying from 0.5 to 5 m, minimizing side effects due to differences in sunlight exposure). Glass discs were covered with 10 to 30 cm ofrunning water at the beginning ofcolonization. After 2-3 weeks ofcolony growth, the colonizedplates were removed and placed into plastic bags filled withriver water. The filled plastic bags and separate water samples were transported in an insulatedbox to the laboratory. After a 24- hour acclimationperiod in the lab to a small plastic aquariacontaining river water (temperature constantbutnotreported, light cycle 14:8, light intensity ca. 80**~Mol** 

### photon m2 S-I), 

### three to five glass discs were taken from the same plates, fixed with 5% formaldehyde and used to identify the periphyton underthe light microscope. Another set ofcolonized discs were sonicated with methanol:0.5 M ammonium acetate (98:2; v:v). The extractedmatterwas analyzed by reverse-phase HPLC to determine lipophilic pigments. The periphyton biomass was estimated as**~g** 

### chI 

### a perunit ofsubstrate surface, and the percentage ofeach dominant algal group was estimated according to the method ofWilhelm etal. (1991). The ratio ofeach diagnostic pigmentidentified (fucoxanthine, lutein, and zeaxanthin) versus total chI a in the sample was used to estimate the abundance ofdiatoms, chlorophyceae, and cyanobacteria, respectively.

The colonized glass discs (afterthe 24-hour acclimationperiod) were prepared for fluorescence testing by being removed from the Plexiglas plate and cleared ofall visible debris, including living animals. The prepared colonized glass discs were placed into the wells of24-well polystyrene microplates which were also large enough for the armed fiberoptics from the fluorescence system. The PAM 101-103 fluorometer (PulseAmplitude Modulated) was equipped with ahalogen lamp which provided actinic light. Fluorescence measurements were taken using two methods. In the first method, the periphyton samples were exposedto prepared atrazine solutions for atleast 5 hours; PAM measurements were initiated after 20 minutes ofdark-adaptation. A single saturation pulse (ca. 1200**~Mol** 

### photon m2 S-I) 

### was applied to estimatepotentially photosynthetic efficiency F jF max 

### for each concentration (accordingto prior groups, such as Samson and Popovic, 1988). In the second method, the periphyton samples were combined with 900**~L** 

### ofWhatman GF/F filtered water from the algae sample site. After 20 minutes of dark-adaptation, the fluorescence measurements were performed by measuring F v 

### then determining 

### F max, 

### 80 seconds later, by a 600 ms saturating pulse. A non-saturating pulse program was initiated (ca. 160**~E** 

### m2 S-I

### pulses every 60 seconds). Afterthe first pulse, 100**~L** 

### ofprepared atrazine solution was injected. Fluorescence measurements were taken every 60'seconds for 20 minutes.

The prepared atrazine solutions (using high-grade atrazine standard) were a concentration series prepared by diluting a 20 mM stock solution with Whatman GF/F filtered water from the river sample site. Final concentrations were 0.01,0.03,0.1,0.3,1,3.1,10,31.6 and 100**~M.** 

### Method ofapplication was not stated. Atrazine was dissolved in acetone at 0.5% ofthe total volume. Acetone was also added to each control.

2The study authors used the method ofConrad 

### etal. (1993) to calculate fluorescence yield: 

### R = 1 - 

### (y/Ymax), where 

### R (relative yield) is the value used to draw the doseresponse curve,

### y is the herbicide sensitive fluorescence yield, and

### Ymax is the value obtained whenthe herbicide effectis maximal and so the background fluorescence is maximal. Relative yield values were between 1 (control, healthy periphyton sample) and 

## o(photosynthesis totally inhibited, maximum fluorescence yield, with no electron flows throughphotosynthesis). The data were fitted to a logistic equationusing least squares method for determining EC50 values for atrazine.



### In orderto measure the photosynthetic activity in terms of14C incorporation, colonized glass discs were placed into 20-mL scintillation vials with 2 mL ofWhatman GF/F filtered water from the same river sample site. Aliquots (unspecified volume) ofthe prepared atrazine solutions (final concentrations of0.01, 0.03, 0.1, 0.3,1.0,3.1,10,31.6, and 100 

### 11M) were addedto the samples. After one hour ofpreincubation (temperature 8- 23°C depending onthe in situ watertemperature ofthe sampling station, PAR 

### ca. 70 IlMol photon m-2 S-1), 20 ilL ofNaH14C03 (specific activity 0.4 IlCi) was addedto each vial. Afterphotosynthesis was allowed to proceed underthose conditions for two hours, the reaction was stopped by adding formaldehyde (final concentration 3%) then 200 ilL ofglacial acetic acid (in a fume hood) in orderto remove inorganic carbon. After one hour, the supernatantwas removed. The discs were dried for six hours at 60°C under a stream ofair. The labeled organic matter was extracted using 1 mL ofdimethyl sulfoxide (one hour at45°C). Radioactivity was measured using 15 mL ofscintillation cocktail and LSC analysis. The dose-response curves were traced using gross radioactivity values as percent ofradioactivity ofthe control at eachherbicide concentration.



### Results: Results from HPLC analysis showed that all ofthe stations showed a seasonal shiftin the taxonomic composition: diatoms were the dominant group 

### (Figure 1). Oz#6, the most upstream station, was the leastcontaminated. Biomass (chI a) increased downstream 

### (Table 1).

3

## 100%



## 80%
(J) 

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## c: eu 

## 60% 

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## 20%

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## san-piing nunt>er 

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Figure 1. Relative abundance (percentage oftotal biomass) ofthe three major groups ofperiphytic species, obtained by HPLC analysis, during the whole survey, Black: cyanobacteria; white: chlorophyta; striped: diatoms,



### Fluorescence (FrlF max

### vs Conradrelativeyield 'R') Results from fluorescence yield measurements 

### (F dF max) ofperiphyton samples indicated that this measurement was insufficiently sensitive to ensure a satisfactory estimate ofthe effects ofatrazine in this test system. No important changes in 

### FdFmax were revealed using this method, even after 24 hours ofincubation with atrazine. For atrazine concentrations of0.03, 0.1, 0.3,1,3.1,10,31.6, and 100 J.lM, the relative response 

### FdFmax (% ofcontrols) were 

### ca. 103%, 

### ca. 105%, 

### ca. 80%, 

### ca. 90%, 

### ca. 75%, 

### ca. 

### 70%,
ca. 75% and 

### ca. 70%, respectively, in Oz#6 on one date, June 2000 (estimated values from 

### Figure 2). The modified version ofthe method ofConrad 

### etai. (1993) produced relative yield, 

### R, values based on the short-term effects ofatrazine. This method allowed for the calculation ofan EC50 (0.67 J.lM) for station Oz#6 (June 2000). For atrazine concentrations of0, 0.03, 0.1, 0.3,1,3.1,10,31.6, and 100 J.lM, the relative yield, R, (% ofcontrols) were 

### ca. 100%, 

### ca. 65%, 

### ca. 95%, 

### ca. 50%, 

### ca. 30%, 

### ca. 35%, 

### ca. 5%, 

### ca. 15% and 

### ca. 0%, respectively (station Oz#6 on one date, June 2000; estimated values from 

### Figure 2). The modified version ofthe method ofConrad 

### etai. (1993) generated dose-response curves which allowed the calculation ofEC50 values. Dose-response
curves for all other station samples were not illustrated for atrazine; 

### F dF max values were also notprovided.

4

## 120



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## 10 

## 100 [atrazine]**(~M)** Figure 2. Compared dose-response curves obtained on samples from OZ#6 (June 2000) with atrazine. Closed circles were relative yield, R, fory measured according to Conrad etal. (1993). Open squares were fluorescence yield, F,IF max , linked to photosynthetic efficiency, measured after preincubation with atrazine in the dark. Data are expressed as percentage ofcontrol value (R = 1 with Y= 61.2; = 0.46).



### EC50 (fluorescence measurements (Conradmethod) vs l4

### Cincorporation) ForRiver Ozanne station Oz#1.1 (average chlorophyll 

### a concentration 20.5 Jlg cm2

, average known concentration atrazine 6.48 nM), the EC50 values averaged 3.55 JlM for the PAM method measurements and 0.5 JlM for the 14C method measurements. For River Ozanne station Oz#3 (average chlorophyll 

### a concentration 12.6 Jlg cm2

, average known concentration atrazine 19.14 nM), the EC50 values averaged 3.16 11M for the PAM method measurements and 1.45 JlM for the 14C method measurements. For River Ozanne station Oz#4 (average chlorophyll 

### a concentration 4.6 Ilg cm2

, average known concentration atrazine 5.33 nM), the EC50 values averaged 1.61 JlM for the PAM method measurements and 1.06 JlM for the 14C method measurements. ForRiver Ozanne station Oz#5/6 (average chlorophyll a concentration 3.9 Ilg cm2

, average known concentration atrazine 0.23 nM), the EC50 values averaged 0.84 JlM for the PAM method measurements and 0.75 JlM for the 14C method measurements. Detailed results
are reported in Table 1 (mean and range ofvalues represent five sampling periods at each site).

5Table 1. Minimal, maximum and mean values (values in brackets) for known atrazine concentrations in the R. Ozanne station samples and the average chlorophyll a content, EC50 obtained from PAM measurements ('Conrad') and 14C incorporation.

Known atrazine EC50 (11M) Station Chlorophyll a (Ilg cm-z ) concentration (nM) PAM 14C
Oz#l.l 1.3-53.8 [20.5] 0.19-21.32 [6.48] 1.39-4.48 [3.55] 0.16-1.0 [0.5] Oz#3 9.3-20.4 [12.6] 0.23-78.8 [19.14] 1.92-5.18 [3.16] 0.24-2.65 [1.45] Oz#4 1.2-8.4 [4.6] 0.33-19.94 [5.33] 1.0-3.34 [1.61] 0.62-1.53 [1.06] Oz#5/6 0.3-9.7 [3.9] 0.14-0.46 [0.23] 0.26-1.36 [0.84] 0.42-0.97 [0.75]

The EC50 values which were calculated by the study authors were much higher than the known maximal atrazine contamination in Ozanne (78.8 nM). However, the EC50 results ofthe study authors were in agreement with those ofprior literature ofthe sensitivity offreshwater periphyton to atrazine: EC50 range 0.21 to 3.32 

### 11Musing photosynthesis-irradiance curves (Guasch & Sabater, 1998).
Overall, the study authors determined that the fluorescence method investigated in the study was adaptable for use in natural phytoplankton samples (using detached algae on a glass fiber filter), provided that enough biomass can be concentrated on a small surface to produce a readable fluorescence signal.



### Data Summary Table:
Time Nominal Weighted Magnitude Atrazine Day of Average Did Effect of Effect Concen- Study Effect Atrazine Recovery Comments relative to tration Occurred Concentration Occur-if controls (p.gIL/ fromDayOto so, when? When Effect

Occurred No clear 6.81 1* Not able to Not able to Results for only effect determine. determine. Oz#6 station, June No clear 21.56 1* Not able to Not able to 2000. Increased! effect) determine. determine. Atrazine decreased**~20%** Not able to Not able to concentrations efficiency of (decreased 68.15 1* determine. determine. were not measured. photosynthesis efficiency) Magnitude of (decreased**~1O%** Not able to Not ableto effects estimated /increased (decreased 215.69 1* determine. determine. from Figure 2. fluorescence efficiency) yield, F,/F 

### ma:J**~25%** Not able to Not able to No statistical (decreased 681.58 1* determine. determine. comparison of efficiency) treatment to control**~30%** 2156.90 1* Not able to Not able to or experimental

6Time Magnitude Nominal Weighted Atrazine Day of Average Did of Effect Atrazine Effect relative to Concen- Study Effect Recovery Comments tration Occurred Concentration Occur- if controls (pg/L/ from Day Oto so, when? When Effect

Occurred (decreased determine. determine. variability data efficiency) presented.

25% (decreased 6815.80 1* Not able to Not able to Observations at 6.8 efficiency) determine. determine. and 21.6 ug/L were

30% consideredto not (decreased 21569.00 1* Not able to Not able to likely be treatment efficiency) determine. determine. related.**~35%** 6.81 1* Not able to Not able to Results for only determine. determine. Oz#6 station, June**~5%** 21.56 1* Not able to Not able to 2000. determine. determine. Atrazine**~50%** 68.15 1* Not able to Not able to concentrations determine. determine. were not measured. Decreased**~70%** 215.69 1* Not able to Not able to Magnitude of efficiency of determine. determine. effects estimated photosynthesis**~65%** 681.58 1* Not able to Not able to from Figure 2. (increased determine. determine. relative**~95%** 2156.90 1* Not able to Not able to No statistical fluorescence determine. determine. comparison of yield):Conrad**~85%** 6815.80 1* Not able to Not able to treatmentto control method determine. determine. or experimental variability data presented.**~100%** 21569.00 1* Not able to Not able to All effects determine. determine. observed may have



# . been relatedto atrazine exposure. EC50 is lowest of all sampling sites Decreased (4) and sampling efficiency of dates (5). EC50 photosynthesis values ranged from (increased 50% (ECso) 56.07 Not able to Not able to 56.07 to 1117.27 relative 1* /lgIL for these determine. determine. fluorescence experiments. The yield):Conrad average EC50 of method2 all sites and dates
was 539.22 /lgiL (reportedby study authors).



### 7Umts ill paperpresented as uM.

2 Results are same as those presented in Dorigo et al. 2004.



### * Time ofperiphyton exposure to atrazine was reported as "at least 5 hours" in the study Method section and appeared to last for more than 24 hours for some samples due to statements in the Results section ofthe studyreport. The primary reviewer assumed 1 day (24 hours) for all samples. Time Nominal Weighted Magnitude Atrazine Day of Average Did Effect of Effect Concen- Study Effect Atrazine Recovery Comments relative to tration Occurred Concentration Occur-if controls (/lgIL/ from Day Oto so, when? When Effect

Occurred EC50 is lowest of all sampling sites (4) and sampling dates (5). EC50 Decreased values for these primary samples ranged Not able to Not able to from 34.51 to productivity 50% (ECso) 34.51 3 hours determine. determine. 571.57 /lg/L for 

## C

4Cincorporation) these experiments. The average EC50 ofall sites and dates was 245.88 /lgiL (reportedby study authors). I

Reviewer Comments: 

### The study was atest ofthe use ofPulse-Amplitude Modulated (PAM) fluorometry to
measure 

### in vivo chlorophyll 

### a fluorescence offreshwaterperiphyton exposedto atrazine and isoproturon. The study authors used the standard 14

### C method for determining photosynthetic activities to validate the EC50 results produced by the PAM method.

The authors used fluorescence as ameasure ofphotosynthetic activity.

The PAM method described in the study was intended to detectthe changes in apparent short-term toxicity ofthe test compound under field conditions.

The authors determined thatthe F**~F** max

### parameterwas less sensitive than the Conrad parameter (R). Only the Conradparameterpermitted calculation ofECsos.

The method for obtaining fluorescence measurements from periphyton communities presented inthis study was a modified version ofthe protocol designed by Conrad etal. 

### (1993) for unialgal phytoplankton studies.

8The Method section ofthe study was unclearwith regard to the amount oftime thatthe periphyton was exposed to atrazine priorto PAM measurements. The exposure time was reported as "at least 5 hours" inthe study Method section and appeared to last for more than 24 hours for some samples due to statements inthe Results section ofthe study report. Additionally, the study authors reported thatthey tested a secondprotocol with fluorescence measurements performed every 60 seconds for 20 minutes; therefore, the exposure duration for this protocol appears to be 20 minutes. The primary reviewer assumed I day (24 hours) exposure for all samples submittedto the fluorescence protocols when completing the Data Summary Table ofthis review. The exposure was 3 hours for the 14C-incorporationprotocol.

The authors reported thatthe sampling locations were contaminated with both atrazine and isoproturon and thatthere was variability inthe degree ofcontamination among sampling stations and overtime atthe same sampling station. The source water is located in an agricultural watershed so it is possible thatthe water was contaminated with other pesticides as well.

Toxicity ofatrazine treatments to the natural communities was loosely associated with the degree ofbackground contamination. That is, communities exposed to greater background concentrations ofatrazine tendedto be less sensitive to the addition of atrazine (experimental treatment) than communities exposed to lowerbackground concentrations ofatrazine.

There is no indicationthat atrazine concentrations were measured inthe experimental treatments.

Atrazine was dissolved in acetone (also added to the controls) priorto exposing the periphyton communities. The concentration ofacetone in the final volume was 0.5% (5 mlll). Because acetone is commonly used to extract chlorophyll it is unclear what ifany effects the acetone had onthe results.

EC50 values reported in this study for fluorescence yield using the Conrad method were the same results reported by the same group in Dorigo et al. 2004.



### Description ofUse 

### in Document: Invalid for the purpose ofsetting an LOC for atrazine.



### Rationale for Use: Acetone was used as a solvent at a concentration (5 mlll), which is 10 times higherthan the criteriafor study acceptance (0.5 

### mIll, static testing), thus the toxic effects ofatrazine may have been confounded by the addition ofacetone. The

9exposed organisms were taken from sites and times with variable levels ofatrazine and isoproturon contamination. In some cases the contamination was greaterthan the atrazine treatment. Exposure to otherpesticides may have also occurred. River water
was also used as dilution waterto make the test solutions, and atrazine concentrations
were not analytically confirmed. Therefore, the exposure concentrations are uncertain. The test concentrations may have been higherthan nominal values because contaminated river water was used as dilution water.



### Limitations ofStudy: Some ofthe results have good applicability to mesocosms or natural aquatic systems; however, the study was designed to evaluate the potential utility ofPulse-Amplitude Modulated (PAM) fluorometry. Therefore, the presentation and discussion ofresults ofthe study were focused on illustrating the strengths and deficiencies ofthis method. Acetone was used as a solvent at arelatively high concentration (5 

### mIll). Very little raw data was provided. Forthe EC50s based on fluorescence data, it was unclearhow long the samples were exposed to atrazine. There
was no indication thatthe study design included replication oftreatment groups. The organisms were obtained from sites with history ofexposure to numerous pesticides. There is uncertainty about exposure concentrations.



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


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

# j/t.~/'.'rt i

... 

# jh I 

### Tertiary Reviewer: BrianAnderson, Biologist, EPA 

# /'i !' // i//( /

10