Open Literature Review Summary
Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Dorigo, D., X. Bourrain, A. Berard, C. Leboulanger. 2004. Seasonal changes in the sensitivity ofrivermicroalgae to atrazine and isoproturon along a contamination gradient. The Science ofthe Total Environment. 318(1-3): 101-114. (MRID 48261109) 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: 

### 9/4/09
Purpose ofStudy: This study used Pulse-Amplitude Modulated (PAM) fluorometry to measure in vivo chlorophyll 

### a fluorescence in orderto assess the short-termtoxicity effects ofatrazine and isoproturon on freshwater periphyton and phytoplankton obtained from a riverthat has apesticide-contamination gradient.
Summary ofStudy Findings: The study evaluated effects ofatrazine and isoproturon. This review focuses onthe effects ofatrazine.

Methods: The study site was the River Ozanne and its tributaries which is located in central France (latitude 48°12'40'; longitude 1°9'49'). The river drained an area ofintensive agriculture [colza (oil seedrape), com (maize), and winterwheat crops] and contained agricultural runoffcontaminated with urea and triazine herbicides, mainly atrazine and isoproturon. Forperiphyton studies, river samples were collected from established stations chosen from upstreamto downstream (Oz#617, Oz#5/6, Oz#4, Oz#3 and Oz#I). The study authors did notpresentperiphyton data for station Oz#617. Forphytoplankton studies, river samples were collected from established stations chosen from up to downstream (Oz#617, Oz#2 and Oz#l; lentic sites). The river was sampled five times for periphyton studies and six times for phytoplankton studies. Sampling was performed every two weeks from May to June 2000 (mid-herbicide applicationperiod), once in September

1 1••••

# 1.111 20878222000 (post-herbicide applicationperiod) and once inJanuary 2001 (pre-herbicide applicationperiod). Sampling sites were chosento have similar environmental conditions (watervelocity, canopy cover, and light exposure). The water was tested for 77 herbicides by an external laboratory (Loire Bretagne WaterAgency. In addition to atrazine, measurements were made for six othertriazines and degradation products. Atrazine was extracted using dichloromethane and was vacuum-concentratedprior to analysis via gas-chromatography with nitrogen-phosphorous detection (GCINPD). Mass spectrometry was employed when necessary.

Forperiphyton sampling (previously described by Dorigo and Leboulanger, 2001), small glass discs (1.5 cm2 area) were glued onto Plexiglas plates, which were adhered to concrete blocks. The concrete blocks were placed in the center ofthe river. After 2-3 weeks ofcolony growth, the colonizedplates were removed and were transported in coolboxes to the laboratory. Forphytoplankton sampling, the communities were collected using 2-L plastic bottles which were pre-cleaned with dilute HCI and rinsed withriver waterthree times before sampling. Separate river water samples were also taken for WQ analysis (pH and nutrients) and as matrix for the ecotoxicity tests.

The experimental treatmentused ahigh-grade pesticide atrazine standard, dissolved in acetone. A stock solution of20mM atrazine was diluted serially (1005). A solventblank was used for the control. Acetone was added to 0.5% ofthe total volume.
Periphyton andphytoplankton fluorescence assays were performed after a 24-hour acclimation period (rivertemperature; light cycle 14:8, light:dark; light intensity 

### ca. 80 j.lMol photon m-2 S-l).

The periphyton assay was a slightly modified method ofConrad 

### etai., 1993 as described in detail in Dorigo and Leboulanger, 2001. The colonized glass discs were prepared for periphyton fluorescence testing by being placed into the wells of24-well polystyrene microplates with 900 j.lL ofWhatman GFIFfiltered water from the algae sample site and were dark adapted for 20 minutes. Then 10 j.lL ofthe prepared atrazine solutions were added to the microplate wells (the 2001 publication states that 100 j.lL was added). These atrazine solutions were a concentration series preparedby diluting a 20 mM stock solution with Whatman GFIFfiltered waterfrom the river sample site. Final concentrations were 0.01 0.03, 0.1, 0.3,1.0,3.1,10,31.6, and 100 j.lM. The fluorescence signals were monitored using aPAM 101-1 03 fluorometer (Pulse-Amplitude Modulated). The measured increase in fluorescence yield, compared to the control community, was expressed as the EC50, according to Seguin

### etai. (2001).

Forthe phytoplankton assay, 20-mL subsarnples ofthe collected sample was placed into Coming glass tubes with 10 j.lL ofan atrazine stock solution (prepared using high-grade atrazine standard). The atrazine test concentrations ranged from 1 to 10000 nM. Six

2replicates were used for the controls; three replicates were used for each atrazine concentration. After I-h incubation at acclimation conditions, fluorescence signals were measured following a 30-min dark-adaptation period. The fluorescence instrument was a Turner fluorometer. The measured increase inthe in vivo fluorescence was identified as the EC125 (toxic concentration that increases fluorescence 25% relative to the control samples), according to Seguin etal. (2002). According to the study authors, increased fluorescence indicates decreased photosynthetic efficiency.

For determining periphyton community structure, three glass discs were taken from the same plates, fixed with 5% formaldehyde and used to identify the periphyton under the lightmicroscope (640x magnification for non-diatoms and 1600x magnification for diatoms). Diatom-based indices, the biological diatom index (BDI) and the pollutionsensitivity index (PSI), were calculated using Omnidia 8.0 software. Another set of colonized discs were sonicated with methanol:0.5 M ammonium acetate (98:2; v:v). The extracted matter was analyzed by reverse-phase HPLC to determine lipophilic pigments. Each lipophilic pigment was identified from its retention time and absorption spectrum using DAD, according to SCOR (Jeffery etal., 1997). Standard curves were established for the pigments. The periphytonbiomass was expressed as Ilg ChI a per cm2 based on a glass disk surface area of 1.5 cm2

, an extraction volume of4 mL per glass disk and an injection volume of20 ilL. The percent contribution ofeach algal group (a.g.) was calculated according to: %a.g. = (%Ad.p x 100)/(%AcHLa x rw), where d.p. is the diagnostic pigment, A is the area ofthe peak and rw is a published ratio for monocultures.

For determining phytoplankton community structure, the Lugol-preserved cells and filaments were counted using an inverted microscope at 400x magnification. The Shannon-Weaver diversity index (H') was calculated for each sample. Chlorophyll 

### a was extracted in 90% acetone for 24 h and measured according to Strickland and Parsons (1968). The dry weight was determined after filtering across I-11mpore Nucleopore filters and drying at 105°C.



### Results: Water residue analyses Atrazine was measured above the detection limit in all ofthe samples from River Ozanne. Concentrations ofatrazine varied among sampling locations and overtime. Atrazine levels were generally highest inthe May and June sampling dates and were lowest in the September and January sampling dates. The highest value for atrazine was found in June at station Oz#3 (17 Ilg/L). Oz#1 and Oz#3 were generally the most atrazine contaminated stations, especially during May and June. The site furthest upstream (Oz#5/6) typically had the lowest atrazine levels. Atrazine concentrations were similar across all sampling stations in September and January. Measured atrazine concentrations for each sampling station in May, June, September, and January are provided in Figure 1.

3

## 100

10



# ...-.. OJ) ::1. '-' 

## 1 

# = 

# .- N


## 0.1

0.01**------------......j~** 

## upstream 

# --B--may**~jun** 

## ..•.. sep



# .. -G

.. jan



# eo...--.__**~.** 

# .._........

. . 

# . . 

# . - : 

# . . 

# O ... .. .. III



# - ---

# -.

# -- .. 

# -

..

OZ 1 OZ 3 

## OZ4 station 

## OZ 5/6

Figure 1. Contamination by atrazine in IlglL measured at the four sampling stations in May (0), June (_), September (e) and January (0). (Figure 2 ofDorigo et al. 2004)

4

### Ecotoxicity tests Forperiphyton, EC50 values for atrazine from short-term fluorescence tests ranged from 0.26**~M** for Oz#6 (January 2001) to 5.18**~M** for Oz#3 (May 2001). EC50s for each site (the average ofthe five sampling dates during the study period) are presented in Table 1. EC50s were typically highest atthe sites with the highestmeasured levels oftriazines (seven triazines including atrazine and degradationproducts were measured). EC50 results for atrazine ateach sampling station during each sampling month are provided in 

### Figure 2.

Table 1. Sensitivity ofperiphyton samples from the four sites on River Ozanne. EC50s are expressed in IJ.M and were obtained using in vivo fluorescence. Values are averages and standard deviation (in parenthesis) ofsamples obtained over 5 sampling dates. Some analyses are missing for the January sampling (outlined by an asterisk). Symbols for dates are shown in Figure 1. (Table 2 ofDorigo et al. 2004)

Station Triazines (p.g/D EC50 (atraziDe) Urea$ (11gfi) EC50 (isoproturoc) Oll 1.93 <2.29) 3.55 (1.88) 0.26 W.29)* 3.06 0.3) Ol3 4.16 (7.56) 3.16**(1.2~)** 0.21 W.02)**O.~** (1).22) Ol4 1.5 (1.92) 1.61 (0.99) 0.12 (0.08) 0.39 (0.25) Ol5/6 0.09 (0.06) 0.84 (0.57) 0.05 (0.10)* 0.86 (0.51)

5

### -------------~ 

# upstream 10 

# •••

.. ... 

# ..

..

"" 

## 1

.... 

# -&-may 

# 0
0.1**~jun** 

# •••• 'sep 

# ·G· 

# .

. 'Jan
0.01 OZ 1 OZ 

## 3 OZ4 station OZ 5/6

Figure 2, Sensitivity ofperiphyton samples from the four sites on River Ozanne, espressed as EC50 in liM, obtained using in vivo fluorescence, for atrazine. Symbols for dates as in Figure 1. (Figure 3 ofDorigo et al. 2004)

Forphytoplankton, EC125 values for atrazine from short-term fluorescence tests ranged from 37 nM for Oz#6/7 (September) to 153 nM for Oz#l (June). The highest mean EC125 value (the average offive sampling dates during the study period) was site OZ#l, followed by Oz#2 and Oz#6/7, respectively. The sixth sampling date, in January, was excludedbecause ofpoor fluorescence readings due to high levels ofparticulate matter and low levels ofphytoplanktonbiomass. On a given sampling date, the EC125 values ofOz#6/7 were generally lowerthan those ofOz#1 and Oz#2. EC 125 results for atrazine at each sampling station on each sampling date are provided in Table 2.

6Table 2. Structure and sensitivity ofphytoplankton sampled in River Ozanne waterbed. Biomass, diversity, dry weight and EC125 (fluorescence endpoint) for atrazine. Biomass Dry Weight Date Station Diversity (H') EC125 ()lM) ()lg chI aperL) (mglL) Oz#1 3.48 n.a. n.a. 0.07 4 May 2000 Oz#2 2.49 n.a. n.a. 0.08 Oz#6/7 16.98 n.a. 1.98 0.05

Oz#1 21.47 17.35 1.90 0.07 22 May 2000 Oz#2 11.19 3.5 n.a. 0.07 Oz#6/7 25.84 21.9 2.06 0.08

Oz#1 4.05 19.7 2.09 0.15 5 June 2000 Oz#2 14.59 20.5 2.33 0.10 Oz#6/7 22.3 17.9 n.a. 0.08 Oz#l 2.57 10.6 n.a. 0.13 19 June 2000 Oz#2 22.53 11.1 n.a. 0.10 Oz#6/7 31.57 45.15 2.33 0.05

Oz#1 4.52 10.05 2.76 0.08 4 September Oz#2 20.54 11.0 1.70 0.09 2000 Oz#6/7 74.14 42.7 1.55 0.04

Community structure Measurements ofcommunity structure were made on samples priorto the experimental addition ofatrazine.

Forthe periphyton, diatoms (33 species and 12 genera), green algae (7 species), and cyanophytes (3 species) were identified. The mostcommon diatom generawere Navicula, Nitzschia, Gomphonema, Achnanthes, andAmphora. The most species-rich samples were taken in September (Oz#5/6, 29 species) and inJanuary (Oz#4, 39 species). Both Shannon's index, appliedto diatoms, and the total number ofspecies, calculated for each sampling date and site, showed no correlation with the related EC50 values (data was notprovided inthe study). Diatom-based indices (BDI, Biological Diatom Index) showed a clearpattern ofincreasing upstream. The BDI was notwell characterized in the study report. There was no cleartread for the pollution-sensitivity index (PSI), which was used to estimatethe pollution-sensitivity ofthe established community. A large amount ofvariability between different sites and dates was observed. DBI and PSI values for each station and sampling date are provided in Figure 3. Chlorophyll a ranged from 0.049 to 52 Ilg/cm2 for all the periphyton samples, and accounted for ca. 50% ofthe total pigmentin those samples. Overall, diatoms were the most abundant group inthe

7periphyton flora. When other groups were presentthey were most abundant atthe upstream stations.



# • upstream 

# • upstream**I--·-~-------'** IS 

# I-e-=y 18**~juni**

... -- sep

# j 1-· -0-.

## ja~ 13 .-'0 15
0 ell S 

# - 

# •• 0 II 12 

# .. .···0



# .... --

# .•

. 

# • . 9 9 OZ 1 OZ3 OZ4 OZ S/6 OZI OZ3 OZ4 OZS/6 station station Figure 3. Diatom indices obtained for periphyton samples from the four sites on River Ozanne, biological-diatom index (BDI) and pollution-sensitivity index (PSI). Each index is based on a 0-20 scale, 20 being the higher rate related to unaffected biological communities. Symbols for dates as in Figure 1. (Figure 5 ofDorigo et at. 2004)

Forthe phytoplankton, a total of119 phytoplankton taxawere identifiedto at leastthe genus level. Phytoplankton were represented by diatoms (26 species and 11 genera), green algae (58 species), cyanophytes (4 species), cryptophytes (unreported species/genus number), chrysophytes (unreported species/genus number), conjugatophycea (4 species), and euglenophytes (8 species). Chlorophyll a ranged from 2.49 (Oz#2, May) to 74.14 Ilg/L (Oz#6/7, September) for all sampling sites and dates. Dry weight ofthe phytoplankton ranged from 3.5 (Oz#2, May) to 45.15 mg/L (Oz#6/7, June) for all sampling sites and dates. Data for individual sampling sites and dates is shown in Table 2.

8Data Summary Table:

Umts mpaper presented as**~M** (convertedto**~gIL** above)

2 Results are same as those presented in Dorigo and Leboulanger2001

* Methods described in Dorigo andLeboulanger 2001. 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 inthe Results section ofthe study report. The primary reviewer assumed 1 day (24 hours) for all samples. Time Nominal Weighted Magnitude Atrazine Day of Study Average Did Effect of Effect Concen- Effect Atrazine Recovery Comments relative to tration Occurred Concentration Occur-if controls (",giLl fromDayOto so, when? When Effect

Occurred EC50 is lowest ofall sampling sites (4) and sampling dates Decreased per site (5); EC50 efficiency of values ranged from photosynthesis Not able to Not able to 56 to 1117**~gIL.** (increased 50% (ECso) 56 1* determine. determine. Range ofvalues fluorescence reported in text ofthe yield): study report. periphyton2 Endpointwas also reported in Dorigo and Leboulanger, 2001. EC125 is lowest of Decreased all sampling sites (4) efficiency of and sampling dates photosynthesis 25% Not able to Not able to per site (5); EC125s (increased (EC125) 8.6 1.5 hours determine. determine. for these sites ranged fluorescence from 8.6 ug/L to 32 yield): ug/L. Values phytoplankton obtained from Table 2. I

Reviewer Comments: The study applied Pulse-Amplitude Modulated (PAM) fluorometry to measuring in vivo chlorophyll 

### a fluorescence offreshwater periphyton and phytoplankton exposed to atrazine and isoproturon. The study authors performed dose-effect experiments on natural communities obtained from sites that varied in atrazine concentrationto determine ifatolerance or "memory" effect could be observed. In general, the study authors observed increases inthe EC50 (periphyton communities) and EC125 (phytoplankton communities) values at sites that were more highly contaminated and lowerEC50 or Ee125 values at sites with lower levels ofcontamination.

The study authors reported thatthe sampling locations were contaminated with both atrazine and isoproturon and thatthere was variability in the degree ofcontamination

9among sampling stations and overtime atthe same sampling station. The source water is located in an agricultural watershed and the authors reported that atotal of24 pesticides were detected in the water at least once during the study period.

The pesticide contamination levels (including atrazine) for the phytoplankton stations Oz#2 and Oz#6/7 were notreported in the study. Additionally, the contamination levels for Oz#1 were reported inregards to periphyton sampling; however, it was unclear ifthis contamination data was applicable to the phytoplankton sampling as well.

Results reported in this study for EC50 values ofthe periphyton (Table 1 above) were the same results reported from aprevious paperby the same group (Table 1, p. 513; Dorigo and Leboulanger, 2001). There is no difference between the EC50 mean values reported in the two studies; however, the River Ozanne station which was reference for the most downstream station was Oz#I.1 inthe 2001 publication versus Oz#1 in this study
According to the study authors, samples were obtained for periphyton on five dates. Based on statements in the methods section, it is implied thatperiphyton was sampled on three dates between May and June, yet in the results only two datapoints were presented for this time period, that is, "May" and "June" as in Figure 1. Eitherthe "May" or "June" datamay have represented an average oftwo sampling dates or may have represented a single sampling date. The reviewer assumedthatthe average EC50 values (Table 1) were based on five sampling dates.
Toxicity ofatrazine treatments to the natural communities was loosely associated with the degree ofbackground contamination. That is, communities exposedto greater background concentrations ofatrazine tended to be less sensitive to the addition of atrazine (experimental treatment) than communities exposedto lowerbackground concentrations ofatrazine.

There is no indicationthat atrazine concentrations were measured in the experimental treatments.

Atrazine was dissolved in acetone (also added to the controls) priorto exposing the periphyton and phytoplankton communities. The concentration ofacetone in the final volume was 0.5% (5 mlll). Because acetone is commonly used to extract chlorophyll it is unclearwhatifany effects the acetone had onthe results.
Description ofUse in Document: Invalid

10Rationale for Use: Acetone was used as a solvent at a concentration (5 mlll), which is 10 times higherthanthe criteriafor study acceptance (0.5 mlll, static testing), thus any toxic effects ofatrazine may have been confoundedby the addition ofacetone. Although a solventcontrol was included in the study design, datawas notpresentedto allow for an evaluation ofpotential effects ofthe solvent on the study results. The exposed organisms were taken from sites and times with variable levels ofatrazine and isoproturon contamination. In some cases the atrazine contamination was greaterthan the experimental atrazine treatments. Exposure to otherpesticides also occurred. River waterwas 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 mayhave beenhigherthan 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 focus ofthe study was the short-term effects (EC50 and EC 125) ofherbicide contamination on fluorescence measurements ofnatural periphyton and phytoplankton communities, respectively, which were obtained from locations withknown contamination ofatrazine and otherpesticides. Data showing the response at eachtreatment level was not provided, instead EC50 orEC125 values were provided. Acetone was used as a solventat a relatively high concentration (5 mlll). For the EC50s based on fluorescence data, itwas unclearhow long the samples were exposed to atrazine. There was no indicationthatthe study design included replication of treatment groups for the periphyton experiments. The test organisms were obtained from sites withknown contamination ofnumerous pesticides. There is uncertainty about exposure concentrations.
Primary Reviewer: LisaMuto, M.S., StaffScientist, CambridgeEnvironmental Inc Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA

# ~~M..I 

# ;;{wd
TertiaryReviewer: BrainAnderson, Biologist, EPA

# /f~~ 

# .1-/ Is //;

11j j j j j j j j I

j j

I I