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 of photosystem II herbicides on freshwater periphyton.

Journal of Applied Phycology. 13(6): 509-515. (MRID 48261108).

Purpose of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant

communities and associated determination of a Level ofConcern (LOC).

Date of Review: 9/4/09

Purpose of Study:

This study examines the use of Pulse-Amplitude Modulated (PAM) fluorometry to

measure in vivo chlorophyll a fluorescence in order to assess the short-term toxicity

effects of atrazine and isoproturon on freshwater periphyton in rivers.

Summary of Study Findings:

Data were presented on both isoproturon and atrazine. This review focuses on the effects

of atrazine.

Methods:

The study site was the River Ozanne which is located in the center ofFrance. The river

drained an area of intensive colza crops and, therefore, contained agricultural runoff

composed partly of herbicides, 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 of each herbicide.

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For periphyton sampling, approximately 120 small glass discs (1.5 cm2 area) were glued

onto a Plexiglas plate (18 x 22 cm, 4 mm thickness) 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 of colonization. After 2-3 weeks of colony growth, the colonized plates were

removed and placed into plastic bags filled with river water. The filled plastic bags and

separate water samples were transported in an insulated box to the laboratory. After a 24-

hour acclimation period in the lab to a small plastic aquaria containing river water

(temperature constant but not reported, 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 under the light microscope. 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. The periphyton biomass was estimated as ~g chI a per unit ofsubstrate

surface, and the percentage of each dominant algal group was estimated according to the

method of Wilhelm et al. (1991). The ratio of each diagnostic pigment identified

(fucoxanthine, lutein, and zeaxanthin) versus total chI a in the sample was used to

estimate the abundance of diatoms, chlorophyceae, and cyanobacteria, respectively.

The colonized glass discs (after the 24-hour acclimation period) were prepared for

fluorescence testing by being removed from the Plexiglas plate and cleared of all visible

debris, including living animals. The prepared colonized glass discs were placed into the

wells of 24-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 a halogen lamp which provided actinic light.

Fluorescence measurements were taken using two methods. In the first method, the

periphyton samples were exposed to prepared atrazine solutions for at least 5 hours; PAM

measurements were initiated after 20 minutes of dark-adaptation. A single saturation

pulse (ca. 1200 ~Mol photon m2 S-I) was applied to estimate potentially photosynthetic

efficiency F jFmax for each concentration (according to prior groups, such as Samson and

Popovic, 1988). In the second method, the periphyton samples were combined with 900

~L of Whatman GF/F filtered water from the algae sample site. After 20 minutes of

dark-adaptation, the fluorescence measurements were performed by measuring Fv then

determining Fmax, 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). After the first pulse,

100 ~L of prepared 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 of application was not stated. Atrazine was dissolved in acetone at

0.5% ofthe total volume. Acetone was also added to each control.

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The study authors used the method ofConrad et al. (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 when the herbicide effect is 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

through photosynthesis). The data were fitted to a logistic equation using least squares

method for determining EC50 values for atrazine.

In order to measure the photosynthetic activity in terms of 14C incorporation, colonized

glass discs were placed into 20-mL scintillation vials with 2 mL of Whatman GF/F

filtered water from the same river sample site. Aliquots (unspecified volume) ofthe

prepared atrazine solutions (final concentrations of 0.01, 0.03, 0.1, 0.3,1.0,3.1,10,31.6,

and 100 11M) were added to the samples. After one hour of preincubation (temperature 8-

23°C depending on the in situ water temperature ofthe sampling station, PAR ca. 70

IlMol photon m-2 S-1), 20 ilL ofNaH14C03 (specific activity 0.4 IlCi) was added to each

vial. After photosynthesis was allowed to proceed under those conditions for two hours,

the reaction was stopped by adding formaldehyde (final concentration 3%) then 200 ilL

of glacial acetic acid (in a fume hood) in order to remove inorganic carbon. After one

hour, the supernatant was removed. The discs were dried for six hours at 60°C under a

stream of air. The labeled organic matter was extracted using 1 mL of dimethyl sulfoxide

(one hour at 45°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 each herbicide concentration.

Results:

Results from HPLC analysis showed that all ofthe stations showed a seasonal shift in the

taxonomic composition: diatoms were the dominant group (Figure 1). Oz#6, the most

upstream station, was the least contaminated. Biomass (chI a) increased downstream

(Table 1).

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Figure 1. Relative abundance (percentage oftotal biomass) of the three major groups of periphytic

species, obtained by HPLC analysis, during the whole survey, Black: cyanobacteria; white:

chlorophyta; striped: diatoms,

Fluorescence ( FrlFmax vs Conrad relative yield 'R ')

Results from fluorescence yield measurements (FdFmax) of periphyton samples indicated

that this measurement was insufficiently sensitive to ensure a satisfactory estimate ofthe

effects of atrazine in this test system. No important changes in FdFmax were revealed

using this method, even after 24 hours ofincubation with atrazine. For atrazine

concentrations of 0.03, 0.1, 0.3,1,3.1,10,31.6, and 100 J.lM, the relative response

FdFmax (% of controls) 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 of Conrad et ai. (1993) produced

relative yield, R, values based on the short-term effects of atrazine. This method allowed

for the calculation of an EC50 (0.67 J.lM) for station Oz#6 (June 2000). For atrazine

concentrations of 0, 0.03, 0.1, 0.3,1,3.1,10,31.6, and 100 J.lM, the relative yield, R, (%

of controls) 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 et ai. (1993) generated

dose-response curves which allowed the calculation ofEC50 values. Dose-response

curves for all other station samples were not illustrated for atrazine; FdFmax values were

also not provided.

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Figure 2. Compared dose-response curves obtained on samples from OZ#6 (June 2000) with atrazine.

Closed circles were relative yield, R, for y measured according to Conrad et al. (1993). Open squares

were fluorescence yield, F,IFmax , linked to photosynthetic efficiency, measured after preincubation

with atrazine in the dark. Data are expressed as percentage of control value (R = 1 with Y = 61.2; =

0.46).

EC50 (fluorescence measurements (Conrad method) vs l4C incorporation)

For River 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. For River 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 of values represent five sampling periods at each

site).

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Table 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 of prior literature ofthe

sensitivity of freshwater periphyton to atrazine: EC50 range 0.21 to 3.32 11M using

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 ofEffect 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 able to

effects estimated

/increased (decreased 215.69 1* determine. determine. from Figure 2.

fluorescence efficiency)

yield, F,/Fma: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

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Time

Magnitude Nominal Weighted

Atrazine Day of Average Did

ofEffect 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% considered to 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 treatment to 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 related to

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

(reported by study

authors).

7
Umts ill paper presented as uM.

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

* Time of periphyton 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

study report. The primary reviewer assumed 1 day (24 hours) for all samples.

Time

Nominal Weighted

Magnitude Atrazine Day of Average Did

Effect ofEffect 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 C4

Cincorporation) these experiments.

The average EC50

of all sites and

dates was 245.88

/lgiL (reported by

study authors). I

Reviewer Comments:

The study was a test ofthe use of Pulse-Amplitude Modulated (PAM) fluorometry to

measure in vivo chlorophyll a fluorescence offreshwater periphyton exposed to atrazine

and isoproturon. The study authors used the standard 14C method for determining

photosynthetic activities to validate the EC50 results produced by the PAM method.

The authors used fluorescence as a measure of photosynthetic activity.

The PAM method described in the study was intended to detect the changes in apparent

short-term toxicity ofthe test compound under field conditions.

The authors determined that the F~Fmax parameter was less sensitive than the Conrad

parameter (R). Only the Conrad parameter permitted calculation ofECsos.

The method for obtaining fluorescence measurements from periphyton communities

presented in this study was a modified version ofthe protocol designed by Conrad et al.

(1993) for unialgal phytoplankton studies.

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The Method section ofthe study was unclear with regard to the amount oftime that the

periphyton was exposed to atrazine prior to PAM measurements. The exposure time 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 study

report. Additionally, the study authors reported that they tested a second protocol 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 submitted to the fluorescence

protocols when completing the Data Summary Table ofthis review. The exposure was 3

hours for the 14C-incorporation protocol.

The authors reported that the sampling locations were contaminated with both atrazine

and isoproturon and that there was variability in the degree of contamination among

sampling stations and over time at the same sampling station. The source water is located

in an agricultural watershed so it is possible that the water was contaminated with other

pesticides as well.

Toxicity of atrazine treatments to the natural communities was loosely associated with

the degree of background contamination. That is, communities exposed to greater

background concentrations of atrazine tended to be less sensitive to the addition of

atrazine (experimental treatment) than communities exposed to lower background

concentrations of atrazine.

There is no indication that atrazine concentrations were measured in the experimental

treatments.

Atrazine was dissolved in acetone (also added to the controls) prior to exposing the

periphyton communities. The concentration of acetone in the final volume was 0.5% (5

mlll). Because acetone is commonly used to extract chlorophyll it is unclear what if any

effects the acetone had on the 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 of Use 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 higher than the criteria for study acceptance (0.5 mIll, static testing), thus the

toxic effects of atrazine may have been confounded by the addition of acetone. The

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exposed organisms were taken from sites and times with variable levels of atrazine and

isoproturon contamination. In some cases the contamination was greater than the

atrazine treatment. Exposure to other pesticides may have also occurred. River water

was also used as dilution water to make the test solutions, and atrazine concentrations

were not analytically confirmed. Therefore, the exposure concentrations are uncertain.

The test concentrations may have been higher than nominal values because contaminated

river water was used as dilution water.

Limitations of Study: Some ofthe results have good applicability to mesocosms or

natural aquatic systems; however, the study was designed to evaluate the potential utility

of Pulse-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 a relatively high

concentration (5 mIll). Very little raw data was provided. For the EC50s based on

fluorescence data, it was unclear how long the samples were exposed to atrazine. There

was no indication that the study design included replication oftreatment groups. The

organisms were obtained from sites with history of exposure to numerous pesticides.

There is uncertainty about exposure concentrations.

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc

'" Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA j/t.~/'.'rt i ...

jh I

Tertiary Reviewer: Brian Anderson, Biologist, EPA /'i !' // i//(

/

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