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 ofriver microalgae to atrazine and isoproturon along a contamination gradient.

The Science ofthe Total Environment. 318(1-3): 101-114. (MRID 48261109)

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 used 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 and phytoplankton obtained from a river that

has a pesticide-contamination gradient.

Summary of Study Findings:

The study evaluated effects of atrazine and isoproturon. This review focuses on the

effects of atrazine.

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 seed rape), com (maize), and winter wheat crops] and contained agricultural

runoff contaminated with urea and triazine herbicides, mainly atrazine and isoproturon.

For periphyton studies, river samples were collected from established stations chosen

from upstream to downstream (Oz#617, Oz#5/6, Oz#4, Oz#3 and Oz#I). The study

authors did not present periphyton data for station Oz#617. For phytoplankton 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 application period), once in September

1

1••••1.111

2087822
2000 (post-herbicide application period) and once in January 2001 (pre-herbicide

application period). Sampling sites were chosen to have similar environmental

conditions (water velocity, canopy cover, and light exposure). The water was tested for

77 herbicides by an external laboratory (Loire Bretagne Water Agency. In addition to

atrazine, measurements were made for six other triazines and degradation products.

Atrazine was extracted using dichloromethane and was vacuum-concentrated prior to

analysis via gas-chromatography with nitrogen-phosphorous detection (GCINPD). Mass

spectrometry was employed when necessary.

For periphyton sampling (previously described by Dorigo and Leboulanger, 2001), small

glass discs (1.5 cm

2 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 of colony growth, the colonized plates were removed and were transported in coolboxes to the laboratory. For phytoplankton sampling, the communities were collected

using 2-L plastic bottles which were pre-cleaned with dilute HCI and rinsed with river

water three 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 treatment used a high-grade pesticide atrazine standard, dissolved in

acetone. A stock solution of 20mM atrazine was diluted serially (1005

). A solvent blank

was used for the control. Acetone was added to 0.5% ofthe total volume.

Periphyton and phytoplankton fluorescence assays were performed after a 24-hour

acclimation period (river temperature; 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 et ai., 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 of 24-well polystyrene

microplates with 900 j.lL ofWhatman GFIF filtered 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 prepared by diluting a 20 mM stock

solution with Whatman GFIF filtered water from 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 a PAM 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 et ai. (2001).

For the phytoplankton assay, 20-mL subsarnples ofthe collected sample was placed into

Coming glass tubes with 10 j.lL of an atrazine stock solution (prepared using high-grade

atrazine standard). The atrazine test concentrations ranged from 1 to 10000 nM. Six

2
replicates 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 in the in vivo fluorescence was identified as

the EC125 (toxic concentration that increases fluorescence 25% relative to the control

samples), according to Seguin et al. (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

light microscope (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 et al., 1997). Standard curves were established

for the pigments. The periphyton biomass was expressed as Ilg ChI a per cm

2 based on a

glass disk surface area of 1.5 cm

2

, an extraction volume of 4 mL per glass disk and an

injection volume of 20 ilL. The percent contribution of each algal group (a.g.) was

calculated according to: %a.g. = (%Ad.p x 100)/(%AcHLa x r w), where d.p. is the

diagnostic pigment, A is the area ofthe peak and r w 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-11m pore 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 of atrazine varied among sampling locations and over time.

Atrazine levels were generally highest in the 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 of Dorigo et al. 2004)

4
Ecotoxicity tests

For periphyton, 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 at the sites with the highest measured levels oftriazines

(seven triazines including atrazine and degradation products were measured). EC50

results for atrazine at each sampling station during each sampling month are provided in

Figure 2.

Table 1. Sensitivity of periphyton 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 of Dorigo 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 of periphyton 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

of Dorigo et al. 2004)

For phytoplankton, 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

excluded because of poor fluorescence readings due to high levels of particulate matter

and low levels of phytoplankton biomass. On a given sampling date, the EC125 values

of Oz#6/7 were generally lower than those of Oz#1 and Oz#2. EC125 results for atrazine

at each sampling station on each sampling date are provided in Table 2.

6
Table 2. Structure and sensitivity of phytoplankton 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 a per L) (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 of community structure were made on samples prior to the experimental

addition of atrazine.

For the periphyton, diatoms (33 species and 12 genera), green algae (7 species), and

cyanophytes (3 species) were identified. The most common diatom genera were

Navicula, Nitzschia, Gomphonema, Achnanthes, and Amphora. The most species-rich

samples were taken in September (Oz#5/6, 29 species) and in January (Oz#4, 39 species).

Both Shannon's index, applied to diatoms, and the total number ofspecies, calculated for

each sampling date and site, showed no correlation with the related EC50 values (data

was not provided in the study). Diatom-based indices (BDI, Biological Diatom Index)

showed a clear pattern of increasing upstream. The BDI was not well characterized in the

study report. There was no clear tread for the pollution-sensitivity index (PSI), which

was used to estimate the pollution-sensitivity ofthe established community. A large

amount of variability 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 pigment in those samples. Overall, diatoms were the most abundant group in the

7
periphyton flora. When other groups were present they were most abundant at the

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 of Dorigo et at. 2004)

For the phytoplankton, a total of 119 phytoplankton taxa were identified to at least the

genus level. Phytoplankton were represented by diatoms (26 species and 11 genera),

green algae (5 8 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.

8
Data Summary Table:

Umts m paper presented as ~M (converted to ~gIL above)

2 Results are same as those presented in Dorigo and Leboulanger 2001

* Methods described in Dorigo and Leboulanger 2001. 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 ofStudy Average Did

Effect ofEffect Concen- Effect Atrazine Recovery Comments relative to tration Occurred Concentration Occur-if

controls (",giLl fromDayOto so, when?

When Effect

Occurred

EC50 is lowest of all

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 of values

fluorescence reported in text ofthe

yield): study report.

periphyton2 Endpoint was 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 concentration to

determine if a tolerance or "memory" effect could be observed. In general, the study

authors observed increases in the EC50 (periphyton communities) and EC125

(phytoplankton communities) values at sites that were more highly contaminated and

lower EC50 or Ee125 values at sites with lower levels of contamination.

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

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

9
among sampling stations and over time at the same sampling station. The source water is

located in an agricultural watershed and the authors reported that a total of 24 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 not reported in the study. Additionally, the contamination levels

for Oz#1 were reported in regards 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 a previous paper by 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 in the 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 that periphyton was sampled on

three dates between May and June, yet in the results only two data points were presented

for this time period, that is, "May" and "June" as in Figure 1. Either the "May" or

"June" data may have represented an average oftwo sampling dates or may have

represented a single sampling date. The reviewer assumed that the average EC50 values

(Table 1) were based on five sampling dates.

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 and phytoplankton 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.

Description of Use in Document: Invalid

10
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 mlll, static testing), thus any toxic

effects of atrazine may have been confounded by the addition of acetone. Although a

solvent control was included in the study design, data was not presented to allow for an

evaluation of potential effects ofthe solvent on the study results. The exposed organisms

were taken from sites and times with variable levels of atrazine and isoproturon

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

experimental atrazine treatments. Exposure to other pesticides 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 focus ofthe study was the short-term effects (EC50

and EC125) of herbicide contamination on fluorescence measurements of natural

periphyton and phytoplankton communities, respectively, which were obtained from

locations with known contamination of atrazine and other pesticides. Data showing the

response at each treatment level was not provided, instead EC50 or EC125 values were

provided. Acetone was used as a solvent at a relatively high concentration (5 mlll). 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 of

treatment groups for the periphyton experiments. The test organisms were obtained from

sites with known contamination of 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 ~~M..I;;{wd

Tertiary Reviewer: Brain Anderson, Biologist, EPA/f~~ .1-/Is/ /;

11
j

j

j

j

j

j

j

j

I

j

j

I

I