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 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:

October 2, 2009

Purpose of Study:

This study examines the individual and combined effects of three photosystem II

inhibitors (atrazine, isoproturon, and diuron) on their inhibition of photosynthesis of

freshwater phytoplankton studied in outdoor mesocosms.

Summary of Study 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.

For the 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. For the mixture,

1/3 ofthe HC30 was used for each herbicide.

1 11.1.11111

2087852
For short-term dose-response relationship testing (to determine if chosen HC30 resulted in

similar effects on photosynthetic activity ofmesocosm phytoplankton), 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 of methanol (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.

For mesocosm 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. Prior to 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-week pre-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 in the

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

treatment to avoid cross-contamination.

For chemical analysis and biological sampling, depth-integrated water samples were

taken from the four quadrants of each mesocosm using a polyethylene tube (120 cm long,

4.5 cm diameter). Samples were taken close to the sediment to avoid contamination due

to sediment perturbation (it was not stated what the authors meant by 'contamination, i.e.,

the sediment itself or 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

in the laboratory (a 50 ml sample from each pond was centrifuged at 3000 rpm for 5

minutes at room temperature). The algal pellet was resuspended in 10 mL ofthe pond

water (40 ml ofthe supernatant was 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

2
atrazine was added as an internal standard. Analysis was performed with online solidphase extraction-liquid chromatography-tandem mass spectrometry. The water sample

was emiched using a strata X column eluted with methanol using an online system.

Separation and detection of atrazine was performed using HPLC with the following

conditions: Xbridge C18 column (2 mm i.d. x 50 mm), room temperature, flow rate 200

J.!Llmin, gradient mobile phase consisting of water 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 of desethylatrazine was performed using the internal standard of atrazine.

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 of phytoplankton (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

transferred to a quartz glass cuvette and analyzed. The PSII quantum yield was

determined according to the following equation: Y= (Fm' - F) I (Fm'), where F is the

momentary fluorescence yield measured between the pulses and Fm' is the maximum

fluorescence yield induced by a saturation pulse leading to full inhibition of energy

conversion at PSI! reaction centers. After the arithmetic mean of Y for the three

replicated ponds was determined, the inhibition of photosynthetic activity (1) was

calculated using the following equation: 1(%) =:' [1 - (Yapplication I Ycontroz)] X 100%.

Standard deviations were estimated according to Gaussian error propagation.

Fluorescence data from the short-term tests were analyzed using linear regression with

GraphPad Prism 4.0, generating the equation I (c) = m log (c) + n, where I is the

inhibition of photosynthetic activity (%), c is the atrazine concentration, m is the slope

parameter and n is the y-axis-intercept. For the 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 of all measuring data points

for each treatment. The dissipation of atrazine 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 (TUi) which were calculated using the following equation: TUj = (ci)/(HC30i),

where Ci was the measured concentration ofthe herbicide i in the water column and the

HC30i was the 30% hazardous effect concentration ofthe respective herbicide i obtained

from SSD curves. The study author presented the results in TV units. The reviewer used

the previously described formula to convert from TV to J.!g atrazine/L.

3
Results:

Herbicide (Atrazine) Concentrations in the Water Column

In order to maintain the 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 of 0.95 to 1.22 TU for days 0-34 (see Figure 1 and estimates from

primary reviewer in Table 1 for details). In the postexposure period (days 34-173), the

half-life of atrazine 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 degradation products desethylatrazine and desisopropylatrazine were less

than 11% and 6% respectively ofthe target concentration ofthe parent. Estimates of TU

measurements and the inhibition of photosynthetic activity for atrazine are reported in

Table 1 and are based on results illustrated in Figure 1.

4
Table 1. Mean relative herbicide concentrations in the water phase and inhibition of photosynthetic

activity of phytoplankton 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

of atrazine

Time post first treatment Atrazine single treatment

(days) Atrazine (TV) Inhibition of photosynthetic activity (%)

01

1.2 60

31

1.18 50

i 1.15 40

101

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.

5
A 1.1, 160

1.4 140 5'

__.t...._._._n......"'_n.......n_.n...___ ....._u_____ c a: .Sl 1.2 120 3: -l! 1.0 100 0 .. IIlo ::I

41

I:

f,l~ 0.8 80 aa

c::l ~~

otu- 0.6 60 ![ CD 0.4 40 .e2 I: -oc

·s 0.2 20 :::I

< l

0.0 0 =.

B tl

r: 1.4 140 :;

:7 .2 1.2 120 C' i f -

c '1.0 100 CI :::I

8 tl 0

c::~ 0.8 80 !:.; 0::::2

ut- 0.6 &0 «'::I' c~ ..... 0

e 0.4 40 ;;elI

i -'<

D.2 20 ::s

;. I 0.0 * 0 :l.

0

C 5" 1.4 + 140 =r

c 1.2 120 .2 ---_.~ .. _.. _._-----------.,-_._ ..-------------------_._--------_._--_._- f -! 1.0 100 I» = I: 0.8 80 ~a 8s ~~ Ct- 0.6 60 .;t:r 0- .... i 0 ;p.o c

2 0.4 40 -~

.i 0.2 20

Q

0.0 0 i

n

0

1.4 140 5'

c

0 1.2 - 120 a:

= a: l! 0

- 1.0 100 lit ::::I I: as. II ..... 0 !!!: 0.8 ...--_ ....-....-- _..---_ ....--....----... ""--~-_ ....... -_ ..........-..... 80 C e ~" 8::::2 0.6 eo .:z::r

lit:. .-.~ t:. J:! 0." 40 ... --~

:e 0.2- 20 i

:7

:! 0.0 0 3:

l"J

.0.2 -20

0 28 56 84 '12 140 168 196

Time post first treatment (d)

'lilY 'June AUII ,hpt I &t I

Figure 1. Relative herbicide concentrations in the water phase (expressed as TV) (.) and inhibition of

photosynthetic activity of phytoplankton (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 TVmix were 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 of the herbicides. Inhibition of

photosynthetic activity of phytoplankton that was not significantly different from control level is

indicated by an asterisk (two-tailed, unpaired t test, p > 0.05).

6
Effects on Photosynthesis -Laboratory Short-Term Toxicity Tests

In short-term laboratory toxicity tests on the 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 of photosynthetic activity, respectively (inhibition

ranges estimated by the primary reviewer based on Figure 2). The HC30 of atrazine (70

Ilg atrazine/L) caused 43.0 ± 7.6% inhibition of photosynthesis. It is unclear what the

data points plotted in Figure 2 represent (i.e., subsamples ofmesocosms, laboratory

replicates, etc.)

• Atrazine • Isoproturon • Diuron

80

'#.- 70

- 60

> 0t; 50 em.2(,) 40 Ii

- .t::. 30 ~t! 20

j 10

a.

0

..(J.s 0.0 0.5

.

• ..

1.0

loge

1.5 2.0 2.6

Figure 2. Inhibition of photosynthetic activity (%) of phytoplankton 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 of the three herbicides.

Effects on Photosynthesis - Mesocosm Experiment

Prior to application (day -8), photosynthetic activity of phytoplankton 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 of photosynthetic activity of phytoplankton (51.4 ± 10.8% with 1.15 ± 0.07

TU, n = 3) (Figures 1 and 3). Over the 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 of photosynthetic activity during the

constant exposure period was 45.6 ± 9.3 (n = 7). The mean effects induced by the HC30

of atrazine in the mesocosm experiment were also similar to the effects ofthe HC30 of

7
atrazine from the short term 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 linear trend 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 significant reductions of

photosynthetic activity compared to the controls.

t!-

-

>- ao .... ~! eo r-=c- u

I'll r-=r=-

Ll

.. :J ;:) :::l

.2! 40- ... l- I-

'"' ... ..-

e (;I 0 ... d _d Q d >. + 0+-1 ." +-: Q 20- a ...

f~ .. g f:!l .... Ii ... 0 N . e...: i~ ,.I; gr. g, 011 .:(u t) .. 5

FIGURE 3. Herbicide-induced inhibition of photosynthetic activity (%) determined on day 2 after

application. Each bar represents mean and standard deviation of the three replicated mesocosms.

Statistical analysis of the 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.

8
8:0.-----------------.......

a

•

•

"

"

•

•.~

• C • ..

diI

••• •

•

D •

.

.a

•

.-

"

•

II Alrazine

• lsaproharon

.. Ojuron

D Mbcture

•

y .a.1

Dc...

.... • y

0.0 0.2 0.4 0.6 0.8 1.0

Herbicide co~ntrations (TU)

1.2 1.4

Figure 4. Concentration-response relationship between herbicide concentrations (expressed as TV)

and inhibition of photosynthetic activity of phytoplankton (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 of photosynthetic activity by atrazine over time, the

study authors concluded that the decline in inhibition was related to the decrease in

atrazine water concentrations and that community tolerance likely played a minor role

due to the fact that 0.4 TU of atrazine at experiment end (day 173) resulted in a 25%

inhibition of photosynthetic activity.

9
Data Summary Table:

Time

Nominal Weighted

Average Did Magnitude of Atrazine Day of Atrazine Recovery Effect Effect relative 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.

10
Time

Nominal Weighted

Magnitude of Atrazine Day of Average Did

Atrazine Recovery Effect Effect relative 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 TV at

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 reviewer usmg equatIOn proVided by study author. TV = c/HC30 where c IS

the measured concentration of atrazine in the water column and HC30 is the 30% hazardous effect

concentration of atrazine (70 /lg/L as reported by the study authors). c (/lg/L) = TV x 70 /lg/L.

The study authors calculated the half-life of atrazine as 107 days.

11
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 order to

relate the different herbicides to one another. The reviewer used a formula provided by

the study authors to calculate concentrations of atrazine as Ilg/L.

The study authors reported that other previous literature studies concurred with the halflife findings of freshwater microcosm studies of other laboratories.

The subsequent study report, Knauert et. ai., 2009, was an expansion ofthis study in

which effects on phytoplankton 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 data reported in the 2009 study for the mesocosm experiment was

the same as the value reported in the 2008 study, except that it was reported in units of

Ilg/L. .Furthermore, the calculated half-life of atrazine was the same in both studies.

The authors do not provide information about background concentrations of atrazine 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-term toxicity tests (0.1 %) was higher than

that used in the mesocosm experiments «0.002%). Methanol was included in the

controls for the short-term tests. For the 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 contain methanol.

Description of Use in Document: Qualitative (except results from short-term toxicity

tests, which are invalid).

Rationale for Use: Mesocosm experiments contribute to the weight of evidence

regarding effects of atrazine on aquatic plant communities.

Limitations of Study: The results have applicability to mesocosms or natural aquatic

systems since experiments were performed in the field, as well as the laboratory. The

study authors used toxicity units (TU) in the place of standard 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-term toxicity testing of 0.1 % (1 mIlL)

12
exceeded the criteria for study acceptance «0.5 mlll, static-renewal testing); therefore,

the toxic effects of atrazine may have been confounded by the addition ofsolvent. In

addition, the type ofreplication used in the short-term toxicity testing was unclear. With

respect to the mesocosm studies, water chemistry was not adequately monitored either

prior to or following treatment. The study authors did not report if atrazine or other

contaminants were present in the controls; however, in the 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: Lisa Muto, M.S., Staff Scientist, 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/ \

13