Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Knauert, S., U. Dawo, J. Hollender, U. Hommen, and K. Knauer. (2009). Eflects of

Photosystem II Inhibitors and Their Mixture on Freshwater Phytoplankton Succession in

Outdoor Mesocosms. Environmental Toxicology and Chemistry. 28(4): 836-845. (MRID

48261120)

Purpose ofReview:

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 (LaC).

Date ofReview:

October 2, 2009

Purpose of Study:

This study examines the individual and combined effects ofthree photosystem II

inhibitors (atrazine, isoproturon, and diuron) on the community structure of freshwater

phytoplankton studied in outdoor mesocosms.

Summary of Study Findings:

This review focuses on atrazine-only experimental treatments.

Methods:

The data in this study appear to have been collected from the same seven-month

mesocosm experiment as reported in Knauert et al., 2008. The experiment dates and site

were the same and the measured atrazine concentration data reported in Knauert et al.,

2008 was the same as reported in this study. In the prior study, the analysis focused on

the inhibition of phytoplankton photosynthesis whereas this study focused on effects to

phytoplankton community structure.

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

1 11111••111

2087800
selected based on species sensitivity distribution (SSD) curves from laboratory

experimental gro\\-1:h ECso data on a broad range of phototrophic organisms (described in

more detail in Knauert et al., 2008). The HC30 for atrazine was 70 ~g/L.

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 composed of28 highdensity polyethylene tanks (3 m diameter, ca. 15 cm soil-sediment layer, ca. 130 cm

'va1-"'- ",~1.'m'" ~/" If),.,...3 .,,,,1..,.,...,,,\ <"J..:",J.. <.,~-", J...._:~;t :~ +J..", ~_",..~;t +", ~+",J..:I:~", +J..", v\, "'"'~ \..IVlU. lU.L, \,u. V U.J. VV1U-l11\.1),> VY1.11'--'U. YY\.IL\,.; UUJ.J.\....u I.U UJ.\..t 5J.VUlJ,U l.V .;,t.Q.U1I1L\..' Ul"'-'

temperature. Phytoplankton, zooplankton, macroinvertebrates and other organisms were

introduced into the mesocosms via water and sediment from a nearby man-made supply

pond (ca. 500 m

3 volume, 4 m depth), as well as by aerial colonization during the year.

Mesocosms were dominated by Elodea canadensis (Hydrocharitaceae) populations,

which were planted in the sediment.

During March to April 2006, water was circulated among the mesocosms and the supply

pond after which water circulation stopped and the mesocosms were considered isolated.

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. Atrazine was dissolved in methanol; the final concentration of

methanol in the ponds was < 0.002%. Methanol was not added to the control ponds.

During the constant exposure period, atrazine was maintained within ± 20% ofthe target

concentration. Atrazine (as well as diuron and isoproturon) was applied to the water

surface using a commercial sprayer. 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, 55~m mesh size). Phytoplankton samples (100 mL) were taken from each

mesocosm, stained and preserved with approximately 2 mL of Lugol-iodine solution; the

samples were stored in the dark for taxonomic determination. 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. Water

samples were stored frozen at 4°C until analysis. Biological sampling ofthe

ph~ytoplankton was performed weekly from days -8 to 54 and then biweekly from days 54

to 173.

2
For atrazine analysis, the water samples were filtered (cellulose acetate, 0.45 Ilm).

Eighteen (18 mL) aliquots were adjusted to pH 4 with acetate buffer, and radiolabelled

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

was enriched using a strata X column (20 mm length, 2.1 mm Ld.) 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 Ld. x 55 mm), room

temperature, flow rate 200 ilL/min, 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 indicative transitions for

atrazine were described in Knauert et ai., 2008. The limits of quantification were 3 nglL.

Quantification was performed using the internal standard. The intraday precision ofthe

method used for the pesticides applied in this study was 1-3%, and the recovery ofthe

enrichment step was 92-115% (spiking level, 100 ng/L).

For the taxonomic determination, aliquots of the fixed phytoplankton samples were

taken; cells were allowed to settle for at least 24 hours in a sedimentation chamber.

Quantitative evaluation was done according to the method ofUtermohl as described by

Schwoerbel using an inverted microscope (400x magnification). Taxa were identified at

the species level or to the lowest possible taxonomic level. Cell counts were expressed as

individuals/mL for each taxon.

The dissipation of atrazine in the post-exposure period (day 34-173) was described by

calculation ofthe half-life (tv,) using first-order kinetics. Phytoplankton data were

summarized by determining total number of algal cells (total abundance), absolute and

relative abundances ofsingle algal groups and species, and the number of different taxa

found per sample. On the taxon level, mean abundances per treatment were tested for

significant differences using Duncan's multiple t test. The Shannon index, which

considers species number and distribution ofindividuals across species, was used as a

measure for diversity ofthe phytoplankton community in a sample. Community data

were further analyzed using ordination techniques, such as PCA (Principal Component

Analysis) and PRC (Principal Response Curves; the constrained PCA based on

redundancy analysis), performed using CANOCO 4.5. The ordination diagram of PCA

was created for each sampling data and indicated the dissimilarity ofthe community in

these samples by the distance between the two sample scores. The ordination diagram of

PRC displays the deviation ofthe community structure ofthe treated ponds from that of

the controls over time. Only log-transformed abundances were used for the ordinations.

3
Results:

Chemical (Atrazine) Analysis

In order to maintain the target HC30 (70 Jlg atrazine/L) in the constant exposure period,

atrazine was reapplied one time at day 20. Time-weighted average atrazine

concentrations in the constant exposure period were maintained within ± 20% ofthe

target HC3o. The primary reviewer estimated a range of 69-84 IlgiL for days 0-28 based

on Figure 1 (see Table 1 and Figure 1 for details). In the ,post-exposure period (days 34- 17~\ thP h~lf..llfp of Qt,.Q71np ~'''>" I'Ql1'1l1Qtp,i Q" 1/17 r!"""" (,.'- = /1 0')\ h"""pr! An f'i,.d_A,.,i",,. -A. # '"")' ...... Ji._ ......_ .... -.L ...... A.._ .......... _ ....... _""""........... _ ... ~~ w_... __.... _~_~ _u ... ...., t --Ju \..1.- ....,./_j ....,_u v ................. u?o- _ .... _ .........

kinetics. The atrazine concentration declined to 30 Ilg/L (reviewer-estimated) at the end

ofthe experiment (day 173; 42.4% ± 2.4% ofthe HC30, n 3; mean standard deviation

calculated by the study authors). In the controls, the atrazine concentrations ranged from

6 to 28 ng/L throughout the study. The results ofthe atrazine concentration

measurements (in Jlg/L) are reported in Table 1; the results ofthe herbicide concentration

measurements for atrazine, diuron, isoproturon and the herbicide mixture are illustrated

in Figure 1.

4
n Ica es va ue a er reapPlIea Ion 0 a razme

I Time post first treatment (days) Atrazine single treatment (JigIL) I

01

84

Y

7 1

101 76

141

74

171

70

21 1

69 (77)*

28 1

77 I

352

74

i

42t

70

49k

562 I

i

702

i

842 i

Table 1. Mean atrazine concentrations in the water phase over time for atrazine single treatment.

All mean values, including time, were estimated by the primary reviewer from Figure 1. Asterisk

i d' tIft r f f t

1 Constant exposure

2 Post-exposure period.

5
•

I

A 100 •

·r 75 "'\i;;:~---------------------------- f s. ---------ll1--iT~--~-~--------­ 1

:( 25

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10

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.tt

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••

--.. -i----------------------------- I.

ii

E 0'

10.0

.-

"-J 7.5

ff

I:

e 5.0

,i

e

Cl.

0 2.5 .!!

F 0.0

4

Ii o~~.,......................~.,....... ...............~.,....... ............... 10.0r---------------.

I:e 10

Do j 5

.-

...:... 15

!

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B OJ.,!..........................................,............ ....................................,..............l

20'.,..----------------, .H

~;;-;~~~~~~~~~~~~~~:~~~~~~~~~~~~:

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Ht ~;l~------------------------------

-----~-.Jr.--------------------------- •

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•••

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2

Q

1

• • •

• • •

0.0 0 20 40 60 80 100 120 140 160 180 200 0 0 20 40 60 80 100 120 140 160 180 200

Days pre- and post first application Days pre- and post first application

Figure I. Actual measured herbicide concentrations in ""gIL for the different treatments: Atrazine

(A), isoproturon (B), diuron (C), atrazine in the mixture (D), isoproturon in the mixture (E), and

diuron in the mixture (F). Dashed lines indicate the target concentration ± 20%. Each data point

represents the mean ± standard deviation of three replicate ponds. Arrows indicate reapplications of

the herbicides.

Total abundance

Prior to application (day -8), total abundance in all mesocosms was similar (one way

ANOVA,p> 0.05). During the five-week constant exposure, mean total abundance

reached approximately 6000 individuals/mL in the control on day 26. The total

abundance in the atrazine treatment was reduced approximately three-fold to 2500

individuals/mL at day 26 (statistical significance not stated). From day 40 to study

termination, atrazine treatments were not significantly different from the control. The

study authors report that significant differences occurred on day 110 (one-way ANOVA

followed by Duncan's multiple t test,p > 0.05); however, they do not specify ifthe

6
significant difference was for the atrazine-only treatment. The total abundance results for

control and various herbicide treatments are illustrated in Figure 2.

,

/' "...------.....l

-

...I

105

:i

E 1

II

.~

:52

.~ 104

:i

""-

CD

U

c

-B 103

c

.B

CI ! 102.................-+-r-~..-.-r""l1""W""'l""'-..................-.r-..........-r-~'I""'r'"'I""1""W""'I""'~~ ........

o 20 40 60 80 100 120 140 160 180

Time (days post application)

Figure 2. Total abundance (individuals/ml) determined in the control (*), atrazine (0), diuron (e),

isoproturon (0), and mixture (.) treatment over the entire course of the experiment. Dashed lines

indicate the minimum and maximum values ofthe control community. The v.ertical dashed line

indicates the day of the first application (day 0).

.Community structure and succession

On day 5, control phytoplankton communities were dominated mainly by Chrysophyceae

and Cryptophyceae (each accounting for 40% oftotal abundance). At days 19-26, the

cryptophytes comprised up to 80% oftotal abundance, whereas chrysophytes had been

reduced to only 10%. At the end ofJune, the abundance ofthe chlorophytes increased,

yielding more than 80% oftotal phytoplankton because of a bloom of Choriocystis cf.

minor and Monoraphidium circinate. During August (days 82-110), Chrysophyceae and

Cyanophyceae were the dominant algae, comprising 80% ofthe phytoplankton. During

September and October (days 140-173), the Chlorophyceae, dominated by Oocystis

naegelii (ca. 3,000 individuals/mL), made up 60% oftotal phytoplankton.

Before application (day - 8) and a few days after application (day 5), the species

compositions ofthe atrazine-treated communities were not significantly different (p >

0.05) from those ofthe control communities. On every sampling date from days 12 to

173 (study termination), statistically significant differences in species composition were

observed between the control and the atrazine-treated mesocosms (one-way ANOVA

7
followed by Duncan's multiple t test, p < 0.05); these comparisons were based on sample

scores from the first axis ofthe principal component analysis. Atrazine treated

communities showed differences in major algal classes from day 12 to 173. For instance,

Cryptophyceae abundance in atrazine treated communities was lower than in controls

from day 19 to 40. Differences were also apparent at the species level. For example, the

abundances ofChroomonas acuta, the prasinophyte Nephroselmis cf. olivaceae (data not

shown) and Ankyra judayi were also reduced in the atrazine treatment compared to the

control (generally starting on day 12). During the post-treatment period (days 34-173), """"1 1 . 1 ; 1 4 1 • .. 1 1 .",..... • • \.."IllUrupny\;cat: werc lIlt: InU:Sl UUIIllnaIll algal gruup 1Il lilC alHlzmt: lrcalIIlCIll, l;urnpn:smg

40 to 90% oftotal abundance. The bloom ofCyanophyceae and Chrysophyceae

observed in the control ponds during August was nearly completely suppressed in the

atrazine-treated pond because ofthe sensitivity of Chroococcus sp. (days 82-140) and

Chromulina cf. obconica (day 110) toward atrazine. In contrast, growth ofChoriocystis

cf. minor was significantly enhanced in the atrazine treatment compared with the control

(days 82-110).

The relative abundance of major algal classes for control and various herbicide treatments

are illustrated in Figure 3. The abundances ofselected species for the control and

various herbicide treatments are illustrated in Figure 4. The PCA results for control and

various herbicide treatments are illustrated in Figure 5.

8
Control

-8 5 12 19 26 40 54 82 110 140 173

Time (daYli post application)

Diuron

-8 5 12 19 26 40 54 82 110 140 173

11me (daYli post application)

Atrazine

-8 5 12 19 26 40 54 82 110140 173

• Chrysophyceae

lS.'S1 Bacillariophyceae

CJ Cyanophyceae

mD Cryptophyceae

Iaa ZygnenuHophyceae

b.cl Prasiiioptiyceae

m Chlorophyceae

Isoproturon

-8 5 12 19 26 40 54 82 110140 173

Time (days post application)

Mixture

.. 5 12 19 26 40 54 82 110140 173

Time (days post application) lime (days post application)

Figure 3. Relative abundance of major algal classes in the control and the various treatments.

9
Cryptomonas erosa fit ovata

Chromulina of. obconica

Oocystis naegeli

..... Control

-_. Control minimum

--. Control maximum

-e- Atmzlne

... DiI.ron

-It- lsoproturon

- Mixt...-e

o ~"~1001~1~1"1~

Time (days postapplcatlon)

Ankyra judayi

Chroomonas aeula

ChroOCOCCU8 spec.

Chodocystis cf. minor

o ~"~1001~1~1"1~

Time (days post application)

10 5

10"

103

102

101

10

104

-

I 103

•

1i

:::J 102 :2

.2:

.=: 101

"

c:::

=-

CD 10° to)

C

II

i 10"

:::J

.a

II 103

10

101

10

10·

103

102

101

10°

Figure 4. Absolute abundances of the two cryptophytes, Chroomonas acuta and Cryptomonas erosa et

ovata; the cyanophyte Chroococcus sp.; the chrysophyte Chromulina cr. obconica; and the three

chlorophytes, Choriocystis cf. minor, Oocystis naegelii, and Ankyra judayi in the different treatments.

Data points localized on the x axis indicate that the abundance of the respective species was below the

detection limit of microscopic analysis on these sampling days. The vertical dashed line indicates the

day ofthe first application (day 0).

10
Constant Exposure Post Treatment

I

i day 110

I,

, r,,=r:-r----~---- , -----------------------

day 12

.. --_.. ..._-

- ------~-----------

1.0

0.0

1.0

0.0

0.5

1.0

0.5

0.5

0.0

-0.5

-0.5

1.5.....----,or----------......------------.

f i I day 5 ! day 54

I I f\ --#._._-- ·-··-···~fi-~--- I

I

I

I -1.0................. ............- ............._r_~........ ........'T"I""....,...r_1""'I~...._r........'""""'",...,..~....

..Q.5

-0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 1.5

-1.0........----.........,.........,~ ........--.....................-.Y'.................-+-t.................,.........,.........,..............-.......................................

-1.0

Figure 5. Plots of principal component analysis sample scores for the phytoplankton data from three

different sampling days. * - control; 0- atrazine; • - diuron; 0 - isoproturon; _ - herbicide mixture.

In contrast to the PCA, which coordinates the data for each sampling date separately and

with a priori infonnation on the treatment ofthe samples, the PRC attempts to explain the

data by considering time and treatment. Therefore, the PRC does not correspond exactly

to the sample scores ofthe first PCA axis. According to the study authors, the PRC

indicates the communities in atrazine treatments could recover within three to five weeks

during the post-treatment period (this seems to conflict with the results of PCA which

11
show no recovery of atrazine treated communities at study termination). The PRC also

indicates, however, that later on, depending on the substance, the community structure

also differed from the controls: some species, especially Chroomonas acuta, reached

higher abundances in the atrazine mesocosms compared with controls. For some algae,

such as the green alga Scenedesmus ecornis, an opposite pattern is predicted by the PRC:

an increase during the treatment period, followed by lower abundances in the atrazine

mesocosms than in controls during the post-treatment period. The PRC results for

control and various herbicide treatments are illustrated in Figure 6. It is not clear how

+1..0. n~,o.n.~~ ..... ~"r",,;r":h+n _ ...rJol"'< ....._+~rl ~_ v.:_.... _~ I:. _,.,.1 ... +,..... +_ ...+_.........;_............ _1 .... +_..........+_ ....._+.... n_.:J +t..._ U.J.V.::11-'",,\.I1\.1,;:) ",V\.l15.lU"U: t'iv'::>vlU.vU U ..l ~'J.SU." U .lv.lUL\.I \.V <.U..1GlL.l.l.l\.l-VJ..UY "'t\.tGU...1J.J.\.I.lJ.....:> a.Uu '-11\.1

results that the study authors state in the text.

1.0

0.5

-0

u

-0.5

-1.00 00 001001~1~100100

Time (days post applicBtion~

Chroomonas Bcura

ChOliocystis cf. minor

NeDhroseltJiis cf. oIlvacea

MonOOJphidium circinsle

Nitzschia lea Ankyra PS: . CryptomonBs

C uIa ~jN8tOri8 tfNUJis

Chromulina cf. oboonica

C06Iastrom mictOporum

C~=$ ~vatfans OszillBtanB lacustris

Chroococcus spec. ScenedeSfflus ecomis

-4 -2 0 2 4 6 8 10

Species weight

Figure 6. Principal response curves (PRCs) representing a significant part of variance in the

phytoplankton community data set (p - 0.002). Time (x axis) and treatment could explain 36% and

27%, respectively, ofthe total variance. The PRC (y axis) captured 16%. The y axis represents the

difference in community structure between the atrazine (0), diuron (e), isoproturon (0), and mixture

(.) treatments and the control expressed as regression coefficients (Cdt) ofthe PRC model. Taxa

with species weights of less than' - 1 or greater than 1 are presented on the right. The species weight

can be interpreted as the affinity ofthe taxon to the PRe.

Number ofdifferent taxa and Shannon diversity

Before application (day - 8), the average number of different taxa in the ponds was 30 ±

3. A few days after application (day 5), 23 ± 6 different species were in the control.

During constant exposure, the number oftaxa in the atrazine treatments were comparable

to those ofthe control (one-way ANOVA, p > 0.05), with an exception on day 5 (38

different species, reviewer-estimated from Figure 7, unclear ifthis time point was

significantly different from the control). During the post-treatment period, the number of

species in the atrazine treatments continued to be comparable to those ofthe control (oneway ANOVA,p > 0.05).

Before application (day - 8) and few days after application (day 5), the Shannon indices

ofthe control and the atrazine-treated communities were similar (one-way ANOVA, p >

0.05). However, in the following three exposure weeks, the Shannon index ofthe

12
atrazine-treated communities remained constant, while those ofthe control decreased by

a factor oftwo (indicating an unbalanced distribution ofthe organism among the various

taxonomic groups in the control - the secondary reviewer believes the lower Shannon

index also reflects the decreased species richness shown on the same dates); it is unclear

ifthe atrazine treatments and controls were significantly different. During the post

treatment period, in general, no significant differences in the Shannon index were

observed (one-way ANOVA,p > 0.05), however, atrazine treatments were higher than

controls on days 110, 140, and 173 and the authors do not specify whether these were

significant differences.

The species number data and Shannon index results for control and various herbicide

treatments are illustrated in Figure 7.

13
A

40

35

30

20

25

15

•

~. ... ,..... ~~i ~- .., ., ~, ,.~" :..\ .,. "'/ ·., :

• II. ,

·

,

··,

·

!

10 ·!

! B 3.Oi-+-r-t!""T"T-.,...,.......'T""'t"'T..,....,...,~,..,....,.~..,....,..,r-T"T"',...,...,..'"rr'T.....,...,I"'T""'I"",...,...,..~

0.5

)(

Q)

"'0 2.0 .s:

c

o 1.5 c

c

1.0

0.O~...-;0--2""r"0............... 40r-r-r-""6-r-OT"'"'r""'l""8...... 0 ..,...,1,.....,..00.........,..

12

r"'1"O......1.....,..40---

16

T'""1'O'-"""1!W"'TS""f"""lO

Time (days post application)

Figure 7. Diversity of control and exposed phytoplankton communities. Total number of algal species

(A) and Shannon index (B) as determined for the control (*), atrazine (0), diuron (e), isoproturon

(0), and mixture (.) treatment are presented. Dashed lines indicate the minimum and maximum

values of the control community. The vertical dashed line indicates the day of the first application

(day 0).

Overall, the study authors concluded that atrazine treatments substantially influenced

phytoplankton community structure due to the sensitivity of a few algal species for

endpoints including abundance, diversity and species composition. However, the

majority ofthe taxonomically identified algal species in the mesocosm were unaffected

by atrazine (data not reported).

14
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 fromDayOto so, when? (pgIL) When Effect

Occurred i

bstimate of

magnitude

based on

Figure 2.

It is unclear if

effect is

statistically

significant.

Abundance in

50% reduction atrazine

Total (transient 77~glLl,2 No treatment was abundance effect - see 84 ~glL Day 19 greater than (phytoplankton) comments) control on day

5, then equal

to control on

day 12. Given

the variability

in the dataset

over time the

reviewer

considered this

a transient

effect.

Statistically

significant (p <

0.05):

comparison

based on

sample scores

on first axis of

principal Community component structure (based Not able to 84 ~gIL Day 12 78 ~glLJ No analysis. on species determine

composition) Effects on

individual

species and

algal groups

I i !

I ~·15\.Ll ""

15
I

Time

Nominal Weighted

Magnitude of Atrazine Day of Average Did

Effect Effect relative Concen- Study Effect Atrazine Recovery Comments

to controls tration Occurred Concentration Occur-if

(pgIL) from Day Oto so, when?

When Effect

Occurred

Estimated

from Figure 7.

It is unclear if

this was a

statistically

significant

difference

between

control and

treatment.

The reviewer

discounts the

Community increased

structure ~50%higher 84 f..lg/L Day 5 81 f..lg/L1 No species

(species number as a

number) treatment

related effect.

Species

richness in the

atrazine

treatment

community

substantially

increased

(25%) from t=

oto t=5 (see

reviewer notes

i

for further

discussion).

Magnitude of

effect

Community

Unclear estimated from

given Figure 7. It is

structure ~30%higher 84 f..lglL Day 12 78 f..lglLJ temporal unclear ifthis

(Shannon

diversity index) variability was a

in data. statistically

significant

difference.

1 Based on the reviewer-estimated values reported ill Table l.

2 Value based on the atrazine measurement prior to adjustment.

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

16
Reviewer Comments:

Atrazine was present in the control ponds (6 to 28 ng/l) throughout the study period.

Although statistical analysis was performed, the results were not always clear regarding

specific comparisons (e.g. atrazine treatments on specific dates). This is because the

authors generally made statements regarding trends in the data and typically did not

mention or present statistical results for individual comparisons. Statements about

statistical signiticance in this review are based on statements made by the study authors.

The PRe results were difficult to interpret as presented.

The study authors reported that the control ponds were not dosed with methanol because

the methanol final concentration ofthe test ponds was less than 0.002%, and the study

authors believed that no effects to the aquatic community would be observed at that

methanol concentration.

A previous study (Knauert et aI., 2008) investigated the inhibition of photosynthesis by a

mixture ofthese three photosystem II inhibitors versus the single herbicides in the same

seven-month mesocosm experiment which was described in this study.

It is unclear why the average species richness increased from t=O to t=5 in the atrazine

treatment ponds. This effect is unlikely to be treatment related (i.e. atrazine treatment

causing speciation). One explanation for the increase in species richness in the atrazine

treatment ponds is that the species were initially present, but undetected. Ifthis is the

case, it is likely that there was a high degree of non-treatment related variability in the

dataset. Furthermore, the variability in the temporal trends throughout the experiment

(fluctuations in increasing/decreasing richness) suggests that there was a high degree of

error in sampling and species counting. Another explanation is that the species were

introduced between t=O and t=5 through aerial colonization, contamination, or some other

means. Species richness of each replicate mesocosm was not provided; therefore, it

cannot be determined if all or just a single replicate showed this increase. Species

richness data from each ofthe replicates would be useful in evaluating the importance of

the apparent increase in the number ofspecies.

Description of Use in Document: Qualitative

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

regarding effects of atrazine on aquatic plant communities.

17
Limitations of Study: The results have good applicability to mesocosms or natural

aquatic systems given that the experiments were performed in the field. However,

atrazine was present in the control ponds (6 to 28 ng/l) throughout the study period.

While this level of atrazine has not been shown to cause adverse effects to aquatic plants

based on the available data, it is unclear if background levels of atrazine in the control

ponds may have altered the phytoplankton community structure to more tolerant species

in all ponds prior to the addition of atrazine. Water chemistry ofthe microcosms was not

adequately monitored either prior to or following treatment. See the Reviewer's

Comments for further limitations associated with this study.

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

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA,ftiU/~

Tertiary Reviewer: Anita Pease. M.S., Senior Biologist, EPA !1'lt"}t (l3 {l \

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