Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Berard, A. and C. Benninghoff. 2001. Pollution-induced community tolerance (PICT) and

seasonal variations in the sensitivity of phytoplankton to atrazine in nanocosms. Chemosphere.

45: 427-437. (MRID 48261101)

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 7, 2009

Purpose of Study:

This study examines the potential effects of atrazine on phytoplankton in experimental outdoor

microcosms.

Summary of Study Findings:

Several other reviewed studies by the same authors reported data from the same set of

microcosm experiments. The studies related to this review are Berard et al., 1999a; Berard et al.,

1999b; Leboulanger et al., 2001; and Seguin et al., 2001 b. Among those studies there is both

overlap and difference in terms ofthe experimental dates presented, the endpoints measured, the

level of detail used in the presentation ofthe data, and the statistical analyses. Each ofthe

individual DERs for the aforementioned studies attempts to discuss similarities and differences

in study designs and reported endpoints.

Methods:

Although the study authors refer to the test systems used in these experiments as "nanocosms",

the same systems were referred to as "microcosms" in other reviewed papers by the lead author

(Berard et al., 1999a; Berard et al., 1999b; Leboulanger et al., 2001; Seguin et al., 2001 b) and

will be referred to as such in this review. Eight experiments were conducted during the spring,

summer, and fall of 1997 and 1998 (Table 1). Source water was obtained from the euphotic

zone in the center of Lake Geneva. The water was filtered (200-J..lm mesh) to remove large

- 1 -

1111111111

2087804
zooplankton, with remaining zooplankton killed by oxygen depletion. Each microcosm

consisted of a 5-L Pyrex bottle containing 2.5 L ofthe phytoplankton suspension plus 2.5 L of

filtered (0.8-llm polycarbonate membrane) lake water. For each experiment, five replicates were

used for atrazine treated and control microcosms. The microcosms were placed in Lake Geneva

at a depth of 1.5 m in an area oflow turbulence and were constantly aerated with filtered air

(pore size not specified). Water samples containing phytoplankton were withdrawn from the

microcosms through a silicon cap via glass and silicon tubing. The study authors cite Berard et

al., 1999a for a complete description ofinstallation ofthe microcosms. Any acclimation ofthe

rnlf'rOf'o"rn" nnor to trp<:ltrnpnt UI<:l" not rpnorlprl ........... _ ... "-"_ .................... y ...... ...,. ... ......... "'... __"'......... _a...a. .. ~~_.... ..... ""' ..... ""'1:"'"' ....._--.

Atrazine (purity 98%) was dissolved in double-distilled water with continuous stirring for 48

hours to make a stock solution of 10 mg/mL. No organic solvent was used. Microcosms were

treated with atrazine at 10 Ilg/L. Experiments were conducted for 11-23 days depending on the

experimental month; experiments were stopped when the relative growth rate ofthe control

cultures declined, just before the stationary phase of phytoplankton growth. Growth was

monitored by making daily in vivo fluorescence measurements using a Turner fluorometer.

Nutrient concentrations were determined at the beginning of each experiment (Table 1). No

details were provided regarding methods used to measure nutrients. Measurements ofwater

temperature and surface irradiance were not described (data presented in Table 1). Atrazine

concentrations were determined at the end of each experiment by HPLC analysis (no details

provided).

Biomass and taxonomy. Chlorophyll a content was determined at the beginning and end of each

experiment (method was not described or referenced). No methods were reported for the

primary production measurements that are presented in Table 2. Phytoplankton community

composition was determined at the beginning and end of each experiment. Samples (50 mL)

were treated with Lugol's solution (concentration not specified) to kill and stain the cells. Cells

were allowed to settle for 24 hours in an Utermohl chamber, then counted (counting accuracy ca.

± 10%) and identified using a reverse-phase microscope. A minimum of400 individuals per

dominant species (defined as > 100 individua1s/ml) were counted. Biomass per species was

estimated using bio-volume calculations.

Short-term (tolerance) bioassay. Development oftolerance was assessed through re-exposure of

the microcosm phytoplankton to atrazine for a short interval and assessing photosynthetic

activity. Tolerance (primary production) was measured at 1 day post-treatment and at the end of

each experiment. A pooled sample was created for each treatment group (atrazine and control

microcosms) which consisted of a 60 mL sample from each ofthe five replicate microcosms.

Then 9 mL ofthe pooled sample was combined with 1 mL atrazine (1-2 mg/L) solution (final

concentration 100-200 Ilg/L) in a 10 ml Pyrex-glass vial. There were duplicates for each

atrazine concentration and four replicates for controls. Heterotrophic assimilation was measured

in duplicate dark vials. Following one hour ofatrazine exposure (conditions not described),

- 2 -
samples were treated with 14C03 (concentration not specified) and incubated for two hours at the

same water depth as the microcosms (no additional details were provided). Following

incubation, the samples were filtered (0.45-llm, cellulose nitrate), then the filters were acidified

(O.IN HCI) and analyzed for total radioactivity using LSC. ECso values were determined by log

linear interpolation ofthe dose-response curves.

Statistical analyses. Diversity (H') of algal communities was estimated at the beginning (pooled

';":lrYlt"llp f'lf ",11 tpn rYll,.,.t'f'I""f'lCrYlC\ <>n~ pn~ (rYlp<>n tlpnclt" n = " npt' tt'P<>hnpnt arf'llln\ f'lf p<>,.,.h ..... _ 1"" _ _ -1'- .. _ _.__ " -... - _ - , _~ -_ J'....... .....- .t"'- --_ _ O _J:"') "'- --_ ....

experiment using the Shannon-Weaver equation. Differences between atrazine treated and

control microcosms were assessed using the Bray-Curtis dissimilarity indices as used by Dahl

and Blanck (1996) and Berard et at. (1999a). The Bray-Curtis index (BCI) was determined for

the mean density ofthe five replicates in each treatment group (n = 5). At experiment

termination, the density of each dominant species in the treated microcosms was compared to the

controls using the Mann-Whitney nonparametric test (p-value not reported). Short-term

(tolerance) ECso values were log transformed to obtain normal distribution. Student's t-test was

used to compare data (ECsos) at the beginning and end ofthe experiments as well as treated vs

control samples. Correlation tests were done using CPAnalysis (Pearson correlation matrix) with

ADE software.

Results:

At 11-23 days post treatment (experiment termination depending on experimental month),

atrazine concentrations were 90-95% ofthe initial nominal 10 Ilg/L treatment concentration (data

not provided).

Initial phytoplankton community composition at the beginning of each experiment is shown in

Figure 1. Table 1 shows the initial nutrient concentrations, water temperature and surface

irradiance throughout the experiments, and duration of each experiment. Table 2 shows

community biomass (as chlorophyll a) and diversity (H') just prior to contamination and at the

end of the experiments. Primary production (photosynthetic activity) in the atrazine treated

microcosms (expressed as % of controls) is presented in Table 2 for one day after contamination

and at the end of the experiments. Overall, chlorophyll a, diversity, and primary productivity in

the atrazine treated microcosms increased, decreased, or did not change relative to the control

microcosms depending on the month ofthe experiment.

In each experiment, atrazine treatment significantly affected the density of at least two dominant

species (dominant defined as > 100 individuals/ml) compared to the controls (Table 3).

Individual species response to atrazine treatment (i.e., inhibition, stimulation, or no change

compared to control) was highly variable among the different experimental dates. Bray-Curtis

indices (BCI) were lower at the end of each experiment than they were one day after

contamination of the respective experiment. According to the study authors, the mean BCI of

all experiments at 1 day post-treatment (0.87) was significantly higher (p=O.05, n=8) than the

mean BCI of all experiments at experiment termination (0.80). The study authors indicate that

- 3 -
the BCI is represented as similarity in Table 4. Depending on the experiment, the atrazine

treated communities were 1% to 21% less similar to the controls at the end of the experiment

(BCI 1 day prior to exposure - BCI end of experiment).

Short-term toxicity (tolerance) experiments. Results from the short-term experiments are shown

in Table 4. The EC50 values (based on photosynthetic activity) of control microcosm

communities were highly variable among the experimental dates, ranging from 38 to 829 ~g/L.

A +.......... ....,.~_ ...... +_...... £0> ........ ....1 _:..... _ "' .... _ __.......... ~+~ (~ ...... __-=.-.._ -.. ... ~_,....""'" ....... oi- ..... 1 f\ • ..-- ..... oi....................;_ ..... /T '\ ......."..,_.. ~1_ ....... rU1~lH'--Ll'-aL'-u 1111'-1 V V~111 V111U1UlULl \}.,-. P1IVl I;.;hpV::'UU;; LV 1 V POe Q.llaLlUl;,/ 1J) Wl;,ll;.; Q.l::'V

highly variable (37 to 589 Jlg/L) and unlike the controls showed consistently higher ECsos at the

end of the experiments compared to one day after microcosm contamination. There was a

significant correlation between short-term toxicity (ECsos) at the beginning and end of the

experiments for atrazine-treated microcosms (Figure 2b) but not the control microcosms (data

not shown). Atrazine-treated microcosm communities were significantly (p=0.03, n=6,

comparison based on all experimental months) less sensitive to atrazine than the controls at the

end of the experiments (Table 4). However, the controls were less sensitive than the atrazine

treated microcosm communities at the end ofsome experiments (i.e., April 97 and August 97).

Short-term toxicity (sensitivity) was likely associated with factors other than atrazine alone. For

instance, the study authors present correlations in chlorophyll a, diversity (both p=O.OOI, Figure

3) and sensitivity of algae to atrazine (p 0.09) between treated and control microcosms at the

end of the experiments (Figure 3).· Additionally, diversity (W) indices determined at the end of

the experiments correlated with sensitivity to atrazine at the beginning of the experiments for

both control and atrazine-treated phytoplankton (Table 5). Table 5 shows additional correlations

between environmental parameters, community characteristics, and short-term response of

primary production to atrazine (EC50).

- 4 -
ID Xamhophyccac

ID ZYlll)cm1l1op/1~e-._

[J ChlOlophYl:~a~

II Badlluricph)Wile (~nl1~l"','

D Raci1l ilriol'h) <:C<lr ("",rl;r;d~~1

[J (.1I1)osl'pll)I:I,:i"

EI er)pl'''I,bi<:eac

all )illllllh~L"'1Io!

• r}';lnono.:lcti3

Figure 1. Initial algal class composition of the natural phytoplankton microcosms. (study figure 1)

- 5 -
1000

•

•

100

control nanocosms beglning of experiment: [C50 (IJQ/l atrOllne)

r=0,89 p=0,01 n=7

1O-t""'---,---,----,--r--..----r-,--...,.,..----,-----,---.--,--,--,-.,---,-1

10

1000

¢

E

-~

Q.

x..--

_(J) Q) c:

0--

-g g100

(1)0

.. --J E-

.. 01

() .::J,

~2OUCLlJ

\J

C!J

o(J)

;::

(a)

100 1000

treaied nanoosms begining of experiment: EeSO (j.Jg/L airazine)

10 -¥'----.,----,--,--.--.-,.-,-,-.,.-----..---,--y-~··.,.--,__.___r_.,._i

10

1000

(b)

Figure 2. Correlations between sensitivities to atrazine (EC50) of phytoplankton microcosm communities

(CPAnalysis, Pearson correlation matrix). (a) Treated communities at the end of experiment vs. control

communities at beginning of experiment. (b) Treated communities at end of experiment vs. treated

communities at beginning of experiment. (study figure 2)

- 6 -
1000

55

u

UJ

(a)

20

0

15

-

0)

::L 10 "-'

Vl

Q+-c 5 0

0

o 0

(b)

sensitivity to atrazine (EC50): r=0.74 p=0,09 n=6

•

•

ii' iii

100

EC 50 treated nanocosms (~g/I ATZ)

chlorophyll a r::;0.95 p=O, 00 1 n=8

5 10 15

treated nanocosms (jJg/1 chi a)

•

i •

1000

20

5

0 diversity H': r::;0,97 p=O,OOl n=8

::J 4 1J

:~

1J

c 3

"-en

:B-...... 2 III

2

-c

0

u

0

(C) 0 2 3 4 5

treated nanocosms (bits/individual)

Figure 3. Correlations between sensitivities to atrazine (EC50), diversity and biomass of phytoplankton

microcosm communities at end of experiments, control vs. treated communities (CPAnalysis, Pearson

correlation matrix) (a) Sensitivity to atrazine (EC50), (b) Biomass (chlorophyll a), (c) Diversity (H'). (study

figure 3).

- 7 -
Table 1. Initial nutrient concentrations!, water temperature and surface irradiance throughout the

Orthophosphatcs (mg PIL) 0.006 0.012 0.003 0.001 0.003 0.001 0.001 0000

Inorganic nitrogen (mg NiL) 055 0.52 0.38 0.023 0,42 0,45 0.037 0.16

Silicon (mg Si02/L) 0.12 0.81 0.70 1.63 0.12 0.28 0.54 0.14

1\Ap,>:)r"I t~'t'rH"'l;':>1"'qtl1'rJ> H'~~" __"'Y~'_'~'~ 9.0 W.3 19.3 23.9 1'1.2 17.5 21.1 1:.1

Minimum temperature Cc) 5.6 5.5 18.1 21.2 10.8 15.3 18.8 153

Maximum temperature eC) 13.6 13.4 20.5 25.2 20.7 20.6 22.0 20.7

Mean surface irradiance (Ilmo!/m2/s) 800 880 1,039 883 1,241 1.009 916 546

Minimum irradiance (Ilmolim2/s) 259 280 466 305 862 255 107 223

Maximum irradiance (~moVm2/s) 1,108 1,325 1,485 1.097 1,405 1,456 1.274 906

Duration of experiment (days) 23 15 II 14 21 16 12 14

Integrated sample from the 10 microcosms

Beginning of Experiments End of Experiments mm==l,

Prior to Treatment 1 Day PT1 Untreated Controls Treated Microcosms

Table 2. Phytoplankton biomass (as chlorophyll a), diversity (Shannon-Weaver), and primary production

(photosynthetic activity) in the microcosms at the beginning and end of the experiments. Primary production

is that in the atrazine treated microcosms expressed as the percentage of primary production in the control

microcosms. (study table 2)

Chi a Diversity pp2 response ChI a Diversity Chla Diversity PP response index H' index H' index H'

Experiment (llglL) (bits/ind.) (% of control) (llglL) (bits/ind.) tug/L) (bits/ind.) (% of control)

March 1997 4.70 0.88 90 7.06 1.49 7.06 1.60 92

April 1997 2.33 1.10 77 7.73 1.72 9.18 1.69 119

June 1997 1.15 2.20 101 12.93 2.53 12.69 2.27 56

August 1997 2.05 1.80 85 4.59 2.76 4.64 2,46 85

May 1998 3.23 Ll4 96 11.09 1.52 11.70 1.22 97

June 1998 1.57 2.11 102 7.50 2.14 7.73 2.03 92

July 1998 2.01 2.94 95 1538 2.25 20.74 2,46 109

September 1998 6.83 1.90 86 13,70 3.87 13.80 3.74 72

Posttreatment.

2 Primary production.

- 8 -
Table 3. Response of dominant species (i.e. >100 individuals/ml) to 10 J.tg/L atrazine in natural phytoplankton

microcosms.(results from Mann-Whitney nonparametric test at experiment termination). (study table 3)1.2

nob' ~",,·d.., MJ,c1l AI";] luf~' Au!..,1 MJy Jw", July !>.."k.1ttl>.-, t~7 1m 19'91 1991 199~ l-Nll l-;1j~ J99~

0, liMtrlic';;J -{95~t: -N~'J (l .. ':')*>i-)

(y.. Il~>lUo..14't U: lJ.~aB$JiruL'J (I

JL mi,.,,, .. ~SS(;-1:' (l (\ (l (l

<'-.1;' l,tOI>h)'-\:.k' 9.. mJ'",'liJ 'f.j1 .....nN. (l (I t i'~"".i a -t ('l~••,)

(h-hro"""""",s". +(~~l..;. .. ~'J..~~l.j ,j _,:~~IlJ~,) (\ + (~']O/l.j (I

1). h}'~:it:..clJJ.·:,-,J, t (91',; -t .:llS~'i) a -\~""'iJ -~'l4~:'J + \.)~-;~ a

0 .. y,\01'Itl"',':.: Di!t(Jj'''....m 31'. II -N4Cji1

.<.",,11 n..8'-·nJI~ "'u" t I'll",.) -+ {'~5':l) (I (l -\\}')"..;, -('Il'J\'j t ('lil'.'.)

X,at lI1~).pk)..x·~t: 1""iirJlUtmtJ i'JM....'p:mu IJ -~'}~':..) ('I

S. ";pi!"" -{9ol'.•)

S. mim;(!t!..\' (\ -\~lt:t:l -\';J:l";'i) (I -,:9~i,:,

.~. fo"""", ([

D. t:i~"'wfsm: (I (I II

El.s:.. il14ri";')pk)',-v..:h': r. ,~rownL7l\i\- -+ \'}5~.-) (J II

.'\'. wc·i4-'W';,vi.s

N i(zs..'1t£i ,\ I), -t N7~i)

S. at'.\ (I (J II

.f.J• .w'~' ... o'J~(jRoim;J

,"nAyr..l ian,'e.ol:,uw ()

CiliIMyJ~ ...ux h I)f', -:iS~,) -\~,~',,; (l (\ q II

C. !tlJJgurifo' --';%~t.) a -\YS%.;1 (I (l (I -\~9<.') (I

(1ku-:)I)lt)..,"'.j~ C(,lt"i1JilJ:l.amAI)" a b1JJc:JlIJthi'U gt'lut';'.u,\u II

,\1it'NJ<·iru"wn pklillAm (l

.<.",,11 o..!!,"31~ ......u., (I -,'l6"i,; II -{'N'.,,; (I

};(ullrJ.\1mm ~\Il.

Zyg'''''''''k'l'lly......J\· .w. gr:J1ia'il'l't;J a -.~S,~(j,i) a "';~S~i,)

1 (+) = greater density ofindividual than in controls, (-) fewer individuals than in controls, (0) = no significant

effect, and "blank" = not present «100 individuals/mL).

2 percentages were not defined but may be l-p value.

The taxonomic class associated with each species differed among the papers in which the study

author's present this dataset (this study, Berard et aL 1999a, Berard et aL 1999b). The reviewer

presented data in the DERs as it was presented in the individual publication.

- 9-
Table 4. Short-term effects of atrazine on primary production (photosynthetic activity) of phytoplankton

previously exposed to atrazine at 10 r-tglL (expressed as r-tg atrazine/L; 95%CL < EC50 < 95%CL) and BrayCurtis indices calculated between atrazine treated and control microcosm communities. (from study table 4)

ofExperiments End ofExperiments

1 day after contamination 11 to 23 days atler contamination

Experiment EC50 Controls1 EC50 Treatedl BCe EC50 Controls1 EC50 Treated] BCl2

March 1997 34 < 38 < 42 48 < 52 56 0.97 NDJ 42 < 53 < 66 0.90

April 1997 134 < 161 < 193 108 < 139 178 0.83 280 < 308 < 338 218 < 237 < 258 0.79

June 1997 105 < 115 < 126 48 < 57 < 68 0.90 69 80 93 157 192 < 236 0.87

August 1997 41 <46<52 33 37 <42 0.92 52 < 56 < 60 43<47<51 0.71

May 1998 43 < 49 < 56 38 < 43 < 48 0.79 93 < 105 < 118 135 < 168 208 0.77

June 1998 202 269 < 358 117 < 127 < 138 0.84 44 51 <60 ND3 0.83

July 1998 64 < 68 < 72 66 < 72 < 78 0.84 42 47 < 53 69 78 < 87 081

September 1998 565 < 829 < 1,218 449 < 556 < 687 0.86 109 < 149 204 421 < 589 < 823 0.74 I

I Based on an integrated sample from the five replicate microcosms by treatement treated)

2 Calculated for the mean density ofthe five replicate microcosms by treatment (control, atrazine treated)

3 Not determined due to protocol error.

- 10-
Table 5. Correlation coefficients between environmental parameters and characteristics of phytoplankton

communities in microcosms; CPAnal sis, Pearson correlation matrix. stud Table 51

L L Parameter H' Aend "og og og

EC50CI EC50AI EC50Cend

Diversity (H') at start r = -0.71

n=7

p = 0.05

Diversity (H') Cend r = 0.68 r 0.63

n 8 n=8

p =0.05 P 0.09

Diversity (H') Aend r 0.68 r =0.68

n 8 n=8

p = 0.05 p 0.05

ChI a Aend

Start irradiance (day I of experiment) r = 0.84 r 0.81

n 7 n=7

p = 0.05 P 0.05

Mean irradiance during experiment r = -0.76

n 8

p = 0.05

Mean temperature r = -0.83

n=7

p 0.05

Initial concentrations orthophosphates r = 0.77

n 7

p = 0.05

C = controls, A = treated at 10 1!g!L atrazine, Log EC50C I short-term sensitivity of control microcosm

phytoplankton to atrazine after 1 day of untreated incubation, Log EC50A I = short-term sensitivity oftreated

microcosm phytoplankton at I day posttreatment, Log EC50Cend short-term sensitivity of control microcosm

phytoplankton at end of experiments (11-23 days of incubation).

- 11 -
Data Summary Table'.

I Time Weighted I

Magnitude of Nominal Day ofStudy Average Atrazine Did Recovery Atrazine Concentration

Effect Effect relative to ,Concentrati Effect from Day Oto Occur - ifso, Comments

controls

on (p,gIL)1 Occurred When Effect when?

I

Occurred

Magnitude of

effects

calculated

from results

in Table 2.

Reviewer

assumes data

End of is based on

experiments mean values,

no statistics

Change in (11-23 days): No (effects provided. Up to 35% 12 (35% phytoplankton increase and up to 10 flglL increase, July Not calculable observed at

biomass end of Effect

(chlorophyll a) 2% decrease 98) and 11 experiment) (increase, (2%

decrease, decrease, no

June 97) change)

relative to the

control

varied

depending on

experimental

month.

Maximum

values are

reported here.

Magnitude of

effects

calculated

from results

in Table 2.

End of

experiments Based on

(11-23 days): No (effects mean values,

Change in Up to 9% increase 12 (9% observed at

no statistics

phytoplankton and up to 20% 10 flglL increase, July Not calculable end of provided.

diversity (H') decrease 98) and 21 experiment) (20% Effect

decrease, (increase,

May 98) decrease, no

change)

relative to the

control

varied

depending on

- 12 -
Time Weighted

Magnitude of Nominal Day ofStudy Average Atrazine Did Recovery Atrazine Concentration Effect Effect relative to IConcentral/

Effect from Day 0 to Occur - ifso, Comments

controls

on (llgIL/ Occul'I'ed When Effect when?

I

I

Occul'I'ed

i

I i experimental

month.

MuxluilHil

values are

reported here.

Magnitude of

effects

calculated

from results

in Table 2.

Reviewer

assumes data

is based on

mean values,

no statistics

provided.

Varied from Effect

Varied (up to Unknown 1 day or end Yes and No (increase,

Change in primary 19% increase and (reviewer of Not calculable (depends on decrease, no

productivity up to 44% assumes 10 experiments experimental change)

decrease) flglL) (11-23 days) month) relative to the

control

varied

depending on

experimental

month.

Atrazine

treatments in

March 97,

Aug 97, and

Sept 97

showed

I I I

decreased

primary

- 13 -
IMag.aude of Nominal I Time Weighte~ 1

Average Atrazme D'd R

Atrazine Day ofStudy • I ecovery

Effect Effect relative to Effect ConcentratIOn 0 if Comments Concentrati fi DOt ccur- I so,

controls Occurred rom ay 0 ?

on (p.gILl When Effect when.

Occurred

productivity

compared to

the control on

day after

contaminatio

n and final

day of

experiment.

Species

specific

effects shown

Change in No (effects in Table 3.

End of phytoplankton Unknown 10 flg/L experiments Not calculable observed at p-value not community species (11-23 days) end of reported. composition experiment) Percentages

reported in

Table 3 may

i

be I-p value.

i

- 14 -
Time Weighted

Magnitude of Nominal Day ofStudy Average Atrazine Did Recovery Atrazine Concentration Effect Effect relative to Concentrati Effect fromDayOto Occur- ifso, Comments

controls

on ()lglLl Occurred When Effect when?

Occurred I

Magnitude of

effects

c~lcu!3.ted

from results

in Table 4

(BCI after

one day of

exposure -

BCI at end of

experiment).

Based on

mean values.

No statistics

provided for

Change in End of (l) individual

phytoplankton experiments No (effects experiments:

comparison community Up to 21% 10 IlgiL (11-23 days): Not calculable observed at ofBCIone

composition (Bray- 14 days (21% end of day after

Curtis indices) increase, Aug experiment) contaminatio 97) n to BCI end

of

experiments

or (2)

comparison

ofBCI

calculated

between

treatment and

control to

BCI

calculated

within

controls (data

not provided)

I I

- 15 -
ECso is

lowest of all

(.\Ynp-rirnp-nt",J

---r -- ~~---~-*~~

co=l Did Recovery

Occur - ifso,

when?

Not able to

determine 3 hours Unknown

J._.. Time Weighted

Nominal Average Atrazine

Atrazine ~;} orStudy Concentration

Concentrati .0 ec d from Day 0 to

on (figIL/ . ccurre When Effect

r-f_ccurred

I i

Magnitude of

Effect relative to

controls

50% (ECso)

Effect

Decreased primary

productivity

dates (8)

measured

twice for

each (one day

after

microcosm

treatment and

at the end of

the

experiment).

I

i ECsos ranged

! from 37 to

I

·829 Jlg

. atrazine/L

..~._ I!(table 4) I --~

At 11-23 days post treatment (experiment termination), atrazine concentrations in the water were 90-95% ofthe

initial nominal 10 !J.gL concentration.

2 The ECso value of37 f.lg/l was the lowest value from the August 1997 experiment at the beginning ofthe

experiment (measured as the endpoint of 3-hour short term atrazine sensitivity by a log linear interpolation ofthe

dose-respose curves). Exposure concentrations are uncertain for the short-term toxicity testing on communities

from microcosms that were pre-exposed to atrazine (nominal = 10 f.lg/L). For these tests, 9 ml water samples from

the atrazine treated microcosms were added to Iml of an atrazine concentration ranging from 100-200 f.lg/L.

Therefore, the actual concentrations would have been higher than the nominal values reported for the short-term

experiments (i.e. 100 and 200 JlgIL, other concentrations not reported).
Reviewer Comments:

The microcosms were filled with water from Lake Geneva previously reported (Blanc et al.,

1997,1998,1999) to contain atrazine at 0.03-0.04 !1g/L in the euphotic zone ofthe lake water

from 1996 to 1998. It is not stated whether the lake water used for the experiments was tested to

establish that it was not contaminated with any pollutants. If atrazine was present in the

microcosms, then it could confound the results ofthis study. Moreover, water used for

individual experiments was collected at different times during the period from 1997 to 1998, thus

:mv";.1. temnoral variabilitv.. in the backl!round _ concentration of atrazine would further confound the

results. Atrazine was measured at the end ofthe microcosm experiments and was 90-95% of

nominal, thus it is likely that any background contamination of atrazine was probably low.

A complete description of installation ofthe microcosms prior to use was referenced (Berard et

al., 1999a), but not presented in this article. Any acclimation ofthe microcosms prior to use was

not reported.

The microcosms did not contain a sediment layer.

As a microcosm study, there may be little relevance ofthe results to natural systems despite the

natural source ofthe communities. The small size ofthe containers (5 L) may have introduced

container edge effects and lessened mixing.

Some of the methods were not reported, including those used to monitor water temperature and

surface irradiance, quantification of chlorophyll a, and measurement of atrazine. Additionally,

methods used to determine primary production in the microcosms was not reported (i.e. the

results presented in Table 2). It is unclear whether the methodology was similar to or different

than that reported for the short-term toxicity testing. The reviewer assumes that the results

presented in Table 2 represent primary production at a single atrazine concentration, not the

concentration series of exposure used in the short-term toxicity testing. The reviewer assumes

that exposure was the same as the exposure in the microcosm (10 !1g atrazine/L).

Only a single atrazine concentration was tested for the microcosm experiments (10 !1g1L).

The study authors indicate that the experiments were conducted for 11-23 days with termination

occurring when relative growth rate of control cultures declined; just before the stationary phase

of phytoplankton growth (duration of each experiment is reported in Table 1).

The Mann-Whitney results are particularly difficult to interpret. It is unclear whether the

reported values are one minus the p-value or the percent change in the species density. More

complete explanations ofthe methods and statistical analyses would have been helpful.
The Bray-Curtis index (BCI) was not well-described, which may lead to misinterpretation of the

results. The BCI was used as a metric to compare community structure; however, it is not clear

exactly what the BCl value represented because the BCI was not defined with an equation and

the set ofspecies or taxonomic groups used to calculate the BCI were not clearly described.

Quantitative results, either tabular or plotted, were not presented to support the reported ECso

values for the short-term effects of atrazine on primary productivity (photosynthetic activity).

For short-term toxicity tests, only two of the concentrations used were reported and the number

of concentrations tested was not stated. The range of concentrations tested was 100 to 200 flg

atrazine/L.

Exposure concentrations are uncertain for the short-term toxicity testing on communities from

microcosms that were pre-exposed to atrazine (nominal 10 Jlg/L). For these tests, 9 ml water

samples from the atrazine treated microcosms were added to 1ml of an atrazine concentration

ranging from 100-200 Jlg/L. Therefore, the actual concentrations would have been higher than

the nominal values reported for the short-term experiments (i.e. 100 and 200 Jlg/L, other

concentrations not reported). Additionally, the actual exposure for the short-term experiments

would not have been the same for communities from atrazine-treated mesocosms and those from

control mesocosms.

Description of Use in Document:

,Qualitative (microcosm experiments), Invalid (Short-term toxicity tests)

Rationale for Use:

Qualitative - Microcosm experiments contribute to the weight of evidence regarding effects of

atrazine on aquatic plant communities.

Invalid - Actual exposure in short-term tests is uncertain; confounded by the addition of atrazine

from water used in the treated microcosms, number oftest concentrations is unknown, and only

two ofthe test concentrations were reported (lowest and highest). In addition, a narrow range of

concentrations was tested (100-200 Jlg atrazine/L) and most ofthe reported ECso values would

have been extrapolated as they were outside the range of concentrations tested.

Limitations of Study:

There were numerous deficiencies regarding the presentation ofthe methods and results. No

effort was made to document whether other chemicals (including atrazine) were present in the

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treatment and/or control microcosms prior to experiment initiation, although atrazine was

measured in the treated microcosms at the end ofthe experiments and was ~90-95% of nominal.

Installation ofthe microcosms and any acclimation interval prior to use were not sufficiently

detailed. There was potential for bottle effects due to the small size ofthe microcosms. The

microcosms did not contain a sediment layer. Methods used to quantify atrazine and chlorophyll

a were not reported. Methods used the measure primary production ofthe microcosm

communities were not reported. There is uncertainty about p-values used for 'significant' MannWhitney test results. For the short-term toxicity tests, no results were presented to support the _,.,.__.-+".;1 L71'""' .....,,1, ... ""r. ,-.\.., ...... ,..~_~ +,.,._ +_~,.: ....~.;.,. .. 4-~ .....+ ........ro> .... t.. ...... ..,_,3 __ l~~:+..... A ........r __........ "'T ..._ ['2 t..."""'~ ..._ ..... \ ,..._...1 IvpUl L\.... u L\".....5U v aU.l""L). Ill\.,; .::UIVlt.--t.-\,,;1111 l.VAl\,.;lt..y \.\.I.:')\. vvu.,:, UQ.,:)\.tU VII llllu"u",..u. \,;APVL)U1\.< \-' l.1.VU.1.L)} Q..I.lU

was performed on communities either pre-exposed or not pre-exposed to atrazine. Actual

exposure in short-term tests is uncertain; confounded by the addition of atrazine from water used

in the treated microcosms, number oftest concentrations is unknown, and only two ofthe test

concentrations were reported (lowest and highest).

Primary Reviewer: Lynne Binari, M.S., Subcontractor to Cambridge Environmental Inc.

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 1fj;l~~4

Tertiary Reviewer: Anita Pease, M.S. Senior Biologist, EPA ~(I~/l \

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