### 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 ofphytoplankton to atrazine in nanocosms. Chemosphere. 45: 427-437. (MRID 48261101) Purpose ofReview: 

### Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associated determination ofa Level ofConcern (LOC).
Date ofReview: 

### October 7, 2009 Purpose ofStudy: 

### This study examines the potential effects ofatrazine on phytoplankton in experimental outdoor microcosms. Summary ofStudy 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., 2001b. Among those studies there is both overlap and difference in terms ofthe experimental dates presented, the endpoints measured, the level ofdetail 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 referto 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., 2001b) 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 ofLake 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 ofa 5-L Pyrex bottle containing 2.5 L ofthe phytoplankton suspension plus 2.5 L of filtered (0.8-llm 

### polycarbonate membrane) lake water. Foreach 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 ofphytoplankton growth. Growth was monitored by making daily in vivo 

### fluorescence measurements using a Turner fluorometer.
Nutrient concentrations were determined at the beginning ofeach experiment 

### (Table 1). 

### No details were provided regarding methods used to measure nutrients. Measurements ofwater temperature and surface irradiance were not described (datapresented in 

### Table 1). 

### Atrazine concentrations were determined at the end ofeach experiment by HPLC analysis (no details provided).
Biomass and taxonomy. Chlorophyll a 

### content was determined at the beginning and end ofeach 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 atthe beginning and end ofeach 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 ofa 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 atthe 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') ofalgal communities was estimated atthe 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:"') "'- --_ .... experimentusing 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 eachtreatment group (n = 5). At experiment termination, the density ofeach 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. Correlationtests 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 ofeach 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 % ofcontrols) is presented in 

### Table 2 for one day after contamination and at the end ofthe 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 ofat 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 ofthe 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 ofall 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 ofthe experiment (BCI 1 day prior to exposure - BCI end ofexperiment).
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 atthe 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 ofthe experiments (Table 4). However, the controls were less sensitive than the atrazine treated microcosm communities atthe 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 ofalgae to atrazine (p 0.09) between treated and control microcosms at the end ofthe experiments (Figure 3).· 

### Additionally, diversity (W) 

### indices determined at the end of the experiments correlated with sensitivity to atrazine at the beginning ofthe 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 ofthe 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,

### ~2 OU CLlJ \J C!J 

### o (J) ;:: 

## (a)

1000 100 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) ofphytoplankton microcosm communities (CPAnalysis, Pearson correlation matrix). (a) Treated communities at the end ofexperiment vs. control communities at beginning ofexperiment. (b) Treated communities at end ofexperiment vs. treated communities at beginning ofexperiment. (study figure 2)



### - 6

-1000



## 55 u UJ 

## (a)
20 0 15



# - 0) 10 ::L "-' Vl 

### Q +- 5 c 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 diversity H': r::;0,97 p=O,OOl n=8 0 ::J 4 1J**:~** 1J 3 c 

### "- 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 ofphytoplankton microcosm communities at end ofexperiments, 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!, watertemperature 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>** 9.0 W.3 19.3 23.9 1'1.2 17.5 21.1 1:.1**H'~~"** __**"'Y~'_'~'~** 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 ofexperiment (days) 23 15 II 14 21 16 12 14

Integrated sample from the 10 microcosms

Beginning ofExperiments End ofExperiments 

# mm==l,
Prior to Treatment 1 Day PT 1 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 ofthe experiments. Primary production is that in the atrazine treated microcosms expressed as the percentage ofprimary 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.) (% ofcontrol) (llglL) (bits/ind.) tug/L) (bits/ind.) (% ofcontrol) 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 ofdominant species (i.e. >100 individuals/ml) to 10 J.tg/L atrazine in natural phytoplankton microcosms .(results from Mann-Whitney nonparametric test at experimenttermination). (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" = notpresent «100 individuals/mL). 2 percentages were not defined butmay be l-p value. 

### The taxonomic class associated with each species differed among the papers in which the study author's presentthis dataset (this study, Berard et aL 1999a, Berard et aL 1999b). The reviewer presented data in the DERs as itwas presented in the individual publication.



### - 9-Table 4. Short-term effects ofatrazine on primary production (photosynthetic activity) ofphytoplankton 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 Controls 1 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 ofphytoplankton 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

Startirradiance (day I ofexperiment) 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 ofcontrol microcosm phytoplankton to atrazine after 1 day ofuntreated incubation, Log EC50AI = short-term sensitivity oftreated microcosm phytoplankton at I day posttreatment, Log EC50Cend short-term sensitivity ofcontrol microcosm phytoplankton at end ofexperiments (11-23 days ofincubation).



### - 11

-

### Data Summary Table' 

# . 

# I Time Weighted I Magnitude of Nominal Day of Study AverageAtrazine DidRecovery Atrazine Concentration Effect Effectrelative to
, Concentrati Effect from Day Oto Occur

- if so, 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 of Study AverageAtrazine DidRecovery Atrazine Concentration Effect Effectrelative to 

# IConcentral/ Effect from Day 0 to Occur

- 

### if so, 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 AverageAtrazme D'dR Atrazine Day of Study • I ecovery Effect Effectrelative 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 of Study AverageAtrazine DidRecovery Atrazine Concentration Effect Effect relative to Concentrati Effect fromDayOto Occur

- 

### if so, 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 ofall (.\Ynp-rirnp-nt",J ---r --**~~---~-*~~** 

# co=l DidRecovery Occur

- 

### if so, when?

Not able to determine Unknown 3 hours 

# J._.. Time Weighted Nominal AverageAtrazine Atrazine

### ~;} orStudy Concentration Concentrati

.0 ec d from Day 0to on (figIL/

. ccurre When Effect r-f_ccurred

I 

## i Magnitude of Effectrelativeto controls

50% (ECso) Effect

Decreased primary productivity dates (8) measured twice for each (one day after microcosm treatment and atthe end of the experiment). I 

### iECsos 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 of3-hour shortterm atrazine sensitivityby 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). Forthese tests, 9 ml water samples from the atrazine treated microcosms were added to Iml ofan atrazine concentration ranging from 100-200 f.lg/L. Therefore, the actual concentrations would have been higher thanthe 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 Genevapreviously reported (Blanc 

### etal., 1997,1998,1999) to contain atrazine at 0.03-0.04 !1g/L inthe euphotic zone ofthe lakewater from 1996 to 1998. It 

### is not stated whetherthe lake waterused for the experiments was testedto establishthat it was not contaminated with any pollutants. Ifatrazine was present in the microcosms, then it could confoundthe results ofthis study. Moreover, water used for individual experiments was collected at different times during the period from 1997 to 1998, thus :mv temnoral variabilitv inthe backl!round concentration ofatrazine would further confound the ";.1.

.. _ 

### results. Atrazine was measured atthe end ofthe microcosm experiments and was 90-95% of nominal, thus it is likely that any background contamination ofatrazine was probably low.
A complete description ofinstallation ofthe microcosms priorto use was referenced (Berard 

### et al., 1999a), but notpresented in this article. Any acclimation ofthe microcosms priorto use was notreported.
The microcosms did notcontain a sedimentlayer.
As amicrocosm 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 ofthe methods were not reported, including those used to monitor water temperature and surface irradiance, quantification ofchlorophyll 

### a, and measurement ofatrazine. 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 ofexposure used in the short-term toxicity testing. The reviewer assumes that exposure was the same as the exposure inthe 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 ofcontrol cultures declined; just before the stationary phase ofphytoplankton growth (duration ofeach experiment is reported in Table 1).


### The Mann-Whitney results are particularly difficult to interpret. It 

### is unclear whetherthe reported values are one minus the p-value orthe 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 ofthe 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 ofatrazine on primary productivity (photosynthetic activity).
For short-term toxicity tests, only two ofthe concentrations used were reported and the number ofconcentrations tested was not stated. The range ofconcentrations 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 ofUse in Document: ,Qualitative (microcosm experiments), 

### Invalid (Short-termtoxicity tests)


### Rationale for Use: Qualitative

- Microcosm experiments contribute to the weight ofevidence regarding effects of atrazine on aquatic plant communities.
Invalid

- Actual exposure in short-term tests is uncertain; confounded by the addition ofatrazine from waterused 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 ofconcentrations tested.


### Limitations ofStudy: 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 priorto experiment initiation, although atrazine was measured inthe treated microcosms at the end ofthe experiments and was**~90-95%** 

### ofnominal. Installation ofthe microcosms and any acclimation interval priorto use were not sufficiently detailed. There was potential for bottle effects due to the small size ofthe microcosms. The microcosms did not contain a sedimentlayer. Methods used to quantify atrazine and chlorophyll 

### a were not reported. Methods used the measure primary production ofthe microcosm communities were notreported. There is uncertainty about p-values used for 'significant' MannWhitney test results. For the short-termtoxicity tests, no results were presented to supportthe

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_ ['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 eitherpre-exposedornot pre-exposedto atrazine. Actual exposure in short-term tests is uncertain; confounded by the addition ofatrazine 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., Subcontractorto Cambridge Environmental Inc. Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA

# 1fj;l~**~4** Tertiary Reviewer: Anita Pease, M.S. SeniorBiologist, EPA

# ~(I~ 

# /l\

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