Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Berard, A., 1. Peite, and J. Druart. 1999. Seasonal variations in the sensitivity of Lakt:

Geneva phytoplankton community structure to atrazine. Arch. Hydrobiol. 145(3):277-

295. (MP"tP i.t(~1..bH63)

Purpose of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this

study were also used to parameterize the Comprehensive Aquatic System Model (CASM)

and establish the model-associated LOC for atrazine.

Date of Review:

December 18, 2008

Purpose of Study:

This study examines the potential effects of atrazine to aquatic plants in lake enclosures

(outdoor microcosms).

Summary of Study Findings:

Methods: Both outdoor (microcosm) and laboratory (monoculture) studies were

performed. The laboratory monoculture experiments will be briefly mentioned, but

details ofthose methods will not be reproduced here, in order to focus on the microcosm

experiments.

Twelve (12) experiments were conducted on phytoplankton communities in Lake

Geneva, Switzerland in outdoor microcosms treated with 10 )..tg/L atrazine, during the

spring and summer of 1995, 1996, 1997, and 1998. The authors reference 1996 and 1998

reports that documented background atrazine concentrations of 0.03 to 0.04 )..tg/L in the

euphotic zone ofthe whole lake. For microcosms, phytoplankton communities were

sampled from the middle of Lake Geneva, from the euphotic layer, using a hand pump to

avoid cell damage. Samples were filtered through a 200 )..tm mesh and remaining

zooplankton were killed by oxygen depletion via bubbling pure nitrogen through the

samples for 1 hour. A preliminary experiment (results not shown in the paper) showed

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2087805
that the oxygen depletion did not affect the phytoplankton growth. The resulting samples

represented phytoplankton inocula for the microcosms.

Microcosms were 5 L Pyrex bottles. They were initialized with 2.5 L of plankton

suspension (inocula) and 2.5 L of natural lake water from the same sampling location,

that had been filtered through a 0.9 11m polycarbonate membrane filter. Nutrient

concentrations were measured at the beginning ofthe experiments. Water samples were

laken lhruugh a silil;ull l;CtlJ <ina glass ana silic0il tubing. The ii1iCtGC0SiilS 'vv'crc

continuously aerated to prevent sedimentation and depletion of carbonates. Microcosms

were placed in a quiet area of Lake Geneva at a depth of 1.5 m, under natural temperature

and irradiance conditions. See Figure I for the experiment design. Experiments ran for

10-21 days, and were stopped when the relative growth rate of controls declined.

Atrazine (98%, Greyhound/Chern service, UK) was dissolved in double-distilled water

with continuous stirring for 48 hours to achieve a 10 mg/mL stock solution. No solvents

were used. The effective concentration ofthe stock was checked by high performance

liquid chromatography. Solution was added to test microcosms to achieve 10 I1g/L

atrazine, with either 3 or 5 replicates for tests and controls, depending on the experiment.

Plankton biomass was estimated daily by in vivo fluorescence measurements taken with a

Turner Fluorimeter. Community composition was measured by cell counting in initial

and final samples from each experiment, using the Utermohl inverted microscope

technique and counting a minimum of 400 individuals per dominant species (more than

100 individuals per mL), which gave an accuracy of about ± I0%. Changes in cell

densities for each species were the parameters ofinterest.

Initial and final algal community diversities were estimated using the Shannon-Weaver

equation. The relative densities (percentages) ofthe inocula algal classes (except for

Bacillariophiceae, which was separated into pennate and centric diatoms) were analyzed

by correspondence analysis. The density of each dominant species in the contaminated

communities was compared to control using the Mann-Whitney non-parametric test (also

called the Wilcoxon rank-sum test, which tests the null hypothesis that the samples are

from the same underlying population). Differences between contaminated and control

communities were described with the Bray-Curtis dissimilarity index (BCI), calculated

for the mean density over replicate communities. Significance of BCI differences was

assessed using the Mann-Whitney test (comparing to within-control differences).

Four ofthe dominant algal species were tested for sensitivity to atrazine in laboratory

monocultures: Chlorella vulgaris Beij. SHL 108b, Nitzschia sp. SHL 139/1, Fragilaria

crotonensis Kitton, and Oscillatoria limnetica Lemmermann. The former two were part

ofthe authors' existing laboratory culture collection isolated from Lake Geneva, and the

latter two were obtained from outside culture collections, isolated from unpolluted

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aquatic ecosystems. Details will not be reproduced here, but are provided in the original

paper.

Results: Both outdoor (microcosm) and laboratory (monoculture) studies were

performed. The laboratory monoculture results will be briefly mentioned, but details of

those methods will not be reproduced here.

The 12 microcosm experiments were performed at different times of year and under

different environmental conditions in Lake Geneva. Figure 2 shows the seasonal pattern

of Secchi transparency, which is routinely monitored in the lake by the lead author's lab,

and the PEG-model plankton succession phase. Figure 3 shows the results of

correspondence analysis on the species composition of algal inocula (the first three

directions explained over 74% ofthe variance). Table 1 shows the initial chlorophyll a,

Shannon diversity index, and nutrient concentrations, as well as duration and range of

temperature and irradiance conditions, for each experiment.

The four March and April experiments were characterized by the spring bloom

phytoplankton succession stage - in particular, the experiment of April 1997 began

during the first days of zooplankton grazing just after the time ofmaximal phytoplankton

biomass. The diversity in these inocula communities were low, since they were

dominated by small fast-growing centric diatoms (Stephanodiscus minutulus,

Stephanodiscus noeastrae and Cyclotella sp.), cryptophytes (Rhodomonas minuta,

Rhodomonas minuta var. nannoplanctica, Cryptomonas sp.) and some pennate diatoms

(Asterionella Formosa, Fragilaria crotonensis and Melosira varians). April 1996 and

March 1997 experiments, which occurred closest to the middle ofthe spring bloom, had

depleted nutrients compared to other experiments. June 1995 and June 1996

experiments started during the "clear water" phase. The inocula communities for these

experiments had low biomass and were dominated by Chlorophyceae (Chlorella vulgaris,

Elakatothrix gelatinosa, and some Eudorina elegans) and cryptophytes. Silica

concentrations were higher in these experiments because of nutrient recycling induced by

intense grazing. June 1997 and July 1995 experiments started at the end ofthe clear

water phase, and had more diverse inocula dominated by pennate diatoms, cryptophytes,

desmids (Staurastrum sp.) and dinoflagellates. These experiments were less well

explained by correspondence analysis (see Figure 3) and June 1997 showed some

closeness to the clear water experiments. August and September experiments started at

the end ofsummer succession, and inocula were dominated by chlorophytes,

dinoflagellates, chrysophytes, and filamentous zygnematophyceae, with low

concentrations ofsilica. Average temperatures for the different experiments ranged from

below 13°C during the spring experiments to above 15°C during the summer and early

autumn experiments. lrradiance was highest during the summer and lowest during the

fall, with intermediate irradiance in the spring.

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The Bray-Curtis index (BCI) was used to compare phytoplankton communities between

atrazine treatment and control. The index was close to one, indicated high similarity, at

the beginning ofthe experiments, but became lower at the end ofthe experiments

because of increased differences between control and test communities (values shown in

Figure 4). (It was implied that the values in Figure 4 are end-of-experiment BCls

between mean treatment densities and mean control densities, but this is not explicitly

stated. Additionally, significant Mann-Whitney results are reported as large percentages,

and it is never stated what they represent.) The clear water (June) experiments showed

the most dissimJlanty trom treatment, and the spring bioom experiments showed the most

similarity. Differences in BCI ofthe spring and summer were significant (MannWhitney 96%). Significant differences were tested by applying a Mann-Whitney test

between BCI within the control group and BCI between controls and treatments. This

found significant treatment effects for all three June experiments, as well as September

1995 and April 1996 (Mann-Whitney 95%) and in August 1995 (Mann-Whitney 90%).

In each experiment, atrazine treatment significantly affected the density oftwo or more

species (see Table 2). In general, atrazine treatment increased the density of

Rhodomonas minuta var. nannoplantica, Ochromonas sp., and Asterionellaformosa, and

decreased the density of Stephanodiscus minutulus and Chlorella vulgaris. Other species

showed different types ofresponses in different experiments: Oscillatoria limnetica

decreased with atrazine treatment in March 1997 and April 1996 experiments, but grew

well with treatment in other experiments, while Desmarella brachycalax and some

pennate diatoms' responses to treatment also varied among experiments.

In the laboratory monocultures, atrazine at 10 ~g/L levels inhibited Chlorella vulgaris

and Fragilaria crotonensis slightly but significantly, and had no effect on Oscillatoria

limnetica or Nitzschia sp. A figure of the results is provided in the original article but is

not reproduced here.

Beginning vs. end of experiment biomass and diversity measures are shown in Table 3

for control and treatment groups. (It is assumed that the values are replicate means, but

this is not explicitly stated.)

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3 to 5 replicates per treatment

(depending on experiment)

quiet zone of lake Geneva

ltatural <:onditions oflighT

mulremperature ,f~~;a~~ l·Sffi

t

GFiC /iller

continuous "" aeration ~~~....

r air outflow

~==~sampling

5 liter Pyrex flask

Figure 1. Design of the outdoor microcosms in Lake Geneva.

5
days

~~ <0 1;;",,,- "'I;; fii (X] to ~l{l '" ~'" IQ

u') ll) '" ~'" '" Q! gj~ U'l ", Ol

~8 '" experiments § ... -.. "" til ",,,,,,, '" --'" "" m 't; ~~ 0> ....

Q~t 0 0 ~~ ..... o~~t;1Q~ 0 ..

w~ i?ig,~ il> ~~~ (i}~ ... ........ q; ~Q; go U'I ..... N O-SJ N ..... ""::! C\I C\l o~!! q;:

&

Figure 2. Secchi transparency measured in Lake Geneva by lnstitut National de Recherche

Agronomique, Station d'Hydrobiologie Lacustre. Starting dates of the twelve microcosm

experiments shown by arrows.

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(20.9% 01 variance)

his 1

(37.9% 01

wuiatlce)

@5prinll bloom

OdeMwaler phase

01" summersuccession

OwiIUIllI'll8I S\lCQIssion

olad SlJCCe$Sion

(15.9% oIvarlance)

1

I 2>1gn8llla!Ol>t>yto.e

2 c:hl<>nlphyoeae

3 Saa"Uadop/lyceu OInI,.18s •

4 BaciUattopl\pae ~nalas

S~a.

6 ClJP\opllyCe.e

1DMfl~e

8 Cyanollat1erfa

9~!lyQOaO .'

Figure 3. Correspondence analysis of inoculum characteristics of the 12 microcosm experiments. A:

projection in the plane defined by axes 1 and 2; B: projection in the plane defined by axes 1 and 3.

7
Bray-Curtis index

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spring bloom expertments

clear waler phesEI experiments

>?:t~ SUnlmer and fall experiments

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~~1;\j ~' ..-,.. ?:~;! : ;:;;':~

fiii:.: 0,4 ~~ $f;,$)

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Figure 4. Bray-Curtis indices of the 12 microcosm experiments. The Bray-Curtis index was

calculated for the mean density of replicate communities, and the significance of difference between

control and treatment was tested with a Mann-Whitney test between indices calculated among

controls (C-C) and indices calculated between controls and treatments (C-A). **: C-C/C-A MannWhitney test was 95%. *: C-C/C-A Mann-Whitney test was 90%.

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Table 1. Biomass and diversity of the inocula used in the 12 microcosm experiments. Nutrient concentrations in the natural water used as culture

medium for each microcosm experiment. Mean temperatures and average surface irradiance of microcosm cultures. Duration of each microcosm

experiment.

Experiment Jun-95 Jul-95 Aug-95 Sep-95 Apr-96 Jun-96 Sep-96 Mar-97 Apr-97 .1un-97 Aug-97 Mar-98

Chlorophyll-a ().lg/L) 0.48 1.83 0.81 2.61 3.54 0.66 3.2 4.7 2.33 1.15 2.05 1.75

Shannon diversity index (H': 1.95 1.7 2.63 2.63 1.73 1.94 2.42 0.88 1.1 2.2 1.8 1.4

bits/individual)

Orthophosphates (mg P/L) 0.002 0.004 0.006 0.001 0.002 0.002 0.004 0.006 0.012 0.003 0.001 0.018

Inorganic nitrogen (mg N/L) 0.35 0.58 0.24 0.27 0.43 0.54 0.24 0.55 0.52 0.38 0.207 0.55

Silica (mg Si02/L) 1.3 1.21 0.13 <0.02 0.34 1.03 0.34 0.12 0.81 0.7 0.1 1.08

Temperature (0C)

Mean 15.9 20.9 22.4 16.8 12.6 19.1 16 9 10.3 19.3 23.9 9.3

Minimum 9.6 15.8 18.9 14.8 10.4 15.9 14.2 5.6 5.5 18.1 21.2 4.4

Maximum 20.8 24.5 25.6 19.6 16.3 25 19.4 13.6 13.4 20.5 25.2 12.4

Surface irradiance ().lmollm2

/s)

Mean 959 1218 1022 552 830 948 590 800 879 1038 883 616

Minimum 197 559 409 109 295 186 176 259 279 466 305 70

Maximum 1501 1480 1361 999 1314 1475 988 1107 1325 1485 1149 892

Duration (days) 21 21 17 21 14 18 18 21 13 I 1 14 10

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Table 2. Response of the dominant species to 10 ~g/L atrazine in microcosms with natural phytoplankton communities. Mann-Whitney test with 3

replicates for 1995 and 5 replicates for 1996-1998, investigated at end of each experiment: +: greater density of individuals than in controls; 0: no

significant effect; -: lower density of individuals than in controls. Blank field: species not considered, <100 individuals/mL.

Class Species Jun-95 Jul-95 Aug-95 Sep-95 Apr-96 Jun-96 Sep-96 Mar-97 Apr-97 J~'n-97 Aug-97 Mar-98

Cyanobacteria Oscillatoria limnetica + (>90%) + (>90%) + (90%) - (97%) 0 0 -(95%)

Pseudanabaena galeata - (95%)

Cryptophyceae Rhodomonas minuta + (85%) - (93%)

Rhodomonas minuta var.

nanno. + (90%) +(97%) + (97%) + (93%) 0 0

Chl)'sophyceae Oehromonas sp. 0 0 0 +(90%) 0 0 + (95%) +(98%) 0 -(98%) +(99%)

Desmarella brachycalyx 0 -(90%) +(96%) + (95%) + (85%) 0 - (88%)

Dinobryon sp. 0 0

small flagellate cells 0 + (95%) + (95%) 0

Xanthophyceae Tribonema ambiguum 0

Stephanodiscus minutulus 0 0 -(91%) - (90%) -(97%)

Asterionella formosa 0 0 +(90%) +(94%) +(95%) 0 0 0

Dialoma elongatum + (98%) 0

Bacillariophyceae Fragilaria crotonensis + (90%) 0 0 0 0 0 + (95%) +195%) 0

Nitzschia acicularis +(90%) 0 0 +(97%) 0

Nitzschia sp. 0 + 197%)

Synedra acus 0 0 0 0 0 0

Synedra acus var.

angustissima + (85%) + (89%) 0

Chlorophyceae Ankyra lanceolata 0

Chlamydomonas spp. 0 0 - (85%) -(98%)

Chlorella vulgaris -(90%) 0 - (90%) -(99%) - (97%) - (96%) 0 - (95%) 0 -(95%) 0 -(93%)

Coelastrum sp. 0

Elakatothrix gelatinosa + (96%)

Monoraphidium minutum 0

small flagellate cells + (90%) 0 0 -(97%) 0 -(96%) 0

Zygnematophyceae Staurastrum sp. +(90%)

Mougcotia gracillima 0 0 0 () +(89%) -_._----

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Table 3. Biomass (chlorophyll a concentration) and diversity (Shannon diversity index in bits per individual) calculated at the end of each of the 12

microcosm experiments.

Experiment Jun-95 Jul-95 Aug-95 Sep-95 Apr-96 Jun-96 Sep-96 Mar-97 Apr-97 Jun-97 Aug-97 Mar-98

Chl-a control (Ilg/L) 72.15 9.03 11.66 36.22 16.94 13.41 16.58 7.06 7.73 12.93 4.59 11.49

Chl-a atrazine (Ilg/L) 10.43 7.42 10.95 38.09 16.28 11.72 24.27 7.06 9.18 12.69 4.64 11.36

H' control (bits/ind.) 0.023 0.69 1.5 2.03 1.73 0.99 1.51 1.49 1.72 2.53 2.76 1.58

H' atrazine (bits/ind.) 0.35 I.2 1.83 2.38 2.37 1.13 1.44 1.6 1.69 2.27 2.46 1.84

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Data Summary Table

Effect Magnitude Nominal Day o/Study Time Weighted Did Recovery Comments

ofEffect Atrazine tlfect Average Occur- !lso,

relative to Concentrati Occurred Atrazine when?

controls on (flg/L) Concentration

Fom Da)' 0 to

When Effect

Occurred

Change in Up to 85% 10 flg/L 18 to 21 NR /10 flg/L No (effects Magnitudes from

phytoplankton days (ends noted at end of June 1995,

biomass of experiments) September 1996,

experiments) and June 1997

experiments

(Table 3)

Change in >1,400% 10 flg/L 21,18, and NR/ 10 flg/L No (effects Magnitudes from

phytoplankton increase, 7% 21 (ends of noted at end of June 1995,

community decrease, experiments) experiments) September 1996,

diversity and 7% and June 1997

(Shannon Increase experiments

index) (Table 3)

Change in NR 10 flg/L oto 21 days NR /10 flg/L No (effects Species-specific

phytoplankton (ends of noted at end of effects shown in

community experiments) experiments) Table 2.

species

composition

Note: NR=Not Reported

Reviewer Comments:

Some aspects of interpretation were made difficult by differences in methods and

presentations used by these French authors that are not common in the United States,

such as the correspondence analysis and the percentages provided as Mann-Whitney test

results. The Mann-Whitney results are particularly difficult to interpret - it may be that

the values cited are one minus the p-value, but they may have some other meaning. More

complete explanations ofthe methods would have been helpful.

Community restructuring varied greatly from one experiment to another. Community

response was to some extent explained by seasonal/successional differences. Many other

factors besides atrazine (temperature, water quality, etc.) affect community structure in a

natural setting, which complicated interpretation of results. Some species, which showed

no reaction to atrazine in the laboratory, were stimulated by indirect effects of atrazine in

situ, namely the unequal stress the contaminant placed on their competitors. ChIarella

vulgaris, on the other hand, almost always significantly declined with atrazine treatment,

and responded similarly in the laboratory.

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The microcosms were affected by artifacts ofthe sealed bottles, as the authors note. In

the bottles, diversity tends to collapse over time. Also, they note that it was difficult to

take uniform samples, particularly for filamentous or colony-forming species.

Description of Use in Document (QUAL, QUAN, INV): QUAL

R IItionlllp for IT",p· Thp"tllrlv rnntrihlltp" to thp wpi(rht of pviclp,ncp, rp'(r::Jrcling effects of ~----~----- -~- - --. - --- ---~--.1 - ------- ._--- -- ~~-- . --0--- - - - -- - .. ' '-' '--' .

atrazine on aquatic plant communities.

Limitations of Study: Study effects were difficult to quantify for the Data Summary

Table due to the wide range of effects observed in the different experiments. The study

design was scientifically sound.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Brian Anderson, U.S. EPA IIt

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