Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Berard, A., C. Leboulanger, and T. Pelte. 1999. Tolerance of Oscillatoria limnetica

Lemmermann to atrazine in natural phytoplankton populations and in pure culture: influence of

season and temperature. Archives of Environmental Contamination and Toxicology. 37:472-

479. (MRID 47543601)

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: September 4, 2009

Purpose of Study:

This study examines the potential effects of atrazine on the cyanobacterium Oscillatoria

limnetica Lemmermann in experimental outdoor microcosms (in monoculture and as part of

natural phytoplankton communities) and in vitro culture.

Summary of Study Findings:

The monoculture experiments will be mentioned, although in limited detail, in order to focus on

the community microcosm experiments.

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 aI., 1999a; Berard and

Benninghoff, 200 I; Lebou1anger et aI., 2001; and Seguin et aI., 200 Ib. 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.

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111111.111

2087807
Methods:

Three types of experiments were conducted: 1) natural phytoplankton communities cultivated in

outdoor microcosms, 2) monocultures of 0. limnetica cultivated in outdoor microcosms, and 3)

in vitro monocultures of 0. limnetica. A total ofthirteen outdoor microcosm experiments were

conducted during the spring and summer of 1995, 1996, 1997, and 1998.

Natural phytoplankton communities in outdoor microcosms. Nine experiments were conducted

during 1995-1998. The source water was obtained from the center of Lake Geneva. The study

authors cite another paper that reported atrazine contamination (0.03-0.04 !lg/L) in this Lake

during 1995-1997. Each microcosm consisted of a 5-L Pyrex bottle containing 2.5 L of

phytoplankton suspension plus 2.5 L offiltered (0.8-!lm polycarbonate filter) lake water from the

same location; three to five replicates for tests and controls were utilized for each experiment.

Outdoor installation ofthe microcosms was described elsewhere (Berard et al., 1999a). Any

acclimation ofthe microcosms prior to use was not reported. Atrazine (purity 98%) was

dissolved in double-distilled water, at 10 mg/mL, with continuous stirring, and each microcosm

was treated with atrazine at 10 !lglL. No organic solvent was used. Experiments were

conducted for 10-21 days, and terminated when the relative growth rate ofthe control cultures

declined; just before the stationary phase of phytoplankton growth. Nutrient concentrations,

biomass (as chlorophyll a), and diversity (H') were determined at the beginning ofthe

experiment (Tables 1 and 2). Water samples containing phytoplankton were withdrawn from

the microcosms through a silicon cap via glass and silicon tubing. Phytoplankton biomass was

estimated daily by in vivo fluorescence measurements using a Turner fluorometer to monitor

growth. Phytoplankton community composition was determined by counting cells and filaments

(counting accuracy ca. ± 10%) in samples at the beginning and end of each experiment using an

Utermohl inverted microscope at a magnification of 200. Phytoplankton identifications were

determined using key figures and nomenclature ofBourreUy (1966-70). Water temperature was

continuously measured in situ using a Hexaparameter (Ponselle) device. Surface irradiance was

recorded by a local meteorological station.

At the beginning of each study, the diversity (H') ofthe algal communities was estimated using

the Shannon-Weaver equation. Specific characteristics ofthe algal inoculum were analyzed by

cluster analysis with ADE software. The inoculum was described in the data matrix by the

percentages oftotal biomass ofthe algal classes, except for Bacillariophyceae, which was

separated into pinnate and centric diatoms.

At experiment termination, the density of each dominant species in the atrazine treated

microcosms was compared to the control microcosms using the Mann-Whitney nonparametric

test. Dominant species were those at a concentration of2: 100 individuals/ml (a minimum of400

individuals of each dominant species wcre counted).

Oscillatoria limnetica monocultures in outdoor microcosms. Four experiments were conducted

in March, April, June and August of 1997, at the same location as the natural phytoplankton

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microcosm experiments. Microcosms (5-L Pyrex bottles) containing 5 L of filtered (0.8-llm)

lake water were inoculated with 33 mL of 0. limnetica (in exponential growth, strain CCAP

1459/18 isolated from unpolluted aquatic ecosystems) to yield an initial biomass of 2.14 Ilg/L

chlorophyll a. 0. limnetica cultivated at 12DC was used for the experiments conducted in March

and April, while cells cultivated at 20DC were used in June and August to alleviate temperature

stress. For each experiment, three microcosms were treated with atrazine at 10 Ilg/L, while three

microcosms were maintained as untreated controls; experiments were terminated after 7-22 days

(Figure 3). 0. limnetica biomass was estimated daily via optical density (OD) at 650 run; OD65o •. .• •. <- - - . •. .•• . - • . . _. was highly correlated tK- = U.'IJ, data not presented) With the number ot U. llmnetica tllaments.

Changes in 0. limnetica biomass were analyzed using the student's t test (p=0.05).

In vitro monocultures of 0. limnetica. "Cold" and "warm" experiments (each performed twice)

were conducted using 0. limnetica strain CCAP 1459/18. Batch cultures (250 mL) of Z medium

(specific for cyanobacteria) were inoculated with 0. limnetica at 2.14 Ilg/L biomass and treated

with atrazine at twenty-two concentrations ranging from 1.153-125 Ilg/L (geometric nominal

concentration series). Three untreated control cultures were also prepared. Cultures were

maintained at either 13 ± 1°C (cold experiment) or 20 ± I DC (warm experiment) on a 16:8 hour

light:dark cycle, using cool-white fluorescent light with an irradiance of 39 /lmol quanta/m2

/s.

Growth was monitored via OD65o immediately after addition ofinoculum and atrazine, then daily

during the exponential grov.1.h phase; days 5-9 for cold experiments, days 3-6 for warm

experiments.

Percent inhibition of grovy1.h was determined by comparison of OD values from atrazine treated

cultures to the mean OD values for the controls on days 7-10 for the cold experiments and days

4-7 for the warm experiments. EC50 values and confidence intervals (95%) were estimated from

linear regression analysis (Splus software) of percent inhibition of growth vs. log dose of

atrazine. EC50 values were determined from ten to twelve atrazine concentrations within the

portion of the curve where changes in growth inhibition were linear. The 8- and 5-day EC50

values determined for the cold and warm experiments, respectively, were the minimal EC50

values determined for each experiment. Those values were used to assess the influence of

temperature on the sensitivity of 0. limnetica to atrazine.

Results:

Outdoor microcosms. Mean water temperatures were ~9-14°C during the spring experiments

(March, April and May), -15-23°C during the summer experiments (June, July and August) and

~16-18°C during the fall experiments (September). Mean global irradiance values were ~16-24

MJ/m2 (spring), -14-23 MJ/m2 (summer), and 10-11 MJ/m2 (fall) (Figure 1). Initial nutrient

levels varied among the monthly experiments within the range of 0.000 to 0.012 mg P/L for

orthophosphates, 0.023-0.58 mg NIL for inorganic nitrogen, and <0.02 to 1.63 mg SlOziL for

silicium (Table 1).

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In the natural phytoplankton microcosms, initial biomass was 0.48-6.83 /lg chlorophyll aIL, with

initial dominant species dependent upon the season (Figure 2 and Tables 2-3). According to the

study authors, cyanobacteria were not the dominant species at the start of the experiments;

however, 0. limnetica was always the dominant cyanobacteria. Based on Mann-Whitney

analyses, 0. limnetica growth in atrazine-treated microcosms was significantly inhibited

compared to the control during the spring (March, April, May) experiments, significantly

stimulated during the summer (June, July, September) experiments, and not significantly

different for the September 1996 and July 1998 experiments (Table 4). Regarding other

dominant phytoplankton species, members of chlorophytes, chrysophytes, bacillariophytes, and

zygnematophytes showed significant inhibition (Mann-Whitney nonparametric test, p-value

uncertain) in at least one of the atrazine treated microcosms, but cryptophytes and xanthophytes

were not inhibited (Table 4). Overall, the effects on species abundance (inhibition, stimulation,

or no effect relative to the controls) varied by experimental month.

In the monoculture microcosms, 0. limnetica growth in atrazine treated microcosms was

significantly inhibited as compared to controls (student test, p 0.05) at the end of the spring

(March, April) experiments. There was no effect on grovvth during the June and August

experiments (Figure 3).

For all microcosm experiments, lower temperature effected growth of 0. limnetica. According

to the study authors, at temperatures <15°C, grmvth was signifIcantly decreased in both the

natural phytoplankton microcosms (p=0.0041, n 9, two-way test) and the monoculture

microcosms (p=0.06, n 4, one-way test), while at temperatures>15°C, growth increased or was

unaffected relative to the controls. The authors state that mean irradiance and initial nutrient

concentration had no effect on the sensitivity of 0. limnetica to atrazine.

In vitro monocultures. Sensitivity of 0. limnetica to atrazine increased with decreasing

temperature, with estimated ECso values of 24.2 /lg atrazine/L (95% confidence interval, 23.7-

24.7 /lg/L) when cultivated at 13°C and 52.3 /lg atrazine/L (95% confIdence interval, 51.1-53.6

/lg/L) when cultivated at 20°C.

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36 MJ/m2

"C

32

28

r 1 r • temperature

24 T Tr

0 global irradiance

20 !

1

1

16 ".

12 :":,'- .~

8 , ). -.'

:.J} fI' -', . f; ,

4 ~. g,

0 ':"~ ' \';:.

l' 'C l' QO ...... l' trl QO l' \Ii 00

0' C' 0' a- 0' 0'. 0\ 0' 0' 0' 0' 0'1

.c " GJ V

" >, '§ ... ... ... .;:: .;:: § § "3 "3 GJ GJ ()

0.. 0.. '.2( ..0 ..Q .0 <:tl <: <: ....,. ...., .... .... t\ll .... E e ::a = c: 0« B .::l

o.. Q.. E- lI) 4> GJ fZl Ul fZl

experiment

Figure 1. Mean water temperature and global irradiance conditions of microcosm (natural phytoplankton

and monoculture). Bars represent minimum/maximum values recorded during each experiment. (study

figure 1)

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Xanthophyceae

Zygnematophyceae

Chlorophyceae

Bacillariophyceae (pennates) I

Bacillariophyceae (centrales) ;

Chrysophyceae

Cryptophyceae

Dinophyceae

Cyanobacteria

,----------------. •

III!

o

:I:!BJ

l~

10

:m

•

•

20%;

10%.

100% ,

90%

80010

70"/0

6004. •

§ 50%; 'n j

-~ 40% 1

30"10 •

r- ..0 00 0- f:}'. '" .= 110

'::: ::! '"

"" Q. '" "3 « --

>D ..... VI 00 C\ 0'

'" S- O'-

e- "" is.. l.t l.t

en Vl Vl

experiment

;l

....

I I

I I

I I

Figure 2. Initial algal class composition of the natural phytoplankton microcosms. Cluster analysis

(Manhattan distance) on the algal class community structure. (study figure 2)

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• • f I

., '" ::: : days

August 1997

..,

00850

() 02

0025

0015

Q ODS

April 1997

0.02

003 00650

001

0025 OD85O March 1997 0.0. 00850 June 1997

0.035

0.02 0.03

0.015 0.025

0.02

001 0.015

0.015

0,025

0.005

Figure 3. Growth of Oscillatoria limnetica in monoculture microcosms, measured by OD65O• Diamond symbol

= atrazine treated (A), square symbol = control (C). Error bars represent confidence interval (n = 3; p=O.05).

(study figure 3)

- 7 .
Table 1. Initial nutrient concentrations in microcosms (natural phytoplankton and monoculture). (study

Experiment

Mar. April April May June June July July Aug. Sept.

Nutrient 97 96 97 98 95 97 95 98 97 95 96 98

Orthophosphates (mg P/L) 0,006 0.002 0.012 0.003 0.002 0.003 0004 0.001 0.001 0.001 0004 0.000

Inorganic nitrogen (mg NfL) 0.55 0.43 052 0.42 0.35 0.38 0.58 0.037 0.023 0.27 0.24 0,156

Silicium (mg SiOiL) 0.12 0.34 0.81 0.12 1.30 0.70 1.21 0.54 1.63 <0.02 0.34

1.90

6.83

Sept.

98

3.20

Sept.

96

2,61

Sept.

95

July

98

2.01

July

95

0,48 1.83

June

95

3.23

May

98

3.54

April

96

4.70

Mar.

IParameter 97

ICblomphyll a (.gIL)

IDiversity index (H' : bits/individu_a_I) 0._8_8__1_.7_3__1_.1_4__1_._95__1._7_0__2_.9_4__2_.6_3__2_.4_2_

Table 2. Initial biomass (as chlorophyll a) and diversity (Shannon-Weaver index) in natural phytoplankton

microcosms. (study table 2)

I

Table 3. Initial dominant species in natural phytoplankton microcosms (from pp. 474~475 ofstudy).

Experiment

_M_ar_c.h_.;...'_A;;....pr..i_I,,_M--:.aY:-..;...{"_9'o/_-'%_Hl'---! ,J Ju_n_e_--"(: I_I19--:.95) +!__~JUly (' 95-' 9_8.;...)__-+-__S._e-,-p_te_mber (' 95-' 98)

Centric diatoms: Chlorophytes: Pennate diatoms Chlorophytes:

Stephanodiscus minufulus Chlamydomonas sp. Chrysophytes: Chlamydomonas sp.

Stephanodiscus noeasfrae ChIarella vulgaris Ochromonas sp. Coelastrum sp.

Cye/ofella sp. Elakatothrix gelatinosa Dinobryon sp. Dinoflagellates:

Cryptophytes: Eudarina elegans Cryptophytes: Ceratium hirundinella

Rhodomonas minuta Cryptophytes R. minuta var.nanno, Chrysophytes

R. minuta vaLnanno. Desmids: Dinobryon sp.

Cryptomonas sp. Sfaurastrum sp. Cyanobacteria:

Pennate diatoms: Cyanobacteria: 0. limnetica

Asterionellaformsa 0. limnetica Filamentous Zygnematophytes

~ragilaria crotonensis Dinoflagellates I Mougeotia gracillima

Me'!osir'",a VU/lurlO

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Table 4. Response of dominant species to 10 J-Lg/L atrazine in natural phytoplankton microcosms (results

from Mann-Whitney nonparametric test at experiment termination). (study table 4i,2

Experiment

Taxon Mar. 97 May 98 June 95 July 95 July 98 Sept. Sept. Sept.

Cyanobacteria

Osci/latoria limnetica -(95%) -(97%) -(95%) +(90%) +(90%) 0 +(90%) 0 +(98%)

Pseudanabaena 0

Cryptophyceae

Rhodomonas minuta 0 0

R. minuta var. 0 0

Chrysophyceae

Ochromonas sp. +(95%) +(90%) 0 0 0 0 0

Desmare/la brachycalyx +(95%) 0 +(95%) 0

Dinobryon sp. 0

Small cells 0

Xanthophyceae

Tribonema 0 0

Bacillariophyceae

Stephanodiscus minutulus 0 0 -(95%)

Asterionellaformsa +(94%) 0 +(90%)

Diatoma elongatum 0 0 +(98%) 0 0

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

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

Nitzschia sp. 0 0

Synedra acus 0 0 0 0 0 0

acus var. 0

Chlorophyceae

Chlamydomonas sp.

Chlorella vulgaris

Small cells

-(85%) 0

-(95%) -(97%) -(90%) o

o

-(95%) -(99%)

o 0

o

o

o

Zygnematophyceae

Staurastrum sp. 1-(90%)

. Mougeolia gracillima 0 0 0 -(85%)

rW greater density of individual 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 I-p value.

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Comments

Species-specific

effects shown in

Table 4.

ts

at Statistical

significance

fits) unclear.

Percentages

reported in Table 4

may be I-p value.

sms

ry

if

?

D t S T bl N; t I h I k d

Measured atrazine

I

concentrations

IL~~

were not determined.

2 NR = not reported

aa ummary a e: a uralJ!!!J!.top an ton commumt,es In out oor mlcroco

Time

Weighted

Magnitude of Nominal Day ofStudy Average Did

Atrazine Atrazine Recove Effect Effect relative to Concentration Effect Concentration Occur- controls (J.lgIL) Occurred from Day Oto so, when I ."T'__ ~_ ~rr~_ ..

I I rr ru::rt £))1::(;1

j Occurred 1 I

I

Change in No (effec phytoplankton End of

community NR1 10 J.lg/L experiments Not calculable observed

end of species (10-21 days) experime composition

I I , II I

As summarized above, the species composition results for the natural phytoplankton

communities in outdoor microcosms are the same as those reported in another reviewed article

by the same lead author (Berard et aI., 1999a). Six of 15 community experiments were reported

in both articles, while the results from experiments reported exclusively in one article did not

differ substantially. The methods in the two studies appear to be identical as they pertain to

experiments on the natural phytoplankton communities, thus study limitations should be

applicable to both studies.

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Reviewer Comments:

This article presented results from outdoor natural phytoplankton microcosms, outdoor

monoculture microcosms, and in vitro laboratory experiments.

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

1998) to contain atrazine at 0.03-0.04 ~glL in the euphotic zone ofthe lake water. It is not stated

whether the lake water was tested for atrazine and/or other contaminants prior to or during the

experimental study. If atrazine was present in the microcosms during the experiments, the

observed effects relative to the controls may be misleading. Moreover, water used for individual

experiments was collected from 1995-1998, thus any temporal variability in the background

concentration of atrazine in the source water would further confound the results.

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

aI., I999a), but not presented in this article. Any acclimation ofthe microcosms prior to

treatment 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 mixing would have been lessened.

The treatment was limited to one test concentration (l0 ~g atrazine/L). The study authors

indicate that the experiments were conducted for 10-21 days with termination occurring when

relative growth rate of control cultures declined, just before the stationary phase of

phytoplankton growth. However, the exact duration ofthe atrazine exposure period for each of

the natural phytoplankton outdoor microcosm experiments is unclear. In addition, although the

number ofreplicates is reported as 3-5 for controls and atrazine treatment groups, the exact

number ofreplicates for each ofthe nine outdoor experiments is not provided.

There is no indication that water in the microcosms was analyzed for atrazine following

treatment.

The study authors state that measurements ofsome microcosm characteristics were made at the

"beginning of each experiment". It is unclear ifthese measurements were made pre- or posttreatment.

The Mann-Whitney results are particularly difficult to interpret. For example, it is unclear

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

More complete explanations ofthe methods and statistical analyses would have been helpful.

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Overall, community structure varied greatly from one experiment to another, depending on the

seasonal variability as well as environmental parameters (e.g., temperature). Growth of 0.

limnetica was inhibited in spring phytoplankton communities exposed to 10 IlgiL atrazine,

whereas exposure to the same level of atrazine in the summer resulted in growth stimulation of

this cyanobacterium. The study authors proposed that the increased growth of 0. limnetica in the

mixed species microcosms during the summer experiments and the lack of enhanced growth in

the monoculture microcosms suggested that the response of 0. limnetica (in the natural

community) to atrazine was influenced by species interactions as well as environmental

conditions. For example, Chlorella vulgaris, a chlorophyte, aimost aiways significantly dec1ined

with atrazine treatment. The inhibition of the chlorophytes may have allowed increased growth

by a more tolerant species, such as, 0. limnetica.

Description of Use in Document: Qualitative

Rationale for Use: The study contributes to the weight of evidence regarding effects of atrazine

on aquatic plant communities.

Limitations of Study: There were numerous reporting and measuring deficiencies. No effort

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

treatment andlor control microcosms prior to experiment initiation. Water used for microcosms

was from Lake Geneva, which has documented atrazine contamination. Installation ofthe

microcosms and any acclimation interval prior to use were not detailed. Atrazine treatment

concentrations were not confirmed or monitored in the microcosms following treatment. There

is uncertainty about p-values used for 'significant' Mann-Whitney results. There was a potential

for bottle effects due to the small size ofthe microcosm. In addition, the exact test duration and

number ofreplicates for each ofthe outdoor microcosm experiments were not reported.

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

,.,o' ,-""'I

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 741:tW;f/1MAf

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA Mt/i'jl. Il3 III

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