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 ofEnvironmental Contamination and Toxicology. 37:472- 479. (MRID 47543601) 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 ofaLevel ofConcern (LOC).
Date ofReview: September4, 2009 Purpose ofStudy: This study examines the potential effects ofatrazine onthe cyanobacterium 

### Oscillatoria limnetica Lemmermann in experimental outdoor microcosms (inmonoculture and as partof natural phytoplankton communities) and 

### in vitro culture. Summary ofStudy Findings: The monoculture experiments will be mentioned, although in limited detail, in orderto focus on the community microcosm experiments.

Several otherreviewed 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, 200I; Lebou1angeret aI., 2001; and Seguin et aI., 200Ib. 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.



### - 1 - 

# 111111.111 2087807Methods: Three types ofexperiments were conducted: 1) natural phytoplankton communities cultivated in outdoormicrocosms, 2) monocultures of

### 0. limnetica cultivated in outdoormicrocosms, and 3) 

### in vitro monocultures of

### 0. limnetica. A total ofthirteen outdoormicrocosm experiments were conducted during the spring and summerof 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 ofLake Geneva. The study authors cite anotherpaperthat reported atrazine contamination (0.03-0.04 !lg/L) in this Lake during 1995-1997. Each microcosm consisted ofa 5-L Pyrex bottle containing 2.5 L of phytoplankton suspensionplus 2.5 L offiltered (0.8-!lmpolycarbonate filter) lake waterfrom the same location; three to five replicates for tests and controls were utilized for each experiment. Outdoor installation ofthe microcosms was described elsewhere (Berard 

### etal., 1999a). Any acclimation ofthe microcosms priorto use was not reported. Atrazine (purity 98%) was dissolved in double-distilled water, at 10 mg/mL, with continuous stirring, and eachmicrocosm 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;justbefore the stationary phase ofphytoplankton growth. Nutrientconcentrations, biomass (as chlorophyll a), and diversity (H') were determined atthe 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 atthe beginning and end ofeach experiment using an Utermohl inverted microscope at amagnification of200. Phytoplankton identifications were determined using key figures and nomenclature ofBourreUy (1966-70). Watertemperature was continuously measured 

### in situ using a Hexaparameter (Ponselle) device. Surface irradiance was recorded by a local meteorological station.

Atthe beginning ofeach study, the diversity (H') ofthe algal communities was estimatedusing the Shannon-Weaver equation. Specific characteristics ofthe algal inoculum were analyzedby clusteranalysis with ADE software. The inoculumwas described in the datamatrix by the percentages oftotal biomass ofthe algal classes, except for

### Bacillariophyceae, which was separated into pinnate and centric diatoms.

Atexperimenttermination, the density ofeach 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 (aminimum of400 individuals ofeach 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

- 2
-microcosm experiments. Microcosms (5-L Pyrex bottles) containing 5 L offiltered (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 of2.14 Ilg/L chlorophyll 

### a. 

### 0. limnetica cultivated at 12D C was used for the experiments conducted in March and April, while cells cultivated at 20D C 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 

### ttest (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) ofZ 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 ± IDC (warm experiment) on a 16:8 hour light:dark cycle, using cool-white fluorescent light with an irradiance of39 /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 ofgrovy1.h was determined by comparison ofOD 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 forthe 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 ofthe 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/m 2 (spring), -14-23 MJ/m2 (summer), and 10-11 MJ/m 2 (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).

- 3

-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 ofchlorophytes, chrysophytes, bacillariophytes, and zygnematophytes showed significant inhibition (Mann-Whitney nonparametric test, p-value uncertain) in at least one ofthe atrazine treated microcosms, but cryptophytes and xanthophytes were not inhibited (Table 4). Overall, the effects on species abundance (inhibition, stimulation, orno 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 ofthe 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 orwas unaffected relative to the controls. The authors state that mean irradiance and initial nutrient concentrationhad 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 of24.2 /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.



### - 4-36 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 

## E- o.. Q.. lI) 4> GJ fZl Ul fZl experiment Figure 1. Mean water temperature and global irradiance conditions ofmicrocosm (natural phytoplankton and monoculture). Bars represent minimum/maximum values recorded during each experiment. (study figure 1)



### - 5 -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 ofthe natural phytoplankton microcosms. Cluster analysis (Manhattan distance) on the algal class community structure. (study figure 2)



### - 6

-• • 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 ofOscillatoria 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 1.83 0,48 June

95 3.23 May

98 3.54 April

96 4.70 Mar.

97 IParameter 

# I Cblomphyll 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_I

19--:.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. Elakatothrixgelatinosa 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

- 8 -Table 4. Response ofdominant species to 10 J-Lg/L atrazine in natural phytoplankton microcosms (results from Mann-Whitney nonparametric test at experiment termination). (study table4i,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%) Asterionella formsa +(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%)

. Mougeoliagracillima 0 0 0 -(85%) 

## rW 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 notdefined but may be I-p value.



### - 9 -Comments

Species-specific effects shown in Table 4. ts at Statistical significance fits) unclear. Percentages reported in Table4 may be I-p value. 

### sms

ry

if ? d I k 

### T bl N; 

### t I h D t S

I

### IL~~

Measured atrazine concentrations were not determined. 2 NR = not reported aa ummary a 

### e: 

### a uralJ!!!J!.top an ton commumt,es In 

### out oormlcroco Time Weighted Magnitude of Nominal Day of Study 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 rrru::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 , I I I

As summarized above, the species composition results for the natural phytoplankton communities inoutdoormicrocosms are the same as those reported in anotherreviewed article by the same lead author (Berardet aI., 1999a). Six of15 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 appearto be identical as they pertain to experiments on the natural phytoplankton communities, thus study limitations shouldbe applicable to both studies.

- 10
-Reviewer Comments: This article presented results from outdoornatural phytoplanktonmicrocosms, outdoor monoculture microcosms, and in vitro laboratory experiments.

The microcosms were filled with water from Lake Genevapreviously reported (Blanc 

### etai., 1998) to contain atrazine at 0.03-0.04**~glL** in the euphotic zone ofthe lake water. Itis not stated whetherthe lake water was tested for atrazine and/orothercontaminants priorto or during the experimental study. Ifatrazine was presentinthe microcosms during the experiments, the observed effects relative to the controls may be misleading. Moreover, waterused for individual experiments was collected from 1995-1998, thus any temporal variability inthe background concentration ofatrazine in the source waterwould further confound the results.

A complete description ofinstallationofthe microcosms priorto use was referenced (Berard et aI., I999a), but notpresented inthis article. Any acclimation ofthe microcosms priorto treatment was notreported. The microcosms did not contain a sedimentlayer.

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 thatthe experiments were conducted for 10-21 days with termination occurring when relative growth rate ofcontrol cultures declined,justbefore the stationary phase of phytoplankton growth. However, the exact duration ofthe atrazine exposure period for each of the natural phytoplankton outdoormicrocosm experiments is unclear. In addition, although the number ofreplicates is reported as 3-5 for controls and atrazine treatmentgroups, the exact numberofreplicates for each ofthe nine outdoorexperiments is not provided.

There is no indication that waterin the microcosms was analyzed for atrazine following treatment.

The study authors state that measurements ofsome microcosm characteristics were made atthe "beginning ofeach experiment". It is unclearifthese measurements were made pre- orposttreatment.
The Mann-Whitney results are particularly difficultto interpret. Forexample, it is unclear whetherthe reportedvalues are one minus the p-value orthe percentchange in species density. More complete explanations ofthe methods and statistical analyses would have been helpful.

- 11
-

### 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 ofatrazine in the summer resulted in growth stimulation of this cyanobacterium. The study authors proposed thatthe increased growth of

### 0. limnetica 

### in the mixed species microcosms during the summer experiments and the lack ofenhanced 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 ofthe chlorophytes may have allowed increased growth by amore tolerant species, such as, 

### 0. limnetica. Description ofUse in Document: 

### Qualitative
Rationale for Use: 

### The study contributes to the weight ofevidence regarding effects ofatrazine on aquatic plant communities.
Limitations ofStudy: 

### There were numerous reporting andmeasuring deficiencies. No effort was madeto document whether otherchemicals (including atrazine) were present inthe treatment andlorcontrol microcosms priorto experimentinitiation. Waterused for microcosms was from Lake Geneva, which has documented atrazine contamination. Installation ofthe microcosms and any acclimation intervalpriorto use were not detailed. Atrazine treatment concentrations were not confirmed ormonitored in the microcosms following treatment. There is uncertainty aboutp-values used for 'significant' Mann-Whitney results. There was apotential for bottle effects due to the small size ofthe microcosm. In addition, the exacttest duration and number ofreplicates for each ofthe outdoormicrocosmexperiments were notreported. Primary Reviewer: 

### Lynne Binari, M.S., subcontractorto Cambridge Environmental Inc. ,.,o' ,-""'I Secondary Reviewer: 

### Michael Lowit, Ph.D., Ecologist, EPA

# 7 4 

# 1:tW;f/1MAf
Tertiary Reviewer: 

### AnitaPease, M.S., SeniorBiologist, EPA 

# Mt/i'jl. Il3

## III

- 12
-